A Makarevich - Academia.edu (original) (raw)

Papers by A Makarevich

Research paper thumbnail of The Role of Oxytocin, Protein Kinase A, and ERK-Related MAP-Kinase in the Control of Porcine Ovarian Follicle Functions

Experimental and Clinical Endocrinology & Diabetes, 2004

The aim of our in vitro experiments was to study the role of oxytocin (OT), cAMP/protein kinase A... more The aim of our in vitro experiments was to study the role of oxytocin (OT), cAMP/protein kinase A (PKA), and mitogen-activated protein kinase (ERKs MAP-kinase) in the control of ovarian cell functions as well as the role of PKA and MAPK in mediating OT effects on these processes. The whole porcine ovarian follicles were cultured in the presence or absence of OT (1, 10, 100 ng/ml), PKA inhibitor Rp-cAMPS (10 nM), MAP-kinase inhibitor PD98059 (1 microg/ml), or their combination. The release of prostaglandins F (PGF) and E (PGE) were determined by RIA, PKA (alpha-cat subunit), the proliferation-associated peptide PCNA and ERK-1, -2 expression in cell lyzates were analysed by Western-blotting. OT stimulated the release of PGF and PGE, and accumulation of PKA, ERK-1/-2, and PCNA in cell lysate. PD98059 decreased the basal PGF and PGE output, as well as reduced both ERK-1 and ERK-2 accumulation in cell lysates. Rp-cAMPS decreased PKA accumulation in cell lysates. Rp-cAMPS prevented the OT-induced stimulation of PKA, ERK-1, ERK-2, PGF, and PGE, PD98059 did so for PKA, PGF, and PGE. However, PD98059 reduced either basal or OT-induced p-ERK level. OT-stimulated PCNA accumulation was only slightly modified by these blockers. These observations suggest that OT, PKA, and ERKs MAPK can be involved in the control of PGs release and proliferation of ovarian cells. The influence of OT on both PKA and MAPK, and the ability of PKA and MAPK blockers to prevent completely or partially OT effects suggest, that effects of OT on PGF and PGE can be mediated by both PKA and MAPK. The role of MAPK and PKA in mediating the proliferative effects of OT seems to be minor assuming the involvement of other intracellular messengers.

Research paper thumbnail of PERSPECTIVE: Rooster Spermatozoa Cryopreservation and Quality Assessment

Cryo letters, 2021

Unsuccessful rooster fertility following cryopreservation may be linked to specific changes in sp... more Unsuccessful rooster fertility following cryopreservation may be linked to specific changes in spermatozoa quality, which can be determined using various methods. These determinations also facilitate the design of improved freezing and thawing processes. Here, we update the current state of methodologies available for the assessment of rooster semen quality after cryopreservation. Computer-assisted sperm analyses (CASA) is one of the main systems used to analyse motion parameters of spermatozoa (total motility, progressive motility and motion parameters). Moreover, fluorescent techniques and flow cytometry can improve the assessment of various aspects of semen quality (viability, acrosome status, mitochondrial potential, lipid peroxidation, DNA damage, lipid peroxidation and cell debris removal) using specific fluorescent markers such as ethidium bromide, Yo-Pro-1, Annexin V, propidium iodide, SYBR-14, PNA, JC-1, BODIPY, acridine orange and DRAQ5. Transmission electron microscopy al...

Research paper thumbnail of Development of a sensitive radioimmunoassay for IGF-I determination in samples from blood plasma and cell-conditioned medium

Research paper thumbnail of Pathological Alterations in Hepatocytes of Dairy Cows With a Tendency to Emaciation and Fattening

Folia Veterinaria

The aim of this study was to demonstrate the histochemical and histopathological alterations in t... more The aim of this study was to demonstrate the histochemical and histopathological alterations in the livers of cows with a tendency to become emaciated (body condition score - BCS1 and 2) and a tendency to become fattened (BCS4 and 5) in comparison to the cows of average body condition (BCS3) presented as a control. The histochemical analysis (PAS reaction) showed that the influence of emaciation and fattening in our study was manifested by a decreased occurrence of glycogen and a decreased level of the PAS-positive matter in the hepatocytes of dairy cows with BCS1, 2, 4 and 5. An abundant accumulation of lipids in the form of large lipid droplets, liposomes and lipoproteins observed in the hepatocytes of emaciated and fattened (BCS1 and 5) cows may be related to moderate-severe steatosis. These observations suggest a relationship between liver steatosis and the occurrence of lipoproteins in cows with a tendency toward emaciation and fattening.

Research paper thumbnail of Are IGF-I, thyroid hormone and metabolite concentrations in calf plasma associated with growth rate, sex and age?

Journal of Animal and Feed Sciences

The aims of our experiments were (1) to study age-dependent changes in bovine plasma concentratio... more The aims of our experiments were (1) to study age-dependent changes in bovine plasma concentrations of insulin-like growth factor I (IGF-1), thyroid hormones (T3, T4), some metabolites (glucose, triglycerides, ketone bodies), calcium, phosphorus and sodium, and (2) to determine whether plasma concentrations of these substances are associated with individual and sex-dependent differences in the growth rates of calves. Eight male and 8 female calves were weighed at the beginning and at the end of the experiment and daily gain and feed consumption (dry matter, crude protein, PDIN and NEL per kg daily gain) were determined. Plasma concentrations of IGF-I, T3, T4, glucose, triglycerides, ketone bodies, calcium, phosphorus and sodium were measured in calves at 2, 4, 6, 8, 10 and 12 weeks of age. There were significant age-dependent increases in the plasma concentrations of IGF-I and glucose. Males had a significantly higher daily gain and lower feed consumption than females, but no sex-dependent differences in plasma constituents were detected. In both sexes, daily gain was positively correlated with plasma IGF-I (coefficients of correlation r=0.630 in females and r=0.614 in males) and glucose (r=0.496 and 0.427, respectively) concentrations, but not with other substances. Our observations show that there are age-dependent changes in IGF-I and glucose concentrations (but not in those of other substances), and that IGF-I and glucose, in contrast with other substances, may be indicators or regulators of individual growth rates.

Research paper thumbnail of Ovarian, metabolic and endocrine indexes in dairy cows with different body condition scores

Journal of Animal and Feed Sciences

Body condition can influence bovine fertility, but the morphological, biochemical and endocrine m... more Body condition can influence bovine fertility, but the morphological, biochemical and endocrine mechanisms of this influence are not fully understood. The aim of the study was to examine the interrelationships between cow body condition, morphological and endocrine state of the ovary, and blood metabolic indexes. Czech Fleckvieh dairy cows at the follicular phase of the ovarian cycle and with a tendency towards emaciation (body condition score-CS2) and cows with an average body condition score (BCS3) were compared. Plasma concentrations of aspartate aminotransferase (AST), non-esterified fatty acids (NEFAs), Ca 2+ , inorganic phosphorus (P i), Mg 2+ , Fe 2+ , Cu 2+ , Zn 2+ (determined using photometrically), leptin and insulin (ELISA), ovarian area, number of visible ovarian follicles, diameter of primary and secondary ovarian follicles and corpora albicantia (macro-and micrometric analysis of ovarian histological sections), as well as the release of progesterone, testosterone, oestradiol and insulin-like growth factor I (IGF-I) by isolated ovarian granulosa cells (RIA) were analysed. No significant differences between BCS2 and BCS3 cows in blood metabolic and endocrine indexes (except for decreased Zn 2+ in BCS3 cows) were found. The ovaries of BCS2 cows, however, showed a lower ovarian area, diameter of both primary and secondary follicles and corpora albicantia, but not the number of visible secondary follicles as compared with BCS3 cows. No differences between the release of progesterone, testosterone and IGF-I by ovarian granulosa cells isolated from BCS2 or BCS3 cows were found, but the granulosa cells of BCS2 animals released more oestradiol than those of BCS3 cows. These results indicate that a slight reduction in BCS (tendency towards emaciation) does not substantially affect ovarian secretory activity or metabolic blood indexes. On the other hand, a tendency towards emaciation is associated with reduced ovarian follicle growth (but not their number) and increased secretion of oestradiol. These observations suggest that a tendency towards emaciation can suppress bovine fertility via alterations in ovarian folliculogenesis and oestrogen release.

Research paper thumbnail of The yield and cell viability of bovine <i>in vivo</i> recovered embryos in relation to season of flushing

Journal of Animal and Feed Sciences

Research paper thumbnail of Quality of Pinzgau bull spermatozoa following different periods of cryostorage

Zygote

Summary The aim of this work was to examine the influence of cryostorage duration of Pinzgau bull... more Summary The aim of this work was to examine the influence of cryostorage duration of Pinzgau bull's insemination doses (IDs) on some sperm traits. The IDs were frozen by a slow freezing method and stored in liquid nitrogen for different periods: less than 8 years (group 1), 8–13 years (group 2) and 14–18 years (group 3). Motility (CASA), pathological sperm rate (Giemsa staining), apoptotic (Yo-Pro-1-positive) and necrotic (propidium iodide-positive) cell occurrence and fertilizing ability (penetration/fertilization test) of spermatozoa were evaluated post-thaw. The average post-thaw sperm motility in all examined groups was over 40%. No significant influence of storage length either on the sperm total motility or progressive movement was revealed. In each tested group the average rate of malformed spermatozoa did not exceed 20%. No effect of cryostorage length on the occurrence of apoptotic or necrotic sperm was noted. Similarly, penetrating/fertilizing ability of sperm did not ...

Research paper thumbnail of State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes

Cryobiology, 2015

This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and d... more This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and development of rabbit pronuclear zygotes. Zygotes were collected from superovulated females and immediately used for 1) slow-freezing in a solution containing 1.5 M 1,2-propanediol and 0.2 M sucrose, or 2) vitrification in a solution containing 42.0% (v/v) of ethylene glycol, 18.0% (w/v) of dextran and 0.3 M sucrose as cryoprotectants. After thawing or warming, respectively, zygotes were evaluated for 1) actin distribution, 2) in vitro or 3) in vivo development to blastocyst. Comparing actin filaments distribution, a significantly higher number of vitrified zygotes with actin distributed in cell border was observed (55 ± 7.7 vs. 74 ± 6.1% for slow-frozen vs. vitrified, respectively). After 24 and 72 h of in vitro development, significant differences in the cleavage and morula rate among the groups were observed (9 ± 2.4 and 3 ± 1.3 vs. 44 ± 3.0 and 28 ± 2.7% for slow-frozen vs. vitrified, respectively). None of the slow-frozen zygotes reached the blastocyst stage, in contrast to the vitrified counterparts (11 ± 1.9%). Under in vivo culture conditions, a significant difference in blastocyst rate was observed between vitrified and fresh embryos (6 ± 1.5 vs. 35 ± 4.4% respectively). Our results showed that alterations in actin cytoskeleton and deteriorated development are more evident in slow-frozen than vitrified pronuclear zygotes. Vitrification method seems to be a more effective option for rabbit zygotes cryopreservation, although pronuclear zygotes manipulation per se resulted in a notable decrease in embryo development.

Research paper thumbnail of Developmental Rate of Rabbit Parthenogenetic Embryos Derived Using Different Activating Protocols

Asian-Australasian Journal of Animal Sciences, 2004

The present study compares development of rabbit embryos generated using different oocyte activat... more The present study compares development of rabbit embryos generated using different oocyte activation protocols and reconstructed with embryonic or cumulus cells as nuclear donor. In vivo matured oocytes were collected from New Zealand White rabbits at 16 h after ovulation treatment and were activated at18 h of post-ovulation treatment. The following schemes of oocytes activation were tested: 1) single electric pulse (EP, 3.2 kV/cm, 3×20 µs, 0.3 M mannitol)+5 min culture in the presence of 5 mM Ionomycin, 2) single electric pulse (EP, 3.2 kV/cm, 3×20 µs, 0.3 M mannitol)+1 h culture in the presence of 2 mM 6-DMAP, and 3) three electric pulses 30 min apart. Cleavage rate, percentage of expanded and hatched blastocysts as well as total cell number of blastomeres of parthenogenetic embryos were significantly higher using either EP+6-DMAP or 3×EP schemes, comparing with EP+Ionomycin. Development rate up to hatched blastocyst stage of cloned rabbit embryos using the EP+6-DMAP for activation of nuclei were 19% for embryonic cell nuclei and 36% for cumulus cell nuclei. The best activation protocol optimalized in this study was the combined treatment "EP+6-DMAP", which may be potentially used for nuclear transfer protocol.

Research paper thumbnail of Effect of growth hormone and inhibitors of protein kinase A on IGF-I, oxytocin and progesterone release by cultured bovine granulosa cells

Annales d'endocrinologie, 2000

In our experiments we studied the action of GH on the release of nonapeptide, steroid hormones an... more In our experiments we studied the action of GH on the release of nonapeptide, steroid hormones and growth factor, in bovine ovarian granulosa cells, as well as the role of cAMP-stimulated protein kinase A (PKA) in the mediation of these GH effects. For this purpose, the effects of exogenous bGH (0.001-10 mg/ml), PKA blockers KT5720 (100 ng/ml) and Rp-cAMPS (1 mmol), alone and in combination, on IGF-I, oxytocin and progesterone secretion were investigated. It was found that GH addition to culture medium strongly (p<0.05) stimulated IGF-I (at a concentration of 0.01-0.1 mgGH/ml medium), oxytocin (0.01-10 mgGH/ml) and progesterone (0.01-1 mgGH/ml medium) secretion into the culture medium. PKA blockers KT5720 and Rp-cAMPS given alone did not affect release of these substances. Rp-cAMPS partially prevented GH effect on IGF-I release, but enhanced GH action on progesterone output. KT5720 did not modify action of GH on oxytocin release. These observations confirm the involvement of GH i...

Research paper thumbnail of Integration and expression of the WAP-hPC gene in three generations of transgenic rabbits

The aim of the present study was to obtain transgenic rabbits expressing human protein C in their... more The aim of the present study was to obtain transgenic rabbits expressing human protein C in their mammary gland. A fusion construct which consisted of 4.2 kb long mouse whey acidic protein (WAP) promoter and 9.4 kb genomic human protein C (hPC) was microinjected into rabbit zygotes. Born animals were subjected to PCR amplification to detect the integration of the

Research paper thumbnail of Effect of Growth Hormone on Porcine Ovarian Cells and its intracellular Mechanisms

Research paper thumbnail of The secretory activity of cultured ovarian granulosa cells as indicator of biochemical activity of porcine growth hormone

Research paper thumbnail of Effect of body condition and season on yield and quality of in vitro produced bovine embryos

Zygote, 2014

SummaryThe aim of our study was to examine the effects of cow's body condition score (BCS; sc... more SummaryThe 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....

Research paper thumbnail of Effects of superovulation, culture and microinjection on development of rabbit embryos in vitro

Theriogenology, 1998

Factors influencing the developmental potencial of cultured rabbit zygotes and their ability to i... more Factors influencing the developmental potencial of cultured rabbit zygotes and their ability to incorporate and integrate the WAP-hPC (human protein C) gene were investigated. Rabbit zygotes (n=1053) were recovered from both superovulated and nontreated New Zealand White females. The hormonal treatment of rabbit donors resulted in a doubling of the number of recovered ova per donor when compared with the nontreated group (18 vs 9 ova). However, the quality of recovered zygotes (presence of both pronuclei) was significantly better in the nontreated group (99 vs 88%, Experiment 1). The effect of various culture media on the development of rabbit zygotes in vitro was evaluated after incubation under CO2-free conditions (Experiment 2). In serum-flee, growth factorsupplemented medium (BSEITS, DME/F12, 1.5% BSA, EGF, insulin, transferrin and sodium selenite) the percentage of morula/blastocyst stage embryos was significantly higher (88%) than in DME/FCS, (DME/F12, 10% fetal calf serum, 59%) or the control group (DME/F12, 1.5% BSA, 25%). In Experiment 3, zygotes were microinjected with the WAP-hPC gene and were examined after 72 h of culture. Zygote cleavage and the percentage of morula/blastocyst stage intact embryos were higher (79 and 58%, respectively) than in microinjected embryos (31.0 and 21.5%, respectively). Summarized data of the PCR assay of microinjected zygotes demonstrated positive signals for the integration of the WAP-hPC gene in 6.6% (34 of 515) of all the microinjected zygotes.

Research paper thumbnail of Evidence that growth factors IGF-I, IGF-II and EGF can stimulate nuclear maturation of porcine oocytes via intracellular protein kinase A

Reproduction Nutrition Development, 2000

The aim of our in vitro experiments was to study the role of growth factors and protein kinase A ... more The aim of our in vitro experiments was to study the role of growth factors and protein kinase A (PKA)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. Oocytes were cultured with or without growth factors (IGF-I, IGF-II, EGF; 10 ng. mL-1 medium) and inhibitors of PKA (Rp-cAMPS or KT5720; 100 ng. mL-1). Stages of meiosis were determined from the structure of chromosomes after staining with Giemza. Intracellular levels of PKA were evaluated immunocytochemically using primary antisera against the PKA regulatory and catalytic subunits and by Western immunoblotting using primary antiserum against the PKA catalytic subunit. It was found that after 24 h culture the majority of oocytes had resumed nuclear maturation (they were at a stage of meiosis after diplotene) and that after 48 h culture the majority of cells had completed maturation (they had reached metaphase II of meiosis). Addition of IGF-I, IGF-II or EGF, or a combination of IGF-I and EGF, significantly increased the proportion of oocytes which resumed and completed meiosis. Immunocytochemistry demonstrated a significant increase in the proportion of cells containing catalytic and, in some cases, the regulatory subunits of PKA after addition of IGF-I, IGF-II and EGF. Immunoblotting showed the presence of 2 forms of the PKA catalytic subunit within the oocytes (MW approximately 52 and 40 kD). EGF, but not IGF-I or IGF-II, increased the content of both isoforms. Inhibitors of PKA, when given alone, did not substantially influence the proportion of oocytes which resumed or completed meiosis. However, Rp-cAMPS and KT5720 both prevented the stimulatory effects of IGF-I, IGF-II and EGF on the resumption and completion of oocyte maturation. The present observations suggest (1) that IGF-I, IGF-II and EGF are potent stimulators of both resumption and completion of porcine oocyte nuclear maturation, (2) that PKA is present in oocytes, and (3) that PKA-dependent intracellular mechanisms can mediate the action of growth factors on porcine oocytes.

Research paper thumbnail of during in vitro embryo production Viability of bovine embryos following exposure to the green filtered or wider bandwidth light

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

Zygote, 2010

SummaryWith the development of embryo technologies, such asin vitrofertilization, cloning and tra... more SummaryWith the development of embryo technologies, such asin vitrofertilization, 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 sev...

Research paper thumbnail of Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro

The Journal of Steroid Biochemistry and Molecular Biology, 1997

The aim of our experiments was to study the influence of genisteln [tyroslne kinase (TK) inhibito... more The aim of our experiments was to study the influence of genisteln [tyroslne kinase (TK) inhibitor with estrogenic activity] and lavendustin A (TK inhibitor without estrogenic activity) on female reproductive processes in domestic animals in vitro. It was found that genistein (0.001-1 #glml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01-10 pglml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa ceils (at 0.01-I0 #glml), estradiol output by rabbit granulosa cells (at 1 #g/ml) and porcine ovarian follicles (at l0 pglml), as well as cAMP production by bovine (at 0.001-1 pg/ml) and rabbit (at 1 #glml) granulosa cells. No effects of genistein (at 10 pg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P < 0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 #glml), as well as the preimplantation development of rabbit zygotes (at 1 #g/ml). Lavendustin A (0.001-1 #glml) increased IGF-I release by bovine (but not by porcine) granulosa cells, cAMP release by bovine granulosa cells, and PGF-2 alpha output by porcine ovarian follicles (at 10 gg/ml). Lavendustin (at 1 #glml) had no significant effect on IGF-I release by porcine granulosa cells, on estradiol and cAMP output by rabbit granulosa cells, or on progesterone secretion by porcine follicles (at 10 #gl hal). Inhibitory actions of lavendustln (at 10 #glml) on estradiol secretion by porcine follicles were also found. Furthermore, lavendustin, like genistein, promoted the reinitiation and completion of meiosis in porcine oocytes. The present study demonstrates a predominantly stimulatory effect of TK inhibition on endocrine and generative processes in domestic animals. The majority of these effects are similar l~or both compounds, indirectly suggesting that their action is due to tyrosine klnase inhibition and protein kinase A-stimulation, rather than estrogenic activity.

Research paper thumbnail of The Role of Oxytocin, Protein Kinase A, and ERK-Related MAP-Kinase in the Control of Porcine Ovarian Follicle Functions

Experimental and Clinical Endocrinology & Diabetes, 2004

The aim of our in vitro experiments was to study the role of oxytocin (OT), cAMP/protein kinase A... more The aim of our in vitro experiments was to study the role of oxytocin (OT), cAMP/protein kinase A (PKA), and mitogen-activated protein kinase (ERKs MAP-kinase) in the control of ovarian cell functions as well as the role of PKA and MAPK in mediating OT effects on these processes. The whole porcine ovarian follicles were cultured in the presence or absence of OT (1, 10, 100 ng/ml), PKA inhibitor Rp-cAMPS (10 nM), MAP-kinase inhibitor PD98059 (1 microg/ml), or their combination. The release of prostaglandins F (PGF) and E (PGE) were determined by RIA, PKA (alpha-cat subunit), the proliferation-associated peptide PCNA and ERK-1, -2 expression in cell lyzates were analysed by Western-blotting. OT stimulated the release of PGF and PGE, and accumulation of PKA, ERK-1/-2, and PCNA in cell lysate. PD98059 decreased the basal PGF and PGE output, as well as reduced both ERK-1 and ERK-2 accumulation in cell lysates. Rp-cAMPS decreased PKA accumulation in cell lysates. Rp-cAMPS prevented the OT-induced stimulation of PKA, ERK-1, ERK-2, PGF, and PGE, PD98059 did so for PKA, PGF, and PGE. However, PD98059 reduced either basal or OT-induced p-ERK level. OT-stimulated PCNA accumulation was only slightly modified by these blockers. These observations suggest that OT, PKA, and ERKs MAPK can be involved in the control of PGs release and proliferation of ovarian cells. The influence of OT on both PKA and MAPK, and the ability of PKA and MAPK blockers to prevent completely or partially OT effects suggest, that effects of OT on PGF and PGE can be mediated by both PKA and MAPK. The role of MAPK and PKA in mediating the proliferative effects of OT seems to be minor assuming the involvement of other intracellular messengers.

Research paper thumbnail of PERSPECTIVE: Rooster Spermatozoa Cryopreservation and Quality Assessment

Cryo letters, 2021

Unsuccessful rooster fertility following cryopreservation may be linked to specific changes in sp... more Unsuccessful rooster fertility following cryopreservation may be linked to specific changes in spermatozoa quality, which can be determined using various methods. These determinations also facilitate the design of improved freezing and thawing processes. Here, we update the current state of methodologies available for the assessment of rooster semen quality after cryopreservation. Computer-assisted sperm analyses (CASA) is one of the main systems used to analyse motion parameters of spermatozoa (total motility, progressive motility and motion parameters). Moreover, fluorescent techniques and flow cytometry can improve the assessment of various aspects of semen quality (viability, acrosome status, mitochondrial potential, lipid peroxidation, DNA damage, lipid peroxidation and cell debris removal) using specific fluorescent markers such as ethidium bromide, Yo-Pro-1, Annexin V, propidium iodide, SYBR-14, PNA, JC-1, BODIPY, acridine orange and DRAQ5. Transmission electron microscopy al...

Research paper thumbnail of Development of a sensitive radioimmunoassay for IGF-I determination in samples from blood plasma and cell-conditioned medium

Research paper thumbnail of Pathological Alterations in Hepatocytes of Dairy Cows With a Tendency to Emaciation and Fattening

Folia Veterinaria

The aim of this study was to demonstrate the histochemical and histopathological alterations in t... more The aim of this study was to demonstrate the histochemical and histopathological alterations in the livers of cows with a tendency to become emaciated (body condition score - BCS1 and 2) and a tendency to become fattened (BCS4 and 5) in comparison to the cows of average body condition (BCS3) presented as a control. The histochemical analysis (PAS reaction) showed that the influence of emaciation and fattening in our study was manifested by a decreased occurrence of glycogen and a decreased level of the PAS-positive matter in the hepatocytes of dairy cows with BCS1, 2, 4 and 5. An abundant accumulation of lipids in the form of large lipid droplets, liposomes and lipoproteins observed in the hepatocytes of emaciated and fattened (BCS1 and 5) cows may be related to moderate-severe steatosis. These observations suggest a relationship between liver steatosis and the occurrence of lipoproteins in cows with a tendency toward emaciation and fattening.

Research paper thumbnail of Are IGF-I, thyroid hormone and metabolite concentrations in calf plasma associated with growth rate, sex and age?

Journal of Animal and Feed Sciences

The aims of our experiments were (1) to study age-dependent changes in bovine plasma concentratio... more The aims of our experiments were (1) to study age-dependent changes in bovine plasma concentrations of insulin-like growth factor I (IGF-1), thyroid hormones (T3, T4), some metabolites (glucose, triglycerides, ketone bodies), calcium, phosphorus and sodium, and (2) to determine whether plasma concentrations of these substances are associated with individual and sex-dependent differences in the growth rates of calves. Eight male and 8 female calves were weighed at the beginning and at the end of the experiment and daily gain and feed consumption (dry matter, crude protein, PDIN and NEL per kg daily gain) were determined. Plasma concentrations of IGF-I, T3, T4, glucose, triglycerides, ketone bodies, calcium, phosphorus and sodium were measured in calves at 2, 4, 6, 8, 10 and 12 weeks of age. There were significant age-dependent increases in the plasma concentrations of IGF-I and glucose. Males had a significantly higher daily gain and lower feed consumption than females, but no sex-dependent differences in plasma constituents were detected. In both sexes, daily gain was positively correlated with plasma IGF-I (coefficients of correlation r=0.630 in females and r=0.614 in males) and glucose (r=0.496 and 0.427, respectively) concentrations, but not with other substances. Our observations show that there are age-dependent changes in IGF-I and glucose concentrations (but not in those of other substances), and that IGF-I and glucose, in contrast with other substances, may be indicators or regulators of individual growth rates.

Research paper thumbnail of Ovarian, metabolic and endocrine indexes in dairy cows with different body condition scores

Journal of Animal and Feed Sciences

Body condition can influence bovine fertility, but the morphological, biochemical and endocrine m... more Body condition can influence bovine fertility, but the morphological, biochemical and endocrine mechanisms of this influence are not fully understood. The aim of the study was to examine the interrelationships between cow body condition, morphological and endocrine state of the ovary, and blood metabolic indexes. Czech Fleckvieh dairy cows at the follicular phase of the ovarian cycle and with a tendency towards emaciation (body condition score-CS2) and cows with an average body condition score (BCS3) were compared. Plasma concentrations of aspartate aminotransferase (AST), non-esterified fatty acids (NEFAs), Ca 2+ , inorganic phosphorus (P i), Mg 2+ , Fe 2+ , Cu 2+ , Zn 2+ (determined using photometrically), leptin and insulin (ELISA), ovarian area, number of visible ovarian follicles, diameter of primary and secondary ovarian follicles and corpora albicantia (macro-and micrometric analysis of ovarian histological sections), as well as the release of progesterone, testosterone, oestradiol and insulin-like growth factor I (IGF-I) by isolated ovarian granulosa cells (RIA) were analysed. No significant differences between BCS2 and BCS3 cows in blood metabolic and endocrine indexes (except for decreased Zn 2+ in BCS3 cows) were found. The ovaries of BCS2 cows, however, showed a lower ovarian area, diameter of both primary and secondary follicles and corpora albicantia, but not the number of visible secondary follicles as compared with BCS3 cows. No differences between the release of progesterone, testosterone and IGF-I by ovarian granulosa cells isolated from BCS2 or BCS3 cows were found, but the granulosa cells of BCS2 animals released more oestradiol than those of BCS3 cows. These results indicate that a slight reduction in BCS (tendency towards emaciation) does not substantially affect ovarian secretory activity or metabolic blood indexes. On the other hand, a tendency towards emaciation is associated with reduced ovarian follicle growth (but not their number) and increased secretion of oestradiol. These observations suggest that a tendency towards emaciation can suppress bovine fertility via alterations in ovarian folliculogenesis and oestrogen release.

Research paper thumbnail of The yield and cell viability of bovine <i>in vivo</i> recovered embryos in relation to season of flushing

Journal of Animal and Feed Sciences

Research paper thumbnail of Quality of Pinzgau bull spermatozoa following different periods of cryostorage

Zygote

Summary The aim of this work was to examine the influence of cryostorage duration of Pinzgau bull... more Summary The aim of this work was to examine the influence of cryostorage duration of Pinzgau bull's insemination doses (IDs) on some sperm traits. The IDs were frozen by a slow freezing method and stored in liquid nitrogen for different periods: less than 8 years (group 1), 8–13 years (group 2) and 14–18 years (group 3). Motility (CASA), pathological sperm rate (Giemsa staining), apoptotic (Yo-Pro-1-positive) and necrotic (propidium iodide-positive) cell occurrence and fertilizing ability (penetration/fertilization test) of spermatozoa were evaluated post-thaw. The average post-thaw sperm motility in all examined groups was over 40%. No significant influence of storage length either on the sperm total motility or progressive movement was revealed. In each tested group the average rate of malformed spermatozoa did not exceed 20%. No effect of cryostorage length on the occurrence of apoptotic or necrotic sperm was noted. Similarly, penetrating/fertilizing ability of sperm did not ...

Research paper thumbnail of State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes

Cryobiology, 2015

This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and d... more This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and development of rabbit pronuclear zygotes. Zygotes were collected from superovulated females and immediately used for 1) slow-freezing in a solution containing 1.5 M 1,2-propanediol and 0.2 M sucrose, or 2) vitrification in a solution containing 42.0% (v/v) of ethylene glycol, 18.0% (w/v) of dextran and 0.3 M sucrose as cryoprotectants. After thawing or warming, respectively, zygotes were evaluated for 1) actin distribution, 2) in vitro or 3) in vivo development to blastocyst. Comparing actin filaments distribution, a significantly higher number of vitrified zygotes with actin distributed in cell border was observed (55 ± 7.7 vs. 74 ± 6.1% for slow-frozen vs. vitrified, respectively). After 24 and 72 h of in vitro development, significant differences in the cleavage and morula rate among the groups were observed (9 ± 2.4 and 3 ± 1.3 vs. 44 ± 3.0 and 28 ± 2.7% for slow-frozen vs. vitrified, respectively). None of the slow-frozen zygotes reached the blastocyst stage, in contrast to the vitrified counterparts (11 ± 1.9%). Under in vivo culture conditions, a significant difference in blastocyst rate was observed between vitrified and fresh embryos (6 ± 1.5 vs. 35 ± 4.4% respectively). Our results showed that alterations in actin cytoskeleton and deteriorated development are more evident in slow-frozen than vitrified pronuclear zygotes. Vitrification method seems to be a more effective option for rabbit zygotes cryopreservation, although pronuclear zygotes manipulation per se resulted in a notable decrease in embryo development.

Research paper thumbnail of Developmental Rate of Rabbit Parthenogenetic Embryos Derived Using Different Activating Protocols

Asian-Australasian Journal of Animal Sciences, 2004

The present study compares development of rabbit embryos generated using different oocyte activat... more The present study compares development of rabbit embryos generated using different oocyte activation protocols and reconstructed with embryonic or cumulus cells as nuclear donor. In vivo matured oocytes were collected from New Zealand White rabbits at 16 h after ovulation treatment and were activated at18 h of post-ovulation treatment. The following schemes of oocytes activation were tested: 1) single electric pulse (EP, 3.2 kV/cm, 3×20 µs, 0.3 M mannitol)+5 min culture in the presence of 5 mM Ionomycin, 2) single electric pulse (EP, 3.2 kV/cm, 3×20 µs, 0.3 M mannitol)+1 h culture in the presence of 2 mM 6-DMAP, and 3) three electric pulses 30 min apart. Cleavage rate, percentage of expanded and hatched blastocysts as well as total cell number of blastomeres of parthenogenetic embryos were significantly higher using either EP+6-DMAP or 3×EP schemes, comparing with EP+Ionomycin. Development rate up to hatched blastocyst stage of cloned rabbit embryos using the EP+6-DMAP for activation of nuclei were 19% for embryonic cell nuclei and 36% for cumulus cell nuclei. The best activation protocol optimalized in this study was the combined treatment "EP+6-DMAP", which may be potentially used for nuclear transfer protocol.

Research paper thumbnail of Effect of growth hormone and inhibitors of protein kinase A on IGF-I, oxytocin and progesterone release by cultured bovine granulosa cells

Annales d'endocrinologie, 2000

In our experiments we studied the action of GH on the release of nonapeptide, steroid hormones an... more In our experiments we studied the action of GH on the release of nonapeptide, steroid hormones and growth factor, in bovine ovarian granulosa cells, as well as the role of cAMP-stimulated protein kinase A (PKA) in the mediation of these GH effects. For this purpose, the effects of exogenous bGH (0.001-10 mg/ml), PKA blockers KT5720 (100 ng/ml) and Rp-cAMPS (1 mmol), alone and in combination, on IGF-I, oxytocin and progesterone secretion were investigated. It was found that GH addition to culture medium strongly (p<0.05) stimulated IGF-I (at a concentration of 0.01-0.1 mgGH/ml medium), oxytocin (0.01-10 mgGH/ml) and progesterone (0.01-1 mgGH/ml medium) secretion into the culture medium. PKA blockers KT5720 and Rp-cAMPS given alone did not affect release of these substances. Rp-cAMPS partially prevented GH effect on IGF-I release, but enhanced GH action on progesterone output. KT5720 did not modify action of GH on oxytocin release. These observations confirm the involvement of GH i...

Research paper thumbnail of Integration and expression of the WAP-hPC gene in three generations of transgenic rabbits

The aim of the present study was to obtain transgenic rabbits expressing human protein C in their... more The aim of the present study was to obtain transgenic rabbits expressing human protein C in their mammary gland. A fusion construct which consisted of 4.2 kb long mouse whey acidic protein (WAP) promoter and 9.4 kb genomic human protein C (hPC) was microinjected into rabbit zygotes. Born animals were subjected to PCR amplification to detect the integration of the

Research paper thumbnail of Effect of Growth Hormone on Porcine Ovarian Cells and its intracellular Mechanisms

Research paper thumbnail of The secretory activity of cultured ovarian granulosa cells as indicator of biochemical activity of porcine growth hormone

Research paper thumbnail of Effect of body condition and season on yield and quality of in vitro produced bovine embryos

Zygote, 2014

SummaryThe aim of our study was to examine the effects of cow's body condition score (BCS; sc... more SummaryThe 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....

Research paper thumbnail of Effects of superovulation, culture and microinjection on development of rabbit embryos in vitro

Theriogenology, 1998

Factors influencing the developmental potencial of cultured rabbit zygotes and their ability to i... more Factors influencing the developmental potencial of cultured rabbit zygotes and their ability to incorporate and integrate the WAP-hPC (human protein C) gene were investigated. Rabbit zygotes (n=1053) were recovered from both superovulated and nontreated New Zealand White females. The hormonal treatment of rabbit donors resulted in a doubling of the number of recovered ova per donor when compared with the nontreated group (18 vs 9 ova). However, the quality of recovered zygotes (presence of both pronuclei) was significantly better in the nontreated group (99 vs 88%, Experiment 1). The effect of various culture media on the development of rabbit zygotes in vitro was evaluated after incubation under CO2-free conditions (Experiment 2). In serum-flee, growth factorsupplemented medium (BSEITS, DME/F12, 1.5% BSA, EGF, insulin, transferrin and sodium selenite) the percentage of morula/blastocyst stage embryos was significantly higher (88%) than in DME/FCS, (DME/F12, 10% fetal calf serum, 59%) or the control group (DME/F12, 1.5% BSA, 25%). In Experiment 3, zygotes were microinjected with the WAP-hPC gene and were examined after 72 h of culture. Zygote cleavage and the percentage of morula/blastocyst stage intact embryos were higher (79 and 58%, respectively) than in microinjected embryos (31.0 and 21.5%, respectively). Summarized data of the PCR assay of microinjected zygotes demonstrated positive signals for the integration of the WAP-hPC gene in 6.6% (34 of 515) of all the microinjected zygotes.

Research paper thumbnail of Evidence that growth factors IGF-I, IGF-II and EGF can stimulate nuclear maturation of porcine oocytes via intracellular protein kinase A

Reproduction Nutrition Development, 2000

The aim of our in vitro experiments was to study the role of growth factors and protein kinase A ... more The aim of our in vitro experiments was to study the role of growth factors and protein kinase A (PKA)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. Oocytes were cultured with or without growth factors (IGF-I, IGF-II, EGF; 10 ng. mL-1 medium) and inhibitors of PKA (Rp-cAMPS or KT5720; 100 ng. mL-1). Stages of meiosis were determined from the structure of chromosomes after staining with Giemza. Intracellular levels of PKA were evaluated immunocytochemically using primary antisera against the PKA regulatory and catalytic subunits and by Western immunoblotting using primary antiserum against the PKA catalytic subunit. It was found that after 24 h culture the majority of oocytes had resumed nuclear maturation (they were at a stage of meiosis after diplotene) and that after 48 h culture the majority of cells had completed maturation (they had reached metaphase II of meiosis). Addition of IGF-I, IGF-II or EGF, or a combination of IGF-I and EGF, significantly increased the proportion of oocytes which resumed and completed meiosis. Immunocytochemistry demonstrated a significant increase in the proportion of cells containing catalytic and, in some cases, the regulatory subunits of PKA after addition of IGF-I, IGF-II and EGF. Immunoblotting showed the presence of 2 forms of the PKA catalytic subunit within the oocytes (MW approximately 52 and 40 kD). EGF, but not IGF-I or IGF-II, increased the content of both isoforms. Inhibitors of PKA, when given alone, did not substantially influence the proportion of oocytes which resumed or completed meiosis. However, Rp-cAMPS and KT5720 both prevented the stimulatory effects of IGF-I, IGF-II and EGF on the resumption and completion of oocyte maturation. The present observations suggest (1) that IGF-I, IGF-II and EGF are potent stimulators of both resumption and completion of porcine oocyte nuclear maturation, (2) that PKA is present in oocytes, and (3) that PKA-dependent intracellular mechanisms can mediate the action of growth factors on porcine oocytes.

Research paper thumbnail of during in vitro embryo production Viability of bovine embryos following exposure to the green filtered or wider bandwidth light

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

Zygote, 2010

SummaryWith the development of embryo technologies, such asin vitrofertilization, cloning and tra... more SummaryWith the development of embryo technologies, such asin vitrofertilization, 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 sev...

Research paper thumbnail of Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro

The Journal of Steroid Biochemistry and Molecular Biology, 1997

The aim of our experiments was to study the influence of genisteln [tyroslne kinase (TK) inhibito... more The aim of our experiments was to study the influence of genisteln [tyroslne kinase (TK) inhibitor with estrogenic activity] and lavendustin A (TK inhibitor without estrogenic activity) on female reproductive processes in domestic animals in vitro. It was found that genistein (0.001-1 #glml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01-10 pglml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa ceils (at 0.01-I0 #glml), estradiol output by rabbit granulosa cells (at 1 #g/ml) and porcine ovarian follicles (at l0 pglml), as well as cAMP production by bovine (at 0.001-1 pg/ml) and rabbit (at 1 #glml) granulosa cells. No effects of genistein (at 10 pg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P < 0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 #glml), as well as the preimplantation development of rabbit zygotes (at 1 #g/ml). Lavendustin A (0.001-1 #glml) increased IGF-I release by bovine (but not by porcine) granulosa cells, cAMP release by bovine granulosa cells, and PGF-2 alpha output by porcine ovarian follicles (at 10 gg/ml). Lavendustin (at 1 #glml) had no significant effect on IGF-I release by porcine granulosa cells, on estradiol and cAMP output by rabbit granulosa cells, or on progesterone secretion by porcine follicles (at 10 #gl hal). Inhibitory actions of lavendustln (at 10 #glml) on estradiol secretion by porcine follicles were also found. Furthermore, lavendustin, like genistein, promoted the reinitiation and completion of meiosis in porcine oocytes. The present study demonstrates a predominantly stimulatory effect of TK inhibition on endocrine and generative processes in domestic animals. The majority of these effects are similar l~or both compounds, indirectly suggesting that their action is due to tyrosine klnase inhibition and protein kinase A-stimulation, rather than estrogenic activity.