Transition of cell numbers in bovine preimplantation embryos: in vivo collected and in vitro produced embryos

The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0.16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos.     

Growth and metabolism of murine and bovine embryos in bovine uterine flushing-supplemented culture media.

The aim of this study was to compare the development and metabolic activity of cultured murine and bovine embryos in 2 standard media (HAM F-10 and RPMI) in the presence or absence of bovine uterine flushings. Murine morulae (n = 653) and day 7 bovine embryos (n = 273) were cultured for 18 h or 36 h in either HAM F-10 or RPMI in the presence or absence of bovine uterine flushings. After culture, the development, quality, and metabolic activity (glucose utilization or methionine uptake and incorporation) of embryos was assessed. It was found that HAM F-10 (without uterine flushings) was a more suitable medium than RPMI for optimal development and metabolism of murine and bovine embryos. Poor quality and development, as well as decreased metabolism, were evident after culture of murine embryos in RPMI; in contrast, this medium had no adverse effects on bovine embryos in culture. Supplementation of HAM F-10 with bovine uterine flushings improved the growth of murine embryos and the protein synthesis (as measured by an increased methionine incorporation) for both murine and bovine embryos. However, supplementation with bovine uterine flushings could not overcome deficiencies of an inappropriate medium (RPMI) for murine embryos. Supplementation of a well-defined culture medium with uterine flushings increased metabolism of embryos in culture, and thus might help to increase pregnancy rates after transfer of such embryos to recipient cows.     

Stage-specific effect of growth hormone on developmental competence of bovine embryos produced in-vitro

Many efforts have been made to develop effective culture conditions for the production of bovine blastocysts. Growth hormone (GH) and glucose are known to affect in vitro embryo development. To improve in vitro culture conditions, the culture medium containing fetal calf serum (FCS) or bovine serum albumin (BSA) was supplemented with GH at various periods of development, and the effects of GH on the rate of development and the quality of the blastocysts were studied. Then, starting at the morula stage, the effect of glucose and GH on the rate of development was studied. In all experimental periods, FCS was more effective than BSA at improving the development rate and increasing the cell number of blastocysts. Adding GH to the culture medium between 18 and 48 h after fertilization (1-8 cell stage embryo) did not affect either the rate of blastulation or the cell number regardless of the serum protein (FCS or BSA). From 48 to 120 h after fertilization (5-cell to morula stage) GH increased the cell number of the blastocysts in the presence of BSA, but not in the presence of FCS. From 120 to 192 h after fertilization (morula to blastocyst stage), GH improved the developmental rate and cell number in the presence of FCS, although there was no significant difference when BSA was used instead of FCS as the serum protein. When cows were implanted with blastocysts developed in the presence of GH from the morula stage, their pregnancy rate did not differ from that of the control. Increasing the glucose concentration in the medium from 1.5 mM to 3 mM starting at the morula stage (120 h after fertilization) slightly decreased the rate of development, but on the other hand, decreasing the glucose concentration to 0 mM did not affect either the rate of development or the cell number. Also, then GH had no effect on the developmental rate or the cell number in the absence of glucose. In conclusion, when the medium was supplemented with serum, GH improved embryo development from the morula stage, but an increased concentration of glucose decreased embryo development. Furthermore, GH did not improve the pregnancy rate of blastocysts developed in vitro.     

Evaluation of bovine viral diarrhea virus uptake by preimplantation embryos

Bovine embryos were exposed to bovine viral diarrhea (BVD) virus in vitro. An uptake of BVD virus by the embryos could not be detected by several assay systems. A significant decrease in the titer of BVD virus was found to occur when the virus was incubated in saline solution + 5% goat serum or minimal essential medium + 5% goat serum for 24 hours at 37 C. Since there was significant inactivation of the BVD virus during the incubation period, lack of viral infectivity of the embryos may have been due to adverse effects of the experimental environmental conditions on the virus or the embryos or upon viral-embryo interaction.     

Comparison of various cryopreservation media for vitrification of 7-day bovine embryos]

A vitrification medium, attested for cryopreservation of mouse eight-blastomere embryos (6.85 mol/l glycerol as a cryoprotective agent), was checked up with respect to preservation of bovine seven-day morulas and blastocysts. As this medium was not found to be convenient either as to its technical parameters or the reached embryo survival, its composition was modified. The glycerol content was complemented or partly replaced by other cryoprotectives (methanol, saccharose, L-proline). A comparison of technical parameters and viability of rewarmed embryos shows that our requirements (technically simple technique of freezing and rewarming and good survival) are met in the best way by a cryoprotective combination of 4.11 mol/l glycerol and 1.0 mol/l saccharose. The total of 54.5% embryos developed in-vitro conditions following vitrification in the medium of this composition and after rewarming.     

Reduced quality of bovine embryos cultured in media conditioned by exposure to an inflamed endometrium

OBJECTIVE: A suboptimal uterine environment contributes to the bovine repeat breeder syndrome. Subclinical endometritis is a component, so the mechanism by which inflammation affects embryo survival was investigated by assessing the effect of non-cellular products of an inflamed endometrium on embryo development to blastocyst. DESIGN: Endometrial fluid from a lactating dairy cow was collected by lavage using embryo culture medium. Aseptic inflammation was then induced by infusion of glycogen and lavage was repeated 6 h later. The recovered fluid was used to culture Day 5 in-vitro-derived embryos for 2 days. Embryo development and quality were compared for two treatment groups (culture media conditioned by inflamed or non-inflamed endometrium) and two controls (control or control + serum). RESULTS: Development to blastocyst was higher for conditioned media or media + serum (inflamed 42.2%; non-inflamed 49.3%; control + serum 50.9%; control 16.9%; P < 0.001). Blastocyst cell numbers were lower for the conditioned-inflamed group (inflamed, 83.1; non-inflamed, 99.8; control + serum, 100.6; control, 110.1; P < 0.001). Trophectoderm cell number, but not inner cell mass number, was reduced in the conditioned-inflamed group and the inner cell mass:trophectoderm ratio was increased (P < 0.001). CONCLUSION: Altering the embryo culture environment with the products of endometrial inflammation had a subtle effect on embryo quality. An increased inner cell mass:trophoblast ratio is likely to negatively affect embryonic survival. Altered embryo quality is a mechanism for early embryonic failure in cows with subclinical endometritis. Culture of embryos with normal endometrial fluid may improve in vitro embryo production.     

Culture system and long-term storage of culture media in the in vitro production of bovine embryos

The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed.     

Cryopreservation of bovine embryos

The method of cryopreservation of embryos aged seven days, proposed for embryo transfer with cattle by Niemann (1985), was tested under production conditions on three cattle breeding farms and three experimental animal units. The number of donors was 128, and 467 intact embryos were obtained from them and were cryopreserved in semen straw. Following thawing, 455 were recovered, and 439 (96.5 percent) of these were suitable for transfer. A pregnancy rate of 49.0 percent was recorded from 412 transfers. This rate was differentiated by oestric cycle conditions of heifer recipients, which gave percentages of 46.0 among recipients of seven-day old embryos, 45.7 for eight-day recipients, and 65.8 for six-day recipients. Related to pregnancy results recorded on the same units from transfer of freshly collected seven-day embryos, the efficiency coefficient was 0.69 (550 fresh transfers = 65.4 percent and 222 cryopreserved transfers = 48.2 percent). The method is recommended for general field practice.     

Nuclear transplantation in bovine embryos

This study was conducted to develop a method for transplanting nuclei in bovine embryos and to test the development of several stages of donor nuclei transplanted to enucleated pronuclear recipient embryos. Pronuclear embryos were centrifuged to reveal nuclei. Nuclei were removed without penetrating the plasma membrane as membrane-bound karyoplasts, and were inserted into enucleated zygotes by electrically induced cell fusion. The highest rate of fusion (79%) occurred in Zimmerman Cell Fusion medium at 100 V for 20 to 40 microseconds with the fusion membranes oriented parallel to the electrodes. The effect of nuclear transplantation on development was tested in pronuclear embryos in which nuclei were removed and reinserted and the embryos were then transferred to sheep oviducts for 5 d. Of the intact nuclear transplant embryos recovered, 5/29 (17%) developed to morulae or blastocysts compared with 11/30 (37%) of the non-manipulated embryos. Two nuclear transplant embryos were transferred to a recipient cow, and both developed to normal offspring. When nuclei from two-, four-, or eight-cell embryos were transplanted to pronuclear recipient embryos, no development was observed.     

Non-surgical transfer of bovine embryos

Bovine embryos obtained from donors six to nine days after oestrus were transferred non-surgically at a rate of one per recipient using a sterile insemination instrument, protected from contamination by the vagina with a plastic sheath. The percentage of recipients pregnant increased with the age of embryo transferred and for day 6 and 7 embryos was 33% compared to 58% for day 9 and 8 embryos. This difference approached statistical significance. Bacterial contamination of the instrument on withdrawal after transfer was not related to the success or failure of pregnancy. Maintenance of pregnancy to term and calving appeared to be normal. It is suggested that this method could be used for the routine transfer of eight and nine day embryos.     

Cytogenetic study of in vitro-derived bovine embryos

A cytogenetic study of early bovine embryos (2-16 blastomeres) produced in vitro was conducted to determine the incidence of embryos carrying chromosome anomalies. The embryos were produced from immature oocytes matured in vitro and fertilized by sperm prepared using the Percoll density gradient method. Slides were prepared according to an 'air drying' technique and the chromosomal complement of embryos was studied by Giemsa-staining. Approximately 57% of prepared embryos were suitable for analysis. The results revealed that 18% of cytogenetically analysed embryos presented chromosomal anomalies, including haploidy (8%), aneuploidy (2%) and polyploidy (8%). Our results were compared to the results of other studies in cattle and other domestic animals.     

Infection of early bovine embryos with bovine herpesvirus-1

Recently hatched bovine embryos were exposed in vitro to 1 of 4 strains of bovine herpesvirus-1 to determine whether the viruses would replicate in these embryos and, if so, what pathologic consequences would ensue. Exposure to each of the viruses resulted in embryonic infection and death, and replication of the agents was demonstrated by electron microscopy and titration of progeny virus. There were no dramatic differences between virus strains in pathogenicity or in the ultrastructural pathologic findings of infection.     

Evaluation of feeding spray-dried bovine plasma protein on production performance of laying hens exposed to high ambient temperatures

The purpose of this study was to evaluate feeding 2 levels of spray-dried bovine plasma protein (SDP) on production performance of laying hens subjected to high ambient temperatures. Two groups of 96 Hy-Line W-98 hens (38 wk of age) were housed in each of 2 environmentally controlled chambers. At 40 wk of age, all hens were fed 3 diet treatments consisting of (1) a control diet (0% SDP); (2) the control diet supplemented with 0.75% SDP; and (3) the control diet supplemented with 1.50% SDP. Hens in each chamber (8 cages of 4 hens per cage) were ad libitum fed 1 of each diet for 5 wk. The heat stress (HS) chamber was maintained at 21°C (wk 1), 29°C (wk 2), and 35°C (wk 3 to 5). The thermoneutral chamber was maintained at 21°C during wk 1 to 5. A significant main effect of week was observed for hens maintained in the HS chamber for egg production, egg weight, egg mass, and feed consumption, which resulted in acute heat stress causing a reduction in these parameters. Hens fed the 1.50% SDP diet in the HS chamber produced greater (P < 0.05) egg mass on average than hens fed the control or 0.75% SDP diet (wk 1 to 5). During the second week of acute HS (wk 4), hens fed the control and 1.50% SDP diets had greater (P < 0.05) egg production than those fed the 0.75% SDP diet. During wk 5, hens in the HS chamber that were fed the 1.50% SDP diet produced more (P < 0.05) eggs than those fed the control diet. Therefore, based on the results of this study, acute HS negatively affected short-term production performance. In addition, feeding hens an SDP-supplemented diet may have a slight positive effect on production performance when maintained in acute HS conditions.     

Resveratrol–cyclodextrin complex affects the expression of genes associated with lipid metabolism in bovine in vitro produced embryos

Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol–methyl β‐cyclodextrin (RV ‐CD ) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV ‐CD during in vitro oocyte maturation (IVM ) or in vitro embryo culture (IVC ) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV ‐CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT ‐qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV ‐CD and embryo development and blastocysts gene expression by RT ‐qPCR were studied. A group without RV ‐CD (control^?) and a group with cyclodextrin (control⁺) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p  <  .05). Blastocysts produced by IVC with resveratrol showed that RV ‐CD could modify the expression of genes related to lipid metabolism (CYP 51A1 , PNPLA 2 and MTORC 1 ) compared with control groups (p  < .05). RV ‐CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts.     

Susceptibility of bovine naked 2- and 4-cell embryos and hatched blastocysts produced in vitro to infection with noncytopathogenic bovine viral diarrhea virus.

To investigate the susceptibility of early bovine embryos to noncytopathogenic bovine viral diarrhea virus (NCP BVDV), 2- and 4-cell embryos produced in vitro from which zona pellucida had been removed by pronase treatment, and hatched blastocysts were exposed to 10(6) TCID50/m/ of NCP BVDV No. 12 strain. The virus was detected in all embryo samples immediately prior to cultivation but not in the medium. After 24-hr culture, the virus was isolated from four media and two embryo samples in four experiments in the blastocyst group, and the viral antigen was demonstrated in the cytoplasm of the embryo cells by the immunofluorescent technique. By contrast, no virus was recovered from, or viral antigen detected in samples from the 2- and 4-cell embryo group in any of the experiments, even though they were exposed to the virus after removal of the zona pellucida. These findings suggest that 2- and 4-cell embryos are unlikely to be susceptible to NCP BVDV, but that blastocysts are capable of being infected with the virus. hatched blastocyst, noncytopathogenic bovine viral diarrhea virus.     

Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro

The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (P<0.05 and P<0.01). In conclusion, both FCS and BSA supplementation can be useful for repairing cultures of bovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.     

Proteomic profile of pre-implantational ovine embryos produced in vivo

The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan ( http://www.targetscan.org ) and mIRBase ( http://www.mirbase.org ) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.