The morphological and functional ultrastructure of cells in pre-implantation embryos.

Cells of pre-implantation embryos are equipped with many morphological and functional systems through which they can synthesize specific proteins and effectively ensure the protection of early embryonic development. Here we present evidence for the existence of these systems in morphologically normal and abnormal bovine blastocyst stage embryos in vivo at the ultrastructural and actin cytoskeleton levels. The appearance of organelles in the trophectoderm (TE) and inner cell mass (ICM) cells, responsible for their synthetic activities and their role in the development of early bovine embryos are described. We point out the importance of endocytic processes and the participation of extracellular vesicles in the formation of intercellular contacts and homeostasis of the embryo microenvironment. Several changes in the ultrastructural morphology of embryos produced by different methods (ICSI, parthenogenetic AC/DC electrical activation, IVF with separated sperm) and freezing/thawed embryos are described. We also show alterations occurred in the organelles after viral contamination of embryos with BHV-1 and BVDV viruses, and in embryos from over-conditioned cows. Recorded changes in organelles and appearance of cellular autophagic structures (vesicles, multivesicular bodies and autophagolysosomes) may negatively affect embryo metabolism and lead to the emergence of pathological processes in TE and ICM cells of preimplantation embryos.

Factors affecting rabbit sperm cryopreservation: a mini-review.

Rabbits are an important animal species for meeting the nutritional requirements of the world's growing population due to the high conversion rate of feed. In most countries, the rabbit industry currently relies on artificial insemination with fresh or chilled and frozen-thawed spermatozoa. Various factors during the freezing process, including diluents, sperm preparation and freezing techniques, antioxidants, sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for the poor sperm quality post thaw. Despite the extensive progress reached in the field of rabbit sperm cryopreservation, new methodological approaches that could overcome problems in sperm cryopreservation are necessary. The aim of this review was to describe the factors that affect the cryopreservation of rabbit sperm.

Methodological approaches for vitrification of bovine oocytes.

Numerous factors affect vitrification success and post-thaw development of oocytes after in vitro fertilization. Therefore, elaboration of an optimal methodology ensuring higher cryotolerance of oocytes and subsequent blastocyst yield is still of great interest. This paper describes and evaluates critical factors affecting the success of oocyte vitrification. In particular, an appropriate oocyte stage such as maturation status (germinal vesicle stage, metaphase II stage), presence/absence of cumulus cells before vitrification, and the effect of follicle size, as well as different culture systems and media for in vitro production of embryos, the types and concentrations of cryoprotectants, and cooling and warming rates at vitrification are considered. Special attention is paid to various cryocarriers used for low-volume vitrification, which ensures safe storage of oocytes/embryos in liquid nitrogen and their successful post-thaw recovery. At the end, we focussed on how age of oocyte donors (heifers, cows) influences post-thaw development. This review summarizes results of recently published studies describing different methodologies of cryopreservation and post-thaw oocyte development with the main focus on vitrification of bovine oocytes.

Development and ultrastructure of bovine matured oocytes vitrified using electron microscopy grids.

The aim of this study was to establish a methodology of cryopreservation of cattle oocytes and the quality assessment of oocytes and subsequent embryos produced in vitro under our laboratory conditions. Previously in vitro matured (IVM) oocytes were vitrified in minimum volume by ultra-rapid cooling technique. The oocytes were put into the equilibration solution (3% ethylene glycol in M199-HEPES + 10% foetal bovine serum) for 12 min, transferred to vitrification solution (30% ethylene glycol + 1 M sucrose in M199-HEPES + 10% foetal bovine serum) at room temperature for 25 s, then placed onto nickel electron microscopy grids and plunged into liquid nitrogen. After warming 75% of the oocytes were assessed as viable. Part of viable oocytes was taken for electron microscopy, the remaining oocytes were fertilized in vitro, and the presumptive zygotes were cultured until the blastocyst stage. Embryo cleavage and blastocyst rates in vitrified group after warming were 64.98% and 17.3%, resp. versus 70.72% and 25.54% in the control group (P < 0.05). No significant differences were found in the blastocyst total cell number, TUNEL and dead cell indexes between both groups. Ultrastructure of vitrified oocytes showed damages in smooth endoplasmic reticulum (SER) vesicles and lipid droplets as well as irregular arrangement of solitary cortical granules. Several mitochondria were damaged and the microtubules around the chromosomes were less occurred compared to the control group. However, the extent of injuries was lower than reported by other authors studying the ultrastructure of vitrified bovine oocytes, what is also supported by the better development of our oocytes after IVF. In conclusion, the designed oocyte vitrification technique ensures obtaining the blastocysts of the quality comparable to the fresh oocytes.

Relationship between the animal body condition and reproduction: the biotechnological aspects.

The aim of this review was to evaluate the relationship between the body condition of cows and reproduction. Reproduction was evaluated from the viewpoint of animal husbandry traits, ovarian activity and embryo transfer. Main emphasis was given to the review of articles from the area of biotechnical methods (in vitro embryo production, embryo transfer). Most authors agree on the opinion that the worsening of the reproduction traits of cows is a result of changes in the body condition score (BCS) either under or over their average value. Worsening of reproduction traits was presented not only from a zootechnical viewpoint (e.g., calving interval, 56 d nonreturn rate, etc.) but also in term of ovarian activity, oocyte recovery and in vitro embryo production. In general, the body condition of cows is an important factor affecting female reproduction ability at the ovarian level.

Relationship between body conditions and environmental contaminants in bovine ovarian cells.

The effect of body condition and environmental contaminants on reproductive processes is known; however, it is not known whether basic ovarian cell functions and their response to these contaminants depend on body condition. This study aimed to understand the interrelationships between body conditions and environmental contaminants on ovarian cells. For this purpose, we compared ovarian granulosa cells isolated from cows with an emaciation tendency (body condition score, BCS2 on a scale from 1 to 5) and cows with average body condition (BCS3); proliferation, apoptosis, secretory activity and the response to environmental contaminants were all assessed in the cells. In the 1st series of experiments, ovarian granulosa cells isolated from BCS2 and BCS3 cows were cultured with and without benzene, xylene, and toluene (0.1%). The accumulation of nuclear and cytoplasmic markers of apoptosis (p53 and bax, respectively), a proliferation marker (PCNA), progesterone (P4), and insulin-like growth factor I (IGF-I) was evaluated by Western blot and radioimmunoassay (RIA) experiments. In the 2nd series of experiments, the groups of granulosa cells were cultured with and without mycotoxine deoxynivalenol (DON, 0, 10, 100, or 1000 ng/ml). The secretion of P4 and testosterone (T) was measured by RIA. In comparison to cells from BCS2 animals, ovarian cells isolated from BCS3 cows accumulated higher levels of bax and PCNA but not p53, and they secreted higher amounts of IGF-I but not P4 or T. In cells from BCS2 animals, benzene and xylene promoted p53 accumulation, and toluene reduced the accumulation. In the BCS2 group, all treatments promoted bax and PCNA expression. However, in cells from BCS3 animals, all environmental pollutants inhibited p53, toluene inhibited PCNA but not bax, and xylene did not affect the expression of proliferation or apoptosis markers. In the BCS2 group, P4 was inhibited by xylene, and IGF-I was stimulated by xylene but not by benzene or toluene. Low-dose exposure to DON (10 ng/ml) promoted P4 release from cells from both BCS2 and BCS3 animals, but high-dose exposure to DON (1000 ng/ml) reduced P4 secretion from the cells from BCS2 animals but not from the cells from BCS3 animals. The release of T was inhibited by high-dose exposure to DON (1000 ng/ml), irrespective of the BCS. An emaciation tendency reduces proliferation, apoptosis, and IGF-I release, and it induces or reverses the action of environmental contaminants on ovarian functions. Taken together, these observations demonstrate the effect of body condition and the direct influence of environmental contaminants on basic bovine ovarian functions. Furthermore, they demonstrate for the first time that the response of ovarian cells to environmental contaminants can be regulated by cow body condition.

Histological characteristics of ovarian follicle atresia in dairy cows with different milk production.

Follicle atresia in mammals is a universal phenomenon characteristic by degenerative morphological changes in granulosa and theca cells. The unfavourable effect of milk production in relation to fertility has been studied starting from the 70s of the last century; however, there is no unambiguous and persuasive data on association of ovarian atresia with milk yield of dairy cows. The aim of this study was to define histological signs of ovarian follicle atresia in dairy cows in relation to their milk production. The ovaries were recovered from slaughtered Holstein dairy cows assigned into two groups according to average level of annual milk production: Group 1 (n = 25)-low (≤8,000 kg/year) and Group 2 (n = 23)-high (≥8,000 kg/year). Atresia of antral follicles was evaluated on the basis of histopathological image (staining with basic fuchsine and toluidine blue) of nonovulated follicles, classified into five categories: an initial atresia, cystic atresia, obliterated atresia, atresia with luteinization of the granulosa and follicle structures of the fibrous body-corpus fibrosum. We found that the histopathological image of follicle atresia in groups of low-milk- or high-milk-producing cows is essentially similar. Prevalent form of atresia in follicles of all experimental cows was the formation of fibrous bodies and obliterated atresia. The occurrence of fibrous bodies was significantly higher (55.44%) in low-milk-producing cows compared with high-milk-producing cows (34.61%). In the same way, the higher incidence of obliterated atresia was recorded in ovarian follicles from cows with the lower milk production (36.96%) compared to the cows with the higher milk production (25.48%). In contrast, ovaries from lower milk-producing cows showed lower (p < 0.05) incidence of initial (p < 0.001) and cystic (p < 0.05) follicle atresia than ovaries from the higher milk-producing cows. Our results show that cows in the higher lactation group showed more initial and cystic atresia, what may adversely affect the fertility of dairy cows.

Effect of body condition and season on yield and quality of in vitro produced bovine embryos.

The aim of our study was to examine the effects of cow's body condition score (BCS; scale 1-5) and season on the quality of bovine in vitro produced embryos. The proportion of good quality oocytes (Q1 and Q2) was higher (P < 0.05) in the BCS 2 (57.60%) and BCS 3 (60.90%) groups compared with the BCS 1 (43.60%) group. There were no statistical differences in embryo cleavage and blastocyst rate among the BCS groups. The highest total cell number (TCN, DAPI stain) of blastocysts (P < 0.05), recorded in BCS 1 (122.27 ± 6.90) in comparison with BCS 2 (101.8 ± 3.60) or BCS 3 (105.44 ± 3.70) groups, was related to higher dead cell (DCI, TUNEL) index in this group (7.07%) when compared with BCS 2 (6.54%) or BCS 3 (6.06%), respectively. The yield of good quality oocytes during spring was lower (P < 0.05) compared with the summer season. There were significant differences (P < 0.05) in maturation and cleavage rates between autumn and summer (73.42%, 76.2% vs. 85.0%, 41.8%, respectively). The highest (P < 0.01) blastocyst rate was noted during spring and summer months. Significant difference (P < 0.05) in the TCN among spring (99.38 ± 3.90), autumn (110.1 ± 4.58) or summer (108.96 ± 3.52) was observed. The highest proportion of embryos with the best (grade I) actin cytoskeleton (phalloidin-TRITC) quality was noted during the summer months. Our results indicate that body condition affects the initial quality of oocytes, but does not affect embryo cleavage, blastocyst rate and actin quality. This finding may suggest that development in vitro can mask the influence of BCS. The season affects yield and quality of blastocysts in the way that the autumn period is more favorable for embryo development.

The cryoprotective effect of Ficoll on the rabbit spermatozoa quality.

The aim of this study was to evaluate the effect of the addition of Ficoll 70 into the cryopreservation medium containing sucrose and dimethyl sulfoxide (DMSO) on rabbit spermatozoa characteristics following freezing/thawing. This large molecular weight polymer elevates the viscosity of medium and, therefore, could better protect spermatozoa during the freezing process. Only ejaculates of good initial motility (>80%) were used in the experiments. Heterospermic pools were diluted in a freezing medium composed of commercial diluent, 16% dimethyl sulphoxide (DMSO) and 2% sucrose (control) or in the same medium enriched with 4% Ficoll 70 (Ficoll) and frozen in liquid nitrogen vapours for 10 min before being plunged in liquid nitrogen. The quality of fresh and frozen/thawed spermatozoa samples was evaluated in vitro using the Computer Assisted Semen Analysis (CASA) system, fluorescent probes (peanut agglutinin (PNA)-Alexa Fluor®; annexin V-FLOUS) and by electron microscopy. Better cryoprotective effect was observed when Ficoll 70 was added, compared with the semen cryopreserved with sucrose and DMSO only. The higher values (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing and at 30 min following incubation at 37°C were obtained in the Ficoll group. Moreover, the higher number (P < 0.05) of acrosome intact sperm was found in the Ficoll compared with the control group. Furthermore, no significant differences in kindling rates and number of pups born between frozen/thawed and fresh semen group were found. In conclusion, our study showed that the addition of Ficoll 70 might improve several characteristics of rabbit spermatozoa measured in vitro following freezing/thawing.

Effect of insulin-like growth factor I on functional parameters of ram cooled-stored spermatozoa.

The aim of the study was to examine the effects of insulin-like growth factor I (IGF-I) on ram sperm traits after hypothermic storage. Sperm ejaculates from Lacaune rams were diluted in a Tris extender, pooled, divided into groups of IGF-I doses tested (0, 10, 100 or 200 ng.ml-1) and stored (0-5°C) for 96 h. IGF-I elevated whole sperm motility as measured by a Computer-assisted Sperm Analyser (CASA) system, by 24 h (10 ng.ml-1) and 48 h (200 ng.ml-1) of storage, and by progressive movement on each day of storage. After 72 h the sperm samples were analysed for plasma membrane integrity (peanut agglutinin-fluorescein isothiocyanate), membrane stability (annexin V-Fluos) and apoptosis (Yo-Pro®-1) using fluorescence microscopy. The addition of IGF-I (at 100 or 200 ng.ml-1) reduced the ratio of sperm with disrupted membranes and the ratio of annexin V-labelled sperm. The ratio of apoptotic sperm was reduced by IGF-I given at 10 or 100 ng.ml-1 compared with control. Sperm fertilizing ability, determined at 48 h by an in vitro fertilization (IVF) test on bovine oocytes, was increased by IGF-I given at 100 ng.ml-1 from 47.0 to 67.7%. In conclusion, IGF-I maintained ram sperm functions following cooling storage and its effects were reflected in sperm fertilizing ability in vitro.

Post-thaw culture in presence of insulin-like growth factor I improves the quality of cattle cryopreserved embryos.

The goal of this study was to examine the effect of insulin-like growth factor I (IGF-I; added during post-thaw culture (48 h)) on the preimplantation viability and quality of cryopreserved bovine in vivo recovered embryos. The morula stage embryos, non-surgically recovered from superovulated dairy cows of Czech Fleckvieh cattle breed, had previously been cryopreserved by a slow freezing technique and stored in liquid nitrogen since 1989-1990. Following thawing, the embryos were cultured for 48 h either alone (no IGF-I) or in the presence of IGF-I (10 or 100 ng/ml); non-cultured embryos served as a control. Thereafter, the embryos were analyzed for cleavage to the blastocyst stage, apoptosis (TUNEL), embryo cell number and quality of actin cytoskeleton. Following post-thaw culture 41% of embryos developed to advanced blastocysts. IGF-I increased this per cent and, at a higher dose, essentially reduced the per cent of degenerated embryos. In cultured embryos, IGF-I at both doses elevated the cell number compared with non-cultured embryos. However, in comparison with embryos cultured without IGF-I, only the higher IGF-I dose resulted in elevating the embryo cell number. The TUNEL index was significantly lowered by IGF-I treatment. Thawed embryos were mostly of the grade III actin type and fewer (12%) had grade II actin, whilst no grade I actin embryos were noted. The addition of IGF-I resulted in the appearance of grade I actin embryos (8.33 and 6.9% for 10 and 100 ng/ml, respectively). These observations indicate that the addition of IGF-I during post-thaw culture can improve the quality of bovine cryopreserved embryos.

Functional characteristics of ram cooling-stored spermatozoa under the influence of epidermal growth factor.

The aim of the study was to examine the effects of epidermal growth factor (EGF) on ram sperm traits following hypothermic storage. Fresh ram ejaculates were diluted in Triladyl extender, pooled and divided into groups according to EGF doses added (0, 100, 200 or 400 ng/ml). Following 72-96 h storage at 4ºC, the spermatozoa were stained for a plasma membrane integrity (PNA-FITC), membrane phosphatidylserine (PS) translocation (annexin V-Fluos) and apoptosis (Yo-Pro-1), and analyzed by fluorescent microscopy. Sperm motility was measured using computer-assisted sperm analysis (CASA) and sperm fertilizing ability was tested using in vitro penetration/fertilization test on bovine prematured oocytes. EGF increased sperm motility at all doses tested, decreased the proportion of spermatozoa with damaged plasma membrane (at 200 or 400 ng/ml), and decreased the proportion of apoptotic (Yo-Pro-1) spermatozoa when given at 200 or 400 (but not 100) ng/ml. The proportion of spermatozoa with PS translocations (8.5%) was affected by neither of the EGF concentrations tested. However, fertilizing ability of ram sperm in the in vitro test was not improved by EGF (200 ng/ml). In summary, EGF when given at higher concentrations improved sperm viability and motility after cooling storage, but these effects were not reflected in sperm fertilizing ability in vitro.