scholarly journals Effect of vitrification on apoptotic changes in feline embryos

2018 ◽  
Vol 63 (No. 4) ◽  
pp. 144-151 ◽  
Author(s):  
M. Ochota ◽  
W. Niżański
Keyword(s):  
Embryo Development ◽  
Annexin V ◽  
Blastocyst Stage ◽  
Expansion Rate ◽  
Control Group ◽  
Morphological Appearance ◽  
Further Development

The aim of this study was to evaluate the influence of a vitrification-warming procedure on the viability of cat embryos and blastocysts and the incidence of apoptotic changes in blastocysts subjected to vitrification and blastocyst that developed from vitrified embryos. In the first part of the experiment, post-thaw embryo development and blastocyst viability were evaluated based on morphological appearance and the ability to develop (embryos) or re-expand (blastocyst) compared to control. In the second part, blastocysts that were viable after vitrification-warming and blastocysts that developed from vitrified-warmed embryos were stained with Annexin-V and TUNEL to evaluate apoptotic changes. Most of the vitrified-warmed embryos were viable after thawing, 36.3% developed to morula, and 14.7% to the blastocyst stage. The overall re-expansion rate of blastocysts that were vitrified on day 7 was 55.6%. Vitrification significantly increased apoptotic and necrotic changes in blastocysts, but did not influence late apoptotic and necrotic changes in blastocysts that developed from vitrified-warmed embryos. The total number of blastomeres in blastocysts was similar in blastocysts that developed from vitrified-warmed embryos (99.1 ± 23.1), but lower in blastocysts vitrified on day 7 (82.1 ± 16.8), if compared to the control group (107.9 ± 24.2). These results show that the vitrification-warming procedure returns embryos capable of further development to a good quality blastocyst in vitro. Whereas, vitrification of blastocysts caused the progression of apoptotic changes in some blastomeres, however it did not affect the overall blastocyst viability.

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

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
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.

27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
I. Hiriart ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Blastocyst Stage ◽  
Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. 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 ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 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 (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. 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. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

scholarly journals 296 LEUKEMIA INHIBITORY FACTOR INFLUENCES SHEEP OOCYTE PARTHENOGENETIC DEVELOPMENT DURING THE TRANSITION FROM GERMINAL VESICLE TO EARLY PRONUCLEAR STAGE

2005 ◽  
Vol 17 (2) ◽  
pp. 298
Author(s):  
G. Ptak ◽  
F. Lopes ◽  
P. Loi
Keyword(s):  
Embryo Development ◽  
Leukemia Inhibitory Factor ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Inhibitory Factor ◽  
Control Group ◽  
Blastocyst Development ◽  
Human Follicular Fluid ◽  
Oocyte Competence

Leukemia inhibitory factor (LIF) is an indispensable cytokine for female fertility. The influence of LIF on embryo development and particularly implantation has been recently confirmed; however, the effect of this cytokine on the oocyte has not been studied. The presence of LIF in human follicular fluid implies its possible role in the acquisition of oocyte competence. Furthermore, the up-regulation of LIF by steroid hormones in sheep makes entirely feasible the hypothesis that ovulatory estradiol peak plays a role in the preparation of female gamete for fertilization. With this in mind, we studied the effect of LIF during in vitro development of sheep oocytes mimicking the physiological expression of LIF induced by the ovulatory peak of estradiol in mice. GV stage oocytes matured and chemically activated in the presence of LIF and anti-LIF antibody were cultured to the blastocyst stage in our standard media. To eliminate the effect of the putative presence of LIF in heat inactivated fetal calf serum used for oocyte maturation, aliquots of LIF were treated at 56°C for 30 min and added to the maturation medium. The proportion of embryos that reached the blastocyst stage in vitro was significantly higher (P < 0.001) for oocytes matured and activated with LIF (36/93; 39%) than for the group incubated with antibody against LIF (6/68; 9%). The significant effect of anti-LIF antibody (P < 0.001) was also observed when compared with blastocysts developed from the control group of oocytes matured without LIF addition (31/106; 29%). Although the beneficial influence of LIF treatment on embryo development demonstrated with those preliminary data was not confirmed statistically, due to low number of oocytes involved, the proportion of embryos reaching the blastocyst stage in vitro was about 10% higher for those incubated with LIF than for either those cultured without the cytokine or those, matured in the presence of heat-treated LIF (15/55; 27%); however, the rate of blastocyst development appeared very similar to that of the control group. This study revealed for the first time a role of LIF in determining oocyte competence. Further investigation to determine how LIF achieves its effects on the oocyte are ongoing in our laboratory. This work was supported by FIRB RBNE01HPMX, COFIN 2002074357, COFIN 2003073943 002, and British Council 2004.

Effect of nano-selenium and nano-zinc particles during in vitro maturation on the developmental competence of bovine oocytes

2018 ◽  
Vol 58 (11) ◽  
pp. 2021 ◽  
Author(s):  
B. R. Abdel-Halim ◽  
Nermeen A. Helmy
Keyword(s):  
Dna Damage ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Expansion Rate ◽  
Control Group ◽  
Nano Zinc ◽  
Bovine Oocytes

The objectives of the current study were to evaluate the effects of supplemental nano-selenium (NSe) and nano-zinc oxide (NZn-O) particles during in vitro maturation (IVM) on DNA damage of cumulus cells, glutathione (GSH) concentration in bovine oocytes, subsequent embryo development and re-expansion rate of vitrified warmed blastocysts. The current study was conducted on bovine ovaries obtained from a local abattoir and transported to the laboratory in sterile phosphate buffer saline with antibiotics at 37°C, within 1 h after slaughter. Ovaries were pooled, regardless of stage of the oestrous cycle of the donor. Only cumulus-intact complexes with evenly granulated cytoplasm were selected for IVM. Experimental design included the following: Experiment 1 studied the effect of addition of 1.0 µg/mL NSe or NZn-O to IVM medium on DNA damage of cumulus cells; Experiment 2 evaluated the effects of NSe or NZn-O on intracellular glutathione in oocytes and cumulus cells; in Experiment 3, the development of oocytes matured in IVM medium supplemented with 1.0 µg/mL NSe or NZn-O was investigated; and in Experiment 4, the effects of adding 1.0 µg/mL NSe and NZn-O to in vitro fertilisation media on vitrified oocytes and embryos were investigated. The DNA damage in cumulus cells decreased with supplemental NSe and NZn-O at concentration of 1 µg/mL in the IVM medium (180.2 ± 21.4, 55.8 ± 4.3 and 56.6 ± 3.9 for the control and NSe and NZn-O groups respectively). Total GSH concentrations increased following supplementation with 1 µg/mL NSe and 1 µg/mL NZn-O, compared with the control group. Re-expansion rate of vitrified warmed blastocysts in experimental media containing NSe and NZn-O with ethylene glycol was higher than that of the control. In conclusion, providing NSe and NZn-O during oocyte maturation significantly increased both intracellular GSH concentration and DNA integrity of cumulus cells. Optimal embryo development was partially dependent on the presence of NSe and NZn-O during IVM. NSe and NZn-O during oocyte maturation act as a good cryoprotective agents of vitrified, warmed blastocysts.

Effect of cyanocobalamin on oocyte maturation, in vitro fertilization, and embryo development in mice

Zygote ◽  
2020 ◽  
pp. 1-8
Author(s):  
Tamana Rostami ◽  
Fardin Fathi ◽  
Vahideh Assadollahi ◽  
Javad Hosseini ◽  
Mohamad Bagher Khadem Erfan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Control Group ◽  
Vitro Fertilization ◽  
Maturation In Vitro

Summary The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus–oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

251 SELECTION OF PREPUBERTAL SHEEP OOCYTES USING BRILLIANT CRESYL BLUE TEST

2011 ◽  
Vol 23 (1) ◽  
pp. 223 ◽  
Author(s):  
M. G. Catalá ◽  
D. Izquierdo ◽  
R. Romaguera ◽  
S. Hammami ◽  
M. Roura ◽  
...  
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Control Group ◽  
Oocyte Diameter ◽  
Oocyte Growth ◽  
Indirect Measure ◽  
Brilliant Cresyl Blue ◽  
Exact Test ◽  
Cresyl Blue

The aim of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth to select competent prepubertal sheep oocytes for in vitro embryo production. The BCB stain allows the determination of glucose–6-phosphate dehydrogenase (G6PD) activity, an enzyme with decreased activity in oocytes that have finished their growth phase. Oocytes were obtained after slicing the surface of lamb ovaries (2–5 months old) obtained from a local abattoir. Oocytes with more than 3 compact cumulus layers and homogeny cytoplasm were selected and stained with different concentrations of BCB diluted in PBS (13-, 26-, 39-, and 52-μM BCB) during 60 min at 38.5°C in a humidified air atmosphere. Oocytes were classified into groups depending on their cytoplasm coloration: oocytes with blue cytoplasm or grown oocytes (BCB+) and oocytes without blue coloration or growing oocytes (BCB–). Oocytes were matured in an enriched TCM-199 medium for 24 h at 38.5°C and 5% CO2 in a humidified atmosphere. Oocyte diameter was also measured. Matured oocytes were partially denuded and transferred to fertilization medium (SOF) supplemented with 10% of oestrous sheep serum. Fresh semen was kept at room temperature (25°C) for 1 h. Highly motile spermatozoa were selected by using Ovipure density gradient (Nidacon EVB S.L.), and oocytes were fertilized with 1 × 106 sp mL–1. After 20 h postinsemination, presumptive zygotes were cultured for 8 days in SOF with 10% of fetal bovine serum at 38.5°C, 5% CO2, and 90% N2. Data was analysed by performing Fisher’s exact test for blastocyst production and ANOVA with Tukey’s post-test for oocyte diameter. Table 1 shows the percentage of BCB-stained oocytes and their embryo development. In this study oocytes exposed during 60 min to 13-μM BCB showed a higher percentage of embryos reaching blastocyst stage than did those in the control group (≤0.01). In other species such as goats (Rodriguez-Gonzalez et al. 2002 Theriogenology 57(5), 1397–1409) and cattle (Alm et al. 2005 Theriogenology 63(8), 2194–2205), the best protocol for the oocyte selection was the use of 26-μM BCB during 90 min. Oocyte diameter showed significant differences between BCB– with BCB+ and control group (110 μm, 134 μm, and 121 μm, respectively, ≤0.001). In conclusion, using 13 μM of BCB during 60 min is a suitable technique for increasing embryo blastocyst rates using lamb oocytes. Table 1.Effect of BCB1 concentration on embryo development of lamb oocytes The grant sponsor was the Spanish Ministry of Science and Innovation, Code: AGL2007-60227-CO2-01.

scholarly journals Cysteamine, glutathione and ionomycin treatments improve in vitro fertilization of prepubertal goat oocytes

Zygote ◽  
2004 ◽  
Vol 12 (4) ◽  
pp. 277-284 ◽  
Author(s):  
Aixa Urdaneta ◽  
Ana Raquel Jiménez ◽  
María-Teresa Paramio ◽  
Dolors Izquierdo
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Control Group ◽  
Sperm Capacitation ◽  
Vitro Fertilization ◽  
Maturation Medium

The aim of this study was to improve in vitro embryo development of prepubertal goat oocytes by studying the effect of adding cysteamine to in vitro maturation medium, glutathione (GSH) to in vitro fertilization medium and ionomycin to the sperm capacitation medium. In experiment 1, we analysed the effect of 1 mM GSH added to fertilization medium of oocytes matured with 400 μM cysteamine. The control group were oocytes without cysteamine and GSH. In experiment 2, oocytes matured and fertilized in the presence of 400 μM cysteamine and 1 mM GSH, respectively, were inseminated with spermatozoa treated with ionomycin or heparin. In experiment 1, the percentages of total and normal fertilized oocytes were significantly higher for oocytes supplemented with cysteamine and GSH (40.26% and 30.20%, respectively) than for oocytes from the control group (16.66%, and 10.61%, respectively). The percentage of total embryos obtained after 7 days of culture was significantly higher in the group supplemented with cysteamine and GSH (30.62%) than in the control group (8.09%) . In experiment 2, percentages of total and normal fertilized oocytes were significantly higher for the group of spermatozoa capacitated with ionomycin (52.21% and 37.17%, respectively) than with heparin (38.62% and 28.35%, respectively). After 7 days of culture, total embryo rate was significantly higher in the group of sperm capacitated with ionomycin (44.91%) than with heparin (38.69%) . However, the percentage of embryos developed to the blastocyst stage was not affected by any of the treatments studied.

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.

Bovine non-competent oocytes (BCB–) negatively impact the capacity of competent (BCB+) oocytes to undergo in vitro maturation, fertilisation and embryonic development

Zygote ◽  
2015 ◽  
Vol 24 (2) ◽  
pp. 245-251 ◽  
Author(s):  
M.B. Salviano ◽  
F.J.F. Collares ◽  
B.S. Becker ◽  
B.A. Rodrigues ◽  
J.L. Rodrigues
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
The Other ◽  
Control Group ◽  
Cleavage Rate ◽  
Morphological Evaluation ◽  
Group 3

SummaryCompetent oocyte selection remains a bottleneck in the in vitro production (IVP) of mammalian embryos. Among the vital assays described for selecting competent oocytes for IVP, the brilliant cresyl blue (BCB) test has shown consistent results. The aim of the first experiment was to observe if oocytes directly submitted to IVM show similar cleavage and blastocyst rates as those obtained with oocytes maintained under the same in vitro conditions as the oocytes that undergo the BCB test. Bovine cumulus–oocyte complexes (COCs) were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised grouped into three groups: (1) directly submitted to IVM; (2) oocytes submitted to the BCB test without the addition of BCB stain (BCB control group); and (3) submitted to the BCB test. The results showed that oocytes directly submitted to IVM reached similar cleavage (48/80 – 60%) and embryonic development rates to the blastocyst stage (10/48 – 21%) as the results obtained with the BCB control group oocytes (45/77 – 58% and 08/45 – 18%, respectively). The aim of the second experiment was to determine the cleavage and blastocyst rates obtained from BCB+ oocytes undergoing IVM in the presence of BCB– oocytes at a ratio of 10:1. COCs were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised into two groups that were submitted to IVM either directly (1: control group) or submitted to the BCB test prior to IVM. After the BCB test, the COCs were classified as either BCB+ (blue cytoplasm) or BCB– (colourless cytoplasm) and then divided into four experimental groups: (2) BCB+; (3) BCB–; and (4) BCB+ matured in same IVM medium drop as (5) BCB– at a ratio of 10:1. After IVM (24 h), oocytes from the different experimental groups were submitted to in vitro fertilisation (IVF) and in vitro culture (IVC) under the same culture conditions until they reached the blastocyst stage (D7). With regards to the cleavage rate (48 h after IVF), only group 3 (102/229 – 44%) differed (P < 0.05) from the other groups [1 (145/241 – 60%); 2 (150/225 – 67%); 4 (201/318 – 63%) and 5 (21/33 – 63%)]. On day 7, the embryos from group 2 (BCB+) achieved the highest blastocyst rate (46/150 – 31%) (P < 0.05) when compared with the embryo development capacity of the other experimental groups (1: 31/145 – 21%; group 3: 17/102 – 17%; group 4: 46/201 – 23%; and group 5: 2/21 – 10%). In conclusion, submitting BCB+ oocytes that were separated from BCB– oocytes to IVM increases the rate of embryonic development to the blastocyst stage when compared to the control group, BCB– oocyte group, BCB+ paracrine group and BCB– paracrine group. The presence of non-competent oocytes during IVM, even in low proportion (1:10), reduces the capacity of competent oocytes to undergo embryo development and achieve blastocyst stage during IVC.

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

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 932
Author(s):  
Arkadiusz Matuszewski ◽  
Monika Łukasiewicz ◽  
Jan Niemiec ◽  
Maciej Kamaszewski ◽  
Sławomir Jaworski ◽  
...  
Keyword(s):  
Calcium Carbonate ◽  
Embryo Development ◽  
Bone Quality ◽  
Chicken Embryo ◽  
Broiler Chickens ◽  
Selection Procedure ◽  
High Specificity ◽  
Control Group ◽  
In Ovo

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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