High temperature impairs rabbit viability, feed consumption, growth and fecundity: examination of endocrine mechanisms.

The aim of the present study was to examine the effect of high ambient temperature on rabbit feed consumption, growth, viability, and fecundity, as well as the morphology and endocrine function of gonadal and adrenal cells. Adult does and their offspring were kept at either a comfortable (20°C; control) or high (36°C) temperature throughout pregnancy and up until weaning of pups. Doe mortality and fecundity, and plasma concentrations of hormones were evaluated. In addition, granulosa cells were cultured with and without FSH to assess progesterone production. In the offspring, we assessed mortality, total feed consumption, feed efficiency, growth, plasma hormone concentrations, as well as the microstructure in ovarian granulosa cells, testicular Leydig cells, and adrenocortical cells. We observed greater mortality of both adult animals and offspring at the higher ambient temperature compared with the control. The higher ambient temperature suppressed feed consumption, feed efficiency, and growth of pups. Adult and young females exposed to a high temperature had lower circulating concentrations of progesterone, but not of estradiol, compared with controls. Young males exposed to a high ambient temperature had greater circulating concentrations of testosterone, but not progesterone, compared with controls. High ambient temperature reduced circulating IGF-I concentrations in all the animals. Corticosterone level was increased in plasma of young but not of adult animals. Granulosa cells isolated from the ovaries of does subjected to high temperatures released less progesterone, and they had poorer response to the stimulatory action of FSH than the cells from control does. High temperatures induced fragmentation of nucleoli in ovarian granulosa cells, but they did not alter the state of other organelles in ovarian, testicular, or adrenocortical cells. A negative influence of high temperature on rabbit feed consumption, growth, viability, and fecundity was observed. Taken together, these changes could be due to a decrease in IGF-I and/or progesterone secretion, destruction of ovarian cell nucleoli, and/or impaired ovarian cell response to FSH.

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.

Effect of oxytocin, IBMX and dbcAMP on rabbit ovarian follicles.

Oxytocin (OT) and protein kinase A (PKA), a possible intracellular mediator of hormone action in the ovary, can be potent activators of ovarian functions and fertility. Nevertheless, action of OT on ovarian follicle atresia has not been studied yet. Only single administration of PKA activators [3-isobutyl-1-methyl-xanthine (IBMX) and dibutyryl cyclic adenosine monophosphate (dbcAMP)] on ovarian follicle atresia was studied previously. The aim of this study was to examine the effect of OT (single treatment per one reproductive cycle, multiple treatments for three cycles), IBMX and dbcAMP (multiple treatments) on folliculogenesis and follicular atresia in rabbit. The ovarian cycle in control females was induced only by gonadotropins. Experimental females received co-administration of gonadotropins with OT, IBMX or dbcAMP (at 50 µg/female). All females were artificially inseminated. Single-treated females were euthanized after 18-19 h. Multiple-treated females were euthanized after the third reproductive cycle. Histological sections of the ovaries were prepared and evaluated by a light microscopy. The follicles were divided into four classes according to the structure of granulosa and theca cells as follows: none or small atresia, cystic atresia, obliterative atresia and atresia associated with luteinization. The ovaries from the control and experimental females, treated during one reproductive cycle or three cycles, were compared. Single OT co-administration increased proportion of follicles with atresia associated with luteinization, but not other types of atresia. No influence of multiple OT co-administration on follicular atresia was recorded. Multiple IBMX and dbcAMP co-administration decreased the proportion of atretic follicles and increased the proportion of healthy follicles without atresia.

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.

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.

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 (

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.

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.

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.

[Morphological-functional characteristic of periarticular tissue after total hip arthroplasty: histological, cytochemical and electron microscopy aspects]. / Morfologicko-funkcná charakteristika periartikulárneho tkaniva po endoprotéze bedrového klbu: histologické, cytochemické a elektrónmikroskopické aspekty.

PURPOSE OF THE STUDY Analysis of the tissue harvested around the total hip replacement isolated from re-operated patients in order to: (1) characterize complexity of structural processes developing in the region of the total hip replacement and, (2) to define the role and significance of histological structures of this tissue, mainly in relation to implant loosening, from the viewpoint of formal and causal pathogenesis. MATERIAL AND METHODS Biopsied material isolated from periarticular tissue of re-operated patients (n=19) after THR was analyzed using the methods of light optic, fluorescent (TUNEL), and transmission electron microscopy. RESULTS Histological analysis revealed fibroproliferaive processes and epithelioid granulomatosis cell reaction around the implant with the formation of giant multinuclear syncythial (osteoclastlike) structures as a response to foreign bodies. These structures phagocyte fragments of foreign material (polyethylene particles from the implant, cement fragments). All the used methods revealed a range of regressive changes in the layers of foreign microparticles (inside giant multinuclear cells) typical of fibrinoid necrosis in collagen fibres and apoptosis. In certain cases, progressive changes as chondroid and synovial differentiation (metaplasia) were observed. DISCUSSION Total hip replacement, despite all positive aspects for patients, may cause permanent inflammatory processes in its surrounding. This may result in an extensive fibroproduction of a differently thick layer of connective tissue around the implant. An important factor of loosening of THR is probably osteoclastic resorption in the area of "bone-implant" interface, as a result of the interaction between the inflammatory mechanisms around the implant. CONCLUSION In the postoperative period, there occur fibroproliferative changes in the periarticular tissue and large population of multinuclear cells. In our view, these cells play a role in the production of wear particles from the implant and microparticles of bone tissues and bone cement. Fibroproliferative process may be considered as an immune response to the implanted foreign material.

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.

Development and viability of bovine preimplantation embryos after the in vitro infection with bovine herpesvirus-1 (BHV-1): immunocytochemical and ultrastructural studies.

The aim of our study was to examine whether: (1) the exposure of bovine embryos to the BHV-1 virus in vitro can compromise their further development and alter the ultrastructural morphology of cellular organelles; (2) whether the zona pellucida (ZP) can be a barrier protecting embryos against infection; and (3) whether washing with trypsin after viral exposure can prevent virus penetration inside the embryo and subsequent virus-induced damages. The embryos were recovered from superovulated Holstein-Friesian donor cows on day 6 of the estrous cycle. Only compact morulas or early blastocysts were selected for experiments with virus incubation. We used the embryos either with intact ZP (either with or without trypsin washing) or embryos in which the ZP barrier was avoided by using the microinjection of a BHV-1 suspension under the ZP. ZP-intact embryos (n = 153) were exposed to BHV-1 at 10(6.16) TCID(50)/ml for 60 min, then washed in trypsin according to IETS guidelines and postincubated in synthetic oviduct fluid (SOF) medium for 48 h. Some of the embryos (n = 36) were microinjected with 20 pl of BHV-1 suspension under the ZP, the embryos were washed in SOF medium and cultured for 48 h. Embryo development was evaluated by morphological inspection, the presence of viral particles was determined both immunocytochemically, using fluorescent anti-IBR-FITC conjugate and by transmission electron microscopy (TEM) on the basis of the ultrastructure of the cellular organelles. It was found that BHV-1 exposure impairs embryo development to higher preimplantation stages independent of the presence of the ZP or the trypsin treatment step, as most of the embryos were arrested at the morula stage when compared with the control. Immunofluorescence analysis confirmed the presence of BHV-1 particles in about 75% of embryos that were passed through the trypsin treatment and in all the BHV-1-microinjected embryos. Ultrastructural analysis, using TEM, revealed the presence of virus-like particles inside the BHV-1-exposed embryos, where the trypsin washing step was omitted. Conversely, in trypsin-treated BHV-1-exposed embryos, TEM detected only the envelope-free virus-like particles adhered to pores of the ZP. The embryos that were microinjected with BHV-1 suspension showed the presence of BHV-1 particles, as well as ultrastructural alterations in cell organelles. Taken together these findings may suggest that BHV-1 infection compromises preimplantation development of bovine embryos in vitro and therefore the ZP may not be enough on its own to prevent virus-induced damage, unless it is not accompanied with trypsin washing.

Female reproductive toxicology of cadmium.

The aim of this study was to determine effects of Cd on the structure of ovary, oviduct and uterus after an experimental administration. Animals were divided into three groups. In group A rabbits received cadmium i.p. and were killed after 48 h. In group C Cd was administered p.o. for 5 month. The group K was the control. Decreased relative volume of growing follicles and increased stroma after Cd administration were detected. The number of atretic follicles was significantly higher after administration of Cd. The most frequent ultrastructural alterations observed were undulation of external nuclear membrane, dilatation of perinuclear cistern and endoplasmic reticulum. In all studied types of cells mitochondria with altered structure were found. In the oviduct the highest amount of epithelium in the group with long-term Cd administration was found. Microscopic analysis showed oedematization of the oviduct tissue, caused by disintegration of the capillary wall. An electron microscopic analysis showed dilatation of perinuclear cistern. The intercellular spaces were enlarged and junctions between cells were affected. Mainly after a long-term cadmium administration nuclear chromatin disintegration was present. In the uterus a significant change was determined in the relative volume of glandular epithelium. Increase of stroma was a sign of uterus oedamatization caused by damage in the wall of blood vessels and subsequent diapedesis. After Cd administration alteration in uterus were less expressed, in comparison with ovary and oviduct. Alteration of nuclear chromatin contain following Cd administration suggests degenerative functional changes.

The effect of hyperthermia in vitro on vitality of rabbit preimplantation embryos.

The aim of our study was to test the influence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 degrees C or 42.5 degrees C) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 degrees C) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 degrees C or 42.5 degrees C) or standard temperature (control 37.5 degrees C), post-incubated overnight (16-20 h) at 37.5 degrees C and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 degrees C did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 degrees C. Western-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 degrees C) and embryos (exposed to 41.5 degrees C). Following the elevation of temperature to 42.5 degrees C embryo development was dramatically compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis.

Ultrastructural morphometry of mammary gland in transgenic and non-transgenic rabbits.

The mammary gland of transgenic animals has been used for the production of recombinant proteins of therapeutic and nutraceutical use. The objective of this study was to compare the ultrastructure of transgenic and non-transgenic rabbit mammary gland tissue. New Zealand White transgenic rabbits were obtained by breeding non-transgenic rabbits with transgenic founder rabbits containing a whey acidic protein-human factor VIII (WAP-hFVIII) transgene integrated into their genome. Samples of mammary gland tissue from lactating rabbit females were isolated by surgical procedures. These samples were examined by optical and electron microscopy and photographs were taken. Measurements of ultrastructural organelles were made from digital images of the mammary cells. No differences were found in the cellular structure of mammary tissue, but significant differences t((0.001)) in the relative volume of mitochondria and vacuoles between transgenic and non-transgenic mammary gland epithelium were observed.

Preimplantation development and viability of in vitro cultured rabbit embryos derived from in vivo fertilized gene-microinjected eggs: apoptosis and ultrastructure analyses.

Microinjection (Mi) of gene constructs into pronuclei of fertilized eggs is a widely used method to generate transgenic animals. However, the efficiency of gene integration and expression is very low because of the low viability of reconstructed embryos resulting from cell fragmentation and cleavage arrest. As a consequence, only a few viable embryos integrate and express transgene. Since cellular fragmentation and cleavage stage arrest in embryos may be associated with apoptosis, we aimed to test the hypothesis that the low viability of Mi-derived eggs is caused by a high rate of apoptosis in embryos, as a result of the detrimental effect of Mi. Pronuclear stage eggs (19-20 hours post-coitum, hpc) were microinjected with several picolitres of DNA construct into the male pronucleus (gene-Mi); the intact eggs (non-Mi) or eggs microinjected with phosphate-buffered saline (PBS-Mi) served as controls. Epidermal growth factor (EGF; 0, 20 and 200 ng/ml) was added to the culture medium and the embryos were cultured up to 94-96 hpc. Apoptosis was detected using the TUNEL assay, and the ultrastructure was analysed using electron microscopy of Durcupan ACM thin sections of the embryo. Gene-Mi embryos had significantly lower (p < 0.05) blastocyst yields and a higher percentage of cleavage-arrested embryos than those in the non-Mi group. In gene-Mi groups, approximately 40% of all cleavage-stage-arrested embryos had fragmented blastomeres. Both gene-Mi- and PBS-Mi-derived blastocysts had a significantly higher TUNEL index (p < 0.001) and lower total cell number (p < 0.05) than the non-Mi embryos. Comparison of the quality of gene-Mi embryos with that of PBS-Mi embryos indicated that the deleterious effect of Mi on the embryo was caused by the Mi procedure itself, rather than DNA. EGF (at 20 ng/ml) had beneficial effects on the quality of gene-Mi-derived embryos, eliminating the influence of the Mi procedure on apoptosis and embryo cell number. Ultrastructural analysis confirmed a higher occurrence of apoptotic signs (nuclear membrane blebbing, areas with electron-dense material, numerous apoptotic bodies) in Mi-derived cleavage-arrested embryos compared with untreated or Mi-derived normal-looking embryos. These findings suggest an association between embryo cleavage arrest and apoptosis in Mi-derived embryos. Inclusion of EGF in the embryo culture medium can eliminate the detrimental effect of Mi on embryo quality.

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification.

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p < 0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not significantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.

Ultrastructural morphometry of precompacted bovine embryos produced in vivo and in vitro after activation by electric pulse AC/DC.

Early bovine precompacted embryos (at 1- to 8-blastomere stage) were analyzed by electron microscopy. The volume density of cellular components was determined by morphometric analysis to quantify the ultrastructure of early bovine embryos produced either in vivo or parthenogenetically after stimulation of oocytes by electric pulse AC/DC. In embryos obtained in vivo, most of cellular volume was occupied by cytoplasm (82.93%). The relative volume of lipids, vacuoles, mitochondria was relatively low (5.46; 5.07; 3.78%, respectively), and the relative volume of Golgi apparatus and cell inclusions was the lowest (1.51%). AC/DC-derived parthenogenotes had a relative high area occupied by vacuoles and lipids (18.68 vs. 14.33%) and a significantly lower relative volume was occupied by cytoplasm (60.63%) when compared with the control in vivo embryos. These observations demonstrated that parthenogenetic embryos had significantly altered ultrastructure, indicating extensive subcellular damages. These findings are discussed from the physiological and functional point of view.

Aneuploidy in the transgenic rabbit.

The aim of this study was to determine whether there are differences in the karyotypes between transgenic and non-transgenic or control rabbits. New Zealand White transgenic rabbits (F1 generation) were obtained after breeding of transgenic founder rabbits that were derived from single--SM--or double microinjection--DM--with a WAP-hFVIII transgene. C-metaphase plates were obtained from short-time culture of peripheral blood lymphocytes synchronized by the addition of colcemide. A significantly higher rate of aneuploidy was observed in c-metaphase spreads of transgenic (56-66%) rabbits, as compared to non-transgenic ones (28-38%) (P < 0.05; P < 0.01). The patterns of chromosome banding were identical in both groups of rabbits. No structural aberrations were revealed in either group. These findings demonstrate that transgenic rabbits have a higher frequency of numerical chromosomal aberrations in their peripheral blood lymphocytes than normal rabbits, but without apparent deleterious effects on health or reproduction.