Effect of spermatozoa motility hyperactivation factors and gamete coincubation duration on in vitro bovine embryo development using flow cytometrically sorted spermatozoa

2017 ◽  
Vol 29 (4) ◽  
pp. 805 ◽  
Author(s):  
Luis B. Ferré ◽  
Yanina Bogliotti ◽  
James L. Chitwood ◽  
Cristóbal Fresno ◽  
Hugo H. Ortega ◽  
...  
Keyword(s):  
Embryo Development ◽  
X Chromosome ◽  
Embryo Quality ◽  
Bos Taurus ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Blastocyst Formation ◽  
Spermatozoa Motility

The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30 min before 18 h fertilisation with hyperactivating factors, namely 10 mM caffeine (CA), 5 mM theophylline (TH), 10 mM caffeine and 5 mM theophylline (CA + TH); and untreated spermatozoa (control). In Experiment 2, matured B. taurus oocytes were fertilised using a short (8 h) or standard (18 h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA + TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8 h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8 h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8 h (3%) to 18 h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode’s albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte–sperm coincubation time (8 h) resulted in similar overall embryo performance rates compared with the prolonged (18 h) interval.

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

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.

106 EFFECTS OF BONE MORPHOGENETIC PROTEIN 4 (BMP4) AND ITS INHIBITOR NOGGIN ON BOVINE IN VITRO EMBRYO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 158
Author(s):  
I. La Rosa ◽  
R. Fernandez-Martin ◽  
D. A. Paz ◽  
D. F. Salamone
Keyword(s):  
Bone Morphogenetic Protein ◽  
Embryo Development ◽  
Cell Number ◽  
Control Group ◽  
Bone Morphogenetic Protein 4 ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Nuclear Staining ◽  
Morphogenetic Protein

Bone morphogenetic protein 4 (BMP4) is a member of the BMP family of conserved morphogenes in charge of many events of differentiation (Chen et al. 2004 Growth Factors 22, 233–241) BMP4 is involved in regulation of pluripotency in humans and mice though the role in bovine early embryo development is still undefined. Noggin is a BMP4 inhibitor (Groppe et al. 2002 Nature 420, 636–642) that does not have a specific receptor but functions by directly binding BMP ligands. The objective of this work was to study the effects of BMP4 and Noggin on early bovine embryo development. Cumulus–oocyte complexes (COC) were aspirated from abattoir ovaries and in vitro matured in TCM containing 10% fetal bovine serum (FBS), 2 mM FSH, 20 mM cysteamine, 1% antibiotic- antimycotic (15240, GIBCO, Grand Island, NY, USA) and 0.1 mM sodium pyruvate. Incubation conditions were a 6.5% CO2 humidified atmosphere at 39°C. After 22 h, in vitro fertilization was performed. Briefly, frozen–thawed semen was centrifuged twice at 490 × g and resuspended in B.O. solution to a final concentration of 20 × 106 mL–1 and incubation with COC was performed for 5 h. Presumptive zygotes were randomly cultured in CR2 with 0.3% BSA, free of serum and co-culture (control, n = 217) or supplemented with 100 ng mL–1 of either BMP4 (n = 218) or Noggin (n = 205). Cleavage and blastocyst rates were evaluated at Days 2 and 9 of culture. Blastocysts cell numbers were analysed by nuclear staining with Hoechst 33342. The expression pattern of the transcription factor Oct-4 was studied by immunocytochemistry and confocal microscope analysis in blastocysts. Chi-square tests were applied for cleavage, blastocyst, and hatching rates. One-way ANOVA was used to compare blastocyst cell number and a proportion test was used for Oct-4 expression. For all, P < 0.05 was considered significant. Cleavage rate was significantly lower in the Noggin group compared to control (51.2% v. 62.3%) whereas the BMP group (61.3%) did not differ from control or Noggin groups. Blastocyst rates for the BMP and Noggin groups were statistically lower than control (9.24% and 11.7% v. 20.6%, respectively). Hatching rate for the control group was significantly higher than both BMP and Noggin groups (4.6% v. 1.4% and 0.49%, respectively). Blastocyst cell number did not differ between groups (130, 117, and 128 for control, BMP4, and Noggin groups, respectively). Oct-4 expressing cells over total cell number was lower in BMP (72%; n = 3) and Noggin (72%; n = 3) groups compared to control (83%; n = 3). In our conditions, BMP inhibition with Noggin or addition of exogenous BMP4 negatively affected developmental rates and altered the proportion of pluripotent (Oct-4 positive) cells. Our results demonstrate the importance of a correct balance within the BMP signalling system for proper bovine in vitro embryo development.

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.

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.

238 KNOCKING DOWN OF THYROID HORMONE RECEPTORS INHIBITS DEVELOPMENT OF EARLY BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
N. Y. Rho ◽  
F. A. Ashkar ◽  
T. Revay ◽  
P. Madan ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Reference Genes ◽  
Messenger Rna ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Production ◽  
Mrna Transcripts ◽  
In Vitro Embryo Production

Thyroid hormones (TH) play an important role in the physiology of vertebrates, ranging from the regulation of metabolic processes to cell proliferation, differentiation, and embryo development. We have previously shown a beneficial effect of supplementing TH in in vitro embryo production media. Recently, detection of TH receptors (TR) in oocytes and early stages of pre-implantation embryos indicated a possible regulatory role for TH in these stages (unpublished data). The objective of this study was to investigate the importance of TR expression in the pre-attachment bovine embryo in vitro. Bovine embryos, produced by standard in vitro embryo production procedures, were microinjected at the zygote stage with small interfering RNA (siRNA) specifically designed for knocking down either TR-α or TR-β. In addition, groups of zygotes were microinjected with scrambled siRNA (SI) or were not injected (NI), and these groups served as controls. Embryo developmental rates were assessed using light microscopy for blastocyst formation rates and expression of TR messenger RNA (mRNA) transcripts at the blastocyst stage was assessed by quantitative PCR across all groups. Expression of TR mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase, H2a, and 18S as reference genes. There was a significant decrease in blastocyst formation rates in both embryo groups injected with either TR-α (P < 0.002) and TR-β (P < 0.001) siRNA compared with the NI and SI groups. Moreover, the TR-β knockdown group exhibited a lower developmental rate than the TR-α knockdown group, which indicates a stronger inhibitory role for TR-β. Quantification of the level of TR mRNA expression in four groups normalized with three different reference genes shows a consistent significant reduction in the levels of TR-α (P < 0.05) and TR-β (P < 0.02) mRNA transcripts compared with the NI and SI groups. However, TR-β expression was inhibited more than was TR-α expression. In conclusion, the results indicate that knocking down either TR-α or TR-β restrains embryo development. This suggests that TH play a vital role in the regulation of embryo development through their receptors during bovine early embryogenesis. The specific role of each of these receptors and their mechanism of action in mediating development needs to be further elucidated. Funding was provided by CRC, NSERC, and the EmbryoGENE network.

18 EMBRYO AGGREGATION IN PIG IMPROVES CLONING EFFICIENCY AND EMBRYO QUALITY

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.

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

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 27-32 ◽  
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.

Male-dependent variability of fertilization and embryo development in two bovine in vitro fertilization systems and the effects of casein phosphopeptides (CPPs)

1997 ◽  
Vol 9 (4) ◽  
pp. 465 ◽  
Author(s):  
U. Kreysing ◽  
T. Nagai ◽  
H. Niemann
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Individual Variability ◽  
Bovine Embryo ◽  
Blastocyst Formation ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Casein Phosphopeptides ◽  
Fertilization System

This study investigated the effects of semen from five different bulls and two different ejaculates of the same bull on penetration, cleavage, blastocyst formation, and cell allocation in bovine blastocysts produced in vitro. Casein phosphopeptides (CPPs) were tested for their ability to enhance fertilization and minimize variability among bulls and ejaculates. In Experiment 1, the BO-fertilization system was employed. Penetration and polyspermy both displayed great variation among bulls and between ejaculates, whereas no significant differences were observed in cleavage and blastocyst-formation rates. Similar variability was observed in penetration, polyspermy, cleavage, blastocyst-formation rates and cell allocation and distribution when the two fertilization systems, TALP and BO, were compared in Experiment 2. The BO-system supported penetration and polyspermy better (P < 0·05) than the TALP-system, whereas the TALP-system was superior (P < 0·05) in supporting cleavage and blastocyst formation. Significant interactions existed between bulls and the fertilization system employed. It is concluded that the success of in vitrofertilization is markedly dependent on individual bulls as well as on ejaculates from the same bull. CPPs are able to enhance penetration and embryo development in certain bulls or ejaculates and thus contribute to reducing the degree of individual variability, but they do not generally improve the success of bovine embryo production in vitro.

133 EFFECTS OF ACTIVINA ON mRNA EXPRESSION IN IN VITRO FERTILIZED BOVINE EMBRYOS CULTURED FROM CHEMICALLY DEFINED TWO-STEP CULTURE MEDIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 147
Author(s):  
J. E. Park ◽  
G. Jang ◽  
H. J. Oh ◽  
S. G. Hong ◽  
I. S. Yang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Early Stage ◽  
Activin A ◽  
Developmental Competence ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Defined Culture

During the preimplantation stage, embryo development occurs in a maternal environment within the oviducts and uterine horns. It has been speculated that both the embryo itself and the maternal reproductive tract provide paracrine factors that influence embryo development (Jones et al. 2006 Reproduction 132(5), 799–810). Activins are known for FSH releasers, and several previous studies have reported that activin subunits and activin receptors mRNA were expressed in oocytes, zygotes, and oviduct (Yoshioka et al. 1998 Reprod. Fertil. Dev. 10(3), 293–298; Gandolfi et al. 1995 Mol. Reprod. Dev. 40(3), 286–291). The purposes of the present study were Experiment 1) to evaluate the effects of activin A on developmental competence of bovine embryos derived from two-step defined culture medium (Lim et al. 2007 Theriogenology 67(2), 293–302) and Experiment 2) to analyze the effects of activin A on transcriptional level of the genes in IVF embryos. Cumulus–oocyte complexs were harvested from ovaries obtained from a local slaughter house, matured, and fertilized in vitro. In vitro fertilized zygotes cultured in media supplemented with activin A in the early stage at the concentrations of 0, 10, or 100 ng mL–1 or in the later stage medium at the concentrations of 0, 10, or 100 ng mL–1. Data were analyzed using the Statistical Analysis System (SAS) program. In Exp. 1, although the development competence of embryos that cultured with activin A in the early stage medium was not significantly different, development to blastocysts on day 8 in the later stage medium with 100 ng mL–1 activin A was significantly higher than the control group [22.4% (54/264) v. 34.7% (76/233); P < 0.05]. Hatching rate of blastocyst on day 8 was significantly higher in the presence of 100 ng mL–1 activin A in the later stage culture medium compared with the control group [9.3% (5/54) v. 22.4% (17/76); P < 0.05]. In Exp. 2, the relative expression of 3 genes (Na/KATPase, E-cad, Glut-1) related to blastocyst hatching and implantation was analyzed. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitative reverse transcription-polymerase chain reaction. The expression level of the Na/K ATPase, E-cad, and Glut-1 gene were higher in the presence of activin A in the culture medium compared with the control group. In conclusion, this study suggests that activin A during the later stage of in vitro bovine embryo development can enhance the developmental competence of preimplantation embryos, increase the hatching rate, and affect expression level of genes related to hatching and implantation in defined culture medium. This study was financially supported by KOSEF (grant ? M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

93 POSTCRYOPRESERVATION VIABILITY OF EMBRYOS FROM NELLORE HEIFERS SUPPLEMENTED WITH RUMEN-PROTECTED FAT

2010 ◽  
Vol 22 (1) ◽  
pp. 205 ◽  
Author(s):  
M. M. Guardieiro ◽  
G. M. Machado ◽  
M. R. Bastos ◽  
G. B. Mourão ◽  
L. H. D. Carrijo ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Embryo Development ◽  
Linear Models ◽  
Unsaturated Fatty Acids ◽  
Bos Taurus ◽  
Hatching Rate ◽  
Development Evaluation ◽  
Positive Effect

Polyunsaturated fatty acids seem to exert an extra-caloric positive effect on ruminant reproduction, although the reasons for that are still unclear. Although some studies have detected a positive effect of feeding unsaturated fatty acids on embryo development of superovulated Bos taurus cattle (Thangavelu et al. 2007 Theriogenology 68, 949-957), others have not (Petit et al. 2008 J. Dairy Sci. 91, 1786-1790). Our hypothesis was that, although number and quality of embryos from superovulated heifers would not be affected by diet, supplemental fat would improve embryo cryotolerance. Therefore, this study evaluated superovulatory response and embryo production, as well as cryotolerance of embryos cryopreserved through freezing or vitrification in Nellore heifers supplemented with rumen-protected fat. Forty heifers (24 to 36 mo old) were kept in pasture and randomly divided into 2 experimental groups according to supplemental source [F = concentrate with rumen-protected fat (100 g/d of Megalac-E®) and C = control, without fat supplementation]. Supplements were formulated to be isocaloric and isoproteic. Each female underwent both treatments in a cross-over design with approximately 68 d between replicates. After 50 d of feeding, emergence of the wave was synchronized with the aid of hormones to initiate the superovulation protocol. Recovered embryos were frozen or vitrified, and subsequently in vitro embryo development evaluation was accomplished. Data were analyzed using generalized linear models. There was no difference between F and C groups (P > 0.10) regarding superstimulatory response, number of total embryos/ova, viable embryos, degenerate embryos, or unfertilized oocytes recovered. However, group C had a greater superovulatory response than F (18.0 ± 1.3 v. 15.7 ± 1.2 CL; P = 0.06). Group C embryos presented greater hatching rate, independently of the cryopreservation method, at 48 h (33.1 ± 4.0%; n = 148 v. 17.3 ± 3.3%; n = 137; P = 0.009) and at 72 h (44.3 ± 4.2%; n = 148 v. 30.9 ± 4.0%; n = 137; P = 0.04) of in vitro culture. Under the conditions of the present study, supplementation with protected fat did not affect superstimulatory response and quantity or quality of embryos. However embryos from the F group were less tolerant to cryopreservation. Financial support from FAPESP, EMBRAPA, Arm & Hammer, Integral Produbon, and Pfizer of Brazil.

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