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.

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.

210 COMPARISON OF A RECOMBINANT TRYPSIN v. THE PIG PANCREATIC EXTRACT ON IN VITRO-PRODUCED BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 184
Author(s):  
K. J. Mattson ◽  
B. R. Devlin ◽  
N. M. Loskutoff
Keyword(s):  
Gradient Centrifugation ◽  
Bottom Layer ◽  
Control Group ◽  
Original Research ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Ongoing Research ◽  
Pathogenic Agents

According to the Manual of the International Embryo Transfer Society, trypsin can be used to remove certain pathogenic agents from in vivo-derived embryos. Research is currently in progress to determine whether trypsin can also remove pathogenic agents from semen. The original research on embryos involved the use of trypsin from pig pancreatic extracts. Because of stricter guidelines from international regulatory agencies on the use of animal products, several recombinant serine protease products are now becoming available. TrypZean (Sigma, St. Louis, MO, USA) is a recombinant developed from corn and is the first bovine sequence recombinant trypsin to contain no animal by-products. As part of our ongoing research on the effects of trypsin on sperm, the goal of this investigation was to examine the development of bovine embryos produced from sperm treated with the recombinant TrypZean compared with pig pancreas trypsin (Sigma) and a control (no trypsin). Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 9 replications. Semen was collected and pooled from Bos taurus and frozen in an egg-yolk cryodiluent (Biladyl, Minitube, Verona, WI). The semen was processed using density gradient centrifugation composed of 1 mL of 30% Percoll (Sigma), layered over 2 mL of 45% Percoll containing either 0.25% TrypZean (n = 972 oocytes), 0.25% trypsin (n = 1040 oocytes), or no trypsin for the control group (n = 1024 oocytes). The bottom layer for the 2 treatments and control was 2 mL of 90% Percoll containing 10 µg mL–1 of soy-based protease inhibitor (Sigma). The density gradients were centrifuged at 700g for 30 min, after which time the pellets were washed in 5 mL of prewarmed TL HEPES Solution (Cambrex) and centrifuged at 300g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 � 106 sperm mL–1 for in vitro insemination. The results were compared using one-way ANOVA. There were no statistically significant differences (P > 0.05) between any of the measures of embryonic development for the control and either of the treatment groups. Cleavage rates were measured for TrypZean (n = 689, 70.9%), trypsin (n = 729, 70.1%), and the control (n = 757, 73.9%) groups. More embryos reached the morula to blastocyst stages with the TrypZean (n = 367, 53.3%) and trypsin (n = 389, 53.4%) groups than the control (n = 369, 48.7%) group; however, these differences also were not statistically significant (P = 0.91) because of the large variation within the groups. In conclusion, the TrypZean and pig pancreas trypsin treatments of sperm prior to insemination showed no detrimental effects on IVF-derived bovine embryo development.

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.

293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

2007 ◽  
Vol 19 (1) ◽  
pp. 262 ◽  
Author(s):  
I. Dimitriadis ◽  
E. A. Rekka ◽  
E. Vainas ◽  
G. S. Amiridis ◽  
C. A. Rekkas
Keyword(s):  
Lipid Peroxidation ◽  
Embryo Development ◽  
Antioxidant Properties ◽  
Thiobarbituric Acid ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Embryo Production ◽  
Reactive Material

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples&apos; ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P &lt; 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.

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

137 SPLITTING OF IVP BOVINE BLASTOCYST AFFECTS MORPHOLOGY AND GLOBAL GENE EXPRESSION OF RESULTING DEMI-EMBRYOS DURING ELONGATION

2015 ◽  
Vol 27 (1) ◽  
pp. 161
Author(s):  
A. E. Velásquez ◽  
D. Veraguas ◽  
J. F. Cox ◽  
F. O. Castro ◽  
L. l. Rodriguez
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Average Length ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Control Group ◽  
Molecular Characteristics ◽  
Bovine Embryo ◽  
Developmental Potential

Embryo splitting has been used since the early 1980s to produce identical twins and increase the pregnancy rate per available embryo. However, very little is known about the effect of splitting on embryo development and competence. Indeed splitting could provoke a negative effect on embryo survival and it can be presumed that each demi-embryo might respond differently to the injury. In this sense, even when embryos are genetically and morphologically identical at the moment of splitting, their developmental potential and molecular characteristics might change as a consequence of the intense manipulation or epigenetic differences due to the interaction with the environment. We have proposed an approach to evaluate the effect of blastocyst splitting on the morphological and gene expression in in vivo development up to the filamentous stage. For that, the effect of splitting on bovine embryo development was evaluated during the elongation period by transferring split and nonsplit IVF-derived blastocysts to cattle recipients and collecting them at Day 17 of development. The number of collected embryos, embryo size, and global gene expression was compared between both groups. Collected elongated embryos derived from split blastocyst were compared with time matched collected control embryos. From 14 transferred hemi-embryos, 5 (35.7%) were collected while 9 elongated from 17 controls were recovered (52.9%). Neither the recovery rate nor the average length of the elongated embryos was significantly different between the two treatments. However, when embryos were rated depending on their size, more than 50% of embryos from the control group had a length surpassing 100 mm, while only 33% of the split embryos reached that size. Global gene expression was performed using 2-colour microarray-based gene expression analysis. This was a whole-genome microarray study comparing 10 individual elongated embryos derived from split and nonsplit IVF blastocysts. Genes were considered differentially expressed if the fold change is greater than 2 (up or down-regulation) with P ≤ 0.05. A total of 29 585 transcripts were detected in all embryos. From those, 449 (1.5%) were differentially expressed between elongated embryos derived from split and nonsplit IVF blastocysts, among them, 248 (0.83%) genes were down-regulated and 201 (0.67%) genes were up-regulated in split embryos. Gene ontology analysis identified deregulated genes related with intrinsic component of membrane (ELOVL7, GJA1, LAPTM4B, LDLR, SLC18A2, SLC1A3, SLC38A5, TSPAN13), lipid transporter activity (RBP4, APOA1, MTTP), and organophosphate ester transport (GJA1, GJB1, ATP9B). In conclusion, we showed that splitting affect the in vivo developmental capability and gene expression profile during the elongation period of bovine embryos. However, further studies are needed to determine the long-term effect of this technique to produce viable offspring. This work was partially supported by Fondecyt No. 11100082 and Fondequip No. EQM12113 from the Ministry of Education of Chile.

scholarly journals Progesterone and conceptus elongation in cattle: a direct effect on the embryo or an indirect effect via the endometrium?

Reproduction ◽  
2009 ◽  
Vol 138 (3) ◽  
pp. 507-517 ◽  
Author(s):  
M Clemente ◽  
J de La Fuente ◽  
T Fair ◽  
A Al Naib ◽  
A Gutierrez-Adan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Fourfold Increase ◽  
Uterine Environment ◽  
High Concentrations

The steroid hormone progesterone (P4) plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating P4 in the immediate post-conception period have been associated with an advancement of conceptus elongation, an associated increase in interferon-τ production and higher pregnancy rates in cattle. Using in vitro and in vivo models and ∼8500 bovine oocytes across six experiments, the aim of this study was to establish the route through which P4 affects bovine embryo development in vitro and in vivo. mRNA for P4 receptors was present at all stages of embryo development raising the possibility of a direct effect of P4 on the embryo. Exposure to P4 in vitro in the absence or presence of oviduct epithelial cells did not affect the proportion of embryos developing to the blastocyst stage, blastocyst cell number or the relative abundance of selected transcripts in the blastocyst. Furthermore, exposure to P4 in vitro did not affect post-hatching elongation of the embryo following transfer to synchronized recipients and recovery on Day 14. By contrast, transfer of in vitro derived blastocysts to a uterine environment previously primed by elevated P4 resulted in a fourfold increase in conceptus length on Day 14. These data provide clear evidence to support the hypothesis that P4-induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle and that, interestingly, the embryo does not need to be present during the period of high P4 in order to exhibit advanced elongation.

Effect of butafosfan supplementation during oocyte maturation on bovine embryo development

Zygote ◽  
2019 ◽  
Vol 27 (05) ◽  
pp. 321-328
Author(s):  
Lucas Teixeira Hax ◽  
Joao Alveiro Alvarado Rincón ◽  
Augusto Schneider ◽  
Lígia Margareth Cantarelli Pegoraro ◽  
Letícia Franco Collares ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Nuclear Maturation

SummaryAround 60–80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus–oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

scholarly journals Evaluation of in vitro production rates of bovine embryos using melatonin-supplemented culture medium

2021 ◽  
Vol 10 (6) ◽  
pp. e19010615544
Author(s):  
Ricardo Magalhães ◽  
Carlos Renato de Freitas Guaitolini ◽  
Marcio Luiz Denck Tramontin ◽  
Danielle Andressa Oliveira Sestari ◽  
Bruno Argenton de Barros ◽  
...  
Keyword(s):  
Culture Medium ◽  
Statistical Significance ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Bovine Embryo ◽  
Blastocyst Formation ◽  
In Vitro Production ◽  
Embryo Production ◽  
Vitro Fertilization

In this study, we aimed to evaluate the rate of bovine embryo production by using 50 ng/mL melatonin supplementation in in vitro culture medium. For this, oocytes from slaughterhouse ovaries were matured in vitro in TCM-199 medium with Earle’s balanced salt solution + 10% SFB, FSH, and LH in an atmosphere of 5% CO2. Twenty-four hours after IVM, the oocytes underwent in vitro fertilization in human tubal fluid under the same conditions as above, for 18 h. Semen was fractionated by Percoll gradient centrifugation and the concentration of sperm was adjusted to 1 × 106/mL. Probable zygotes were then divided into two groups: the control group grown in drops of 90 μL SOFaa medium + 0.6% BSA + 2.5% SFB, in an atmosphere of 5% CO2, 90% N2, and a melatonin group (Mel), similarly cultured in 90 μL drops of SOFaa medium + 0.6% BSA + 2.5% SFB + 50 ng/mL melatonin. Cleavage rates were assessed on day 3 (D3). On D7, blastocyst formation rates were evaluated. Eight routines were performed (320 oocytes per routine). Data were analyzed with ANOVA, followed by Tukey’s range test using a general linear model. The level of statistical significance was set at 5%. There were no differences in the rates of cleavage or blastocyst formation between the control and melatonin groups (P > 0.05). Thus, under the conditions used in this study, supplementation with melatonin did not yield benefits in increasing the rate of in vitro bovine embryo production.

173 THE EFFECT OF AMINO ACIDS AND HYALURONAN ON BOVINE EMBRYO IN VITRO DEVELOPMENT AND GENE EXPRESSION PATTERN

2006 ◽  
Vol 18 (2) ◽  
pp. 194 ◽  
Author(s):  
A. T. Palasz ◽  
J. Beltrán Breña ◽  
P. De la Fuente ◽  
M. F. Martinez ◽  
A. Gutiérrez-Adán
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Embryo Development ◽  
Control Group ◽  
Bovine Embryo ◽  
In Vitro Development ◽  
Glut 1 ◽  
Vitro Development ◽  
And Control

We have previously shown that bovine embryos cultured in SOFaa (BME + MEM amino acids) culture medium with hyaluronan (HA) + BSA are of better quality (Guti�rrez-Ad�n et al. 2005 Reprod. Fertil. Dev. 17, 219). Our objective was to examine the effect of essential (BME) or non-essential (MEM) amino acids with or without HA (MAP-5; Bioniche, Inc., Belleville, Ontario, Canada) on bovine embryo in vitro development and mRNA transcription of five developmentally important genes; apoptosis (Bax), growth factor (IGF-II), glucose (Glut-1) and fructose (Glut-5) transport and metabolism, and cell to cell adhesion (Cx-43). A total of 1474 presumptive zygotes (5 replicates) were initially cultured in 40 �L drops in the following groups: Group 1, control, SOFaa; Group 2, SOF-1 (MEM only); and Group 3, SOF-2 (BME only). On Day 4 (~96 h post-insemination (pi) the number of zygotes that had developed to d8 cells was recorded and 10 �L of SOF-1 and SOF-2, each with 2.5 mg/mL HA, was added to half of the embryos from Groups 2 and 3, respectively; the other half of Groups 2 and 3 and control group received 10 �L of corresponding medium without HA. Embryos were cultured under paraffin oil at 39�C and 5% CO2 in humidified air. Cleavage rates were recorded on Day 2 and the number of blastocysts on Days 7, 8, and 9. Five blastocysts from each replicate from each treatment were frozen for determination of gene expression patterns later. Cleavage rates and embryo development 96 h pi were compared among groups by chi-square analysis. The effects of HA and medium on blastocyst rates were analyzed by logistic regression and the data on mRNA expression by one-way repeated-measures ANOVA. Cleavage rates were 81.1% in SOFaa and 79.3% in SOF-1 (P = 0.48) and different from those in the SOF-2 group (72.4%; P < 0.02). The proportion of embryos that developed to d8 cells at Day 4 was higher in the control (46.7%) and SOF-1 (46.8%) groups than in the SOF-2 group (32.6%). The number of blastocysts that developed in SOFaa (37.0%), SOF-1 (37.7%), and SOF-1 + HA (37.8%) were higher (P < 0.001) than those in SOF-2 (19.6%) and SOF-2 + HA (21.8%). The level of expression of Glut-5 was not different among the groups. However, SOF-2 was the only group that had significantly lower expression of Glut-5, Igf II, and Cx43, and higher expression of BAX (P < 0.05) as compared to the control group and the SOF-1 groups with or without HA. Addition of HA to SOF-2 medium increased expression of Glut-1 and Igf II and decreased expression of BAX as compared to the SOF-1 only and control groups and the SOF-2 groups with or without HA (P < 0.05). The level of expression of Cx43 was higher in the control than in four remaining groups, and lower in the SOF-2 than in the SOF-1 group (P < 0.05). Addition of HA increased expression of Cx43 in both SOF-1 and SOF-2 groups but this level of expression was lower than in the control group; the level in the SOF-2 + HA group was lower (P < 0.05) than in the SOF-1 + HA group. We conclude that, within our protocol, MEM amino acids only stimulate embryo development to the blastocyst stage and the addition of HA to the SOF-MEM and SOF-BME media on Day 4 of culture improved embryo quality.

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