226 EVALUATION OF IN VITRO EMBRYO DEVELOPMENT OF BOVINE OOCYTES EXPERIMENTALLY EXPOSED TO MYCOPLASMA BOVIS

2011 ◽  
Vol 23 (1) ◽  
pp. 212 ◽  
Author(s):  
D. Pavao ◽  
M. Alves ◽  
R. Qureiroz ◽  
F. Souza ◽  
M. D. Angelo
Keyword(s):  
Statistical Analysis ◽  
Zona Pellucida ◽  
Control Group ◽  
Mycoplasma Bovis ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Animal Reproduction ◽  
Significant Difference

Reproductive biotechnologies are known to improve the quality and productivity of herds. Research on cattle health and on the conditions of oocytes and embryos produced in vitro and in vivo are carried out worldwide to prevent the transmission of infectious agents, particularly those associated with ovarian tissues, the uterine tubes, and the uterus. Thus, there is concern regarding the possibility of transmitting Mycoplasma bovis through animal reproduction procedures. The aim of this study was to evaluate morphological changes by means of optical microscopy, cleavage rate, and blastocyst formation during the in vitro development of bovine embryos experimentally exposed to M. bovis. Oocytes were aspirated from ovaries of slaughtered cows, and oocytes with an intact zona pellucida were selected and matured. After 20 h, the oocytes were divided in 2 groups: a control group (n = 260), and oocytes exposed to 2.5 × 104 cfu mL–1 of M. bovis (n = 261). The semen was treated by a discontinuous Percoll gradient technique, and the sperm concentration was adjusted to approximately 100 000 sperm for each oocyte. Statistical analysis of the experiments was conducted using Student’s t-test (P < 0.05). Embryos exposed to M. bovis showed a cleavage rate of 21.07% (55/261), whereas the control group showed a cleavage rate of 64.6% (168/260). In relation to blastocyst formation, control embryos had a rate of 39.60% (103/260) v. 12.20% (32/261) for the exposed group. Statistical analysis of the experiments revealed a significant difference in the final results. Regarding morphological aspects, we observed failures in the division and asymmetry of blastomeres, cytoplasmic shrinkage, and degeneration and disruption of the zona pellucida in embryos exposed to the pathogen. These results are important to support the development of parameters for evaluating oocytes and in vitro-produced bovine embryos and to establish more efficient methods to control the spread of disease by animal reproduction biotechnologies. Further studies will be done with oocytes exposed to the pathogen and subsequently evaluated according to the Manual of the International Embryo Transfer Society.

scholarly journals Milled versus moulded mock-ups based on the superimposition of 3D meshes from digital oral impressions: a comparative in vitro study in the aesthetic area

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Francesca Cattoni ◽  
Giulia Teté ◽  
Alessandro Mauro Calloni ◽  
Fabio Manazza ◽  
Giorgio Gastaldi ◽  
...  
Keyword(s):  
Statistical Analysis ◽  
Test Group ◽  
In Vitro Study ◽  
Mean Value ◽  
Digital Impression ◽  
Control Group ◽  
Minimal Invasiveness ◽  
Significant Difference ◽  
The Aesthetic

Abstract Background Aesthetic porcelain veneers proved to be a long-term reliable prosthetic solution, ensuring minimal invasiveness. The use of veneers requires an adhesive cementation technique, so maintaining as much enamel as possible is to ensure lasting success. A diagnostic mock-up is a key tool that allows a preview of the outcome of the aesthetic restoration: it is obtainable both in an analog and digital way. With the recent developments in impression technology and the ever so fast growing use of CAD-CAM technologies it is useful to understand the pros and cons of either one of these techniques (analog and digital) in order to identify the easier and more convenient workflow in aesthetic dentistry. Methods After taking pictures and impressions of the dental arcs of a patient in need of aesthetic rehabilitation, 52 resin models were produced and a digital drawing of the smile was outlined. Both an analog and a digital wax-up were obtained from two of the 52 models: the latter was obtained using digital impressions and a dedicated software. The analog wax-up was then used to produce 25 matrices that have later been used to mould 25 resin mock-ups using a traditional moulding protocol (Control Group - CG). The digital wax-up was used to mill 25 PMMA mock-ups. Each mock-up, both milled and moulded (total 50), was then laid on the other 50 resin models as a digital impression of it was taken. The STL. files of the milled mock-ups were compared with the 3D CAD wax-up made using a specific software. The STL. files of the analog printed mock-ups were compared with the traditional wax-up design. A statistical analysis was carried out to evaluate the difference between the groups. Results The statistical analysis showed a significant difference (P > 0.01) between the mean value of the distance between the points of the overlapping STL. meshes in GC (0.0468 mm) and in TG (Test Group - TG) (0,0109 mm). Conclusions The study showed a difference in accuracy between traditional moulded and milled mock-ups compared to their original wax-up. The data analysis reports that the digital method allows for greater accuracy. Within the limitations of this study, a fully digital workflow is to considered more reliable when it come to creating an esthetic mockup: the digital procedure has been shown to be more accurate than the one made manually which is much more operator dependent and it brings an increase to the chance of error, and that could ultimately affect the final result.

168 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS DURING OOCYTE IN VITRO MATURATION ON BOVINE EMBRYOS (Gyr×HOLSTEIN) QUALITY

2016 ◽  
Vol 28 (2) ◽  
pp. 214
Author(s):  
G. R. Leal ◽  
C. A. S. Monteiro ◽  
H. F. R. A. Saraiva ◽  
A. J. R. Camargo ◽  
P. M. S. Rosa ◽  
...  
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Bovine Embryos ◽  
Tropical Conditions ◽  
Significant Difference ◽  
Number Of Cells

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr × Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Quality determines the oocyte proportion that will develop to blastocyst stage, and although the lipid content is important in oocyte development, a high concentration in embryos is associated with cryotolerance reduction, making this a relevant issue for IVP systems. The in vitro maturation system (IVM) simulated physiological oocyte maturation (SPOM) mimics the physiological maturation events by using cyclic adenosine monophosphate (cAMP) modulators, which promote the increase of oocyte competence. Among the modulators, Forskolin has lipolytic properties. The aim of this study was to evaluate the effect of the SPOM system (Albuz 2010 Hum. Reprod. 25, 12) on bovine embryos (Gyr × Holstein) regarding their total number of cells (TNC) and lipid content. Oocytes were obtained by ovum pick-up from Gyr cows in 5 replications. After selection, they were randomly divided into 2 groups: SPOM (S) and control (C). The IVM lasted 24 h for group C (TCM 199 medium without FBS) in culture oven at 38.5°C, 5% CO2 in atmospheric air and high humidity. In the SPOM system, oocytes were in pre-IVM [TCM 199 medium + 100 µM Forskolin + 500 µM 3-isobutyl-1-methylxanthine (IBMX)] for 2 h and followed for extended IVM (TCM 199 medium + 20 µM cilostamide) for 28 h under the same conditions as control group. After IVM, oocytes were fertilised with semen from a single Holstein bull that was prepared by Percoll gradient method in Fert-TALP medium (Bioklone® Animal Reproduction, São Paulo, Brazil) for 22 h and transfered to culture droplets, where they remained for 7 days (n = 10–13 per group). The lipid content analysis was performed by staining with Oil red and the stained area fraction of each embryo was measured using software ImageJ (NIH, Bethesda, MD, USA). The TNC was measured after being stained with Hoechst 33342 and results were analysed by Student's t-test in Instat GraphPad program, with a 5% significance level. There was no significant difference (P > 0.05) between embryos from both groups on TNC (group S: 88.9 ± 28.0A; group C: 101.6 ± 29.1a) and lipid content (group S: 0.93 ± 12:18A; group C: ±0.15 to 0.96) analysis. Some studies have shown there is a beneficial effect on embryo quality when using this system; however, our results demonstrated that there was no effect on total number of cells using our conditions. Some authors have also demonstrated a reduction in embryo lipid content using Forskolin during in vitro culture. Our results suggest that the time of Forskolin exposure was not enough to ensure lipolytic action on the structures produced from oocytes (Gyr) treated in pre-IVM. It was concluded that the SPOM system had no effect on TNC and lipid content of Gyr/Holstein embryos. Financial support from FAPERJ and CAPES is acknowledged.

143 EFFECTS OF BOAR SEMINAL PLASMA IN IN VITRO CULTURE OF PORCINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 219
Author(s):  
J. H. Moon ◽  
S. J. Kim ◽  
J. T. Kang ◽  
S. J. Park ◽  
J. Y. Choi ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Seminal Plasma ◽  
Harmful Effect ◽  
Developmental Rate ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Significant Difference ◽  
Protective Medium ◽  
La Jolla

Seminal plasma consisting of carbohydrates, proteins, and lipids not only serves as a nutritive and protective medium for sperm cells but also play a pivotal role in inducing the tolerance to pre-existing immune cells as well as improving the intra-uterine conditions for implantation of fertilized embryos (Guerin et al. 2009 Hum. Reprod. Update 15, 517–535). However, the effects of seminal plasma in in vitro culture of fertilized embryos are unknown. In the present study, the seminal plasma was separated from the second fraction of a normal farm boar (n = 1) by centrifugation and filtered seminal plasma was stored at –30°C until use. In a preliminary experiment, the optimal activity of seminal plasma was evaluated by incubating the embryos for different time intervals. To investigate the developmental rates, electrically (EA) (triplicates, n = 490) or chemically (CA) (quintuplicates, n = 599) activated 2-day-old porcine embryos were incubated for 3 h in PZM-5 medium (Funakoshi Co., Tokyo, Japan, Catalog no. IFP0410P) containing 0% (EA: n = 122 and CA: n = 152), 0.1% (EA: n = 123 and CA: n = 148), 0.5% (EA: n = 122 and CA: n = 150), or 1% (EA: n = 123 and CA: n = 149) seminal plasma. Similarly, the developmental rate of chemically activated 2-day-old somatic cell nuclear transferred porcine embryos (quadruplicates, n = 239) was studied after incubation with 0% (n = 119) or 0.1% (n = 120) seminal plasma for 3 h. A significant difference was noticed only in the rate of blastocyst formation in the chemically activated embryos treated with 0.1% seminal plasma (31.7 v. 24.8% in the 0% group, ANOVA; P < 0.05; Prism5, GraphPad Software Inc., La Jolla, CA, USA). None of the treatments showed a significant effect on the cleavage rate and cell numbers of blastocysts. In conclusion, the seminal plasma did not show any harmful effect on early embryos development. Furthermore, the seminal plasma (0.1%) improved the rate of blastocyst formation among the chemically activated nuclear transferred embryos. The results of this preliminary study suggest that the addition of seminal plasma during embryo transfer could increase the rate of pregnancy in pig. This study was supported by MKE (#10033839-2012-21), IPET (#311011-05-1-SB010), the Research Institute for Veterinary Science, and TS Corporation.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

scholarly journals Effect of deslorelin acetate treatment in oocyte recovery and in vitro embryo production in domestic cats

2016 ◽  
Vol 19 (10) ◽  
pp. 1091-1095
Author(s):  
Camila Louise Ackermann ◽  
Eduardo Trevisol ◽  
Leticia Ferrari Crocomo ◽  
Tatiana da Silva Rascado ◽  
Rodrigo Volpato ◽  
...  
Keyword(s):  
Treated Group ◽  
Control Group ◽  
Domestic Cats ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Oocyte Recovery ◽  
Significant Difference ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

Objectives The present study investigated the effect of contraceptive treatment with deslorelin acetate on in vitro embryo production and oocyte recovery in domestic queens. Methods Twenty-one mature domestic cats were used. Eleven queens (treated group) and one tom were kept in an experimental cattery, and 10 queens were privately owned (control group). When in interestrus or diestrus (day 0) a deslorelin acetate implant (Suprelorin, 4.7 mg/animal) was inserted into the subcutaneous tissue of the interscapular region in all queens in the treated group. After 6 months of treatment, all animals were ovariohysterectomized, and the ovaries were used for in vitro embryo production. Percentage of cleavage was determined 18 h after oocyte insemination and blastocyst formation was assessed on the eighth day of culture. The rate of cumulus-oocyte complexes (COCs) recovery was analyzed by an unpaired t-test. The cleavage and blastocyst rates were expressed as percentages and analyzed by Fisher’s exact test. All analyses were performed using GraphPad Prism v5.0, with P <0.05 set as the level of significance. Results In the treated group, we recovered 8.3 ± 1.15 grade I COCs per queen; the cleavage rate was 60% and the blastocyst rate was 36%. In the control group, we recovered 18.4 ± 3.21 grade I COCs per queen; the cleavage rate was 55.97% and the blastocyst rate was 34%. Forty percent of treated females did not produce any blastocysts. In the treated group, we observed a significant decrease in COC recovery. Although there was no significant difference in cleavage and blastocyst rates between groups, 40% of treated females did not produce any blastocysts. Conclusions Recovery of grade I COCs is negatively affected by deslorelin treatment in domestic cats. Regarding embryo production, new studies are still necessary to evaluate the success of this technique owing to the individual effect caused by deslorelin acetate.

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

2010 ◽  
Vol 58 (4) ◽  
pp. 465-474 ◽  
Author(s):  
Tamás Somfai ◽  
Yasushi Inaba ◽  
Yoshio Aikawa ◽  
Masaki Ohtake ◽  
Shuji Kobayashi ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Culture Systems ◽  
Significant Difference

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O 2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O 2 compared to 5% O 2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O 2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O 2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O 2 tension, whereas IVD101 supported blastocyst formation only under low O 2 levels but enhanced the proliferation of ICM cells.

scholarly journals 245RELATIVE ABUNDANCE OF HSP 70 MRNA IN BOVINE EMBRYOS PRODUCED IN VITRO USING DIFFERENT EMBRYO/VOLUME RATIOS IN CULTURE

2004 ◽  
Vol 16 (2) ◽  
pp. 243
Author(s):  
A.T.D. Oliveira ◽  
C. Gebert ◽  
R.F.F. Lopes ◽  
H. Niemann ◽  
J.L. Rodrigues
Keyword(s):  
Relative Abundance ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Gene Transcripts

In spite of in vitro embryo production systems having been greatly improved over recent years, employing a variety of culture conditions (media, protein sources, gas atmosphere, etc.), we still do not know much about the real necessity of embryos to develop under the same conditions as occur in vivo. These differences between in vivo and in vitro culture at preimplantation embryonic stages can produce deviations in gene expression and in normal fetal development (large offspring syndrome). Heat shock proteins (Hsp) are engaged in cell response to regulatory signals or perturbations in the microenviroment and can be used as a sensitive indicator of stress caused by suboptimal culture conditions (Wrenzycki et al., 2001Hum. Reprod. 16, 893–901). Hsp act as chaperones in facilitating protein folding and assembly and stabilize damaged proteins to prevent aggregation of fragments, thereby allowing repair or degradation. The aim of the present study was to investigate the effects of different embryo/volume ratios on bovine embryo development and the relative abundance of Hsp 70.1 gene transcripts. In this experiment, oocytes were isolated from slaugterhouse ovaries and matured, fertilized and cultured in groups of 5, 10, 20 or 30 per each drop of 100μL. The oocytes were matured in TCM 199 supplemented with 0.4% BSA. After maturation, oocytes were fertilized in TALP medium, using frozen/thawed sperm, selected using a percoll density gradient. The zygotes were cultured to the morula or Day 7 blastocyst stage employing SOF supplemented with 0.4 % BSA. Developmental check points were cleavage rate (Day 3pi), blastocyst formation (Day 8pi) and hatching (Day 11pi). A semi-quantitative RT-PCR assay was used to determine the relative levels of gene transcripts in single embryos at morula (Day 6) and blastocyst (Day 7) stages (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317). Data of cleavage, blastocyst formation and hatching rates were analyzed using chi-square test. Relative abundance (RA) of Hsp 70.1mRNA were compared in tested groups using ANOVA followed a Tukey test. Differences at P&lt;0.05 were considered significant. Results show that no significative difference in hatching rate per blastocyst produced was detected among the four groups. Cleavage rate and blastocyst formation were significantly higher in groups with 5, 10 and 20 embryos compared with drops containing 30 embryos. Hsp transcripts were detected in morula and blastocyst stages in all groups. In morula stage, no differences were observed in the RA of Hsp 70.1mRNA among groups with 5, 10, 20 and 30 embryos cultured per drop. However, in blastocyst stage, the RA was significantly increased in the group with 20 embryos per drop as compared to the group with 5 embryos. The results show that different embryo/volume ratios in culture influence not only cleavage rate, blastocyst formation and hatching rate, but also expression of Hsp 70.1 gene. Further studies changing other culture conditions and using in vivo-derived bovine embryos will aid in elucidating which culture systems are ideal to produce bovine embryos in vitro. This research was supported by CAPES/DAAD program and CNPq.

scholarly journals Effect of Rolipram on in vitro maturation, gene expression and embryonic development in bovines

2019 ◽  
Vol 71 (5) ◽  
pp. 1433-1444 ◽  
Author(s):  
B.B. Santana ◽  
G.G. Sobral ◽  
E.T. Gomes ◽  
A.M. Batista ◽  
L.P.R. Teixeira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Group ◽  
Cleavage Rate ◽  
Time Control ◽  
Follicular Aspiration ◽  
Maturation Medium ◽  
Significant Difference ◽  
Group A ◽  
Bovine Oocytes

ABSTRACT The aim of this work was to evaluate the effect of the Rolipram during the maturation of bovine oocytes and gene expression of embryos produced in vitro. Bovine ovaries were collected in slaughterhouse. The COCs were selected and divided into 5 groups: Control 0 time; Control: IVM for 24 hours; Rolipram treatments with IVM blocking for 24 hours in maturation medium containing (100, 150 and 200µM). After 24 hours all groups were reseated in IVM for another 24 hours. Subsequently COCs were subjected to the same IVM system and fertilized, being checked for cleavage post fertilization and for blastocyst. In addition, performed expression of the following genes: Mater, BMP15 and Bax. No difference was found in gene expression. Of oocytes evaluated shortly after follicular aspiration, 79.00% were in GV, GVBD, MI, while 13.40%, were in MII and 7.60%, D/NI. Significant difference was observed in different concentrations (T100, T200 and T150µM) in oocytes that have reached the MII phase compared to control treatments (P= 0.003). Differences were observed in cleavage rate (P< 0.05) between T150 and T200 when compared to the C/24 Group. A high difference was observed on blastocyst rate (P< 0.001) among treatments compared to the control group.

131 Effect of Oviductal Fluid During In Vitro Culture on Bovine Embryo Development and Quality

2018 ◽  
Vol 30 (1) ◽  
pp. 205 ◽  
Author(s):  
R. Emmerstorfer ◽  
K. Radefeld ◽  
V. Havlicek ◽  
U. Besenfelder ◽  
H. Yu ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Specific Effect ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Significant Difference

The aim of this work was to establish an in vitro culture approach using bovine oviducal fluid (OF) to improve embryo quality and to provide an in vitro system to study oviduct function. Bovine oviducts ipsilateral to ovulation were collected at the slaughterhouse, 1 to 4 days after ovulation. The OF was collected by flushing the oviducts with 1 mL of Charles Rosenkrans 1 medium (CR1). Samples from 21 oviducts were pooled and proteins were concentrated using centrifugal filter devices. Aliquots of 3 different protein concentrations, determined by Bradford assay, were prepared and stored at –20°C. Abattoir-retrieved cumulus–oocyte complexes were used for standard in vitro maturation (IVM) and IVF (Day 0). On Day 1, presumptive zygotes (n = 1498) were randomly allocated to 4 different culture groups and cultured up to Day 9. The presumptive zygotes of the control group (n = 364) were cultured in CR1 with 5% oestrous cow serum (OCS) supplemented with 1 mg mL−1 hyaluronan. In the experimental groups, OCS was replaced by OF, resulting in 3 groups with final protein concentrations of 0.1 mg mL−1 (n = 380), 0.5 mg mL−1 (n = 380) or 1 mg mL−1 (n = 374). Cleavage rate was recorded on Day 2 and blastocyst yield on Days 7, 8, and 9 after fertilization. On Day 7, blastocysts were removed and either stained (Hoechst 33342) for cell number or subjected to a slow freezing protocol using 1.5 M ethylene glycol. After thawing, the re-expansion and hatching rate of blastocysts were determined at 24, 48 and 72 h. Eight replicates were carried out and data were analysed by ANOVA. Cleavage rate increased with increasing protein concentration (0.1 mg mL−1: 80.9 ± 4.2%; P > 0.05; 0.5 mg mL−1: 83.4 ± 2.5%; P < 0.1) and was significantly higher in the 1 mg mL−1 group (84.5 ± 4.4%; P < 0.05) compared with the control group (79.7 ± 3.4%). The cumulative blastocyst rate on Day 9 was significantly lower (P < 0.05) in all experimental groups (0.1 mg mL−1: 15.8 ± 8.9%; 0.5 mg mL−1: 18.7 ± 12.0%; 1 mg mL−1: 17.0 ± 11.2%) compared with the control group (34.1 ± 5.4%). The total number of cells was not affected by OF (P > 0.05). There was no significant difference (P > 0.05) in the post-thaw re-expansion rate between the experimental groups (0.1 mg mL−1: n = 26 thawed blastocysts; 0.5 mg mL−1: n = 27; 1 mg mL−1: n = 23) and the control group (n = 58). The post-thaw hatching rate was significantly higher at 24 and 72 h, respectively, in the 0.5 mg mL−1 group (44.4% and 74.1%; P < 0.05) and the 1 mg mL−1 group (47.8%; P < 0.05; and 82.6%; P < 0.01) compared with the control group (18.9% and 44.8%). The replacement of serum with OF during in vitro culture of bovine embryos had a stage specific effect, resulting in higher cleavage rates but lower blastocyst rates. To address this issue, OF will be collected at different stages and applied in the matching in vitro culture phases in future studies. Interestingly, the post-thaw hatching rate was up to twice as high in the experimental groups, indicating better quality of those embryos developing to blastocyst stage.

70 Does the addition of docosahexaenoic acid to in vitro systems during culture improve the quality of bovine embryos?

2019 ◽  
Vol 31 (1) ◽  
pp. 160
Author(s):  
J. A. Sánchez Viafara ◽  
G. Lopez de Vasconcelos ◽  
R. Maculan ◽  
N. Gomes Alves ◽  
J. Camisão de Souza
Keyword(s):  
Docosahexaenoic Acid ◽  
Cell Mass ◽  
Control Group ◽  
Hatching Rate ◽  
Link Function ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Internal Cell ◽  
Fresh Embryos

The aims of this study were to decrease the apoptotic index and increase cryotolerance of bovine embryos produced in vitro with the addition of 1 µM docosahexaenoic acid (DHA). On Day 1, presumed zygotes were cultivated with 1µM DHA (Sigma, St. Louis, MO, USA; n=437), and without the agonist (control group; n=450). The cleavage rate (%) was evaluated on Day 2 and the development of blastocysts on Day 7. Embryos before and after vitrification were fixed for the TUNEL trial. After vitrification, the embryos were heated and re-cultivated to evaluate the hatching rate at 12, 24, 36, 48, 60, and 72h. A sample of re-cultivated embryos at 12h of DHA (n=5), and without the agonist (control group; n=6), was frozen for mass spectrometry (MALDI-MS). Statistical analyses of deviance were carried out considering generalized mixed linear models, and the effect of the collection day (block) was considered as random. For the count variables, the Poisson distribution and the log link function were considered. In the cases of variables represented by rates, binomial distribution and the logit link function were used. In the study of cryotolerance, ANOVA of the hatching rate for each one of the times evaluated was carried out. In cases of significance of the effect of treatments, the Dunnett test was applied to compare treatments. Multivariate and univariate statistical models were used for analysis of MALDI-MS. All analyses were made using the GLIMMIX procedure of the SAS software (SAS Institute Inc., Cary, NC, USA). The cleavage rate was not different between the groups (P&gt;0.05) and the production of blastocysts was lower in the DHA group (P&lt;0.05). The number of cells per embryo was higher (P&lt;0.05) by the addition of 1μM DHA in blastocysts pre- and post-vitrification. The rate of total and internal cell mass apoptosis in fresh embryos (11.73 and 15.98%) increased compared with the control group (9.62% and 11.03%, respectively). The proportion of internal cell mass in fresh embryos decreased in the DHA group (39.93%) compared with the control group (57.00%). Hatching rates at 48, 60, and 72h after devitrification in the group treated with 1μM DHA were not different (P&gt;0.05) compared with the control group. Phosphatidylcholine [phosphatidylcholine (32: 0)+H] was more abundant (P&lt;0.05) in embryos cultured with DHA, and thus was considered as a negative apoptosis biomarker. In conclusion, the use of 1μM DHA in vitrification of bovine embryos impairs embryonic quality and development under the conditions of the present study. Research was supported by CAPES, FAPEMIG, PPGCV/UFLA, EVUFMG, CENATTE EMBRIÕES.

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