Exogenous protein affects developmental competence and metabolic activity of bovine pre-implantation embryos in vitro

1998 ◽  
Vol 10 (4) ◽  
pp. 327 ◽  
Author(s):  
J. Eckert ◽  
P. A. Pugh ◽  
J. G. Thompson ◽  
H. Niemann ◽  
H. R. Tervit
Keyword(s):  
Metabolic Activity ◽  
Production Systems ◽  
Lactate Production ◽  
Developmental Competence ◽  
Morphological Development ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Exogenous Protein ◽  
Before And After

The role of exogenous protein during bovine pre-implantation embryo development in two in vitro production systems was investigated. Morphological development, survival after vitrification and metabolic activity before and after vitrification were recorded in blastocysts generated in vitro in synthetic oviduct fluid (SOF) medium in the presence of either bovine serum albumin (BSA) or polyvinyl-alcohol (PVA). Metabolic activity was determined by measuring oxygen consumption, glucose and pyruvate uptake as well as lactate production. Development to blastocysts and survival after vitrification were reduced significantly in medium lacking protein. Of the metabolic parameters measured, only pyruvate uptake was increased significantly in embryos cultured in medium supplemented with PVA. Whereas in BSA-supplemented medium pyruvate uptake was correlated with lactate production, in PVA-supplemented medium glucose uptake was correlated with lactate production. Lactate production increased significantly after vitrification as compared with fresh embryos. Thus, exogenously added protein significantly alters oxidative metabolism. In medium lacking protein, the additional pyruvate may be used for the maintenance of intracellular amino acid pools. Vitrification appears to alter glycolytic metabolic profiles indicating a stress-response. In conclusion, the perturbed metabolism corresponding to reduced developmental capacity of embryos produced under protein-free conditions emphasizes the ambiguity between maximum develop-ment, technical and hygienic requirements and physiological demands of the early bovine embryo in vitro. The use of well-defined recombinant proteins might assist in closing this gap.

235 PROGESTERONE AFFECTS THE MESSENGER RNA EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 265
Author(s):  
K. Knauer ◽  
H. Stinshoff ◽  
S. Wilkening ◽  
C. Wrenzycki
Keyword(s):  
In Vitro Culture ◽  
Messenger Rna ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Morphological Development ◽  
Bovine Embryo ◽  
Development Rates ◽  
Vitro Model

It is known that the progesterone (P4) provided by the corpus luteum is essential for the maintenance of pregnancy. It has been suggested that supplying external P4 in vivo is beneficial to the establishment and upkeep of pregnancy. The aim of the present study was to assess the effects of supplementation with different concentrations of P4 on either of 2 days of in vitro culture (IVC) on early bovine embryo development in an in vitro model. A total of 5073 cumulus–oocyte complexes were matured and fertilized in vitro. Before culture, they were collected in groups of 30 and allocated to 1 of 9 groups. The groups were supplemented with 10, 20, or 100 ng of P4 on Days 4 or 5 of IVC (IVF = Day 0). Alcohol (ETOH) was used as the solvent, so 8 µL of ETOH was used per supplementation. Therefore, two additional groups were supplemented with only ETOH on Day 4 or 5 of IVC. The presumptive zygotes allocated to group 9 were not supplemented. A culture system without oil overlay was used to prevent the lipophilic P4 from moving into the oil. Embryo cleavage and development rates were determined solely on Day 8 of IVC. Single expanded blastocysts were stored at –80°C for RT-qPCR. Subsequently, the relative amounts of six developmentally important gene transcripts (IGF1R, SLC2A1, HSD3B1, IFNT, PGRMC1, and PGRMC2) were analysed in single embryos of all groups. Statistical analysis was performed using one-way and two-way ANOVA, and the level of significance was set at P ≤ 0.05. Cleavage and development rates did not differ among groups (see Table 1). The relative abundance of IGF1R, SLC2A1, PGRMC1, and PGRMC2 was not affected by either the concentration or the timing of P4 supplementation. Nevertheless, there was a statistically significant interaction between the day of treatment and the concentration used for the expression of HSD3B1 mRNA. When 20 ng of P4 was added on Day 5 of IVC, significantly more HSD3B1 transcripts were detected than if 10 ng, 100 ng, or ETOH alone was added. The expression of IFNT was not affected by the day of supplementation, only by the concentration used. Thus, supplementation with 20 ng of P4 resulted in a significantly higher level of transcripts than when 10 ng or ETOH was supplemented. The results indicate that the amount of P4 present during early embryonic development and the timing of its presence had an impact on molecular developmental competence. However, no effects concerning morphological development up to the blastocyst stage could be detected. Table 1.Cleavage and development rates (± SEM) of embryos supplemented with 10, 20, or 100 ng on Day 4 or 5 of in vitro culture (P ≥ 0.05) The financial support of the FBF e.V. is acknowledged.

86 IMPROVED BOVINE EMBRYO PRODUCTION USING NOVEL IN VITRO CULTURE SYSTEMS

2016 ◽  
Vol 28 (2) ◽  
pp. 172
Author(s):  
J. H. Pryor ◽  
J. F. Hasler ◽  
L. Strøbech ◽  
B. Avery ◽  
N. Hashem ◽  
...  
Keyword(s):  
Production Systems ◽  
Uv Light ◽  
Cumulus Cells ◽  
Tween 20 ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Embryo Viability ◽  
Embryo Production ◽  
Cell Counts

Development and testing of new embryo production components is important to improve the outcome following in vitro production of bovine embryos. The objective of this study was to compare media used in two bovine embryo production systems (control and EmbryoTrans Biotech: ETB). In Exp. 1, abattoir-derived cumulus-oocyte complexes were randomly assigned and in vitro matured (IVM) in either control [Medium 199 with Earles salts (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 1% penicillin/streptomycin (Invitrogen), 0.2 mM sodium pyruvate, 2 mM L-glutamine (Sigma Chemical Co., St. Louis, MO, USA), and 5.0 µg mL–1 of Folltropin®-V (Vetoquinol, Pullman, WA, USA)] or ETB BO-IVM medium for 21 to 24 h. IVF was conducted in 500 µL of pre-equilibrated modified Tyrode-lactate medium for control (Pryor et al. 2011 Theriogenology 75, 24–33) or ETB BO-IVF in Nunclon® 4-well multi-dishes (VWR Scientific, Pittsburgh, PA, USA). Seventeen hours post-insemination, presumptive zygotes were cleaned of cumulus cells and cultured in either Bovine Evolve (Zenith Biotech, Guilford, CT, USA) supplemented with 4 mg mL–1 of Probumin BSA (EMD Millipore, Norcross, GA, USA), under oil (Irvine Scientific, Santa Ana, CA, USA) or ETB BO-IVC medium under BO-oil for 7 days (8 days post-IVF). All cultures were performed at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 using BT37 incubators (Planer Plc, Sunbury, UK). For Exp. 2, all conditions were maintained except a modified ETB BO-IVCA medium was used. On Day 8 of IVC, grade 1 and 2 blastocysts (BL) through hatching blastocysts (HBL) were counted and used to calculate total viable rates. In Exp. 2, these embryos were fixed in cold methanol, washed in PBS/0.1% Tween 20, mounted in 10 μg mL–1 Hoechst 33342/glycerol, and viewed under UV light to count cells (n = 49 and 107 for control and ETB, respectively). Each experiment was replicated 3 times with a total of 425 oocytes in Exp. 1 and 430 in Exp. 2, divided equally between treatments. Percentage data were transformed using arcsine square root function before analysis and means compared using a paired Student’s t-test. For Exp. 1, there were no differences in rates of cleavage or viable embryos between control and ETB systems (81.3% and 42.9% v. 80.5% and 48.4%, respectively). In Exp. 2, ETB was superior to control for percent viable, HBL, and combined HBL/expanded BL (51.9, 23.9, 45.8% v. 29.2, 5.8, 20.5, respectively; P < 0.05). Differences between mean cell counts for viable embryos were significant (control = 127.0 ± 6.7 s.e.m. and ETB = 162.7 ± 5.7; P < 0.0001). Embryo viability decreased in control media between Exp. 1 and 2 (42.9 v. 29.2%; P < 0.05). Seasonal differences may have contributed via heat stress with temperatures ranging from 23.8°C for Exp. 1 to 33.8°C for Exp. 2. Interestingly, embryo development in the ETB media did not decrease under the same conditions. In conclusion, ETB media produced more high-quality embryos than control under varying conditions experienced by commercial IVF companies.

scholarly journals In vitro assessment of histamine and lactate production by a multi-strain synbiotic

2021 ◽  
Author(s):  
Gerrit Stuivenberg ◽  
Brendan Daisley ◽  
Polycronis Akouris ◽  
Gregor Reid
Keyword(s):  
Lactic Acidosis ◽  
Lactate Production ◽  
Fermented Food ◽  
In Vitro Production ◽  
Food Items ◽  
Host Health ◽  
In Vitro Assessment ◽  
Vitro Production ◽  
Significant Production

AbstractRecent studies suggest histamine and d-lactate may negatively impact host health. As excess histamine is deleterious to the host, the identification of bacterial producers has contributed to concerns over the consumption of probiotics or live microorganisms in fermented food items. Some probiotic products have been suspected of inducing d-lactic-acidosis; an illness associated with neurocognitive symptoms such as ataxia. The goals of the present study were to test the in vitro production of histamine and d-lactate by a 24-strain daily synbiotic and to outline methods that others can use to test for their production. Using enzymatic based assays, no significant production of histamine was observed compared to controls (P > 0.05), while d-lactate production was comparable to a commercially available probiotic with no associated health risk. These assays provide a means to add to the safety profile of synbiotic and probiotic products.

341 OXYGEN TENSION AND EGF AFFECT METABOLISM OF IN VITRO-MATURED CUMULUS-ENCLOSED MOUSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 278
Author(s):  
K. A. Preis ◽  
G. E. Seidel Jr ◽  
D. K. Gardner
Keyword(s):  
Glucose Uptake ◽  
Oxygen Tension ◽  
Embryo Development ◽  
Metabolic Activity ◽  
Lactate Production ◽  
Defined Medium ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Exact Test

In vitro maturation of immature oocytes results in limited success in both clinical and research laboratories. Although reduced oxygen concentration is beneficial to embryo development, the optimal concentration for oocyte maturation has yet to be determined. The objective of this study was to determine whether oxygen tension (20% or 5% O2) affects oocyte physiology. Additionally, the effect of epidermal growth factor (EGF) in maturation medium on oocyte metabolic activity and subsequent embryo development was determined. Cumulus–oocyte complexes (COCs; n = 231) were collected from 28-day-old unprimed F1 (C57BL/6 × CBA/ca) mice. COCs were individually matured in defined medium at 37°C in 6% CO2 in one of four groups (Table 1). For the metabolism study, COCs were further divided into two groups: individual maturation in a 2-µL drop of medium for 16 h (n = 131); or individual maturation in 5-μL for 12 h and then placed in a 0.5-μL drop of medium for 4 h (n = 100), the time of greatest metabolic activity of the COC. At 17 h of maturation, COCs were individually fertilized, and zygotes were individually cultured until 96 h, at which time blastocyst development was assessed. Metabolic profiles were analyzed by ANOVA, and blastocyst rates were analyzed by Fisher's exact test. Maturation rates and blastocyst development were not different between groups. However, at 12–16 h of maturation, metabolism of COCs was affected by both oxygen tension and EGF (Table 1). Concerning metabolism over the entire course of maturation, glucose uptake and lactate production were higher in COCs in 5% O2 + 100 ng EGF (P < 0.05) than in the remaining three groups. There was no difference between 5% O2 and 20% O2 + 100 ng EGF, but 20% O2 caused less glucose uptake and lactate production than did the other three treatment groups (P < 0.05). Results of this study are the first to show that oxygen tension alters COC metabolism: COCs matured under 5% O2 were more active metabolically than COCs matured under 20% O2. The effect of oxygen tension is to some extent moderated by the presence of EGF, as metabolic activity of COCs matured under 20% O2 + 100 ng EGF was closer to that of COCs matured under 5% O2 conditions. Although blastocyst rates were similar across the four groups, embryos derived from oocytes matured in different oxygen tensions may exhibit different developmental potential. In conclusion, results of this study have implications for the improvement of maturation conditions in both clinical and research laboratories. Table 1. Carbohydrate metabolism of individual COCs at 12–16 h of maturation

296 EFFECT OF GROWTH HORMONE ON EMBRYO DEVELOPMENT AND ULTRASTRUCTURE OF BOS TAURUS IVP BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 304
Author(s):  
L. M. C. Pegoraro ◽  
M. N. Dode ◽  
C. F. Weissheimer ◽  
F. G. Leivas ◽  
A. Vieira ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Culture Medium ◽  
Production Systems ◽  
Bos Taurus ◽  
In Vitro Production ◽  
Assisted Reproductive Technique ◽  
Development Rates ◽  
Developmental Rates ◽  
Chi Square Analysis

Bovine in vitro production systems are one of the most used assisted reproductive technique. However, this technique has some limitations especially in Bos taurus breeds, because of the low percentage of viable blastocysts produced (around 40% of oocytes inseminated) and higher cryosensitivity due to higher lipid content. Growth hormone (GH) can be a promising additive to increase in vitro embryo production. The aim of this study was to evaluate the embryo developmental rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) and ultrastructural features in Bos taurus embryos produced in SOFaa medium with or without GH. Cumulus oocyte complexes (COC) were recovered from slaughterhouse-derived ovaries (Angus Red crosses) and by ovum pick-up (OPU) from Jersey donors. After IVM and IVF, the presumptive zygotes were allocated in the SOFaa medium without (control) or with addition of GH (100 ng mL-1), for culture at 39°C in an atmosphere of 5% CO2. The cleavage and viable blastocyst rates were recorded 2 and 8 days after initiation of IVF, respectively. The results were compared by chi-square analysis. Similar (P > 0.05) cleavage rates were found in different culture medium and bovine breeds (61 v. 63% for Jersey control and Jersey GH; 71 v. 72% for cross-breed control and cross-breed GH). The development rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) did not differ in culture medium with or without GH within breeds (35 v. 30% for Jersey control and GH; 52 v. 56% for cross-breed control and GH; 21 v. 20% for Jersey control and GH; 36 v. 41% for cross-breed control and GH, respectively; P > 0.05). However, when breeds were compared, higher development rates were observed in cross-breed obtained from slaughterhouses than Jersey donors obtained by OPU (35 v. 52% for Jersey v. cross breed control; 30 v. 56% for Jersey v. cross-breed GH; 21 v. 36% for Jersey v. cross-breed control; 20 v. 41% for Jersey v. cross-breed GH. P < 0.001). The analyses of ultrastructure demonstrated no difference in the lipid proportion and organelle distribution of embryos produced with or without GH. We concluded that GH addition to SOFaa medium did not increase developmental rates for cross-breed or Jersey IVP embryos.

221 EFFECT OF ROCKING ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 209
Author(s):  
Y. Serita ◽  
C. Kubota ◽  
T. Kojima
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Rate Of Development ◽  
Fetal Bovine ◽  
Vitro Production

This study tested whether embryo development yield using in vitro fertilization (IVF) could be improved by rocking cultures. Bovine ovaries were obtained at a slaughterhouse and transported to the laboratory within 6 h. Cumulus–oocyte complexes were collected and 20–25 were transferred in 100-μL drops of TCM-199 containing 10% fetal bovine serum and antibiotics under paraffin oil. Maturation was for 20–24 h at 38.5°C under 5% CO2 and 95% air in a humid atmosphere (IVM). In vitro fertilization was carried out for 6 h using frozen–thawed sperm from a single bull in modified Brackett and Oliphant (BO) medium. Presumptive zygotes were cultured in CR1aa supplemented with 10 mg mL–1 of BSA or 5% FBS for 9 d at 38.5°C under 5% CO2, 5% O2, and 90% N2 in a humid atmosphere (IVC). Rocking was performed to a height of 6 cm every 7 s using a Profile Rocker (New Brunswick Scientific Co., Edison, NJ, USA) in an incubator. Dishes were placed at a 15-cm distance from the fulcrum of the rocker. The conventional method (no rocking) served as a control, and every experiment was replicated 3 times. For Experiment 1, the effect of the period of rocking on developmental competence was examined when COC or zygotes were subjected to rocking for IVM, IVF, or IVC (IVM-move, IVF-move, and IVC-move). There were no significant differences in rates of oocyte maturation, cleavage, and development for IVM-move v. the control, or for rate of development between IVC-move and the control. However, the rate of fertilization for IVF-move was higher than that of the control (88.9 v. 67.5%; P < 0.01), and the rate of development was higher for IVF-move than for the control (39.0 v. 25.7%; P < 0.05). For Experiment 2, the effect of rocking frequency during IVF on development was determined. The IVF cultures were rocked every 7, 3.5, and 1.5 s (IVF-1move, IVF-2move, IVF-3move). The rates of cleavage on IVF-1move, IVF-2move, IVF-3move, and the control were 74.3, 69.8, 68.8, and 60.4%, and the rates of development were 39.0, 48.3, 26.2, and 25.7%, respectively. The rates of development on IVF-1move and IVF-2move were significantly different from the control and IVF-3move (P < 0.01). These results showed that rocking during IVF improved fertilization and embryo yield, and that frequency of rocking affected embryo development.

346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

2007 ◽  
Vol 19 (1) ◽  
pp. 288
Author(s):  
C. Kubota ◽  
T. Kojima ◽  
T. Nagai ◽  
X. Tian ◽  
X. Yang
Keyword(s):  
Subsequent Development ◽  
Industrial Applications ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Bovine Embryo ◽  
Becton Dickinson ◽  
In Vitro Storage ◽  
Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco&apos;s PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus&ndash;oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10&percnt; fetal bovine serum (FBS) or 1 mg mL&minus;1 polyvinyl alcohol (PVA). COCs were collected from follicles (3&ndash;8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5&deg;C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10&percnt; FBS and 20 &micro;g mL&minus;1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281&ndash;286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS&ndash;IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM&ndash;IVF&ndash;IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student&apos;s t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC (bFF (n &equals; 87): 0&percnt;, 0&percnt;; PBS/FBS (n &equals; 72): 84&percnt;, 1&percnt;; and PBS/PVA (n &equals; 81): 89&percnt;, 6&percnt;, respectively) were significantly lower than those of the control group (n &equals; 406; 97&percnt; and 29&percnt;, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS&ndash;IVM&ndash;IVF (M199A/FBS (n &equals; 97): 82&percnt;, 28&percnt;; and M199A/PVA (n &equals; 111): 98&percnt;, 31&percnt;, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

257 DIFFERENCES BETWEEN BOS TAURUS AND BOS INDICUS EMBRYOS: TRANSCRIPTS AMOUNTS

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Production System ◽  
Production Systems ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Peroxiredoxin 1 ◽  
Vitro Production

The Zebu breeds (Bos indicus) are different from European breeds (Bos taurus) in some aspects of their reproductive physiology, including follicle recruitment, number of follicular waves, and oocyte ultrastructure. On the other hand, embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. The aim of this study was to evaluate the effect of breed (Gyr v. Holstein) within embryo production system (in vivo and in vitro), as well as effect of production systems within breeds on relative abundance of transcripts related to formation, survival, and subsequent development of blastocysts, such as those involved in water and small solutes transport (Aquaporins 3 and 11), blastocoel formation (Na+/K+-ATPase a1 and |52), and cellular stress response (Peroxiredoxin 1). For in vivo embryo production, donors were superstimulated with FSH and inseminated, and embryos were recovered 7 days after AI. For in vitro embryo production, oocytes recovered by ovum pickup were in vitro matured and fertilized and then cultured for 7 days in culture medium under 5% CO2 at 38.5°C. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA, USA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA, USA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA, USA). The cDNA were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed by REST© software. To evaluate breed effect within the production systems, 2 comparisons were performed: (1) in vivo: Gyr v. Holstein and (2) in vitro: Gyr v. Holstein, considering Holstein data as 1.00. To evaluate production system effect within breeds, 2 comparisons were performed: (1) Gyr: in vivo v. in vitro and (2) Holstein: in vivo v. in vitro, considering in vivo produced embryo data as 1.00. The results are shown as mean ± SEM. For in vivo comparison between breeds, Aquaporin 3 (1.66 ± 0.77), Na+/K+-ATPase a1 (1.61 ± 0.56), and Peroxiredoxin 1 (1.61 ± 0.66) were up-regulated (P < 0.05) in Gyr embryos when compared with Holstein embryos, whereas for in vitro comparison, no differences (P > 0.05) were found. For comparisons between production systems within breeds, only Peroxiredoxin 1 (0.31 ± 0.39) was down-regulated (P < 0.01) in in vitro produced Gyr embryos when compared with in vivo counterparts. No differences (P > 0.05) were found between production systems for the Holstein breed. In conclusion, these data suggest that there is a difference on gene expression between Bos taurus and Bos indicus blastocysts, but such difference between breeds can be attenuated by the in vitro production system, indicating an embryo adaptation to the in vitro culture conditions. The data also suggest that the in vitro production system can influence the amount of transcripts in Gyr embryos. Other genes should be evaluated for a better understanding of these differences. Financial support was provided by CNPq and FAPEMIG.

204 PERIPHERAL PROGESTERONE CONCENTRATION AFFECTS BOVINE OOCYTE QUALITY AT THE MOLECULAR LEVEL

2013 ◽  
Vol 25 (1) ◽  
pp. 250
Author(s):  
N. Schlüter ◽  
A. Hanstedt ◽  
H. Stinshoff ◽  
K. Knauer ◽  
S. Wilkening ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Relative Abundance ◽  
Clinical Signs ◽  
Follicular Phase ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Glucose Transporter 1 ◽  
Vitro Production

The developmental competence of cumulus–oocyte complexes (COC) used for in vitro production is dependent on several factors including the stage of the oestrus cycle. In a recent study, we were able to show that circulating progesterone (P4) had no effect on follicle number, size, recovery rate, or in vitro production suitability of recovered COC (Schlüter et al. 2012 Reprod. Fertil. Dev. 24, 175–176). The aim of the present study was to determine the influence of circulating P4 concentrations on the molecular quality of bovine COC collected during repeated OPU sessions. The COC were aspirated twice per week for 5 to 6 weeks from 12 Holstein Friesian heifers. The first OPU session took place on Day 7 of the oestrous cycle after spontaneous ovulation (ovulation = Day 0). Blood samples were taken at the time of each OPU session, and P4 concentrations were determined using a radioimmunoassay. All animals showed clinical signs of oestrus and large follicles (≥8.5 mm) during the course of the OPU sessions. Following the aspiration of a large follicle, a CL-like structure (induced CL) could be detected. According to the P4 concentrations, the cycle was divided into 3 phases: CL phase after spontaneous ovulation (oCL; P4: ≥1 ng mL–1), follicle phase 1 (Fp; P4 <1 ng mL–1), and induced CL phase (iCL; P4: ≥1 ng mL–1). The length of the cycle after spontaneous ovulation did not differ significantly from that after induced ovulation (22.4 ± 3.1 days v. 23.8 ± 1.8 days, respectively). During the oCL-phase, blood P4 concentrations were significantly higher than during the iCL-phase (4.9 ± 2.3 ng mL–1 v. 3.0 ± 1.6 ng mL–1). For mRNA analysis, denuded COC were individually frozen at –80°C to analyse the relative transcript abundance using RT-qPCR. The transcripts studied play important roles during oocyte development [growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), glucose transporter 1 (SCL2A1), hypoxia inducible factor 2α (HIF2α), progesterone receptor (PGR), progestin and adipoQ receptor 5 (PAQR5), progesterone receptor membrane component 1 and 2 (PGRMC1, PGRMC2)]. Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey’s test. A P-value of ≤0.05 was considered significant. The relative abundance of all transcripts except SCL2A1 was significantly increased in oocytes collected from follicles of the oCL phase compared with that from oocytes that had been aspirated during the iCL phase. A significant increase in the relative amount of PGR, PGRMC1, PGRMC2, and BMP15 transcripts was detected in oocytes stemming from the follicular phase to those from the iCL phase. No differences in the relative abundance of all transcripts were seen comparing oocytes from oCL phase and oocytes from the follicular phase. In summary, circulating P4 concentrations had an effect on the molecular quality of COC recovered during repeated OPU session, which might affect further development. The financial support of the FBF (Förderverein Biotechnologieforschung) e.V. is gratefully acknowledged.

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