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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 also yields valuable information on spermatozoa ultrastructure. The application of these techniques to rooster spermatozoa is reviewed in relation to specific freezing techniques, the effects of cryoprotective agents (CPAs) and extenders, and the determination of spermatozoa quality after cryopreservation.

The present study investigated whether dietary turmeric (Curcuma longa L.) can improve rabbit reproduction, ovarian function, growth, or viability. Female New Zealand White rabbits were either fed a standard diet (n=15) or a diet enriched with 5 g (group E1) or 20 g (group E2) turmeric powder per 100 kg feed mixture (n=16 or 15, respectively). After 295 days, weight gain, conception and kindling rates, pup and mother viability, ovarian macro- and micro-morphometric indices, release of leptin in response to the addition LH, and the release of progesterone, testosterone and leptin by isolated ovarian fragments were analyzed. Dietary turmeric failed to affect ovarian length and weight but did increase the number of primary follicles (E2: 32.5% greater than control group), as well as the diameter of primary (E1: +19.4%, E2: +21.1%), secondary (E2: +41.4%), and tertiary (E1: +97.1%, E2: +205.1%) follicles. Turmeric also increased the number of liveborn (E1: +21.0%) and weaned (E1: +25.0%) pups and decreased the number of stillborn pups (E2: −87.5%) but did not affect weight gain, conception, or kindling rate. Furthermore, dietary turmeric decreased doe mortality during the first reproductive cycle (13.3% in control; 0% in E1; and 6.7% in E2) but not during the second cycle. In vitro, the ovaries of the turmeric-treated rabbits released more progesterone (E1: +85.7%, E2: +90.0%) and less testosterone (E2: −87.0%) and leptin (E2: −29.0%) than the ovaries of control rabbits. Moreover, LH decreased the leptin output of control rabbits but increased that of experimental rabbits. Therefore, it is likely that dietary turmeric improves pup viability and that it could promote rabbit fecundity by either (1) promoting the production of primary ovarian follicles or (2) stimulating the growth of follicles at all stages of folliculogenesis.

Summary The aim of our study was to compare the hFVIII mRNA expression in different organs, pathological changes and selected haematological and biochemical blood parameters between transgenic and non-transgenic rabbits from F3 generation. Selected physiological parameters of 3- to 4-month-old transgenic rabbits from F3 generation carrying human factor VIII gene (hFVIII) were analysed and compared with those of non-transgenic ones. Before slaughtering, the blood for haematological and biochemical analysis was taken from the central ear artery. Pathological and histological examination of vital organs and RT-PCR analysis of several tissue organs of transgenic and non-transgenic animals were performed after slaughtering. Except for the mammary gland tissue, slight hfVIII mRNA expression in the spleen, lung and brain and none expression in the liver, kidney, skeletal muscle and heart of rabbits were recorded. pathological examination of vital organs showed some pathological changes in both transgenic and non-transgenic rabbits which were confirmed by histological qualitative evaluations. Statistically significant lower values of blood haemoglobin in blood of transgenic (11.86 ± 0.86) animals compared with non-transgenic (12.41 ± 1.02, P

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

The aim of this in vitro study was to test the effect of microinjection (Mi) of foreign gene into the rabbit egg pronucleus and epidermal growth factor (EGF) addition on the blastocyst rate, the cell number and the diameter of embryos, and to determine possible relationships between embryo cell number and embryo diameter. Blastocyst rate was significantly decreased in gene- Mi (G-Mi/E0) group (63.1%) comparing to intact ones (83.5%, p1

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 atl8 h of post-ovulation treatment. The following schemes of oocytes activation were tested: 1) single electric pulse (EP, 3.2 kV/cm, 3x20 μs, 0.3 M mannitol)+5 min culture in the presence of 5 mM lonomycin, 2) single electric pulse (EP, 3.2 kV/cm, 3x20 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 3xEP schemes, comparing with EP+lonomycin. 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.

The aim of our in vitro experiments was to study the effects of EGF on rabbit ovarian cells, as well as the possible mechanisms of these effects. The influence of EGF on steroidogenesis, proliferation, cyclic nucleotides and MAP-kinase in rabbit granulosa cells were studied. Results of RIA showed, that EGF stimulated the release of progesterone (1-100 ng/ml), cAMP (at 100 ng/ml), cGMP (1-100 ng/ml). EGF effect on estradiol output was biphasic: at dose 1 ng/ml it inhibited, whilst at 100 ng/ml it strongly increased estradiol secretion. Immunocytochemical study demonstrated an EGF-induced (10 ng/ml) increase in the proportion of cells revealing proliferating cell nuclear antigen (41% vs 24.7% in control, p < 0.01). EGF (10 ng/ml) increased the proportion of cells with immunoreactivity to ERK-1 (more than two-fold) and ERK-3 (three-fold) members of the MAP-kinase family. Moreover, EGF induced the translocation of ERK-1 to the nucleus, whilst preferentially cytoplasmic localization of ERK-3 was not changed after EGF addition. This can indicate regulation of ERK-1 and -3 by EGF, as well as differential patterns of ERK-1 and ERK-3 expression in response to EGF in cultured granulosa cells. - These results indicate that EGF can be a stimulator of proliferation, steroidogenesis and cyclic nucleotide release by rabbit granulosa cells. Stimulation of cAMP and cGMP release, and activation of ERK-related MAP kinase in granulosa cells after EGF addition indicates the involvement of these intracellular messengers in mediating the EGF action on the ovary.

We described a procedure for multiple genotype analysis (determination of sex and of three genetic markers) from a single cell derived from bovine preimplantation embryo. It consists of primer extension preamplification—polymerase chain reaction (PEP-PCR) and subsequent single assay or multiplex PCR. A single blastomere that was isolated by microaspiration from bovine embryos at the 16- to 32-cell stage then was lysed and was subjected to the PEP-PCR. When testing 75 embryos, efficiency of genotyping by standard PCR for κ-casein, growth hormone (GH) and prolactin (PRL) polymorphic alleles was 91, 88 and 89%, respectively. Sexing efficiency in the multiplex PCR was 91%, based on the amplification of Y-specific locus using κ-casein internal standard. The microaspiration of a single blastomere was shown not be invasive for the embryos. It did not alter their development potential in vitro (P 0.05), as was seen by obtaining a similar percentage of embryos developing further into the blastocyst stage in the group subjected to biopsy ( 44 75 , 59%) and in the control group of embryos ( 30 50 , 60%).

The aim of our studies was to determine, whether difference in GH alleles affect growth Performance in cows and whether these effects may be mediated by IGF-I. The presence of L (leucine-rich) and V (valine-rich) alleles of GH, plasma IGF-I level and changes in body mass were analyzed in 84 bulls of Simmental (meat) and Holstein (milk) breeds at 90 d and 180 d of age using PCR, agarose gel electrophoresis and IRMA The agedependent increase in body mass, daily gain and plasma IGF-I concentration was observed in Simmental bulls. In Holstein bulls the age-dependent rise in plasma IGF-I was also found, whilst Holstein bulls had significantly lover IGF-I level, than Simmental bulls. The proportion of LL, VV and LV genotypes of GH in the randomly selected herd of Simmental bulls was 0.25:0.20:0.55. Animals of VV genotype had lower body mass, daily gain and plasma IGF-I level, than bulls of LL and LV genotypes, whilst no differences in these indexes between LL and LV genotypes were found. The observed association of growth rate, GH genotype and IGF-I level, suggest that GH genotype may affect meat Performance via IGF-I secretion.

Summary The amplification of polymerase chain reaction (PCR)-product for the k-casein (k-CN) C allele using a C-allele specific primer was developed. This method was compared and controlled with the existing technique of restriction fragment length polymorphism. Analysis of the genomic DNA of a known k-CN genotype using an allele-specific primer for k-CN C does not allow discrimination of the C heterozygous of homozygous forms, but discriminates C-carriers and non-C-carriers. Blood and milk samples of Holstein, Slovak Pied, Slovak Pinzgauer and Brown Swiss cattle were analysed. The occurrence of the C allele was found only in three Brown Swiss cows in heterozygous forms (AC). Complete genotypes were identified by subsequent restriction endonuclease digestion. The results were in agreement with milk protein analysis on polyacrylamide gel electrophoresis. The allele specific (AS)–PCR method described is a useful screening method for the pre-selection of C-carriers for subsequent MaeII digestion. Therefore, financial benefit can be achieved. Zusammfassung Eine allelspezifische Polymerase Kettenreaktion (PCR) zum Nachweis des k-Casein (k-CN) C Allels wurde mit Hilfe von C allel-spezifischen Primern entwickelt. Diese Methode wurde mit der existierenden Restriktion Fragmentlangen-Poly-Morphismen (RFLP) Technik verglichen und kontrolliert. Die Analyse von genomischer DNA unter Anwendung von allelspezifischen Primern fur das k-CN C Allel ergab identische Resultate im Vergleich zur RFLP Methode. Die Anwendung von allelspezifischen Primer ermoglicht nicht die Unterscheidung von C Heterozygoten und CC Homozygoten, aber ermoglicht die Unterscheidung der Trager des C-Alles von dem Nicht-Tragern des C-Allels. Blut- und Milchproben der Rassen Holstein, Slowakisches Fleckvieh, Slowakische Pinzgauer, und Brown Swiss wurden analysiert. Das Vorkommen des C Allels wurde nur bei drei Kuhen der Rasse Brown Swiss in heterozygoter Form (AC) beobachtet. Diese Genotypen wurden durch Spaltung mit Restriktionsendonukleasen bestatigt und die Resultate stimmten mit der Analyse von Milchproteinen auf PAGE uberein. Die beschriebene Allel-Spezifischen (AS)–PCR Methode ist eine nutzliche Suchmethode fur die Vorselektion von C-Tragern fur die folgende MaeII Spaltung was einen okonomischen Vorteil darstellt.

Differences in microscopic structure of the femur between 1-month-old transgenic rabbits carrying the hFVIII gene and non-transgenic rabbits were investigated. Bone microstructure was evaluated from the point of view of qualitative and quantitative histological characteristics. We identified fibrolamellar bone tissue only in the transgenic animals. Measured values for area, perimeter of the Haversian canals and minimum diameter of the primary osteons' vascular canals were higher in 1-month-old transgenic individuals (P0.05; P0.001). We also observed lower concentrations of Ca, P, K, solids, and total mineral content in femora of transgenic rabbits. A statistically significant difference was observed for the concentration of Ca (P0.05). Our results indicate evident changes in both qualitative and quantitative histological characteristics of the femur, which result especially in better blood supply and slightly reduced mineralization process in 1-month-old transgenic rabbits.

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16 h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (pN chimeric blastocysts (ppN chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.

The aim of this study was to determine whether there are differences in the karyotypes between transgenic and non-transgenic or control rabbits. New Zealand White transgenic rabbits (F1 generation) were obtained after breeding of transgenic founder rabbits that were derived from single--SM--or double microinjection--DM--with a WAP-hFVIII transgene. C-metaphase plates were obtained from short-time culture of peripheral blood lymphocytes synchronized by the addition of colcemide. A significantly higher rate of aneuploidy was observed in c-metaphase spreads of transgenic (56-66%) rabbits, as compared to non-transgenic ones (28-38%) (P0.05; P0.01). The patterns of chromosome banding were identical in both groups of rabbits. No structural aberrations were revealed in either group. These findings demonstrate that transgenic rabbits have a higher frequency of numerical chromosomal aberrations in their peripheral blood lymphocytes than normal rabbits, but without apparent deleterious effects on health or reproduction.

The aim of our experiments was to study the influence of genistein [tyrosine 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 microg/ml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01-10 microg/ml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa cells (at 0.01-10 microg/ml), estradiol output by rabbit granulosa cells (at 1 microg/ml) and porcine ovarian follicles (at 10 microg/ml), as well as cAMP production by bovine (at 0.001-1 microg/ml) and rabbit (at 1 microg/ml) granulosa cells. No effects of genistein (at 10 microg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 microg/ml), as well as the preimplantation development of rabbit zygotes (at 1 microg/ml). Lavendustin A (0.001-1 microg/ml) 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 microg/ml). Lavendustin (at 1 microg/ml) 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 microg/ml). Inhibitory actions of lavendustin (at 10 microg/ml) 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 for both compounds, indirectly suggesting that their action is due to tyrosine kinase inhibition and protein kinase A-stimulation, rather than estrogenic activity.