Inclusion of bovine lipoproteins and the vitamin E analogue, Trolox, during in vitro culture of bovine embryos changes both embryo and fetal development

2012 ◽  
Vol 24 (2) ◽  
pp. 309 ◽  
Author(s):  
J. A. Rooke ◽  
R. G. Watt ◽  
C. J. Ashworth ◽  
T. G. McEvoy
Keyword(s):  
Vitamin E ◽  
In Vitro Culture ◽  
Fetal Development ◽  
Fatty Acid Content ◽  
Liver Weight ◽  
Fetal Weight ◽  
Interactive Effects ◽  
Bovine Embryo ◽  
Zygote Development

This experiment investigated effects of lipoproteins and Trolox (vitamin E analogue) on bovine embryo and fetal development. The treatments were: in vitro culture (IVC) in synthetic oviducal fluid alone (SOF); with bovine lipoproteins (2% v/v; SOFLP); with Trolox (100 μM; SOFT); and with lipoproteins and Trolox (SOFLPT). In vitro culture with lipoproteins increased fatty acid content of blastocysts (P < 0.001) whereas inclusion of Trolox had no effect (P > 0.05). Whereas lipoproteins reduced zygote development to blastocysts (P = 0.03), Trolox facilitated increased development (P < 0.001) and counteracted the reduction observed with lipoproteins (interaction, P = 0.009). Lipoproteins also compromised (P < 0.001) but presence of Trolox (P > 0.05) had no effect on blastocyst morphological grade. Pregnancy rates resulting from synchronous transfer of IVP embryos were not affected by IVC treatment. At Day 70 of pregnancy, compared with SOF, fetal weight was lower in SOFLP but not SOFLPT (interaction, P < 0.001). Liver weight (g kg–1 fetal weight) was greater (P = 0.03) in treatments containing Trolox. Placentome numbers were greater in SOF and SOFLPT compared with SOFLP and SOFT (interaction, P = 0.002); superior embryo grades were also associated with increased numbers of placentomes (P = 0.024). In conclusion, the interactive effects of lipoprotein and Trolox inclusion on in vitro embryo development were also evident in fetal development at Day 70.

83 VITAMIN K2 SUPPLEMENTATION IMPROVES BLASTOCYST RATE BY RECOVERY OF MITOCHONDRIA IN IN VITRO-CULTURED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 155
Author(s):  
L. Baldoceda ◽  
C. Vigneault ◽  
P. Blondin ◽  
C. Robert
Keyword(s):  
In Vitro Culture ◽  
Fluorescent Microscopy ◽  
Vitamin K2 ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Mitotracker Red

Mitochondria play an important role during early mammalian embryo development through their diverse cellular functions, in particular creating balance between production of ATP by electron transport chain and oxidative stress. Embryonic mitochondria are inherited maternally and independently of the nuclear genome. They show limited activity during the early developmental stages before embryonic genome activation. It has been shown that in vitro culture (IVC) has an adverse effect on mitochondrial function in embryos. So far several attempts have been performed to improve and rescue the impaired mitochondria. It has been shown that vitamin K2 (a membrane-bound electron carrier, similar to ubiquinone) was used to rescue mitochondrial dysfunction and resulted in more efficient ATP production in eukaryotic cells (Vos et al. 2012 Science 336, 1306–1310). Therefore, the aim of the present study was to investigate the effects of supplementation of vitamin K2 on mitochondrial activity and blastocyst rate. Cumulus–oocytes complexes (n = 687) recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures. After fertilization, zygotes were cultured in SOF media supplemented with 10 mg mL–1 BSA. At 96 h post-fertilization, vitamin K2 was added to the culture media (n = 448 oocytes). On Day 7, treatment embryos were compared with untreated controls (n = 239 oocytes). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Differences among groups in blastocyst yield were analysed by ANOVA. Mitochondrial activity data was analysed by unpaired 2-tailed t-tests. Results show that the vitamin K2-treated group had a significantly (P < 0.05) higher blastocyst rate (+8.6%), expanded blastocyst rate (+7.8%), as well as better morphological quality compared with the control group. Furthermore, to evaluate mitochondria activity, pools of embryos of each treatment were labelled with a specific dye for active mitochondria (Mitotracker Red). A significantly higher intensity of Mitotracker Red (P < 0.05) was observed in the vitamin K2 treatment versus control group, as measured by fluorescent microscopy. In conclusion, for the first time, our data prove that supplementation of vitamin K2 during IVC of bovine embryos increases blastocyst rates and embryo quality. Future studies will focus on gene expression to identify targets implicated in impaired mitochondrial activity in in vitro bovine embryo production.

134 CYSTEINE AND CATALASE SUPPLEMENTATION FOR 7 DAYS DURING IN VITRO CULTURE IMPAIRS BOVINE EMBRYO DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 214
Author(s):  
B. C. S. Leão ◽  
N. A. S. Rocha ◽  
M. F. Accorsi ◽  
É. Nogueira ◽  
G. Z. Mingoti
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Cellular Proliferation ◽  
Mitochondrial Respiratory Chain ◽  
Organic Peroxide ◽  
Redox Status ◽  
Culture Period ◽  
Bovine Embryo ◽  
Embryo Survival

The production of reactive oxygen species (ROS), such as superoxide anion (O2–), hydroxyl radical (OH–), hydrogen peroxide (H2O2) and organic peroxide, is a normal process that occur in the cellular mitochondrial respiratory chain (Morado et al. 2009 Reprod. Fert. Dev. 21, 608–614). Supplementation with antioxidants during in vitro culture (IVC) appears to increase the resistance of bovine embryos to the oxidative stress, and consequently improve embryo development and cryotolerance (Rocha et al. 2011 Reprod. Fert. Dev. 23 157–158). This study was conducted to evaluate the effects of period of supplementation with intra (cysteine, CIST) or extracellular (catalase, CAT) antioxidants during IVC on embryo development and cryotolerance. Cumulus–oocyte complexes (n = 1132) were maturated for 24 h in B199 medium, at 38.5°C and 5% CO2 in air. After fertilization (Day 0), zygotes were IVC for 7 days in SOF medium (0.5% BSA + 2.5% FCS) in 7% O2, 5% CO2 e 88% N2 atmosphere, at 38.5°C. The antioxidant supplementation was performed during all of the culture period (from Day 1 to Day 7) or during the first 72 h (from Day 1 to Day 3), with 0.6 mM CIST, 100 UI CAT or without antioxidants (CONTR). The cleavage and blastocyst rates were evaluated, respectively, at 72 and 168 h post-insemination, when expanded blastocysts grade I were vitrified (n = 91) by Vitri-Ingá® protocol (Ingámed®, Maringá, PR, Brazil). Then, they were thawed and cultured for 24 h to evaluate re-expansion rates. The differences between groups were analyzed by ANOVA followed by Tukey’s test, and re-expansion rates by chi-square test (P ≤ 0.05). The cleavage and blastocyst rates were, respectively, 83.52 ± 4.52a/36.19 ± 3.21a (CONTR), 79.16 ± 4.52a/38.08 ± 3.21a (CIST Day 3), 77.74 ± 4.52a/42.09 ± 3.21a (CAT Day 3), 73.57 ± 4.05a/11.15 ± 2.87b (CIST Day 7), 71.83 ± 4.05a/15.07 ± 2.87b (CAT Day 7). The embryo re-expansion rates were 90.00%a (CONTR), 93.33%a (CIST Day 3), 75.00%a (CIST Day 7), 63.64%a (CAT Day 3) and 75.00%a (CAT Day 7). Supplementation with antioxidants for 7 days of IVC impaired embryo development, compared with addition up to Day 3 (P ≤ 0.05). However, it did not affect in vitro embryo cryotolerance (P ≥ 0.05). Supplementation with antioxidants throughout all the IVC significantly impaired blastocyst rate, probably by exerting a toxic effect leading to an arrest of embryonic development. It is believed that prolonged culture in the presence of antioxidants results in excessive reduction of ROS leading to an imbalance of the cellular redox status. It is known that ROS, particularly H2O2, act on signaling pathways involved in the cellular proliferation and differentiation, in gene expression and metabolism during embryo development. Supplementation with antioxidants up to Day 3 did not differ from CONTR, probably due to low O2 tension, and the presence of antioxidants in FBS and BSA. In conclusion, supplementation with cysteine and catalase during all of the culture period impaired embryo development, however this reduction did not affect embryo survival after vitrification. Financial support was provided by FAPESP (#2011/18257-2). The authors acknowledge Ingámed, Alta Genetics Brazil.

111 EQUINE AMNIOTIC EPITHELIAL OR BONE MARROW MESENCHYMAL STEM CELLS DIFFERENTLY SUPPORT IN VITRO EMBRYO DEVELOPMENT IN A BOVINE IN VITRO CULTURE MODEL

2009 ◽  
Vol 21 (1) ◽  
pp. 156 ◽  
Author(s):  
F. Cremonesi ◽  
V. Maggio ◽  
A. Lange Consiglio
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
In Vitro Culture ◽  
Calf Serum ◽  
Biologically Active ◽  
Bovine Embryo ◽  
Epithelial Stem Cells ◽  
Marrow Mesenchymal Stem Cells

There are indications that the culture system and the medium composition can affect embryo quality. In fact, various studies have been shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In light of this, recently, some studies used co-culture with mouse embryonic fibroblasts in the effort to improve the development of bovine and ovine in vitro-derived embryos. Despite the progress in equine IVM and ICSI technologies and the different culture conditions reported for preimplantation development of ICSI fertilized horse oocytes, the yield of blastocysts remained low. In the present study we investigated the benefits of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSC) or equine amniotic epithelial stem cells (AE-SC) on blastocyst development. This study employed the bovine embryo as a model and represents the initial step towards standardization of a protocol for the culture of equine embryos in our laboratory. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and layered over Hystopaque™ 1.080, then centrifuged for 20 min at 400g and 4°C. Cell pellets were resuspended in 10 mL Dulbecco Modified Earle’s Medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, penicillin (100 U mL–1) and streptomycin (100 μg mL–1) and seeded in 24-well plates. Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fractions were incubated at 37°C with 0.05% trypsin for 45 min. Separated AE cells were plated on 25 cm2 flask in standard culture media containing 10 ng mL–1 epidermal growth factor. Seven hundred fifty cumulus–oocyte complexes with a homogeneous cytoplasm and two or more layers of cumulus cells were used. After IVM and IVF cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured for up to Day 7: 1) co-culture with granulosa cells (control); 2) co-culture with BM-MSC; 3) co-culture with AE-SC. The culture medium was TCM 199 + 10% fetal bovine serum, pyruvate and gentamicin at 38.5°C in 5% CO2. Statistical analyses was performed by chi square test. Blastocysts developmental rates were similar among control, AE-SC and BM-MSC (35%, 41% and 30%, respectively), but the co-culture with AE-SC gave a significantly greater percentage of blastocysts compared to BM-MSC (P < 0.05). In conclusion, despite the absence of a significant increment in blastocysts attainment using stem cells as feeders for embryo culture, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSC. It can be suggested that these cells secrete biologically active substances including signaling molecules and growth factors of epithelial nature different from those of the BM cells of mesenchymal origin. Regione Lombardia is acknowledged for the “Dote Ricercatori” fellowship to V.M.

149 EFFECT OF RESVERATROL ANALOGUE ON DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 183 ◽  
Author(s):  
T. A. Patrocínio ◽  
C. A. C. Fernandes ◽  
L. S. Amorim ◽  
J. R. Ribeiro ◽  
G. C. Macedo ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Culture Medium ◽  
Synthetic Analogue ◽  
Bovine Embryo ◽  
High Humidity ◽  
Cleavage Rate ◽  
Antioxidant Agents ◽  
Blastocyst Rate

Oxidative stress is one of the main effects of in vitro culture. Generation of reactive oxygen species (ROS) by embryos can be enhanced by the sub-optimal in vitro culture conditions and are associated with a delay in embryonic development. However, supplementation of culture medium with antioxidant agents can minimize the effects of ROS (Guérin et al. 2001 Hum. Reprod. Update 7, 175–189). Resveratrol is an example of a potent antioxidant, and modifications in its structure can improve its biological activity. This study evaluated the effect of AR33 (formula with patent pending), an analogue of resveratrol with high antioxidant activity, on embryo development. Bovine cumulus-oocyte complexes recovered from ovaries collected at the slaughterhouse were in vitro matured for 24 h and oocytes were in vitro fertilized for 20 h, both at 38.8°C under 5% CO2 in air and high humidity. Partially denuded presumptive zygotes were randomly distributed in 4 treatments (with 6 replicates): 0 µM (control, n = 347), 0.1 µM (n = 337), 0.5 µM (n = 277), and 2.5 µM (n = 343) of AR33. The base medium was SOFaa supplemented with 2.5% FCS and incubation conditions were 38.8°C under 5% CO2 in air and high humidity. Half of culture medium was renewed (feeding) at Day 3 and 5 post-fertilization. Cleavage was evaluated at Day 3 and blastocyst rates at Day 7 and 8 post-fertilization. Data were analysed by logistic regression considering the significance level of P < 0.05. Values are shown as mean ± SEM. Cleavage rate was higher (P < 0.05) for 2.5 µM (69.0 ± 4.4%) than for 0, 0.1, and 0.5 µM AR33 (62.1 ± 2.0%, 60.7 ± 5.9%, and 56.7 ± 5.8%, respectively). At Day 7, the blastocyst rate was similar (P > 0.05) among 0.1, 0.5, and 2.5 µM (18.1 ± 5.4%, 17.5 ± 2.9%, and 19.4 ± 3.3%, respectively) and all of them were higher (P < 0.05) than 0 µM AR33 (12.4 ± 2.5%). At Day 8, there was again no difference (P > 0.05) among 0.1, 0.5, and 2.5 µM AR33 (21.0 ± 5.0%, 18.4 ± 2.1%, and 24.6 ± 3.3%, respectively) but only 0.1 and 2.5 µM showed higher (P < 0.05) blastocyst rate than 0 µM AR33 (15.2 ± 2.5%). In conclusion, the synthetic analogue of resveratrol tested in this study can improve bovine embryo development in culture medium supplemented with 2.5% FCS under 5% CO2 in air. A concentration of 2.5 µM AR33 can be a choice for further studies. This study was supported by Fapemig, CAPES, and CNPq.

Varied approach of using MSCs for bovine embryo in vitro culture

2018 ◽  
Vol 30 (2) ◽  
pp. 105-112 ◽  
Author(s):  
Jolanta Opiela ◽  
Bülent Bülbül ◽  
Joanna Romanek
Keyword(s):  
In Vitro Culture ◽  
Bovine Embryo

Consequences of exposure to serum, with or without vitamin E supplementation, in terms of the fatty acid content and viability of bovine blastocysts produced in vitro

2003 ◽  
Vol 15 (5) ◽  
pp. 275 ◽  
Author(s):  
A. Reis ◽  
J. A. Rooke ◽  
G. J. McCallum ◽  
M. E. Staines ◽  
M. Ewen ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Vitamin E ◽  
Acid Content ◽  
Fatty Acid Content ◽  
Calf Serum ◽  
Pyruvate Metabolism ◽  
Cell Counts ◽  
Serum Supplementation ◽  
Development In Vitro

To determine whether serum supplementation influenced fatty acid content of bovine blastocysts and whether vitamin E addition to culture medium containing serum could improve development in vitro, cleaved eggs were cultured in synthetic oviduct fluid supplemented with bovine serum albumin (BSA, 0.4% w/v, fraction V) (SVBSA), fetal calf serum (FCS, 10% v/v) (SFCS) or FCS (10% v/v) plus 100 μM vitamin E (SFCS + E). Blastocyst yields were recorded and fatty acid composition was determined by gas chromatography. Day 7 blastocysts were incubated with [2-14C] pyruvate for 3 h and then fixed for cell counts. Yields of good quality blastocysts were greatest from cleaved eggs cultured in serum-free conditions (P < 0.01). In the presence of serum, supplementation with vitamin E increased both total and good quality blastocyst yields (P < 0.01). Presence of serum increased fatty acid content (mean ± SEM) of blastocysts (SVBSA v. SFCS = 57 ± 2  v. 74 ± 2 ng embryo−1; P < 0.001). In contrast, pyruvate metabolism was greater in blastocysts produced without serum (27 ± 3 v. 21 ± 3 picomoles embryo−13 h−1; P < 0.01) but, on a per cell basis, no differences were detected. Addition of vitamin E to the serum-supplemented formulation did not alter either the fatty acid content (73 ± 2 ng embryo−1) or pyruvate metabolism index (19 ± 1 pmol embryo−13 h−1) of SFCS + E blastocysts. Thus, despite lipid accumulation, supplementary vitamin E improved blastocyst yields in embryos exposed to serum.

scholarly journals Cytokines That Serve as Embryokines in Cattle

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2313
Author(s):  
Alan D. Ealy ◽  
Savannah L. Speckhart ◽  
Lydia K. Wooldridge
Keyword(s):  
Embryo Development ◽  
Interleukin 6 ◽  
Fetal Development ◽  
Embryo Quality ◽  
Bovine Embryo ◽  
Colony Stimulating Factor ◽  
Bovine Embryos ◽  
The Poor ◽  
Stimulating Factor

The term “embryokine” has been used to denote molecules produced by the endometrium, oviduct, or by embryo itself that will influence embryo development. Several cytokines have been identified as embryokines in cattle and other mammals. This review will describe how these cytokines function as embryokines, with special emphasis being placed on their actions on in vitro produced (IVP) bovine embryos. Embryokines are being explored for their ability to overcome the poor development rates of IVP embryos and to limit post-transfer pregnancy retention efficiencies that exist in IVP embryos. This review will focus on describing two of the best-characterized cytokines, colony-stimulating factor 2 and interleukin 6, for their ability to modify bovine embryo quality and confirmation, promote normal fetal development, and generate healthy calves. Additional cytokines will also be discussed for their potential to serve as embryokines.

213 THE EFFECTS OF SERICIN SUPPLEMENTATION IN IN VITRO CULTURE MEDIUM ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED - IN VITRO-FERTILIZED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 205 ◽  
Author(s):  
Y. Inaba ◽  
M. Hosoe ◽  
H. Teramoto ◽  
M. Geshi
Keyword(s):  
In Vitro Culture ◽  
Survival Rates ◽  
Calf Serum ◽  
Protein Supplement ◽  
Bovine Embryo ◽  
Blastocyst Formation ◽  
Freezing And Thawing ◽  
Exact Test ◽  
Alternative Protein

The objective of this study was to investigate the feasibility of the silk protein sericin as an alternative protein supplement for bovine embryo cultures. The effects of sericin supplementation in in vitro culture (IVC) medium on the development and cryosurvival of bovine IVM-IVF embryos were investigated. Cumulus–oocyte complexes were collected from 2- to 8-mm follicles of ovaries obtained from a local abattoir. They were matured for 20 to 22 h in TCM-199 supplemented with 5% fetal bovine serum (FBS), 0.002 AU mL–1 of FSH, and 1 μg mL–1 of oestradiol-17β at 38.5°C under an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in SOF medium (IVC medium) containing amino acids and supplemented with either 5% FBS (FBS group: n = 400) or sericin at 3 different concentrations (wt/vol; 0.05%: n = 493; 0.1%: n = 419; or 0.15%: n = 520; sericin groups) at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2 for 5 days. They were then transferred into each IVC medium supplemented with 0.1 mM β-mercaptoethanol and cultured for an additional 4 days (9 days in total). Cleavage rates were recorded on Day 2 of IVC. The excellent expanded blastocysts harvested on Days 7 and 8 were used for freezing (FBS group: n = 51, 0.05% sericin group: n = 56, 0.1% sericin group: n = 44, 0.15% sericin group: n = 44). They were frozen in m-PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose, 20% calf serum, and 4 mg mL–1 of BSA. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol under the same atmosphere used for IVC for 72 h. Rates of the embryos that reexpanded and developed to the hatching and hatched blastocyst stages were determined at, respectively, 24, 48, and 72 h after thawing. Rates of cleavage and blastocyst formation were expressed as mean ± SD and were analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by Fisher’s exact test and chi-square test. Four replications were performed. Cleavage and blastocyst formation rates did not differ among the groups (FBS: 61.6 ± 15.1 and 22.1 ± 3.5%; 0.05% sericin: 71.6 ± 12.0 and 19.4 ± 6.7%; 0.1% sericin: 70.3 ± 4.7 and 18.4 ± 5.6%; 0.15% sericin: 66.9 ± 10.3 and 17.9 ± 6.9%, respectively). There were no significant differences in post-thaw survival rates after 24 h among the FBS (88.2%), 0.05% sericin (92.9%), 0.1% sericin (84.1%), and 0.15% sericin (93.2%) groups. However, post-thaw survival rates after 72 h in the 0.05% sericin (83.9%) and FBS (82.4%) groups were significantly higher than those in the 0.1% sericin (56.8%) and 0.15% sericin (61.4%) groups (P < 0.05). These results indicate the feasibility of sericin as an alternative protein supplement for bovine embryo culture. Additionally, in this study, 0.05% sericin was shown to be the best concentration for survival of the resultant embryos after freezing and thawing.

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.

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

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

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

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