Study question Does pergafast 201 (PF201) have harmful effects on selected markers of in vitro maturation of pig oocytes? Summary answer PF201 affects the course of in vitro oocyte maturation, reaching the metaphase II stage, formation of the meiotic spindle and methylation of selected epigenetic markers. What is known already PF201 is a chemical used as a color developer and currently serves as a replacement for bisphenol A and S in related paper and other products. Bisphenol A and S are already proven endocrine disruptors that can released into the environment and from there enter the human body, where they disrupt physiological hormonal functions. In the case of PF201, toxicity to aquatic organisms, persistence in the environment and affinity to some proteins associated with non-infectious diseases have already been confirmed. Therefore, further testing of the biological effect of PF201 is necessary to assess the risks to human health and reproduction. Study design, size, duration Cumulus-oocytes complexes (COCs) from the ovaries of slaughtered prepubertal sows were used. In vitro maturation dynamics of PF201-treated COCs were compared with control COCs (n ≥ 30). The meiotic spindle was assessed under a confocal microscope after fluorescence labeling of meiotic chromosomes and spindle microtubules. Dimethylation of histone H3 at lysine K4 (H3K4me2) and trimethylation at lysine K9 (H3K4me3) were imaged under a fluorescence microscope after fluorescent labeling, the intensity was subsequently evaluated in the ImageJ software. Participants/materials, setting, methods COCs were treated with PF201 in at least five independent experiments using the following concentrations: 300 pM, 30 nM and 3 µM, dissolved in DMSO to a final concentration of 0.05%. A vehicle control was used when COCs were cultured in medium with the same concentration of DMSO. COCs were cultured for 48 hours to metaphase II in 5.0% CO2 at 39 °C. Main results and the role of chance PF201 exposure in vitro affected meiotic maturation of pig oocytes, their ability to reach metaphase II, meiotic spindle formation, and epigenetic histone modifications. Treatment with 30 nM concentration of PF201 in vitro caused a significant accumulation of oocytes in metaphase I stage (35% vs. 7% in controls, p < 0.05), consistent with a non-linear effect. Moreover, according to our results, PF201 also disrupts the formation of the meiotic spindle in pig oocytes and probably affects the organization of the meiotic spindle at multiple levels. After 300 pM (30%) and 30 nM (25% vs. 10% in controls, p < 0.05) in vitro treatment of PF201, a significant increase in abnormalities of the meiotic spindle, most often irregular arrangement of the spindle, reduction of the meiotic figure, faulty attachment of chromosomes to tubulin and elongated poles of the meiotic spindle located far apart. A significant increase in the relative fluorescence intensity of epigenetic markers was also observed after treatment of oocytes with PF201. For histone H3K4me2, a significant increase in relative fluorescence intensity was observed after treatment with 300 pM and 3 µM concentrations, while for histone H3K9me3, a significant increase in relative intensity was observed after treatment with 30 nM and 3 µM concentrations. Limitations, reasons for caution As work with human oocytes encounters a number of ethical and legal regulations, a porcine model was chosen, which is physiologically close to the human oocyte. However, it would be great benefit if the effects of PF201 were evaluated in human discarded oocytes that can be obtained from IVF laboratories. Wider implications of the findings It is increasingly difficult for the human population to avoid exposure to environmentally toxic substances, and this fact could explain the decline in human fertility in recent decades. The finding that PF201 negatively affects meiotic maturation suggests, that it may reduce fertilization rates, thus requiring a re-evaluation of its use. Trial registration number MZE-RO0718

Abstract Study question Does pergafast 201 (PF201) have harmful effects on selected markers of in vitro maturation of pig oocytes? Summary answer PF201 affects the course of in vitro oocyte maturation, reaching the metaphase II stage, formation of the meiotic spindle and methylation of selected epigenetic markers. What is known already PF201 is a chemical used as a color developer and currently serves as a replacement for bisphenol A and S in related paper and other products. Bisphenol A and S are already proven endocrine disruptors that can released into the environment and from there enter the human body, where they disrupt physiological hormonal functions. In the case of PF201, toxicity to aquatic organisms, persistence in the environment and affinity to some proteins associated with non-infectious diseases have already been confirmed. Therefore, further testing of the biological effect of PF201 is necessary to assess the risks to human health and reproduction. Study design, size, duration Cumulus-oocytes complexes (COCs) from the ovaries of slaughtered prepubertal sows were used. In vitro maturation dynamics of PF201-treated COCs were compared with control COCs (n ≥ 30). The meiotic spindle was assessed under a confocal microscope after fluorescence labeling of meiotic chromosomes and spindle microtubules. Dimethylation of histone H3 at lysine K4 (H3K4me2) and trimethylation at lysine K9 (H3K4me3) were imaged under a fluorescence microscope after fluorescent labeling, the intensity was subsequently evaluated in the ImageJ software. Participants/materials, setting, methods COCs were treated with PF201 in at least five independent experiments using the following concentrations: 300 pM, 30 nM and 3 µM, dissolved in DMSO to a final concentration of 0.05%. A vehicle control was used when COCs were cultured in medium with the same concentration of DMSO. COCs were cultured for 48 hours to metaphase II in 5.0% CO2 at 39 °C. Main results and the role of chance PF201 exposure in vitro affected meiotic maturation of pig oocytes, their ability to reach metaphase II, meiotic spindle formation, and epigenetic histone modifications. Treatment with 30 nM concentration of PF201 in vitro caused a significant accumulation of oocytes in metaphase I stage (35% vs. 7% in controls, p < 0.05), consistent with a non-linear effect. Moreover, according to our results, PF201 also disrupts the formation of the meiotic spindle in pig oocytes and probably affects the organization of the meiotic spindle at multiple levels. After 300 pM (30%) and 30 nM (25% vs. 10% in controls, p < 0.05) in vitro treatment of PF201, a significant increase in abnormalities of the meiotic spindle, most often irregular arrangement of the spindle, reduction of the meiotic figure, faulty attachment of chromosomes to tubulin and elongated poles of the meiotic spindle located far apart. A significant increase in the relative fluorescence intensity of epigenetic markers was also observed after treatment of oocytes with PF201. For histone H3K4me2, a significant increase in relative fluorescence intensity was observed after treatment with 300 pM and 3 µM concentrations, while for histone H3K9me3, a significant increase in relative intensity was observed after treatment with 30 nM and 3 µM concentrations. Limitations, reasons for caution As work with human oocytes encounters a number of ethical and legal regulations, a porcine model was chosen, which is physiologically close to the human oocyte. However, it would be great benefit if the effects of PF201 were evaluated in human discarded oocytes that can be obtained from IVF laboratories. Wider implications of the findings It is increasingly difficult for the human population to avoid exposure to environmentally toxic substances, and this fact could explain the decline in human fertility in recent decades. The finding that PF201 negatively affects meiotic maturation suggests, that it may reduce fertilization rates, thus requiring a re-evaluation of its use. Trial registration number MZE-RO0718

The nutritional quality of beef relates to the fatty acid (FA) composition of bovine adipose tissue. Those molecular mechanisms that induce the differing amounts and composition of fat in cattle breeds according to age at maturity and purpose of production remain unclear. Therefore, this study investigated the composition of total FAs, adipocyte size, and expression of some key genes involved in several adipogenesis and lipogenesis pathways measured in distinct adipose tissue depots from bulls of the genetically diverse cattle breeds Aberdeen Angus (n = 9), Gascon (n = 10), Holstein (n = 9), and Fleckvieh (n = 10). The animals were finished under identical housing and feeding conditions until slaughter at a similar age of 17 months. After slaughter, cod adipose tissue (CAT), subcutaneous adipose tissue (SAT), and M. longissimus lumborum (MLL) samples were collected. The saturated FA proportions were higher (P < .01) in CAT than in SAT across all breeds, whereas monounsaturated FA proportions were consistently higher (P < .001) in SAT compared to CAT and MLL. Aberdeen Angus bulls were distinguished from the other breeds in the proportions of mostly de novo synthesized C14:0, C16:0, C14:1n-5, C16:1n-7, and conjugated linoleic acid (P < .05). Adipocyte size decreased in the order CAT > SAT > MLL, and the largest adipocytes were observed in CAT of Holstein bulls (P < .05). Gene expression differences were more pronounced between adipose tissue depots than between breeds. The expression levels of ACACA, FASN, and SCD1 genes were related to tissue-specific, and to a lesser extent also breed-specific, differences in FA composition.

Oxidative stress impairs the correct course of meiotic maturation, and it is known that the oocytes are exposed to increased oxidative stress during meiotic maturation in in vitro conditions. Thus, reduction of oxidative stress can lead to improved quality of cultured oocytes. The gasotransmitter carbon monoxide (CO) has a cytoprotective effect in somatic cells. The CO is produced in cells by the enzyme heme oxygenase (HO) and the heme oxygenase/carbon monoxide (HO/CO) pathway has been shown to have an antioxidant effect in somatic cells. It has not yet been investigated whether the CO has an antioxidant effect in oocytes as well. We assessed the level of expression of HO mRNA, using reverse transcription polymerase chain reaction. The HO protein localization was evaluated by the immunocytochemical method. The influence of CO or HO inhibition on meiotic maturation was evaluated in oocytes cultured in a culture medium containing CO donor (CORM-2 or CORM-A1) or HO inhibitor Zn-protoporphyrin IX (Zn-PP IX). Detection of reactive oxygen species (ROS) was performed using the oxidant-sensing probe 2′,7′-dichlorodihydrofluorescein diacetate. We demonstrated the expression of mRNA and proteins of both HO isoforms in porcine oocytes during meiotic maturation. The inhibition of HO enzymes by Zn-PP IX did not affect meiotic maturation. CO delivered by CORM-2 or CORM-A1 donors led to a reduction in the level of ROS in the oocytes during meiotic maturation. However, exogenously delivered CO also inhibited meiotic maturation, especially at higher concentrations. In summary, the CO signaling molecule has antioxidant properties in porcine oocytes and may also be involved in the regulation of meiotic maturation.

Objective The aim of the study was to find a possible association between the β- and κ-casein and β-lactoglobulin genotypes and important milk physiochemical and technological characteristics such as acidity, alcohol stability, the contents of some minerals and the parameters of acid fermentation ability (FEA) in Czech Fleckvieh Cattle. Methods Milk and blood samples were collected from 338 primiparous Czech Fleckvieh cows at the same stage of lactation. The genotypes of individual cows for κ-casein (alleles A, B, and E) and β-lactoglobulin (alleles A and B) were ascertained by polymerase chain reaction-restriction fragment length polymorphism, while their β-casein (alleles A1, A2, A3, and B) genotype was determined using melting curve genotyping analysis. The data collected were i) milk traits including active acidity (pH), titratable acidity (TA), alcohol stability (AS); calcium (Ca), phosphorus (P), sodium (Na), magnesium (Mg), and potassium (K) contents; and ii) yoghurt traits including active acidity (Y-pH), titratable acidity (Y-TA), and the counts of both Lactobacilli and Streptococci in 1 mL of yoghurt. A linear model was assumed with fixed effects of herd, year, and season of calving, an effect of the age of the cow at first calving and effects of the casein and lactoglobulin genotypes of β-CN (β-casein, CSN2), κ-CN (κ-casein, CSN3), and β-LG (β-lactoglobulin, LGB), or the three-way interaction between those genes. Results The genetic polymorphisms were related to the milk TA, AS, content of P and Ca, Y-pH and Lactobacilli number in the fresh yoghurt. The CSN3 genotype was significantly associated with milk AS (p<0.05). The effect of the composite CSN2-CSN3-LGB genotype on the investigated traits mostly reflected the effects of the individual genes. It significantly influenced TA (p<0.01), Y-pH (p<0.05) and the log of the Lactobacilli count (p<0.05). Conclusion Our findings indicate that the yoghurt fermentation test together with milk proteins genotyping could contribute to milk quality control and highlight new perspectives in dairy cattle selection.

The genetic diversity and genetic relatedness of seven indigenous Czech rabbit breeds included in the National Program of Conservation and Utilization of Genetic Resources was evaluated. The set was completed with a modern hybrid strain Hyplus 19×59 for comparison. Sixteen microsatellite loci compiled from published sources were multiplexed by 3 or 4 in order to rationalize the reactions and capillary electrophoresis. High level of diversity was demonstrated in all breeds, as expressed by polymorphism information content of the markers and allelic richness, in spite of the limited populations of most of the traditional breeds. On the other hand, the ubiquitous presence of private alleles and substantial genetic distances among the breeds indicated considerable distinctness of all the breeds at the molecular level. The microsatellite profiles were sufficiently characteristic to allow breed assignment of most individuals. The differences among breeds allowed for building an interpretable similarity dendrogram, although without statistical evidence for the hierarchical structure of the multi-breed population. Clustering at the individual animal level and probabilistic assignment of animals to the newly delimited groups consistently revealed distinct subpopulations in two breeds. The degree of inbreeding as a threat to the conservation program was evaluated with the heterozygosity coefficient and the subpopulation inbreeding coefficient FIS. While heterozygosity corresponded to the panel of European breeds, it remained lower than the values reported for wild and other domestic populations. In conclusion, the conserved rabbit breeds can serve as a reservoir of genetic variants for future breeding despite small living populations.

Bisphenol A (BPA), a chemical component of plastics, is a widely distributed environmental pollutant and contaminant of water, air, and food that negatively impacts human health. Concerns regarding BPA have led to the use of BPA-free alternatives, one of which is bisphenol S (BPS). However, the effects of BPS are not well characterized, and its specific effects on reproduction and fertility remain unknown. It is therefore necessary to evaluate any effects of BPS on mammalian oocytes. The present study is the first to demonstrate the markedly negative effects of BPS on pig oocyte maturation in vitro, even at doses lower than those humans are exposed to in the environment. Our results demonstrate (1) an effect of BPS on the course of the meiotic cell cycle; (2) the failure of tubulin fibre formation, which controls proper chromosome movement; (3) changes in the supply of maternal mRNA; (4) changes in the protein amounts and distribution of oestrogen receptors α and β and of aromatase; and (5) disrupted cumulus cell expansion. Thus, these results confirm that BPS is an example of regrettable substitution because this substance exerts similar or even worse negative effects than those of the material it replaced.

The cumulus expansion of cumulus-oocyte complex (COC) is an essential regulating process of oocyte maturation and as such it is a possible biomarker of the in vitro maturing oocytes quality. Cumulus expansion is usually assessed by non-invasive methods based on visual evaluation with many inaccuracies. On the other hand, analytical measurement of the quantity of hyaluronic acid (HA), the most abundant compound of expanded cumuli, is one of possible methods to evaluate cumulus expansion precisely. Therefore, this study aimed to verify the applicability of HA analysis for evaluating the cumulus expansion and testing oocyte maturation. The COCs were cultured in modified M199 medium for 8-48 h. The samples for the HA analysis were prepared on an 8-hour time scale, and HA retained in COCs was measured using a spectrophotometric method adapted for this purpose. We observed an increasing quantity of HA during the in vitro cultivation. A comparison with expanded COCs' classification or expansion area proved the proposed method of HA analysis suitable for the evaluation of cumulus expansion in vitro. Our findings consider the quantity of HA-expressed cumulus expansion to be a valuable marker of COC quality enabling an adequate oocyte meiotic stage estimation.

Gibbons of the genus Nomascus have a strong sexual dimorphism and dichromatism. As they mature, both sexes develop sex-specific pelage colour. In combination with physical similarities in the genitalia with both sexes, there are problems with determining the sex of young individuals compared to other genus of gibbons. This is a pilot study applying a multiplex polymerase chain reactions based on a non-invasive method for sex determination of gibbons. The study was conducted on 22 faecal samples from gibbons of the genus Nomascus. The animals were monitored by staff so that the samples were identified correctly and each sample was collected immediately after the defecation. Results confirmed the sex in all adult and juvenile animals with known sex; and 2 females and 5 males in juveniles were determined with unknown sex. The results of direct examination completely corresponded with the PCR results. The PCR reaction with template DNA isolated from faecal material required BSA usage, however, we observed the occurrence of nonspecific fragments. This did not affect the reliability of our results and we confirmed the usability of this method for this genus.

In the Czech Republic, ewe milk is mainly processed to cheese. Milk yield and composition are of great economic importance for dairy sheep industry. Fat acid composition of milk is of great interest because of its implications for human health. More than 60% of the fatty acids in sheep’s milk are saturated fatty acids (SFA), while monounsaturated and polyunsaturated fatty acid (MUFA and PUFA) are present at much lower concentrations. Associations between polymorphism at 3 candidate genes and milk production traits in East Friesian sheep farmed in Czech Republic were investigated in present study. Considered genes were acetyl-coenzyme A carboxylase α (ACACA), the major regulatory enzymes of fatty acid biosynthesis; lipoprotein lipase (LPL), which play a central role in plasma triglyceride metabolism, and fat acid synthase (FASN) is the central enzyme of the de novo fatty acid biosynthesis pathway. Association analysis of the 214 observed animals, confirmed that genotypes (SNPs at position 1168A/G, 1330G/T, 1338C/G and 1430C/T) of ACACA locus were associated with observed milk parameters: milk yield, protein and fat content. Significant statistical a result was implied that locus LPL; genotype combination TTTTTT was associated only with milk yield. Allele T has positive effect on increasing milk yield. Locus FASN (SNP at position 257C/T) was associated only with protein content. The C allele has positive effect on increasing protein content. These results indicate that loci should be used for genetic improvement of East Friesian dairy breed as well as local dairy sheep breeds for increasing milk production. This work was supported by the national Agency of Agriculture of the Czech Republic (NAZV) project no. QJ1310107.

A range of substances that are released into the environment, foodstuffs and drinking water as a result of human activity were originally considered relatively harmless, and it was only later that their adverse effects were discovered. In general the use of such substances is currently restricted, and they are often replaced by other substances. This applies also in the case of a range of endocrine disruptors. These substances have the capacity to disturb the balance of physiological functions of the organism on the level of hormonal regulation, and their pleiotropic spectrum of effects is very difficult to predict. Endocrine disruptors include the currently intensively studied bisphenol A (BPA), a prevalent environmental pollutant and contaminant of both water and foodstuffs. BPA has a significantly negative impact on human health, particularly on the regulation mechanisms of reproduction, and influences fertility. The ever increasingly stringent restriction of the industrial production of BPA is leading to its replacement with analogues, primarily with bisphenol S (BPS), which is not subject to these restrictions and whose impacts on the regulation of reproduction have not yet been exhaustively studied. However, the limited number of studies at disposal indicates that BPS may be at least as harmful as BPA. There is therefore a potential danger that the replacement of BPA with BPS will become one of the cases of regrettable substitution, in which the newly used substances manifest similar or even worse negative effects than the substances which they have replaced. The objective of this review is to draw attention to ill-advised replacements of endocrine disruptors with substances whose effects are not yet tested, and which may represent the same risks for the environment, for the reproduction of males and females, and for human health as have been demonstrated in the case of the originally used substances.

Hydrogen sulfide, one of three known gasotransmitters, is involved in physiological processes, including reproductive functions. Oocyte maturation and surrounding cumulus cell expansion play an essential role in female reproduction and subsequent embryonic development. Although the positive effects of exogenous hydrogen sulfide on maturing oocytes are well known, the role of endogenous hydrogen sulfide, which is physiologically released by enzymes, has not yet been described in oocytes. In this study, we observed the presence of Cystathionine β-Synthase (CBS), Cystathionine γ-Lyase (CTH) and 3-Mercaptopyruvate Sulfurtransferase (3-MPST), hydrogen sulfide-releasing enzymes, in porcine oocytes. Endogenous hydrogen sulfide production was detected in immature and matured oocytes as well as its requirement for meiotic maturation. Individual hydrogen sulfide-releasing enzymes seem to be capable of substituting for each other in hydrogen sulfide production. However, meiosis suppression by inhibition of all hydrogen sulfide-releasing enzymes is not irreversible and this effect is a result of M-Phase/Maturation Promoting Factor (MPF) and Mitogen-Activated Protein Kinase (MAPK) activity inhibition. Futhermore, cumulus expansion expressed by hyaluronic acid (HA) production is affected by the inhibition of hydrogen sulfide production. Moreover, quality changes of the expanded cumuli are indicated. These results demonstrate hydrogen sulfide involvement in oocyte maturation as well as cumulus expansion. As such, hydrogen sulfide appears to be an important cell messenger during mammalian oocyte meiosis and adequate cumulus expansion.

Allelic and genotypic distribution of polymorphisms in diacylglycerol acyltransferase 1 (DGAT1), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN), and peroxisome proliferator-activated receptor-γ coactivator-1α (PPARGC1A) genes were assessed in 679 Fleckvieh bulls. Single-locus genotype effects and the combined effect of the two polymorphisms within the FASN gene were evaluated for association with the intramuscular fat content and fatty acid profile determined in muscle and subcutaneous fat. The FASN (g.16024G>A) and FASN (g.17924A>G) polymorphisms were significantly associated mainly with C14:0, C16:0, and C18:1 n-9 concentrations as well as with the atherogenic index. The proportion of explained phenotypic variation markedly increased when the effect of a combination of the two polymorphisms within the FASN gene was tested, with the highest values of 8.6% and 14.8%, respectively, observed for C14:0 in muscle and subcutaneous fat. With a focus on improving the fatty acid composition of beef, the results of this study provide valuable information about the markers applicable in marker-assisted selection.

The objective of this study was to find QTL for milk production traits in Czech Fleckvieh cattle on chromosomes 6, 7, 11, 14, and 23 where QTL were previously identified in other dairy cattle populations. Sixteen grandsire families were genotyped for 38 micro satellite markers on the selected chromosomes. A QTL mapping model based on variance component analysis was implemented via restricted maximum likelihood (REML) to estimate QTL positions and their effects. A significant QTL affecting fat percentage was found at the beginning of chromosome 14 (0 cM), near marker ILSTS039. Suggestive QTL associated with milk production traits appeared on other studied chromosomes (BTA6, BTA7, BTA11, and BTA23). This first QTL search on five chromosomes in Czech Fleckvieh population showed several suggestive QTL that can be promising for further studies and contribute to better understanding of genetics of milk production in the Czech Fleckvieh cattle.

Genetic variation in the Czech Hucul horse population was analyzed using a sequence analysis of the D-loop region of mitochondrial DNA. One hundred and sixty-five Hucul horses were tested. Sequencing of the 700-base pairs fragment of the mitochondrial DNA D-loop region revealed 38 mutation sites representing 14 haplotypes, which were clustered into six haplogroups. The genetic information obtained from the mitochondrial DNA typing is of utmost importance for the future breed-conservation strategies.

The processes of oocyte growth, acquisition of meiotic competence and meiotic maturation are regulated by a large number of molecules. One of them could be calcineurin consisting of catalytic subunit A (Aα, Aβ, Aγ isoforms) and regulatory subunit B (B1, B2 isoforms). Calcineurin is involved in the meiotic maturation of oocytes in invertebrates or in lower vertebrates. In the mammalian oocytes, the possible role of calcineurin in the regulation of oocyte meiosis has not been clarified to date. In this study, to investigate the role of calcineurin during porcine oocyte growth, acquisition of meiotic competence and meiotic maturation, we analysed the expression and localisation of calcineurin subunits and the mRNA expression of calcineurin isoforms. Calcineurin was expressed in growing porcine oocytes, in fully grown oocytes and during their in vitro meiotic maturation. We found both subunits of calcineurin. Calcineurin A and calcineurin B were localised mainly in the cortex in all porcine oocytes. The changes in the intracellular localisation of separate calcineurin subunits during meiotic maturation were determined. We detected mRNA for calcineurin isoforms Aβ, Aγ, B2 in oocytes and mRNA for calcineurin isoforms Aβ, Aγ, B1, and B2 in cumular cells. To our knowledge, this is the first confirmation of calcineurin presence in porcine oocytes.

The breeding value (EBV) of Holstein cattle milk performance from the first lactation was evaluated using a regular Animal Model or by Single-Step Prediction of the genomic breeding value (GEBV). A total of 838 bulls were genotyped using the Illumina BovineSNP50 Beadchip V2. Two overlapping sets of milk performances were evaluated: calving years 1991-2004, with 729 341 lactations and 1 394 487 animals in the pedigree and calving years 1996-2009, with 808 436 lactations and 1 487 608 animals in the pedigree. The older data set included 526 genotyped bulls, in which the daughters' milk performance was known for 210 individuals. All of the genotyped animals were included in the newer data set. Of the young genotyped bulls from the older set, 279 had more than 50 daughters with performance records in the newer set. Genomic relationship matrices (G) were constructed from the allele frequencies of the current genotyped population or by assuming a constant value of 0.5 for all loci. Using current allele frequencies, the correlation of G with the pedigree relationship (A) was 0.74, while it was 0.77 when the constant value was used. G was blended with A with weights of 80 or 99%. The average EBV of the genotyped bulls exceeded the mean EBV of the entire population by 3 SD. Although the number of reference bulls was small, genotyping resulted in an increase of approximately 0.05 in the correlation of the GEBV of young bulls with their results after progeny testing. Only small differences in correlations were found in dependency on the methods used for the determination of G and in dependency on the weight used in blending G with A. Both EBV and GEBV in the older set showed higher correlations with the GEBV of the newer set than the EBV of the newer set.

The aim of the present work was to determine the relative transcript abundance of the oestrogen receptor alpha (ER alpha) and beta (ER beta) gene in long-term cultures (up to 18 days) of pig granulosa cells (GC) obtained from small (1-2 mm; SF-GC) and large (5-7 mm; LF-GC) follicles. GC were cultured in the presence of 10% foetal calf serum (FCS) and one of the following growth factors: leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), or stem cell factor (SCF). The relative transcript abundance of the investigated genes was measured by real time PCR (RT-PCR). In comparison with the control (medium only), LIF significantly increased (P<0.05-0.01) ER alpha gene expression in SF-GC after 3, 15 and 18 days of culture and after 12 days in LF-GC. The ER beta mRNA level in SF-GC was significantly decreased (P<0.01) by all of the investigated factors after 3, 6, 9 and 12 days of incubation. In conclusion, expression of both types of oestrogen receptors during prolonged culture of pig GC under different conditions suggests the role of oestrogens acting via their receptors in maintaining survivability of porcine granulosa cells in vitro.