Calcium Carbonate Nanoparticles—Toxicity and Effect of In Ovo Inoculation on Chicken Embryo Development, Broiler Performance and Bone Status

The use of intensive selection procedure in modern broiler chicken lines has led to the development of several skeletal disorders in broiler chickens. Therefore, current research is focused on methods to improve the bone quality in birds. In ovo technology, using nanoparticles with a high specificity to bones, is a potential approach. The present study aimed to evaluate the effect of in ovo inoculation (IOI) of calcium carbonate nanoparticles (CCN) on chicken embryo development, health status, bone characteristics, and on broiler production results and bone quality. After assessing in vitro cell viability, the IOI procedure was performed with an injection of 500 μg/mL CCN. The control group was not inoculated with CCN. Hatchability, weight, and selected bone and serum parameters were measured in embryos. Part of hatchlings were reared under standard conditions until 42 days, and production results, meat quality, and bone quality of broilers were determined. CCN did not show cytotoxicity to cells and chicken embryo and positively influenced bone parameters of the embryos and of broilers later (calcification) without negatively affecting the production results. Thus, the IOI of CCN could modify the molecular responses at the stage of embryogenesis, resulting in better mineralization, and could provide a sustained effect, thereby improving bone quality in adult birds.

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136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab136 ◽

2005 ◽

Vol 17 (2) ◽

pp. 219 ◽

Cited By ~ 5

Author(s):

C.E. Ferguson ◽

T.R. Davidson ◽

M.R.B. Mello ◽

A.S. Lima ◽

D.J. Kesler ◽

...

Keyword(s):

Embryo Development ◽

Direct Effect ◽

Blastocyst Stage ◽

Control Group ◽

Bovine Embryo ◽

Bovine Embryos ◽

Direct Role ◽

Treatment Groups ◽

And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

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Improvement of quality of oocytes collected in the autumn by enhanced removal of impaired follicles from previously heat-stressed cows

Reproduction ◽

10.1530/rep.0.1220737 ◽

2001 ◽

pp. 737-744 ◽

Cited By ~ 126

Author(s):

Z Roth ◽

A Arav ◽

A Bor ◽

Y Zeron ◽

R Braw-Tal ◽

...

Keyword(s):

Heat Stress ◽

Embryo Development ◽

Treated Group ◽

Oocyte Quality ◽

Control Group ◽

Delayed Effect ◽

Cleavage Rate ◽

Lactating Cows ◽

Summer Heat

The fertility of dairy cows decreases during the summer and remains low during the cooler autumn although the animals are no longer under heat stress. The aim of this study was to characterize a delayed effect of summer heat stress on oocyte quality in the autumn and to improve oocyte quality by enhanced removal of follicles damaged during the previous summer. Lactating cows (n = 16) were subjected to heat stress during the summer. In autumn, ovarian follicles (3-7 mm in diameter) were aspirated by an ultrasound-guided procedure during four consecutive oestrous cycles. Follicles were aspirated from control cows on day 4 and from treated cows on days 4, 7, 11 and 15 of each oestrous cycle. All cows received PGF(2alpha) and GnRH injections on days 19 and 21, respectively, and maintained cyclicity, as indicated by plasma progesterone concentrations. On day 4 of each cycle, the oocytes recovered were examined morphologically, matured and activated in vitro, and cultured for 8 days. In cycle 1 (early October) both groups showed low percentages of grade 1 oocytes, cleavage, four- and eight-cell embryos, morulae and parthenogenetic blastocysts. Subsequently, the number of grade 1 oocytes increased earlier (cycle 2) in treated than in control cows (cycle 3; P < 0.05). The cleavage rate in the control group remained relatively low throughout (32-58%), whereas in the treated group it increased from 40% (cycle 1) to 75% (cycles 3 and 4; P < 0.05). The number at each stage of embryo development increased slightly but remained low throughout in the control group, whereas in the treated group significant (P < 0.05) increases of all stages were observed in cycles 3 and 4. The results show a delayed effect of summer heat stress on oocyte quality and embryo development in the autumn. Enhanced removal of the impaired cohort of follicles led to earlier emergence of healthy follicles and high quality oocytes in the autumn.

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Diamond Nanoparticles Modify Curcumin Activity: In Vitro Studies on Cancer and Normal Cells and In Ovo Studies on Chicken Embryo Model

PLoS ONE ◽

10.1371/journal.pone.0164637 ◽

2016 ◽

Vol 11 (10) ◽

pp. e0164637 ◽

Cited By ~ 15

Author(s):

Barbara Strojny ◽

Marta Grodzik ◽

Ewa Sawosz ◽

Anna Winnicka ◽

Natalia Kurantowicz ◽

...

Keyword(s):

Chicken Embryo ◽

In Vitro Studies ◽

In Ovo ◽

Normal Cells ◽

Diamond Nanoparticles

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161 EFFECT OF OVIDUCTAL FLUID ON BOVINE OOCYTE ZONA PELLUCIDA HARDENING AND SPERM BINDING, AND ON EARLY EMBRYO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab161 ◽

2016 ◽

Vol 28 (2) ◽

pp. 210

Author(s):

P. Hugon ◽

J. Lamy ◽

E. Corbin ◽

P. Mermillod ◽

M. Saint-Dizier

Keyword(s):

Corpus Luteum ◽

Embryo Development ◽

Zona Pellucida ◽

Developmental Stages ◽

Early Embryo ◽

Developmental Competence ◽

Control Group ◽

Vitro Fertilization ◽

Oviductal Fluid

This study was designed to evaluate the effects of oviductal fluid at different periovulatory times on oocyte maturation, modification of the zona pellucida (ZP), fertilization and embryo development. Bovine oviducts were collected at a slaughterhouse and classified as preovulatory (pre-ov: 1 pre-ov follicle and a regressing corpus luteum) or post-ovulatory (post-ov: a corpus haemorrhagicum or recent corpus luteum; n = 10 cows/stage). Both oviducts were flushed with 1 mL of sterile TCM-199, and oviductal flushes (OF) were aliquoted and stored at –80°C. Abattoir-derived bovine ovaries were aspirated and cumulus‐oocyte complexes (COC) with at least 3 cumulus layers and homogeneous oocyte cytoplasm were in vitro matured for 22 h in standard maturation medium (control group, n = 319) or in standard medium with 2× concentrated additives supplemented (50% v/v) with pre-ov OF (n = 255) or post-ov OF (n = 248). After in vitro maturation (IVM), subgroups of COC were denuded, and the time of digestion of the ZP by pronase 0.1% (v/v in TCM-199) was determined to evaluate ZP hardening. After IVM, COC were fertilised in vitro for 18–20 h at a final concentration of 1.106 million spermatozoa (spz)/mL. After in vitro fertilization (IVF), COC were denuded, washed twice and cultured for 8 days more under standard conditions. After IVM, IVF, and embryo culture, oocytes/embryos were fixed with ethanol, stained with Hoescht, and examined under fluorescence microscopy for determination of (1) maturation and developmental stages, (2) numbers of fertilised and polyspermic oocytes, and (3) spz bound to the ZP. Percentages were compared between groups by chi-square. Times of ZP digestion were compared by Kruskal‐Wallis test. Numbers of spz bound to the ZP were compared by ANOVA on normalised data followed by Newman-Keuls tests. Data are presented as mean ± SEM. A P < 0.05 was considered significant. Addition of OF during IVM had no effect on maturation rates compared with the control. However, the digestion time of the ZP by pronase was reduced after IVM with pre-ov OF (313 ± 21 s; n = 26) compared with post-ov OF (459 ± 23 s; n = 23) but not with the control (416 ± 30 s; n = 25). After IVF, the number of spermatozoa bound to the ZP was increased after IVM with pre-ov OF (57 ± 5 spz/oocyte; n = 67) and decreased after IVM with post-ov OF (34 ± 3 spz/oocyte; n = 76) compared with the control (42 ± 5 spz/oocyte; n = 60). Addition of OF during IVM had no effect on rates of IVF and polyspermia. However, the rate of development to the blastocyst stage was less after IVM with post-ov OF (10%, n = 97 cleaved oocytes) compared with control (24%, n = 130) and pre-ov OF (29%, n = 101). In conclusion, the OF collected before ovulation decreased the resistance of the ZP to protease digestion and increased its ability to bind spz, whereas it was the opposite for the post-ov OF. Furthermore, the post-ov OF decreased the developmental competence of fertilised oocytes.

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7 THE EFFECT OF A NOVEL RECOMBINANT PROTEASE TREATMENT ON BOVINE SPERM FERTILIZING CAPACITY AND EMBRYO DEVELOPMENT IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab7 ◽

2009 ◽

Vol 21 (1) ◽

pp. 104

Author(s):

J. T. Aaltonen ◽

K. J. Mattson ◽

N. M. Loskutoff

Keyword(s):

Embryo Development ◽

Disease Transmission ◽

Bos Taurus ◽

Gradient Centrifugation ◽

Control Group ◽

Bovine Embryo ◽

Human Sequence ◽

Bovine Sperm

As described in the IETS Manual (Stringfellow and Seidel, 1995), and endorsed by the OIE, trypsin can be used (for specific pathogens and livestock) to effectively remove certain infectious agents from in vivo-derived embryos for international transport. Because of the multimillion-dollar AI industry for livestock, the OIE has encouraged more research in developing similar decontamination techniques for semen as an added safeguard to animal quarantine for the prevention of disease transmission. Most or all of the earlier studies on embryos used a porcine pancreatic-derived trypsin. Because of more stringent guidelines from international regulatory agencies on the use of animal products, several serine protease recombinants are now available. Previous experiments comparing the porcine pancreatic extract with a recombinant bovine sequence trypsin developed in corn resulted in no statistical difference in cleavage or morula/blastocyst rates. (Mattson et al. 2008 Theriogenology 69, 724–727). An additional in vivo study treating bovine sperm with a yeast-derived human-sequence trypsin resulted in significantly more transferable-quality embryos after the AI of superovulated cows as compared with sperm not treated with trypsin (Blevins et al. 2008 Reprod. Fertil. Dev. 20, 84). The goal of this experiment was to examine the in vitro development of bovine embryos produced from sperm treated with a recombinant trypsin found in a commercially available density gradient centrifugation (DGC) product (Bovipure, Nidacon, Sweden) compared with DGC without trypsin. Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 5 replications (n = 2220 oocytes). Semen was collected and pooled from 2 Bos taurus bulls and frozen in an egg-yolk cryodiluent (Biladyl, Minitube). The semen was processed using Bovipure DGC composed of 2 mL of 40% colloid of silane-coated silica particles containing either a yeast-derived human sequence recombinant trypsin containing no animal by-products (n = 1126 oocytes) or the same colloid without trypsin as the control group (n = 1094 oocytes). Both 40% concentrations were layered over 2 mL of an 80% concentration of the same colloid without any additives. The density gradients were centrifuged at 300g for 20 min, after which time the pellets were washed in 5 mL of prewarmed TL Hepes solution (Cambrex) and centrifuged at 500g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 × 106 sperm mL–1, to be used for in vitro inseminations. Results were compared using a 2-tailed unpaired t-test. Cleavage rates for the trypsin-treated sperm (n = 969, 35.8%) and the control (n = 950, 44.3%) groups were not statistically different (P = 0.20). Although more embryos reached the morula to blastocyst stages in the control group (n = 421, 61.0%) than in the trypsinized group (n = 347, 54.7%), these differences also were not statistically significant (P = 0.85). In conclusion, trypsinized Bovipure DGC of sperm before insemination showed no detrimental effects on IVF-derived bovine embryo development.

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44 Exploring the use of silver and diamond nanoparticles on sperm cell invitro and chicken embryo in ovo

Reproduction Fertility and Development ◽

10.1071/rdv33n2ab44 ◽

2021 ◽

Vol 33 (2) ◽

pp. 129

Author(s):

M. P. Thavhana ◽

T. L. Nedambale ◽

L. J. Shai ◽

M. L. Mphaphathi

Keyword(s):

Cell Viability ◽

Chicken Embryo ◽

Sperm Cell ◽

Control Group ◽

Dependent Manner ◽

Ag Nps ◽

In Ovo ◽

Diamond Nanoparticles ◽

Immune Related Genes ◽

Dose Dependent

In poultry industry, chick viability is a crucial factor determining profitability from fertilized egg to placement at the farm. However, decreases in fertility and hatchability have been observed. Recently, there has been renewed interest in the use of silver nanoparticles (Ag-NPs) due to their antimicrobial properties and growth-promoting ability, and diamond nanoparticles (D-NPs) due to their biocompatibility properties. The aim of the study was to evaluate the effect of silver and diamond nanoparticles on chicken embryo oxidative status, biochemical indices, and expression of immune-related genes and on sperm cell viability. The experiment was conducted in Ross 308 chicken embryos and Ross 308 cockerels. One hundred and fifty fertilized eggs were divided randomly into 5 groups (5×30). Fertilized eggs were injected with 50 mg/L Ag-NPs at volumes of 100μL (group 1), 200μL (group 2) or 50 mg/L D-NPs at volumes of 100μL (group 3) or 200μL (group 4), or received no nanoparticles (control; group 5) and incubated at 37°C and 55% humidity for 20 days. Then, chicken blood was collected and centrifuged to evaluate alkaline phosphatase (ALP), alanine transaminase (ALT), lactate dehydrogenase (LDH), glucose, urea, and free haemoglobin. Chicken embryo liver was used to evaluate antioxidant capacity (TAC) and chicken embryo spleen was used to evaluate expression of the immune-related genes interleukin-1β (IL-1β), toll-like receptor (TLR)4, TLR2, and TLR15. Semen was randomly divided into 1 control and 8 treatment groups and treated with 50 mg/L Ag-NPs: group A (0.1ppm), group B (1ppm), group C (5ppm), group D (10ppm) or 50 mg/L D-NPs: group E (1ppm), group F (5ppm), group G (10ppm), and group H (20ppm). Sperm viability was analysed using prestoblue metabolic assay. Data were analysed using PROC in GLM procedure of SAS 2014 (SAS Institute Inc.). Decrease in sperm cell viability was recorded in a dose-dependent manner. Sperm cell viability decreased (P<0.005) as the concentration of Ag-NP or D-NP increased. Addition of 100μL of Ag-NPs increased the growth rate of chicken embryo but not 200μL of Ag-NPs or addition of D-NPs. Increases in ALP, ALT, LDH, glucose and urea enzyme were observed in a dose-dependent manner in both Ag-NPs and D-NPs. Addition of 50 mg/L Ag-NPs or 50 mg/L D-NPs increased (P<0.001) TAC of chicken embryo as the volume increased. Additions of 200μL of Ag-NPs, 100μL of D-NPs, and 200μL of D-NPs were haemolytic (P<0.001) but addition of 100μL of Ag-NPs was not. Additions of 100 or 200μL of Ag-NPs or 100μL of D-NPs downregulated IL-1β and 200μL of D-NPs upregulated IL-1β compared with the untreated control group. Additions of 100 or 200μL of Ag-NPs or 200μL of D-NPs induced expression of TLR4 and TLR15. Furthermore, addition of Ag-NPs did not result in expression of TLR2. We concluded that administration of 50 mg/L Ag-NPs and 50 mg/L D-NPs in ovo improve immune status and administration of 100μL of Ag-NPs improved the growth rate of chicken embryo. However, toxicity associated with 50 mg/L Ag-NPs and 50 mg/L D-NPs remains a concern and need to be addressed before use.

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18 EMBRYO AGGREGATION IN PIG IMPROVES CLONING EFFICIENCY AND EMBRYO QUALITY

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab18 ◽

2016 ◽

Vol 28 (2) ◽

pp. 139

Author(s):

C. Buemo ◽

A. Gambini ◽

L. Moro ◽

R. F. Y. Martin ◽

D. Salamone

Keyword(s):

In Vitro Culture ◽

Dna Fragmentation ◽

Embryo Development ◽

Embryo Quality ◽

T Test ◽

Control Group ◽

Reconstructed Embryo ◽

Student’S T ◽

Student’S T Test

In this study, we analysed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst size and cell number, DNA fragmentation levels by TUNEL assay, and the relative expression of genes associated with pluripotency, apoptosis, trophoblast markers, and DNA methylation in the porcine. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation; staining was performed with Hoëchst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% polyvinyl alcohol) by a DC pulse of 1.2 kVcm for 80 μs. Then, embryos were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of zona-free embryos was achieved in a well of wells system in 100 μL of SOF medium. Two experimental groups were used, one control group with a single reconstructed embryo per microwell (1×) and the other group placing 3 reconstructed embryo per microwell (3x aggregation group). Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality. Our results showed that aggregation of 3× embryos increased blastocyst formation rate and blastocyst size of pig cloned embryos (Fisher’s test P < 0.05 and Student’s t-test P < 0.05, respectively). The DNA fragmentation levels in 3× aggregated cloned blastocysts were significantly decreased compared to 1x blastocyst (Student’s t-test P < 0.05). Levels of Oct4, Klf4, Igf2, Bax, and Dnmt1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially nondetectable (Student’s t-test P < 0.05). Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

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Effect of trichostatin A on fertilization and embryo development during extended culture of mouse oocyte

Zygote ◽

10.1017/s0967199410000547 ◽

2011 ◽

Vol 20 (1) ◽

pp. 27-32 ◽

Cited By ~ 4

Author(s):

Byung Chul Jee ◽

Jun Woo Jo ◽

Jung Ryeol Lee ◽

Chang Suk Suh ◽

Seok Hyun Kim ◽

...

Keyword(s):

Histone Deacetylase ◽

Embryo Development ◽

Trichostatin A ◽

Formation Rate ◽

Control Group ◽

Blastocyst Formation ◽

Developmental Potential ◽

Extended Culture ◽

Mouse Oocytes

SummaryWe performed this study to investigate the effect of histone deacetylase inhibition during extended culture of in vitro matured mouse oocytes. In vitro matured mouse (BDF1) oocytes were cultured in vitro for 6, 12, and 24 h, respectively, and then inseminated. During in vitro culture for 6 and 12 h, two doses of trichostatin A (TSA), a histone deacetylase inhibitor, were added (100 nM and 500 nM) to the culture medium and the oocytes were then inseminated. During the 24-h in vitro culture, two doses of TSA were added (100 nM and 500 nM) to the medium and the oocytes were activated with 10 mM SrCl2. After the 6-h culture, the fertilization rate was similar to that of the control group, but the blastocyst formation rate was significantly decreased. After the 12-h culture, both the fertilization and blastocyst formation rates were significantly decreased. After the 24-h culture, total fertilization failure occurred. In the oocytes cultured for 6 and 12 h, the fertilization and blastocyst formation rates did not differ between the TSA-supplemented and control groups. Although extended culture of the mouse oocytes significantly affected their fertilization and embryo development, TSA supplementation did not overcome their decreased developmental potential.

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Effect of storage tube material and resveratrol during liquid storage of matured bovine oocytes on subsequent development

Acta Veterinaria Hungarica ◽

10.1556/004.2017.053 ◽

2017 ◽

Vol 65 (4) ◽

pp. 546-555

Author(s):

Tayita Suttirojpattana ◽

Tamás Somfai ◽

Satoko Matoba ◽

Takashi Nagai ◽

Rangsun Parnpai ◽

...

Keyword(s):

Embryo Development ◽

Subsequent Development ◽

Control Group ◽

Tube Material ◽

Blastocyst Development ◽

Liquid Storage ◽

Glass Tubes ◽

Bovine Oocytes

This study determined the optimum storage vessel and the effects of resveratrol for the storage of in vitro matured (IVM) bovine oocytes. After IVM, the oocytes were kept in a Hepes-buffered medium at 25 °C for 20 h in different containers including Eppendorf tubes (ET) made of polypropylene (PP) and polystyrene (PS), and tissue culture tubes (TCT) made of PP, PS, and glass. Then oocytes were subjected to IVF and subsequent in vitro embryo development was compared among the groups and to that of a control group without storage. The percentage of blastocyst development in the control group was significantly higher than in the stored groups (P < 0.05). Among oocytes stored in TCT, the percentage of blastocyst development of oocytes stored in glass TCT was significantly higher than that of oocytes stored in PP and PS TCT (P < 0.05); however, it did not differ from that of oocytes stored in ET. The quality of blastocysts did not differ among the control and stored groups. Embryo development was not affected when 0.1, 1 or 10 μM resveratrol was added to the medium during oocyte storage. In conclusion, glass tubes were optimal for oocyte storage and resveratrol did not improve the development of stored oocytes.

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Graphene oxide nanofilm and the addition of L-glutamine can promote development of embryonic muscle cells

10.21203/rs.3.rs-19279/v2 ◽

2020 ◽

Author(s):

Marlena Zielińska-Górska ◽

Anna Hotowy ◽

Mateusz Wierzbicki ◽

Jaśmina Bałaban ◽

Malwina Sosnowska ◽

...

Keyword(s):

Graphene Oxide ◽

Surface Properties ◽

Satellite Cells ◽

Chicken Embryo ◽

Culture Medium ◽

Muscle Cells ◽

Control Group ◽

Protein Levels ◽

Myogenic Potential

Abstract Background: Formation of muscular pseudo-tissue depends on muscle precursor cells, the extracellular matrix (ECM)-mimicking structure and factors stimulating cell differentiation. These three things cooperate and can create a tissue-like structure, however, their interrelationships are relatively unknown. The objective was to study the interaction between surface properties, culture medium composition and heterogeneous cell culture. We would like to demonstrate that changing the surface properties by coating with graphene oxide nanofilm (nGO) can affect cell behaviour and especially their need for the key amino acid L-glutamine (L-Glu).Results: Chicken embryo muscle cells and their precursors, cultured in vitro, were used as the experimental model. The mesenchymal stem cell, collected from the hind limb of the chicken embryo at day 8 were divided into 4 groups; the control group and groups treated with nGO, L-Glu and nGO supplied with L-Glu (nGOxL-Glu). The roughness of the surface of the plastic plate covered with nGO was much lower than a standard plate. The test of nGO biocompatibility demonstrated that the cells were willing to settle on the nGO without any toxic effects. Moreover, nGO by increasing hydrophilicity and reducing roughness and presumably through chemical bonds available on the GO surface stimulated the colonisation of primary stromal cells that promote embryonic satellite cells. The viability significantly increased in cells cultured on nGOxL-Glu. Observations of cell morphology showed that the most mature state of myogenesis was characteristic for the group nGOxL-Glu. This result was confirmed by increasing the expression of MYF5 genes at mRNA and protein levels. nGO also increased the expression of MYF5 and also very strongly the expression of PAX7 at mRNA and protein levels. However, when analysing the expression of PAX7, a positive link was observed between the nGO surface and the addition of L-Glu.Conclusions: The use of nGO and L-Glu supplement may improve myogenesis and also the myogenic potential of myocytes and their precursors by promoting the formation of satellite cells. Studies have, for the first time, demonstrated positive cooperation between surface properties nGO and L-Glu supplementation to the culture medium regarding the myogenic potential of cells involved in muscle formation.

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