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.

The present study on German Shepherd bitches bred in the same environment of a private kennel and producing offspring for working purposes aimed to ascertain if any of the parameters: litter size, number of live puppies, stillbirth and mortality of puppies is determined by the month of whelping, parity, number of whelpings for one bitch over the course of one year, pregnancy length or bitch age at the time of whelping. Reproductive records of 73 bitches of German Shepherd breed (GSB) documented since 2001 till 2013 were processed. A total of 298 whelpings resulted in 2075 puppies; mortality of puppies was evaluated within the first 3 weeks of life. A significant seasonal influence on litter size and number of live born puppies, but not on stillbirth or mortality, was found. The parity of GSB significantly influenced litter size, live born puppies, and the incidence of stillbirths. The number of whelpings in one bitch over the course of one year significantly influenced litter size, while puppy losses were the same when bitches were bred once or twice a year. Litter size and number of live born puppies had a significant negative effect on pregnancy length. Furthermore, the number of stillbirths significantly increased when pregnancy was prolonged. Bitch age did not influence litter size, number of live born puppies and stillbirths, however it significantly affected mortality.

Abstract The progress of reproductive biotechnology is dependent on the amount, quality, and availability of female gametes – oocytes. The proper selection of a suitable model organism is vital to ensure effective research of the signal pathways that regulate oogenesis and meiotic maturation. Many factors are involved in meiosis regulation and some of them are evolutionarily conserved. Xenopus laevis is a traditional model for cell cycle research, which has become a background for a more detailed study of models that are similar to humans. In contrast to mammalian models, water-living vertebrates are appropriate models for studying effects of environmentally occurring pollutants such as endocrine-disrupting chemicals (EDCs). The triploid gynogenetic Prussian carp is a unique biological model for reproduction studies. The ability of clone production in combination with alternative sexual mode of reproduction brings advantages for the testing of sensitiveness to the effects of EDCs in terms of studying the alternative molecular pathways in meiosis regulations. The aim of this review is to compare meiosis regulating pathways among various animal models, and to suggest the possible utilization of these models in researching EDCs. A comparison of the currently recognized oocyte signalization and the endocrine disruptor effect points out the need for their molecular target identification and introduces some in water living vertebrates as suitable study models.

Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H2S) in the process of porcine oocyte aging. H2S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H2S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H2S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H2S from a donor (Na2S.9H2O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H2S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H2S on MPF and MAPK activities were detected and the intracellular mechanism underlying H2S activity remains unclear, our study clearly demonstrates the role of H2S in the regulation of porcine oocyte aging.

Abstract Cumulus expansion of the cumulus-oocyte complex is necessary for meiotic maturation and acquiring developmental competence. Cumulus expansion is based on extracellular matrix synthesis by cumulus cells. Hyaluronic acid is the most abundant component of this extracellular matrix. Cumulus expansion takes place during meiotic oocyte maturation under in vivo and in vitro conditions. Quantification and measurement of cumulus expansion intensity is one possible method of determining oocyte quality and optimizing conditions for in vitro cultivation. Currently, subjective methods of expanded area and more exact cumulus expansion measurement by hyaluronic acid assessment are available. Among the methods of hyaluronic acid measurement is the use of radioactively labelled synthesis precursors. Alternatively, immunological and analytical methods, including enzyme-linked immunosorbent assay (ELISA), spectrophotometry, and high-performance liquid chromatography (HPLC) in UV light, could be utilized. The high sensitivity of these methods could provide a precise analysis of cumulus expansion without the use of radioisotopes. Therefore, the aim of this review is to summarize and compare available approaches of cumulus expansion measurement, respecting special biological features of expanded cumuli, and to suggest possible solutions for exact cumulus expansion analysis.

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.

The aim of this study was to evaluate the effect of social stress on milking parameters in dairy ewes raised in Slovakia. Twenty-seven cross breed ewes of Tsigai and Improved Valachian breeds with Lacaune were included into the experiment. Ewes were selected from the flock on the basis of their milk flow type during the two pre-experimental milkings. According to milk flow types, they were divided into three groups representing three patterns of milk flows - bimodal group (GB; 11 animals), non-bimodal group (NG; 8 animals) and plateau group (PLC; 8 animals). Before milking, the following treatment to each ewe was applied: social isolation (free movement in parlour alone) in dairy parlour for at least 4min, and afterwards, the ewe was fixed into a milking stall and disturbed by unknown person standing in front of the head who was gently touching it during whole milking (milking under stress). Parameters of milkability and milk flow type were measured using electronic collection jar. For statistical evaluation, data from last pre-experimental (control) and milking under stress were compared. The significant decline of total and machine milk yield was observed during milking under stress as compared to control milking in all three groups. The machine stripping yield and milking time were reduced in BG and peak flow rate in NG. During milking under stress, the clear increase of non-bimodal milk flow types was recoded compared to control milking in BG and PLG. In conclusion, milk flow kinetic can serve an important parameter for evaluation of physiological response of ewes to milking and for further improvement of milking conditions and increasing the milk performance of ewes. (C) 2015 Published by Elsevier B.V.

Hydrogen sulfide (H2S) has been revealed to be a signal molecule with second messenger action in the somatic cells of many tissues, including the reproductive tract. The aim of this study was to address how exogenous H2S acts on the meiotic maturation of porcine oocytes, including key maturation factors such as MPF and MAPK, and cumulus expansion intensity of cumulus-oocyte complexes. We observed that the H2S donor, Na2S, accelerated oocyte in vitro maturation in a dose-dependent manner, following an increase of MPF activity around germinal vesicle breakdown. Concurrently, the H2S donor affected cumulus expansion, monitored by hyaluronic acid production. Our results suggest that the H2S donor influences oocyte maturation and thus also participates in the regulation of cumulus expansion. The exogenous H2S donor apparently affects key signal pathways of oocyte maturation and cumulus expansion, resulting in faster oocyte maturation with little need of cumulus expansion.

Abstract The aim of the study was to determine the influence of different concentrations of phytoestrogens biochanin A (BIO A; 20, 40, 50μg ml-1) and daidzein (DAI; 10, 20, 40, 50μg ml-1) on the course of meiotic maturation of pig oocytes. After a 24-hour cultivation, a stage of nuclear maturation was achieved and the areas of cumulus-oocyte complexes (COCs), as an indicator of cumulus expansion, were evaluated. The effects of both contaminants on oocytes were mani - fested from the lowest concentration used. Nuclear maturation was inhibited in a dose-dependent manner in the case of BIO A. Effects of DAI reached a plateau at a concentration of 20μg ml-1.Possible relationship to limited solubility of DAI was excluded because limits of DAI solubility in culture medium were confirmed at 50 μg ml-1.The cumulus expansion was also influenced in a different manner - reduction of the COC’s area by BIO A was dose-dependent, whereas DAI had the strongest effect on CCs in the lowest and highest concentrations used. Both phytoestrogens negatively influence the meiotic maturation of porcine oocytes but there are significant differences in their concrete effects which could relate to the diverse mechanisms of their acting on target cells.

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.

Pesticides can significantly harm reproduction in animals and people. Pyrethroids are often used as insecticides, and their toxicity for mammals is considered to be low. However, cypermethrin, deltamethrin and fenvalerate - as potent specific inhibitors of protein phosphatase calcineurin - can influence the meiosis of mammalian oocytes. The objective of this study was to evaluate the effects of these pyrethroids on the in vitro maturation of pig oocytes at different levels of meiotic competence. Under the tested concentrations, cypermethrin, deltamethrin and fenvalerate neither had a significant effect on the viability of oocytes nor did they induce significant degeneration of oocytes. However, these pyrethroids significantly affected meiotic maturation. The effects depended on the stage of meiotic competence of the oocytes. Maturation of growing pig oocytes with partial meiotic competence was induced. On the other hand, in fully grown pig oocytes with full meiotic competence, maturation in vitro was delayed. The specificity of these effects was further supported by the same effect of non-pyrethroidal inhibitors of calcineurin - cyclosporin A or hymenistatin I - on the maturation of oocytes with different levels of meiotic competence. However, pyrethroids, which do not inhibit calcineurin - allethrin or permethrin - had no effect on pig oocyte maturation. We demonstrated a significant effect of pyrethroids on the maturation of mammalian oocytes under in vitro conditions. This indicates that exposure to these substances could affect the fertility of people or animals.

When cultured for an extended time, pig oocytes that matured in vitro to the stage of metaphase II undergo the complex process designated as ageing. Under our conditions, some pig oocytes aged 3 days remained at the stage of metaphase II (22%), but others underwent spontaneous parthenogenetic activation (45%), and still others perished through fragmentation (28%) or lysis (5%). Activation of protein kinases C (PKCs) using phorbol-12-myristate-13-acetate (PMA) protects oocytes from fragmentation. None of the oocytes were fragmented after 3 days of aging in 50 nM of PMA. A similar effect (8% of fragmented oocytes) was observed after a 3-day treatment of aging oocytes with 100 μM of 1-stearoyl-2arachidonoyl-sn-glycerol (STEAR). PMA and STEAR activate both calcium-dependent and calcium-independent PKCs. This combined effect on PKCs seems to be essential for the protection of oocytes from fragmentation. Neither the specific activator of calcium-dependent PKCs 1-oleoyl-2-acetyl-sn-glycerol (OLE) nor the specific activator of calcium-independent PKCs dipalmitoyl-l-α-phosphatidylinositol-3,4,5-triphosphate heptaammonium salt (DIPALM) suppressed the fragmentation of aging pig oocytes. Twenty-one percentage of oocytes fragmented when aged for 3 days in 10 μM OLE and 26% of aged oocytes fragmented in 100 nM of DIPALM. However, fragmentation was significantly suppressed to 7% when the oocytes were exposed to the combination of both 10 μM OLE and 100 nM DIPALM. Aging pig oocytes cultured for 1 day with PMA maintained a high capability of being parthenogenetically activated (86% of activated oocytes), using calcium ionophore with 6-dimethylaminopurine. Ageing oocytes treated with PMA also had high capability of cleavage (82%) after their artificial parthenogenetic activation. However, their ability to develop to the stage of blastocyst (12%) was suppressed when compared with oocytes activated immediately after their maturation (29%).

The nitric oxide donor (+)-S-nitroso-N-acetylpenicillamine (SNAP) is capable of inducing parthenogenetic activation in pig oocytes matured in vitro. However, quite a long exposure to the nitric oxide donor, exceeding 10 h, is necessary for successful oocyte activation. Repeated short-term treatment with 2 mm SNAP significantly increased the activation rates despite the fact that the overall exposure time to the nitric oxide donor did not exceed 4 h. With regard to the activation rate, 12 repeated treatments lasting 10 min each were found to be the most efficient regimen (63.3%). The continuous exposure to the nitric oxide donor for the same overall time induced parthenogenetic activation in 12.5% oocytes (2-h continuous treatment with 2 mm SNAP). The development of parthenogenetic embryos increased after repeated short-term treatment with SNAP. After continuous treatment with 2 mm SNAP for 10 h, only 6.7% of the oocytes cleaved, and none developed beyond the 4-cell stage. Thirty-minute treatment repeated four times with 2 mm SNAP induced cleavage in 37.5% of the oocytes, 18.3% developed to the morula stage, and 6.7% reached the blastocyst stage. Based on the results, it is concluded that pulsatile treatment can significantly improve parthenogenetic activation rate when compared with the continuous treatment using nitric oxide donors.

In this paper we assessed: (i) the change in nitric oxide synthase (NOS) isoforms' expression and intracellular localization and in NOS mRNA in porcine oocytes during meiotic maturation; (ii) the effect of NOS inhibition by N(omega)-nitro-l-arginine methyl ester (l-NAME) and aminoguanidine (AG) on meiotic maturation of cumulus-oocyte complexes (COC) as well as denuded oocytes (DO); and (iii) nitric oxide (NO) formation in COC. All three NOS isoforms (eNOS, iNOS and nNOS) and NOS mRNA (eNOS mRNA, iNOS mRNA and nNOS mRNA) were found in both porcine oocytes and their cumulus cells except for nNOS mRNA, which was not detected in the cumulus cells. NOS isoforms differed in their intracellular localization in the oocyte: while iNOS protein was dispersed in the oocyte cytoplasm, nNOS was localized in the oocyte cytoplasm and in germinal vesicles (GV) and eNOS was present in dots in the cytoplasm, GV and was associated with meiotic spindles. l-NAME inhibitor significantly suppressed metaphase (M)I to MII transition (5.0 mM experimental group: 34.9% MI, control group: 9.5% MI) and at the highest concentration (10.0 mM) also affected GV breakdown (GVBD); in contrast also AG inhibited primarily GVBD. The majority of the oocytes (10.0 mM experimental group: 60.8%, control group: 1.2%) was not able to resume meiosis. AG significantly inhibited GVBD in DO, but l-NAME had no significant effect on the GVBD of these cells. During meiotic maturation, NO is formed in COC and the NO formed by cumulus cells is necessary for the process of GVBD.

Summary In fully grown pig oocytes, meiotic maturation in vitro is retarded by inhibition of histone deacetylases by trichostatin A (TSA). In growing oocytes with partial meiotic competence, culture with TSA has no significant effect on the meiotic maturation. Growing oocytes treated with TSA mature mainly to metaphase I. The ratio of oocytes that mature to metaphase II is very limited. After transient exposure to TSA, the maturation of growing oocytes with partial meiotic competence takes a different course. When these oocytes are first cultured in a TSA-free medium, then cultured for another 24 h with 100 nM TSA and finally again in a TSA-free medium for 24 h, the ratio of oocytes that mature to metaphase II significantly increases reaching 59%. When oocytes were cultured for the same length of time without transient exposure to TSA, only 19% matured to metaphase II. Those oocytes that matured to metaphase II after transient exposure to TSA were successfully activated using calcium ionophore. However, the subsequent cleavage was very limited. We can conclude that transient exposure of growing pig oocytes with partial meiotic competence to TSA increases oocyte meiotic competence, but it does not enhance developmental competence after parthenogenetic activation.

The objective of this study was to determine how the concentration of milk urea (MU) and FPCM production affect reproduction in selected Holstein dairy herds in the Czech Republic. A retrospective, observational study comprising analyses of individual cow records from monthly dairy herd improvement (DHI) milk tests including milk urea (MU) concentration was conducted in six commercial Holstein dairy herds in cows that were bred from July 2000 to August 2003. A total of 1 333 cows with available breeding date, pregnancy status, and calving to first service interval from 31 to 150 days were selected. The data were evaluated using the mixed linear model and logistic analyses. The effect of MU concentration on the probability of conception at first service was not significant (P = 0.11). The results indicated a nonlinear relationship between MU concentration and the probability of conception. A significant effect of the length of calving to first service interval (P < 0.05) and FPCM (P < 0.05) was determined. The cows with the calving to first service interval longer than 2 months had higher probability of conception (10% higher). In the group with the highest FPCM a markedly lower level of successful pregnancy was determined (38.99%), which significantly differed from the group with average FPCM production (48.53%). The probability of conception at first service in the group with the lowest FPCM is at an average level (43.55%).

The objective of this study was to assess the health status and the most common health problems occurring in beef cattle in the Czech Republic.Thirty five beef herds of different breeds were monitored from 1 January to 31 December 2006. The health status of 4872 animals (2601 cows and 2271 of their calves) was analyzed. Herds of up to 50 cows and those over 50 cows were evaluated both separately and together. Farm management data were collected by means of a questionnaire completed by the farmers. Both geographic and herd characteristics were evaluated, as well as the course of calving, mortality and morbidity in calves, and disease occurrence in cows. Calving was unassisted in about 80% of the cows, and the conception rate was approximately 90%. More difficult courses of calving were reported from the small herds than from the large ones (p < 0.001). Total calf losses were 10% on the average. Most calf deaths occurred during the first week of life (p < 0.01), the most common cause of death being diarrhoea (p < 0.001). The situation was similar in large and small herds. Diarrhoea was also most often diagnosed in those calves that survived the early life stage. At the age of 120 days, the calves that had suffered from diarrhoea showed a lower body weight by 12.6 kg than the calves of the same age from the same herd that did not have diarrhoea. The occurrence of diarrhoea in small and large herds was 12.1% and 6.7% per herd, respectively (p < 0.05). Also, other health disorders were more often reported from small herds, in calves as well as in adult cows (p < 0.001).