Cow body condition affects the hormonal release of ovarian cells and their responses to gonadotropic and metabolic hormones.

The body condition score (BCS) of cows affects their reproductive efficiency, but the underlying mechanism is unclear. We examined the effect of BCS on the basic ovarian cell functions and their responses to gonadotropic and metabolic hormones. We isolated ovarian cells from cows with a tendency toward emaciation (BCS2) and those with an average body condition (BCS3), and we compared their hormonal release and responses to FSH, leptin, ghrelin, and neuropeptide Y (NPY) added at doses of 0, 1, 10, or 100 ng/mL. Progesterone, testosterone, estradiol, and insulin-like growth factor I (IGF-I) release were evaluated by RIA. No differences were found in progesterone or testosterone release between BCS2 and BCS3 cells; however, ovarian cells from BCS2 cows released more estradiol and IGF-I than cells from BCS3 cows. FSH, ghrelin, and NPY promoted progesterone release in BCS2 cells but had no stimulatory or inhibitory effect on BCS3 cells. In contrast, leptin promoted progesterone release in BCS3 cells and inhibited progesterone release in BCS2 cells. FSH also promoted testosterone release in both BCS2 and BCS3 cells but inhibited progesterone at a low dose in BCS3 cells. Leptin inhibited testosterone release in BCS3 cells but not in BCS2 cells. Estradiol release was promoted by leptin and ghrelin in BCS3 cells; however, it was unaffected by leptin and inhibited by ghrelin in BCS2 cells. IGF-I production was promoted by FSH and inhibited by leptin in both groups. Ghrelin suppressed IGF-I release in BCS2 cells and increased IGF-I release in BCS3 cells. NPY promoted IGF-I release in BCS2 cells but not in BCS3 cells. Our results demonstrate the effects of BCS on ovarian cell estradiol and IGF-I (but not progesterone or testosterone) release, as well as on the responses of ovarian cells to FSH, leptin, ghrelin, and NPY.

Ultrastructure of Cell Organelles in Pre-implantation Embryos from Cows with Different Body Condition Score.

Morphology of important cell organelles (mitochondria, lipid droplets, vacuoles, inclusion bodies and apoptotic bodies) in embryos derived from cows with different body condition score (BCS) was analysed by transmission electron microscopy (TEM). Embryos were recovered on 7th day after the insemination by a standard non-surgical flushing of the uterine horns from superovulated Holstein Friesian cows with BCS 2, 3, 4 and 5. Thereafter, the good quality blastocysts were processed for TEM. The electronograms were evaluated by stereological analysis. The relative volume of lipid droplets in BCS4 and BCS5 embryos increased significantly (18.53 and 22.40%) when compared to BCS3 embryos (5.46%). In the embryos from the BCS4 or BCS5 cows, we observed different morphological patterns of mitochondria, as well as the mitochondria containing vacuoles. BCS4 and BCS5 embryo cell nuclei showed the structure typical for low transcription activity (none or very few reticular nucleoli); also dilated inter-cellular spaces were often observed in these embryos. In conclusion, differences in the ultrastructural morphology of embryos from over-conditioned cows (BCS4 and BCS5), particularly the higher lipid content in the cytoplasm, can be a marker of their low quality, and this fact can be a contributing factor to subfertility in over-conditioned cows.

Quality of preimplantation embryos recovered in vivo from dairy cows in relation to their body condition.

This study examined the impact of cow body condition on the quality of bovine preimplantation embryos. The embryos (n = 107) were flushed from dairy cows and classified according to a five-point scale body condition score (BCS2 n = 17; BCS3 n = 31; BCS4 n = 11) on the 7th day after insemination and then analyzed for development, dead cell index (DCI), cell number and actin cytoskeleton quality. The highest embryo recovery rate (P < 0.05) was recorded in the BCS3 group and the lowest in the BCS4 group. More transferable (morula, blastocyst) embryos were obtained from the BCS4 cows (79%), compared with the BCS2 (64%) or BCS3 (63%) animals. However, cell numbers were higher in the BCS2 and BCS3 groups (P < 0.05) compared with the BCS4 embryos. Conversely, the DCI was lowest in the BCS2 (3.88%; P < 0.05) and highest in the BCS4 (6.56%) embryos. The proportion of embryos with the best actin quality (grade I) was higher in the BCS2 and BCS3 cows compared with the BCS4 group. Almost 25% of all embryos showed fragmented morphology and a higher DCI (5.65%) than normal morulas (1.76%). More fragmented embryos were revealed in the BCS2 (28.6%) and BCS4 (31.25%) groups, and less (19.15%) in the BCS3 group. The cell numbers in such embryos were lower in the BCS4 (22.57) than in the BCS2 (46.25) or BCS3 (42.4) groups. In conclusion, the body condition of dairy cows affects the quality of preimplantation embryos. A BCS over 3.0 resulted in a higher incidence of poor (fragmented) embryos.

Expression of adrenergic receptors in bovine and rabbit oocytes and preimplantation embryos.

Catecholamines play an important role in embryogenesis, and data obtained in the rodent model indicate that they can act even during the preimplantation period of development. Using RT-PCR with specific oligonucleotide primers distinguishing among all members of the adrenergic receptor family, we examined expression of adrenergic receptors in bovine and rabbit oocytes, morulas and blastocysts. We found several profiles of adrenoceptor mRNA expression. Transcripts for some receptor subtypes (bovine alpha 2 receptors, rabbit α2A, α2C, ß1 and ß2 receptors) were detected at all examined stages, which suggests receptor expression throughout (or at most stages) the preimplantation developmental period. Expression in oocytes but not at later stages was found in only one adrenoceptor subtype (rabbit α1B). In contrast, mRNA for several adrenoceptors was found in embryos but not in oocytes (bovine beta adrenoceptors and rabbit α1A). Nucleotide sequences of our PCR products amplified in rabbit oocytes, and preimplantation embryos represent the first published mRNA sequences (partial sequences coding at least one transmembrane region) of rabbit α2C, ß1 and ß2 adrenoceptors. Our results suggest that the expression of adrenergic receptors can be a general feature of mammalian oocytes and preimplantation embryos. On the other hand, comparison of three mammalian species (cattle, rabbit and mouse) revealed possible interspecies differences in the expression of particular adrenoceptor subtypes. Our results support the opinion that stress mediators can act directly in cells of preimplantation embryos.

Ultrastructure of vitrified rabbit transgenic embryos.

The aim of the study was to determine the viability of rabbit transgenic (enhanced green fluorescent protein (EGFP)-positive) embryos cultured in vitro and compare with gene-microinjected (Mi) non-transgenic (EGFP-negative) embryos following vitrification. Non-microinjected and non-vitrified embryos were used as the control. Morphological signs of injury to embryo organelles were determined at the ultrastructural level using transmission electron microscopy (TEM). Morphometric evaluation was performed on cellular organelles using microphotographs obtained by TEM. Intact and Mi embryos recovered from in vivo fertilized eggs at 19-20 hours post coitum (hpc) were cultured for up to 72 hpc (morula stage), evaluated for the EGFP gene integration and then vitrified in 0.25 ml insemination straws in modified EFS (40% ethylene glycol + 18% Ficoll 70 + 0.3 M sucrose) vitrification solution. After 1-3 days the embryos were devitrified, a representative selection of embryos was analyzed by TEM and the remaining embryos were subjected to additional in vitro culture. Observations by TEM showed that the vitrified/warmed EGFP-positive and EGFP-negative embryos had a slight accumulation of cellular debris and lipid droplets compared with the control intact embryos. More severe changes were detected in the membrane structures of the treated embryos, mostly in the cytoplasmic envelope, trophoblastic microvilli, junctional contacts and mitochondria. We suggest that the higher proportion of deteriorated cell structures and organelles in the treated embryos may be due to the vitrification process rather than to mechanical violation (the gene-microinjection procedure), as a detailed inspection of ultrastructure revealed that most damage occurred in the cell membrane structures.

[Comparative characteristics of using isoflurane, sevoflurane and target concentration of propofol during reconstructive operations on the carotid arteries].

GOAL OF THE STUDY: To compare effects of isoflurane, sevoflurane and target concentration of propofol on the systemic hemodynamics, cerebral blood flow and cerebral oximetry of the brain during the carotid endarterectomy. MATERIALS AND METHODS: We studied 95 patients. The patients were divided into 3 groups. Group I included 26 patients who received isoflurane (under I MAC), Group II--40 patients who received sevoflurane (under I MAC), Group III--29 patients who received target concentration of propofol (under 4 mkg/ml) according to the method of Schneider Studied parameters were defined at the stages: before the operation (I), after the induction (II), after the intubation (III), during the separation of the carotid artery (IV), after the crossclamping of the carotid artery (V), before starting the bloodstream (VI), after starting of the bloodstream (VII), after the end of the operation (VIII). RESULTS: At the first stages of the operation, the using of isoflurane, sevoflurane and propofol was accompanied with moderate dose-dependent lowering of indicators ofcirculatory dynamics. The linear blood flow velocity (LBFV) in the middle cerebral artery on the affected side in the groups of isoflurane and propofol did not depend on the indicators ofcirculatory dynamics; in the sevoflurane group the correlation was traced During the breakoff of the blood circulation in the reconstructed carotid arteries while using the anesthesia of isoflurane, sevoflurane and propofol hemodynamics was stable. LBFV and cerebral oximetry (CO) in the groups of isoflurane and propofol did not depend on the systemic hemodynamics; in the sevoflurane group--they depended After the reinitiating of the bloodstream in the conditions of the isoflurane andpropofol anesthesia the reperfusion of the brain was moderate; in the conditions of the sevoflurane anesthesia the risk of reperfusion damage of the brain during the uncontrolled hypertension remained. At the stage of finishing the operation LBFV and CO did not depend on the systemic hemodynamics in the isoflurane and propofol groups, in the sevoflurane group the dependence was indicated. Consequently, at all the stages of the operation we indicated the disorder of the mechanisms of the brain blood supply autoregulation in the sevoflurane group.

Ultrastructure of rabbit embryos exposed to hyperthermia and anti-Hsp 70.

The aim of the study was to determine the effect of short-term hyperthermia and Hsp70 blockage on ultrastructural changes in cell organelles and nucleoli of rabbit preimplantation embryos. The embryos were cultured either at 37.5°C (control, C) or 41.5°C (hyperthermia, HT) during 6 h. The antibody against Hsp70 was added into the culture medium (4 µg/ml) of morula stage embryos from C and HT groups. After termination of the culture, the embryos were processed for transmission electron microscopy. The embryos exposed to hyperthermia showed increased volume of lipid droplets, considerable occurrence of cellular debris in the perivitelline space and slight changes in the occurrence of microvilli on the surface of trophoblastic cells. In the embryos exposed to anti-Hsp 70 at 37.5°C, there were considerable changes in mitochondria morphology, decreased volume of dense bodies in the cytoplasm and considerable changes in the occurrence of microvilli on the surface of trophoblastic cells. In the group of embryos exposed simultaneously to hyperthermia and anti-Hsp 70, mitochondria were also expanded and swollen; the volume of flocculent vesicles and lipid droplets was increased and the volume of dense bodies in the cytoplasm was diminished. General organization of the cytoplasm in groups with anti-Hsp70 was characterized by cell organelle segregation. Averaged size of the nucleolar area was significantly increased in the embryos exposed to hyperthermia, whereas in the group exposed to the anti-Hsp70 without hyperthermia it was significantly diminished. Hyperthermia also caused disintegration of compact status of the nucleoli. In presence of anti-Hsp 70, the structural changes, described within the nucleoli during hyperthermia, were not observed. In conclusion, these results document ultrastructural changes in cell organelles of rabbit preimplantation embryo caused by hyperthermia, and also changes in the nucleolar structures, at which presence of Hsp-70 inhibit these changes.

Quality of rabbit vitrified/thawed transgenic embryos.

The aim of our study was to investigate the influence of vitrification on developmental rate and quality (total number of cells, number of blastomeres in inner cell mass (ICM) area, apoptotic index and embryo diameter) of transgenic (carrying an endogenous-hFVIII or exogenous-enhanced green fluorescent protein (EGFP) gene) rabbit embryos. EGFP-positive rabbit embryos were produced under in vitro conditions by the microinjection of foreign genes into the pronucleus of fertilized eggs. The transgenic rabbit embryos with the hFVIII gene were produced by mating homozygous transgenic rabbits and flushing at the single-cell stage. Developmental rate of vitrified/thawed transgenic embryos that reached hatching blastocyst stage (68.00% and 69.00%) and differed significantly (p < 0.001) from those in control embryos (100.00%). Significant difference (p < 0.05) was found in total cell counts between control (117.00 ± 36.00) and vitrified (141.00 ± 34.80) hFVIII-positive embryos. The higher proportion of ICM cells (32.00%) and greatest embryo diameter (130.85 ± 10.90) were found in the control group compared with the transgenic. Ratio of apoptotic cells was significantly higher (p < 0.01) in the control group (2.50%) and vitrified EGFP-positive embryos (2.90%) compared with the vitrified, hFVIII-positive group of embryos (0.70%). Our results demonstrate that neither gene microinjection itself, nor exogenous (EGFP) and endogenous (hFVIII) gene expression interferes with developmental rate and quality of rabbit embryos. However, a combination of microinjection and vitrification significantly decreases (p < 0.001) the survival rate of rabbit embryos.

Histopathological alterations in the antral ovarian follicles in dairy cows with a tendency to emaciation.

The aim of the study was to define interrelationships between histopathological alterations in ovarian antral follicles and body condition in dairy cows with a tendency to emaciation (BCS 1 and 2) compared with dairy cows with normal body condition (BCS 3). The ovaries were recovered from slaughtered cyclic dairy cows (at the luteal phase of the cycle) of Czech Fleckvieh and Holstein breeds at different times of the post-partum period. The animals were estimated as belonging to certain grade of body condition score (BCS) according to a 5-point scale. Only dairy cows with BCS1 (emaciation; n=6), BCS2 (tendency to emaciation; n=5) and BCS3 (optimal body condition status; n=6) were available for the experiment. The ovarian samples were embedded into Technovit 7100 resin; the tissue sections were stained with buffered basic fuchsine with toluidine blue. For acidic mucopolysaccharides (aMPS) a combination of PAS-technique with Alcian blue was used. Histological analysis showed that emaciation was associated with an increased occurrence of late (cystic) and luteinization-related atresia in granulosa and theca cells and increased levels of aMPS in small atretic follicles. Our observations indicate that dairy cows with a tendency to emaciation (BCS 2) or emaciated (BCS 1) have elevated occurrence of late atresia and atresia with luteinization, while initial atresia is less. This expands our basic knowledge of ovarian histopathology providing new insight into the association of antral follicle atresia and body condition status in dairy cows.

Viability and apoptosis in spermatozoa of transgenic rabbits.

The aim of our study was to compare the viability of sperm cells from transgenic (mWAP-hFVIII gene) or non-transgenic (normal) rabbit males as assessed by viability (SYBR-14/PI) and apoptosis (annexin V) tests. These results were evaluated using female conception rates following insemination with the respective sperm samples. No significant differences were found in concentration and motility between transgenic and non-transgenic spermatozoa. Spermatozoa from both transgenic (63.05 ± 20.05%) or non-transgenic (65.75 ± 22.15%) males, stained with SYBR-14 (green), were found to be morphologically normal. In both groups, the highest proportion of annexin V-positive sperm staining was found in the post-acrosomal part of the sperm head (8.66 and 27.53%). The percentage of sperm that stained with SYBR-14/PI or with annexin V/DAPI was correlated with liveborn in transgenic rabbits (R2 = 0.6118 and R2 = 0.2187, respectively) or non-transgenic rabbits (R2 = 0.671 and R2 = 0.3579, respectively). These data indicate that there was no difference in the viability of rabbit transgenic and non-transgenic spermatozoa when determined by both fluorescence assays.

Quality of transgenic rabbit embryos with different EGFP gene constructs.

The aim of this study was to compare the quality of rabbit transgenic embryos obtained upon microinjection of gene constructs containing different promoters and green fluorescent proteins (CMVIE-EGFP, PGK-EGFP and CMVIE-hrGFP). Developmental rate, total cell number in hatching blastocyst stage, number of apoptotic cells, diameter of embryos, transgene integration and transgenic mosaicism were investigated.The rate of rabbit embryos microinjected with the different gene constructs developed up to morula stage was significantly lower (p < 0.05) than that of intact (non-microinjected) rabbit embryos (66-74vs. 98%). The highest efficiency of transgene integration (15%) was found when the CMVIE-EGFP (DrdI) gene construct was used, however a significantly higher transgenic mosaicism (60%) was found in rabbit embryos using this gene. The lowest cell number was counted in rabbit transgenic embryos with CMVIE-rhGFP linearized by ScaI (115.0 ± 8.20), the highest cell number (134.0 ± 35.00) was detected in rabbit transgenic embryos carrying PGK-EGFP (Not I) gene. The highest number of apoptotic cells (2.6 ± 0.33) was recorded in rabbit transgenic embryos with the integrated CMVIE-EGFP (ClaI) transgene.Based on these results a more suitable gene marker for rabbit transgenic embryos production and selection is the CMVIE-EGFP (ClaI) gene construct. Prior to using microinjected embryos (for embryo transfer, vitrification or ESC isolation) it is necessary to pre-select microinjected embryos with evident transgenic mosaicism.

Comparison of effects of protein kinase A, mitogen-activated protein kinase, and cyclin-dependent kinase blockers on rabbit ovarian granulosa cell functions.

The aim of the present study was to define the role of protein kinase A (PKA)-, mitogen-activated protein kinase (MAPK)-, and cyclin-dependent kinase (CDK)-dependent pathways in the control of ovarian cell functions. The effects of PKA, MAPK, and CDK blockers (KT 5720, PD 98059, and olomoucine, respectively), given at doses of 0.001-10.0 µg/ml medium on functions of cultured rabbit granulosa cells were examined. Expression of PKA, MAPK/ERK1,2, secretory activity (IGF-I output), and proliferation (proliferating cell nuclear antigen, PCNA) in these cells were determined by RIA, immunocytochemistry and Western blotting. A PKA inhibitor, KT 5720 suppressed the expression of PKA and MAPK/ERK1,2, the IGF-I release, and the ratio of PCNA-positive cells in granulosa cells. A MAPK blocker, PD 98059 reduced the expression of MAPK/ERK1,2 (but not PKA), the IGF-I release, and percentage of PCNA-positive cells. A CDK blocker, olomoucine, increased the PKA expression, decreased the expression of MAPK/ERK1,2 and PCNA, but did not affect the IGF-I release. These observations confirm the involvement of PKs in control of basic ovarian functions and demonstrate the involvement of PKA in stimulation of ovarian cell proliferation and MAPK (but not CDK) and in promotion of ovarian IGF-I release. Different activity and specificity of the PKA, MAPK, and CDK blockers in their effects on PCNA and IGF-I suggests different biological role of these PKs in control of proliferative and secretory functions of rabbit ovarian cells.

Antibody to Hsp70 alters response of rabbit preimplantation embryos to hyperthermia in vitro.

The aim of the study was to determine a role of Hsp70 in the response of rabbit preimplantation embryos to hyperthermia (HT) in vitro. The embryos were cultured at standard (ST, 37.5 degrees C, control) or elevated (HT, 41.5 degrees C) temperature for 6h. In half of the embryos from both groups, Hsp70 was blocked by the addition of an antibody against Hsp70 into the medium at stages either prior to (

Morphology of testes from transgenic rabbits: histological and ultrastructural aspects.

The aim of this study was to compare morphological characteristics of testes from transgenic (the WAP-hFVIII gene) and non-transgenic rabbits with emphasis on the histological and ultrastructural aspects. Samples of testes from both groups were fixed and embedded into Durcupan ACM for transmission electron microscopy. For histological analysis, semi-thin toluidine blue-stained sections were evaluated under a Jenaval light microscope. Male fertility was tested based on egg fecundity and blastocyst yield; transgene transmission was proved using PCR assay. Spermatogenesis in rabbit testes had not been destroyed both in transgenic and non-transgenic rabbits. No significant differences were found in the occurrence of individual cell organelles of the Sertoli cells in transgenic and non-transgenic rabbits. The ultrastructure of Leydig cells in testes of transgenic and non-transgenic rabbits was rather similar. No differences in the occurrence of individual organelles of Leydig cells between transgenic and non-transgenic males were found. These results were in concert with fertilizing capacity of transgenic spermatozoa. The presented status of organelles in this study indicates functional activity of the analysed cells.

Several aspects of animal embryo cryopreservation: anti-freeze protein (AFP) as a potential cryoprotectant.

With the development of embryo technologies, such as in vitro fertilization, cloning and transgenesis, cryopreservation of mammalian gametes and embryos has acquired a particular interest. Despite a certain success, various cryopreservation techniques often cause significant morphological and biochemical alterations, which lead to the disruption of cell organelles, cytoskeleton damages, cell death and loss of embryo viability. Ultrastructural studies confirm high sensitivity of the cell membrane and organelle membrane to freezing and thawing. It was found that many substances with low molecular weights have a protective action against cold-induced damage. In this concern, an anti-freeze protein (AFP) and anti-freeze glycoproteins (AFGPs), which occur at extremely high concentrations in fish that live in Arctic waters and protect them against freezing, may be of potential interest for cryostorage of animal embryos at ultra-low temperatures. This mini-review briefly describes several models of AFP/AFGP action to preserve cells against chilling-induced damages and indicates several ways to improve post-thaw developmental potential of the embryo.

Characteristics of rabbit transgenic mammary gland expressing recombinant human factor VIII.

The objective of this research was to compare (i) the content of milk protein and recombinant human factor VIII (rhFVIII) in the milk of transgenic and non-transgenic rabbit females at three lactations and (ii) histological structure, ultrastructural morphology and occurrence of apoptosis in rabbit transgenic and non-transgenic mammary gland during third lactation and involution. Significant differences (t(0.05)) in milk protein content were found between transgenic and non-transgenic at all three lactations. The percentage of apoptotic cells was significantly higher (t(0.01)) in non-transgenic ones compared with transgenic mammary gland tissues (6.5% versus 2.4%) taken at the involution stage. Morphometrical analysis of histological preparations at the involution stage detected a significantly higher (t(0.05)) relative volume of lumen in transgenic animals compared with non-transgenic ones (60.00 versus 46.51%). Ultrastructural morphology of the transgenic mammary gland epithelium at the involution stage revealed an increased relative volume of protein globules (t(0.05)); at the lactation stage, a significantly higher volume of mitochondria (13.8%) compared with the non-transgenic (9.8%) ones was observed. These results, although revealing differences in some parameters of ultrastructure and histology, indicate no harmful effect of the mouse whey acid protein-hFVIII transgene expression on the state of mammary gland of transgenic rabbit females.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate.

The aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8-12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8-12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

Post-thaw survival, cell death and actin cytoskeleton in gene-microinjected rabbit embryos after vitrification.

The aim of this study was to compare two vitrification procedures (VPs), using either ethylene glycol (EG) in combination with dimethylsulfoxide (DMSO, vitrification protocol (VPI)) or Ficoll 70 (vitrification protocol II (VPII)), for rabbit embryo cryopreservation based on their post-thaw survival, cell death and actin cytoskeleton. The pronuclear stage eggs were flushed from the oviducts of the slaughtered New Zealand White rabbit does 19-20h post coitum (hpc) and randomly divided into two groups: intact (control) and microinjected (Mi). Mi embryos or intact embryos were cultured for up to 72hpc (morula stage), and then vitrified using either VPI (VPI+Mi, VPI) or VPII (VPII+Mi, VPII). After 2-3 days at -196 degrees C, the embryos were thawed and cultured until 96-100hpc to assess their development to blastocyst, apoptotic rate (TUNEL assay) and state of actin cytoskeleton (phalloidine-TRITC). Mi procedure reduced blastocyst yield, but it was higher than in either vitrified (VPI) or Mi vitrified (VPI+Mi) embryos. VPI compromised, whereas VPII did not significantly affect blastocyst development compared to intact embryos. Mi and VP both affected the embryo quality increasing TUNEL-index and decreasing the ratio of embryos with high quality actin cytoskeleton compared to control. A higher apoptotic index was recorded in VPI group. A combination of Mi and VP induced an increase in apoptotic rate (10.35% and 7.54% for VPI+Mi and VPII+Mi, respectively) as compared to Mi alone (5.7%). Ratio of embryos belonging to best actin quality (grade I) was different among groups and most of the embryos with grade I actin were in intact (84%), Mi (71%) or VPII (70%) groups. A significantly lower number of embryos with grade I actin quality was observed in VPI (58%), VPI+Mi (54%) or VPII+Mi (66%). These observations indicate that of the vitrification schemes tested, the VPII using EG and ficoll 70 as cryoprotectants, was less harmful than VPI (EG combined with DMSO in vitrification medium).

Alteration in ultrastructural morphology of bovine embryos following subzonal microinjection of bovine viral diarrhea virus (BVDV).

The aim of the present study was to evaluate the development and ultrastructure of preimplantation bovine embryos that were exposed to bovine viral diarrhea virus (BVDV) in vitro. The embryos were recovered from superovulated and fertilized Holstein-Friesian donor cows on day 6 of the estrous cycle. Compact morulae were microinjected with 20 pl of BVDV suspension (10(5.16) TCID(50)/ml viral stock diluted 1:4) under the zona pellucida (ZP), then washed in SOF medium and cultured for 24-48 h. Embryos were evaluated for developmental stages and then processed immunocytochemically for the presence of viral particles, using fluorescent anti-BVDV-FITC conjugate. Ultrastructure of cellular organelles was analysed by transmission electron microscopy (TEM).After microinjection of BVDV under the ZP, significantly more (p<0.001) embryos (83.33%) were arrested at the morula stage compared with the intact control (30.33%). Immunocytochemical analysis localized the BVDV-FITC signal inside the microinjected embryos. TEM revealed: (i) the presence of virus-like particles in the dilated endoplasmic reticulum and in cytoplasmic vacuoles of the trophoblast and embryoblast cells; (ii) the loss of microarchitecture: and (iii) abnormal disintegrated nuclei, which lacked reticular structure and the heterochromatin area. In all, the embryo nuclear structure was altered and the microarchitecture of the nucleolus had disappeared when compared with the nuclei from control embryos. Dilatation of the intercellular space and the loss of the intercellular gap junctions were often observed in bovine BVDV-exposed embryos. These findings provide evidence for the adverse effect of BVDV virus on the development of bovine embryos, which is related to irreversible changes in the ultrastructure of cell organelles.

Developmental rate and allocation of transgenic cells in rabbit chimeric embryos.

The objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR< >N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage. All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR< >N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175+/-13.10, of which 58+/-2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24+/-5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage. Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.