Hormonal indexes as predictors of porcine reproductive traits.

The aim of the present study was to examine the applicability of several hormonal indexes for early prediction of puberty and reproductive state in pigs. For this purpose, we have compared the level of hormones leptin, estradiol, progesterone, and IGF-I in the blood of gilts at 150 days of age and their indexes of puberty and ovarian state at the age of 200 days. The association between blood leptin, estradiol, progesterone, and IGF-I and indexes of future reproductive state has been demonstrated. High blood concentrations of leptin and IGF-I levels were associated with relatively low reproductive traits, while high levels of estradiol and progesterone were associated with future high reproductive indexes. These observations are the first demonstration of the applicability of these endocrine indexes for prediction of porcine reproductive traits.

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.

Morphological and functional alterations of the prostate tissue during clinical progression in hormonally-naïve, hormonally-treated and castration-resistant patients with metastatic prostate cancer.

Since commony used tools in oncological practice for the diagnosis of castration-resistent prostatic acinar adenocarcinoma are based on clinical criteria, such as castrate testosterone level, continuous rise in serum prostate-specific antigen, progression of preexisting disease or appearance of new metastases, it is important to identify reliable histopathological markers for the identification of this disease. Therefore, the aim of the present study was to determine the association between results from histological analysis, ultrastructural analysis and apoptosis in the prostate of patients with metastatic acinar prostatic adenocarcinoma (mPC). Patients were treated with androgen deprivation therapy (ADT), abiraterone acetate (Abi) therapy or received no treatment. Prostate tissue samples were divided into four groups as follows: i) Group 1, tissues from patients with benign prostatic hyperplasia (adenocarcinoma negative); ii) group 2, tissues from patients with metastatic hormone naïve prostate cancer; iii) group 3, tissues from patients with mPC treated with ADT; and iv) group 4, tissues from patients with metastatic castration-resistant prostate cancer treated with ADT and Abi. Immunohistochemical, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling (TUNEL) and ultrastructural assays using light, fluorescence and transmission electron microscopy, respectively, were used to analyze prostate tissue samples. The results demonstrated that ADT and Abi therapy caused histological and ultrastructural changes in prostate tissues. In groups 3 and 4, benign and malignant tissues were affected by the hormonal therapy. Histologically, the malignant epithelium after ADT therapy in groups 3 and 4 presented with a loss of glandular architecture, nuclear and nucleolar shrinkage, chromatin condensation and cytoplasmic clearing. At the ultrastructural level, compact hypertrophic and hyperchromatic nuclei with numerous invaginations were observed in groups 2, 3 and 4. In addition, the incidence of abnormal mitochondria in malignant cells of these groups was high. Group 4 was characterized by the presence of malignant mesenchyme-like cells in the prostatic stroma, arranged in small groups surrounded by collagen fibrils. Furthermore, the cytoplasm of these cells contained filaments. A decrease in the number of apoptotic cells using TUNEL assays in the examined samples was observed with increasing disease progression. The findings from the present study suggest that the duration of treatment with ADT and progression of the disease were associated with apoptosis dysregulation.

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.

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.

Yucca schidigera can promote rabbit growth, fecundity, affect the release of hormones in vivo and in vitro, induce pathological changes in liver, and reduce ovarian resistance to benzene.

This study evaluated the effect of Yucca schidigera (YS) extract on the physiological, reproductive, and endocrine indexes of New Zealand White rabbit does. Six-week-old rabbit does were fed a standard diet (control group) or a diet enriched with 5 or 20g of Y powder extract per 100-kg feed mixture for 350days. The does were artificially inseminated after induction of superovulation. Weight gain; conception and kindling rate; viability of pups and mothers; histopathological state of liver and muscle; plasma levels of progesterone (P4), oxytocin (OT), and prostaglandin F (PGF); and the release of P4, insulin-like growth factor I (IGF-I), OT, and PGF by isolated ovarian fragments and their response to the addition of benzene were analyzed. YS extract supplementation promoted weight gain and induced histopathological changes in the liver (creased vacuolization and occurrence of fuchsinophile inclusions in hepatocytes, liver fibrosis, hyperemia, occurrence of Kupffer cells, signs of necrosis and inflammation). YS consumption was not associated with changes in muscle (occurrence of fuchsinophile inclusions and signs of atrophy, interstitial edema, and inflammation), although Y2 increased muscle vascularization. YS supplementation increased conception and kindling rates but did not affect viability of pups or adult animals. Moreover, it enhanced plasma OT and PGF levels; plasma P4 concentration was increased by low-dose YS, but decreased by high-dose YS. Cultured ovarian fragments isolated from YS-fed does released more P4 and PGF and less IGF-I than ovarian fragments of control animals. However, YS supplementation did not affect ovarian OT release. Benzene alone did not influence the release of hormones by ovaries of control does. YS supplementation induced the inhibitory effect of benzene on the release of PGF, but not on other ovarian hormones. Collectively, these results suggest that dietary supplementation of YS extract can stimulate rabbit performance (growth and fecundity), which may be due to the promotion of P4, OT, and PGF release. It could, however, induce some pathological changes in the liver and reduce resistance of ovaries to the environmental contaminant benzene.

Reproductive performance of New Zealand White rabbits after depletion of apoptotic spermatozoa.

The objective of this study was to assess the utility of the magnetic-activated cell sorting (MACS) technique used for improving characteristics and quality of insemination doses by the elimination ofapoptotic rabbit spermatozoa from a heterospermic pool (Experiment 1) as well as from the ejaculates of individual bucks (Experiment 2). Superparamagnetic microbeads conjugated with annexin V eliminated spermatozoa with externalized phosphatidylserine via MACS. The control (untreated) and magnetically separated spermatozoa (in both E1 and E2) were used for artificial insemination of hormonally treated rabbit does. MACS separation of spermatozoa yielded two fractions: annexin V-negative (AnV) and annexin V-positive (AnV+). The CASA analysis after MACS sperm sorting revealed that the proportion of apoptotic spermatozoa in the semen of New Zealand White bucks varied from 7 to 20%. Transmission electron microscopy revealed that MACS treatment might eliminate spermatozoa with membrane damages and released acrosomal matter. However, the MACS separation (in both E1 and E2) did not affect the reproductive parameters of rabbit does.

[Gunshot wounds caused by non-lethal ammunition on the porcine model post-mortem]. / Strelné poranenia spôsobené neletálnym strelivom na modeli osípanej post-mortem.

In this article we focus on the effects of so called non-lethal ammunition. We studied possible mechanism of firearm injury formation as a consequence of using firearm on the body, to present a more comprehensive material in wound ballistics. We pointed out possible actions of a projectile causes on human, respectively other animal organisms, as well as to a manner in which an injury is caused by rifles or shotguns using non-lethal ammunition with rubber projectiles. In the experiment, we have focused on macroscopic analysis of the tissue penetrated by a rubber projectile fired from a long firearm and pump-action shotgun while focusing on the anatomical-morphological analysis of entry wounds to determine the effectiveness respectively, the wounding potential of the projectile. The results of the experiment based on the macroscopic analysis of entry wounds, cavities and exit wounds, show that when a rubber projectile penetrates the body it causes loss of the tissue (i.e. the minus effect) and mechanical disruption of the tissue similar to lethal projectile. Based on the measures and ballistic computations we concluded that in specific cases, like for example in a close range hit, a penetration of vital organs can cause serious or even lethal injuries.

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.

Nickel induced structural and functional alterations in mouse Leydig cells in vitro.

The present study was aimed at investigating effects of nickel (NiCl(2)) on secretion of testosterone (T), cell viability, ultrastructure and apoptosis in mouse Leydig cells. Testosterone release was measured after 48h of culture with 15.67, 31.25, 62.5, 125, 250, 500 and 1000µmol/L NiCl(2) or without NiCl(2) using radioimmunoassay. Cell viability was assessed by a MTT (metabolic activity assay). Quantification of apoptotic cells was performed using TUNEL assay and the ultrastructural changes were analyzed using transmission electron microscopy. The viability was decreased after addition of ≥250µmol/L NiCl(2). A concentration-dependent depression of T production was observed. The percentage of apoptotic cells was significantly increased only after addition of 125, 250 and 1000µmol/L NiCl(2). After addition of ≥250µmol/L NiCl(2) higher incidence of euchromatin was observed. Lipid droplets and vacuoles in cytoplasm were increased after addition of ≥125µmol/L NiCl(2). NiCl(2) induced decrease in numbers of mitochondria and smooth endoplasmic reticulum after treatment with ≥500µmol/L NiCl(2). Our findings suggest a negative effect of NiCl(2) on steroidogenesis, viability, apoptosis and ultrastructure of mouse Leydig cells.

Texture of the zona pellucida of the mature pig oocyte. The mammalian egg envelope revisited.

The zona pellucida (ZP) of mature pig oocytes is believed to consist of a dense filamentous meshwork, less compact on the inner and outer faces. The uneven surface of the ZP is made of unordered and stretched fibrils surrounding deep funnels which are the openings of the radial canaliculi. The topography of the ZP surface may contribute to the initial interplay between male and female gametes. Using cytochemical techniques for transmission electron microscopy (TEM), such as tannic acid and ruthenium red treatments, we found that the ZP of pig oocytes was essentially made of bundles of fibrils distributed in concentric layers (except in the innermost and outer parts). A correlation appears between the dense structure of the core layer of the ZP and its texture: it is constituted of superposed layers of fibril bundles, whereas only a random meshwork is found in a very thin innermost and in the outer layer. The fascicular configuration may control the permeability of the ZP, giving its semi-rigidity and elasticity, and may facilitate sperm penetration. The liquid crystal-like design of the core layer of the ZP is similar to textures found in the the vitelline envelope (zona radiata) of other vertebrates and possibly of all the deuterostomes. Such texture is probably related to the unique ZP protein composition and to a coordinated synthesis.

The extracellular matrix of porcine mature oocytes: origin, composition and presumptive roles.

The extracellular matrix (ECM) of porcine mature oocytes was revealed by transmission electron microscopy (TEM) after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS) and on the surface of the zona pellucida (ZP), it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells) or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine--precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA) bound to proteoglycans--for various times (with or without chase) and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose) and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI) and metaphase II (MII) and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a favourable factor for sperm penetration.

Effect of growth factors on proliferation, apoptosis and protein kinase A expression in cultured porcine cumulus oophorus cells.

The proliferation, apoptosis and protein kinase A (PKA) in porcine cumulus oophorus (CO) before and after 40 h of culture together with oocytes in the presence of IGF-I, IGF-II and EGF (all at 10 ng x mL(-1) medium) were compared. Cellular proliferation, apoptosis and PKA contents were evaluated by immunocytochemistry using specific antibodies against PCNA, TUNEL and catalytic (C-alpha) and regulatory (RI) subunits of PKA. The in-vitro culture of oocyte-CO complexes in a basal medium was accompanied by a decrease in the proportion of PCNA-positive CO cells (from 51 to 36%, p < 0.05). The addition of either IGF-I or EGF to the culture medium prevented this process and increased the proliferation rate (64 and 67% respectively, p < 0.001). During culture, the percentage of apoptotic (TUNEL-positive) CO cells increased from 42 to 57% (p < 0.01). The addition of IGF-I or EGF resulted in the inhibition of apoptosis to 36 and 12% respectively (p < 0.001). IGF-II and EGF reduced the amount of PKA catalytic subunits in the CO (percentage of cells with immunoreactive PKA catalytic subunits (28%, p < 0.05 and 27%, p < 0.05 respectively; versus control -41%), whilst the effect of IGF-I on this index was insignificant (31%). The expression of the PKA regulatory subunit was increased by EGF (51% compared with 29% in the control, p < 0.05), but not by IGF-I or IGF-II (30 and 29%). Our observations demonstrate that 40 h of culture of porcine CO resulted in a decrease in the proliferation and development of apoptosis in CO cells. IGF-I or EGF can stimulate proliferation and inhibit apoptosis. The influence of growth factors on the PKA content of the CO suggests that cAMP/PKA may be a mediator of the action of growth factors on these cells. The differential effects of IGFs and EGF on the regulatory subunit of PKA may indicate differences between their mechanisms of action.