The effect of ensiled paulownia leaves in a high-forage diet on ruminal fermentation, methane production, fatty acid composition, and milk production performance of dairy cows.

BACKGROUND: The use of industrial by-products rich in bioactive compounds as animal feeds can reduce greenhouse gas production. Paulownia leaves silage (PLS) was supplemented to dairy cows' diet and evaluated in vitro (Exp. 1; Rusitec) and in vivo (Exp. 2, cannulated lactating dairy cows and Exp. 3, non-cannulated lactating dairy cows). The study investigated the PLS effect on ruminal fermentation, microbial populations, methane production and concentration, dry matter intake (DMI), and fatty acid (FA) proportions in ruminal fluid and milk. RESULTS: Several variables of the ruminal fluid were changed in response to the inclusion of PLS. In Exp. 1, the pH increased linearly and quadratically, whereas ammonia and total volatile fatty acid (VFA) concentrations increased linearly and cubically. A linear, quadratic, and cubical decrease in methane concentration was observed with increasing dose of the PLS. Exp. 2 revealed an increase in ruminal pH and ammonia concentrations, but no changes in total VFA concentration. Inclusion of PLS increased ruminal propionate (at 3 h and 6 h after feeding), isovalerate, and valerate concentrations. Addition of PLS also affected several populations of the analyzed microorganisms. The abundances of protozoa and bacteria were increased, whereas the abundance of archaea were decreased by PLS. Methane production decreased by 11% and 14% in PLS-fed cows compared to the control in Exp. 2 and 3, respectively. Exp. 3 revealed a reduction in the milk protein and lactose yield in the PLS-fed cows, but no effect on DMI and energy corrected milk yield. Also, the PLS diet affected the ruminal biohydrogenation process with an increased proportions of C18:3 cis-9 cis-12 cis-15, conjugated linoleic acid, C18:1 trans-11 FA, polyunsaturated fatty acids (PUFA), and reduced n6/n3 ratio and saturated fatty acids (SFA) proportion in milk. The relative transcript abundances of the 5 of 6 analyzed genes regulating FA metabolism increased. CONCLUSIONS: The dietary PLS replacing the alfalfa silage at 60 g/kg diet can reduce the methane emission and improve milk quality with greater proportions of PUFA, including conjugated linoleic acid, and C18:1 trans-11 along with reduction of SFA. Graphical abstract of the experimental roadmap.

The metabolic profile of bovine blastocysts is affected by in vitro culture system and the pattern of first zygotic cleavage.

Time lapse monitoring (TLM) is a commercial system of individual embryo culture based on the well-of-the-well system. It allows for collecting a panel of intrinsic parameters of special importance to non-invasive quality assessment. Besides, a combined analysis of TLM and metabolome data provides a deeper insight into the embryo quality. Two questions have been addressed by this study: (i) whether embryo culture in the Primo Vision affects embryo development and quality compared to the classical system and (ii) whether the first zygotic cleavage (FZC) dysmorphisms affect metabolomic profile of blastocysts. Presumptive zygotes from IVM/IVF oocytes were cultured either in classical drops (pools of 30 embryos, control group) or on Primo Vision plates (16 embryos in separate wells, sharing culture medium, Primo Vision group). The two categories of blastocysts were analysed for relative transcript abundance of five genes (real time PCR; FASN, Hsp70, PLIN2, Bax, Slc2a1) and for metabolite profile (mass spectrometry). Besides, basing on retrospective analysis of the TLM files, the Primo Vision blastocysts were allocated into one of two groups depending on the FZC pattern (normal NC or direct cleavage DC) and analysed for metabolite profiles. The Primo Vision embryos showed higher cleavage rate (p < 0.01) but reduced blastocyst rate (p < 0.05) when compared to the control group. Although the culture system (Primo Vision vs classical) did not affect the relative transcript abundance, the metabolomic analysis revealed different activity of five pathways. Two of them, related to beta-oxidation of fatty acids were up-regulated, whereas the remaining three corresponding to ubiquinone synthesis, metabolism of phenyloalanine and tyrosine as well as vitamin B6 metabolism were down-regulated. The metabolite profiles were however particularly affected by the FZC pattern. The NC and DC blastocysts differed significantly in the activity of 16 metabolic pathways which mainly involved pyruvic acid. The DC embryos displayed a higher level of pyruvic acid (p < 0.05) which may imply disturbances in the switch from lipid to glucose metabolism. Besides, in our opinion, embryos cultured in separate wells in the Primo Vision may face increased ROS level which reduces their developmental potential. Summarizing we underline application of metabolome analysis to embryo quality assessment due to providing a wider insight into embryo response to environmental factors.

Dietary Coleus amboinicus Lour. decreases ruminal methanogenesis and biohydrogenation, and improves meat quality and fatty acid composition in longissimus thoracis muscle of lambs.

BACKGROUND: Methane production and fatty acids (FA) biohydrogenation in the rumen are two main constraints in ruminant production causing environmental burden and reducing food product quality. Rumen functions can be modulated by the biologically active compounds (BACs) of plant origins as shown in several studies e.g. reduction in methane emission, modulation of FA composition with positive impact on the ruminant products. Coleus amboinicus Lour. (CAL) contains high concentration of polyphenols that may potentially reduce methane production and modulate ruminal biohydrogenation of unsaturated FA. This study aimed to investigate the effect of BAC of Coleus amboinicus Lour. (CAL) fed to growing lambs on ruminal methane production, biohydrogenation of unsaturated FA and meat characteristics. In this study, the in vitro experiment aiming at determining the most effective CAL dose for in vivo experiments was followed by two in vivo experiments in rumen-cannulated rams and growing lambs. Experiment 1 (RUSITEC) comprised of control and three experimental diets differing in CAL content (10%, 15%, and 20% of the total diet). The two in vivo experiments were conducted on six growing, rumen-cannulated lambs (Exp. 2) and 16 growing lambs (Exp. 3). Animals were assigned into the control (CON) and experimental (20% of CAL) groups. Several parameters were examined in vitro (pH, ammonia and VFA concentrations, protozoa, methanogens and select bacteria populations) and in vivo (methane production, digestibility, ruminal microorganism populations, meat quality, fatty acids profiles in rumen fluid and meat, transcript expression of 5 genes in meat). RESULTS: CAL lowered in vitro methane production by 51%. In the in vivo Exp. 3, CAL decreased methane production by 20% compared with the CON group, which corresponded to reduction of total methanogen counts by up to 28% in all experiments, notably Methanobacteriales. In Exp. 3, CAL increased or tended to increase populations of some rumen bacteria (Ruminococcus albus, Megasphaera elsdenii, Butyrivibrio proteoclasticus, and Butyrivibrio fibrisolvens). Dietary CAL suppressed the Holotricha population, but increased or tended to increase Entodiniomorpha population in vivo. An increase in the polyunsaturated fatty acid (PUFA) proportion in the rumen of lambs was noted in response to the CAL diet, which was mainly attributable to the increase in C18:3 cis-9 cis-12 cis-15 (LNA) proportion. CAL reduced the mRNA expression of four out of five genes investigated in meat (fatty acid synthase, stearoyl-CoA desaturase, lipoprotein lipase, and fatty acid desaturase 1). CONCLUSIONS: Summarizing, polyphenols of CAL origin (20% in diet) mitigated ruminal methane production by inhibiting the methanogen communities. CAL supplementation also improved ruminal environment by modulating ruminal bacteria involved in fermentation and biohydrogenation of FA. Besides, CAL elevated the LNA concentration, which improved meat quality through increased deposition of n-3 PUFA.

Lipid metabolism and developmental potential of mammalian oocytes and embryos / Metabolizm lipidów a potencjal rozwojowy oocytów i zarodków ssaków.

Developmental potential of oocytes and embryos is one of the key factors determining success in reproduction. In vitro produced embryos display reduced quality thus development of non-invasive approaches for quality assessment is a priority. Lipid metabolism belongs to fundamental mechanisms affecting reproductive processes and shaping the quality of gametes and embryos. The cytoplasm of oocytes and embryos contains specialized organelles for lipid storage (lipid droplets) whose number and size is species dependent. The growth and maturation of the oocyte/embryo is accompanied by a great fluctuation in lipid quality and quantity which in turn affects their quality and freezing suitability. There is a possibility to modify lipid parameters both in vivo and in vitro by supplementing fat to diet and culture media. The manuscript presents the current state of knowledge on lipid engagement in the process of quality acquirement by oocytes and embryos of two livestock species ­ cattle and pig.

Lipid Stores and Lipid Metabolism Associated Gene Expression in Porcine and Bovine Parthenogenetic Embryos Revealed by Fluorescent Staining and RNA-seq.

Compared to other mammalian species, porcine oocytes and embryos are characterized by large amounts of lipids stored mainly in the form of droplets in the cytoplasm. The amount and the morphology of lipid droplets (LD) change throughout the preimplantation development, however, relatively little is known about expression of genes involved in lipid metabolism of early embryos. We compared porcine and bovine blastocyst stage embryos as well as dissected inner cell mass (ICM) and trophoblast (TE) cell populations with regard to lipid droplet storage and expression of genes functionally annotated to selected lipid gene ontology terms using RNA-seq. Comparing the number and the volume occupied by LD between bovine and porcine blastocysts, we have found significant differences both at the level of single embryo and a single blastomere. Aside from different lipid content, we found that embryos regulate the lipid metabolism differentially at the gene expression level. Out of 125 genes, we found 73 to be differentially expressed between entire porcine and bovine blastocyst, and 36 and 51 to be divergent between ICM and TE cell lines. We noticed significant involvement of cholesterol and ganglioside metabolism in preimplantation embryos, as well as a possible shift towards glucose, rather than pyruvate dependence in bovine embryos. A number of genes like DGAT1, CD36 or NR1H3 may serve as lipid associated markers indicating distinct regulatory mechanisms, while upregulated PLIN2, APOA1, SOAT1 indicate significant function during blastocyst formation and cell differentiation in both models.

WNT signalling supported by MEK/ERK inhibition is essential to maintain pluripotency in bovine preimplantation embryo.

Capturing stable embryonic stem cell (ESC) lines from domesticated animals still remains one of the challenges of non-rodent embryology. The stake is high, as stable ESCs derived from species such as cattle present high economic and scientific value. Understanding of the processes leading to the embryonic lineage segregation is crucial to provide species-orientated molecular environment capable of supporting self-renewal and pluripotency. Therefore, the aim of this study was to validate the action of the two core regulatory pathways (WNT and MEK/ERK) during bovine embryo development. In vitro produced bovine embryos were obtained in the presence of inhibitors (i), which enable activation of the WNT pathway (via GSK3i, CHIR99021) and suppression of MEK signalling by PD0325901 in the 2i system and PD184325 and SU5402 in the 3i system. We have followed the changes in the distribution of the key lineage specific markers both at the transcript and protein level. Our results showed that WNT signalling promotes the expression of key inner cell mass (ICM) specific markers in bovine embryos, regardless of the MEK/ERK inhibitor cocktail used. MEK/ERK downregulation is crucial to maintain OCT4 and NANOG expression within the ICM and to prevent their exclusion from the trophectoderm (TE). At the same time, the classical TE marker (CDX2) was downregulated at the mRNA and protein level. As a follow up for the observed pluripotency stimulating effect of the inhibitors, we have tested the potential of the 2i and the 3i culture conditions (supported by LIF) to derive primary bovine ESC lines. As a result, we propose a model in which all of the primary signalling pathways determining embryonic cell fate are active in bovine embryos, yet the requirement for pluripotency maintenance in cattle may differ from the described standards. WNT activation leads to the formation (and stabilisation of the ICM) and MEK/ERK signalling is maintained at low levels. Unlike in the mouse, GATA6 is expressed in both ICM and TE. MEK/ERK signalling affects HP formation in cattle, but this process is activated at the post-blastocyst stage. With regard to self-renewal, 2i is preferable, as 3i also blocks the FGF receptor, what may prevent PI3K signalling, important for pluripotency and self-renewal.

Inhibitor mediated WNT and MEK/ERK signalling affects apoptosis and the expression of quality related genes in bovine in vitro obtained blastocysts.

Culture conditions determine embryo quality, which may be affected on many levels (timing of development, blastomere count, transcripts, metabolite content, apoptosis). Molecular interactions of signalling pathways like MEK/ERK and WNT/ß-catenin are critical for cell-to-cell communication and cellular differentiation. Both pathways are important regulators of apoptosis. We have aimed to verify the prolonged effect of MEK/ERK silencing and WNT activation by chemical inhibitors (2i or 3i systems) on bovine IVP embryos. Apoptotic index, total cell count and transcription of embryo quality markers were evaluated. A higher rate of apoptosis was observed in 2i blastocysts, but was not accompanied by changes in transcript content of genes controlling apoptosis (BAX, BCL2, BAK, BAX/BCL2 ratio). Therefore, alternative pathways of apoptotic activation cannot be ruled out. The expression of genes related to embryo quality (HSPA1A, SLC2A1) was not affected. GJA1 transcripts were significantly higher in 3i blastocysts, what indicates a stimulatory effect of the applied inhibitors on cell-to-cell interactions. The lowest mRNA level of the IFNT2 gene was found in 2i embryos. A variation in the SDHA gene transcript was observed (with the highest content in the 3i blastocysts), what may suggest their reduced quality. It may be concluded that the modifications of culture conditions (activation of the WNT and silencing of the MEK/ERK signalling) might alter pathways crucial for embryo development without causing embryonic death.

The consequences of porcine IVM medium supplementation with follicular fluid become reflected in embryo quality, yield and gene expression patterns.

Oocyte and embryo developmental competence are shaped by multiple extrinsic and intrinsic factors. One of the most extensive research areas in the last decade is the regulation of lipid metabolism in oocytes and embryos of different species. We hypothesized that differences in developmental competence of oocytes and embryos between prepubertal and cyclic gilts may arise due to distinct fatty acid profiles in follicular fluid. We found that supplementation of oocyte maturation media with follicular fluid from prepubertal pigs affected quality and development of embryos from prepubertal pigs while embryos of cyclic pigs were not affected. PLIN2, SCD and ACACA transcripts involved in lipid metabolism were upregulated in embryos originating from oocytes of prepubertal pigs matured with autologous follicular fluid. The surface occupied by lipid droplets tend to increase in oocytes matured with follicular fluid from prepubertal pigs regardless oocyte origin. The change into follicular fluid of cyclic pigs increased the efficiency of embryo culture and improved quality, while gene expression was similar to embryos obtained from cyclic gilts. We assume that the follicular fluids of prepubertal and cyclic pigs influenced the quality of oocytes and embryos obtained from prepubertal pigs which are more susceptible to suboptimal in vitro culture conditions.

Changes in chromosome territory position within the nucleus reflect alternations in gene expression related to embryonic lineage specification.

Loss of totipotentcy in an early embryo is directed by molecular processes responsible for cell fate decisions. Three dimensional genome organisation is an important factor linking chromatin architecture with stage specific gene expression patterns. Little is known about the role of chromosome organisation in gene expression regulation of lineage specific factors in mammalian embryos. Using bovine embryos as a model we have described these interactions at key developmental stages. Three bovine chromosomes (BTA) that differ in size, number of carried genes, and contain loci for key lineage regulators OCT4, NANOG and CDX2, were investigated. The results suggest that large chromosomes regardless of their gene density (BTA12 gene-poor, BTA5 gene-rich) do not significantly change their radial position within the nucleus. Gene loci however, may change its position within the chromosome territory (CT) and relocate its periphery, when stage specific process of gene activation is required. Trophectoderm specific CDX2 and epiblast precursor NANOG loci tend to locate on the surface or outside of the CTs, at stages related with their high expression. We postulate that the observed changes in CT shape reflect global alternations in gene expression related to differentiation.

Interactions of bovine oocytes with follicular elements with respect to lipid metabolism.

Among many factors, lipid metabolism within the follicular environment emerges as an important indicator of oocyte quality. In the literature a crucial significance is described concerning follicular fluid (FF) composition as well as messenger RNA (mRNA) expression in follicular cells. The aim of this study was to describe the relationship between oocyte, FF and follicular cells with regard to lipid metabolism. The set of data originating from individual follicles comprised: lipid droplets (LD) number in oocytes (BODIPY staining), mRNA expression of seven genes in cumulus and granulosa cells (SCD, FADS2, ELOVL2, ELOVL5, GLUT1, GLUT3, GLUT8; real time polymerase chain reaction) and fatty acid (FA) composition in FF (gas chromatography). Obtained results demonstrate significant correlation between oocyte lipid droplets number and FA composition in FF. However, gene expression studies show significant correlation between LD number and GLUT1 gene only. Moreover, the present experiment revealed correlations between FA content in FF and expression of several genes (SCD, FADS2, ELOVL5, GLUT8) in granulosa cells, whereas only the SCD gene in cumulus cells. We suggest that the results of our experiment indicate the importance of glucose : lipid metabolism balance, which contributes to better understanding of energy metabolism conversion between oocytes and the maternal environment.

Mitochondria and mitochondrial DNA in porcine oocytes and cumulus cells--A search for developmental competence marker.

The development of mammalian oocytes is dependent on bidirectional signaling with the surrounding cumulus cells. Among the numerous factors that contribute to oocyte developmental competence, the mitochondria and the mitochondrial DNA play pivotal roles. Although these highly abundant organelles have been well-studied in oocytes, their roles, abundance and metabolism remain elusive in cumulus cells. Therefore, the aim of our study was to analyze the correlation between the mtDNA copy number in cumulus cells and oocytes, as well as the mitochondrial distribution patterns in oocytes, using two groups of animals that differ in terms of the developmental competence of their oocytes. We determined a positive correlation between the mtDNA copy number in the cumulus cells and mtDNA copy number in oocytes of prepubertal pigs and negative correlation in cyclic gilts. These opposing correlations may reflect the differences in the developmental competence of the prepubertal and cyclic oocytes. We also hypothesize that observed differences may reflect different metabolism and energy requirements of the cumulus-oocyte complexes from prepubertal and cyclic gilts. The mitochondrial distribution patterns in the prepubertal and cyclic gilts were not different.

Multifactorial analysis of the follicular environment is predictive of oocyte morphology in cattle.

Numerous attempts have been recently made in the search for a reliable, fast and noninvasive assay for selection of oocytes suitable for in vitro embryo production. Potential markers have been described in the follicle such as follicular fluid (FF) or cumulus cells (CCs). However, the reported findings are contradictory, which may reflect the complexity of metabolism of the ovarian follicle. In the present experiment, a data set from individual follicles of known diameter was obtained: cumulus-oocyte complex (COC) morphology, fatty acid composition and glucose concentration in FF as well as apoptotic index in CCs. The obtained data was statistically analyzed either separately (univariate analysis) or simultaneously (multivariate analysis) to examine its predictive value in morphology assessment of bovine COCs. Although the univariate analysis yielded a complex relation system of the selected parameters, no clear outcome could be established. In multivariate analysis, the concentration of the four fatty acids (C16:0, C16:1, C18:1cis9, C22:5n3) and Δ(9)-desaturase (16) as well as elongase activities were selected as covariates. This allowed prediction of the morphology of a COC with an accuracy of 72%, which is the most interesting finding of the experiment. The present study indicates that the multifactorial model comprising of selected parameters related to the follicle appeared more effective in predicting the morphology of a bovine COC, which may improve the effectiveness of in vitro production systems.

Differences in cytoplasmic maturation between the BCB+ and control porcine oocytes do not justify application of the BCB test for a standard IVM protocol.

The Brilliant Cresyl Blue (BCB) test relies on G6PDH activity and a simple protocol for the selection of higher quality oocytes. Although the BCB+ oocytes of all the species that have been investigated are characterized by superior quality when compared to BCB- counterparts, application of the test for embryo production still remains an open issue. The aim of our study was to compare BCB+ and the control oocytes (not subjected to the BCB test) in terms of selected aspects of cytoplasmic maturation (mtDNA copy number, mitochondria distribution, relative transcript abundance of six marker genes). The results of our study revealed more relevant differences within the BCB+ and the control oocytes (before and after IVM) than between the two categories of oocytes. There was no difference in the transcript abundance of the BCB+ and the control oocytes in 5 out of 6 analyzed genes (BMP15, GDF9, ATP5A1, EEF1A, ZAR1) and in mtDNA content (pre-IVM 179609 vs. 176595 and post-IVM 187243 vs. 246984, respectively). With regard to mitochondria distribution in pre- and post-IVM oocytes, there was nonsignificant tendency for a more frequent occurrence of the expected patterns in the BCB+ group. The results of the present study do not support the application of BCB staining in a routine IVM protocol due to relatively high similarity in selected parameters characterizing cytoplasmic maturation of BCB+ and control oocytes. This high similarity may results from the limited amount of less competent BCB- oocytes (10%) still present among nonselected oocytes of proper morphology.

Changes in sub-cellular localisation of trophoblast and inner cell mass specific transcription factors during bovine preimplantation development.

BACKGROUND: Preimplantation bovine development is emerging as an attractive experimental model, yet little is known about the mechanisms underlying trophoblast (TE)/inner cell mass (ICM) segregation in cattle. To gain an insight into these processes we have studied protein and mRNA distribution during the crucial stages of bovine development. Protein distribution of lineage specific markers OCT4, NANOG, CDX2 were analysed in 5-cell, 8-16 cell, morula and blastocyst stage embryos. ICM/TE mRNA levels were compared in hatched blastocysts and included: OCT4, NANOG, FN-1, KLF4, c-MYC, REX1, CDX2, KRT-18 and GATA6. RESULTS: At the mRNA level the observed distribution patterns agree with the mouse model. CDX2 and OCT4 proteins were first detected in 5-cell stage embryos. NANOG appeared at the morula stage and was located in the cytoplasm forming characteristic rings around the nuclei. Changes in sub-cellular localisation of OCT4, NANOG and CDX2 were noted from the 8-16 cell onwards. CDX2 initially co-localised with OCT4, but at the blastocyst stage a clear lineage segregation could be observed. Interestingly, we have observed in a small proportion of embryos (2%) that CDX2 immunolabelling overlapped with mitotic chromosomes. CONCLUSIONS: Cell fate specification in cattle become evident earlier than presently anticipated - around the time of bovine embryonic genome activation. There is an intriguing possibility that for proper lineage determination certain transcription factors (such as CDX2) may need to occupy specific regions of chromatin prior to its activation in the interphase nucleus. Our observation suggests a possible role of CDX2 in the process of epigenetic regulation of embryonic cell fate.

Apoptotic index within cumulus cells is a questionable marker of meiotic competence of bovine oocytes matured in vitro.

Information gained from most human studies indicate a negative correlation between the apoptotic index (AI) in cumulus cells (CC) and the quality of the corresponding oocytes. However, results obtained in other species are not so consistent. The rate of apoptosis-free COCs (cumulus oocytes complexes) subjected to IVM (in vitro maturation) also varies among studies. The aim of the present study was to investigate whether the AI in cumulus cells of post-IVM COCs is related to the morphology of pre-IVM COCs and to meiotic competence of bovine oocytes. COCs of known morphology (four grade scale) obtained from individual follicles were matured in a well-in-drop system. After IVM, the external layers of CC of each COC were analyzed by TUNEL. In order to determine the meiotic stage, oocytes were stained with DAPI. It was found that 25.6% of bovine COCs contained apoptosis-free cumulus cells. Moreover, the majority of COCs with apoptotic cells were characterized by apoptotic index lower than 15%. The level of apoptosis in CC was related neither to COC morphology nor to the oocyte meiotic stage. It is suggested that the extent of apoptosis in cumulus cells is not a reliable quality marker of the corresponding oocyte after IVM.

Altered expression of porcine Piwi genes and piRNA during development.

Three Sus scrofa Piwi genes (Piwil1, Piwil2 and Piwil4) encoding proteins of 861, 985 and 853 aminoacids, respectively, were cloned and sequenced. Alignment of the Piwi proteins showed the high identity between Sus scrofa and Homo sapiens. Relative transcript abundance of porcine Piwil1, Piwil2 and Piwil4 genes in testes, ovaries and oocytes derived from sexually immature and mature animals was examined using Real-Time PCR. Expression of the three Piwi mRNAs was proved to be tissue specific and restricted exclusively to the gonads. In testes of adult pigs the highest relative transcript abundance was observed for the Sus scrofa Piwil1 gene. On the other hand, in testes of neonatal pigs the Piwil1 transcript level was over 2-fold reduced while the level of Piwil2 transcript was higher. As regards the expression of the Piwil4 transcript, its level was 34-fold elevated in testes of neonatal piglet when compared to adult male. In ovaries of prepubertal and pubertal female pigs transcript abundance of the three Piwi genes was significantly reduced in comparison with testes. However, similarly to testes, in ovaries of neonatal pigs the Piwil2 gene was characterized by the highest relative transcript abundance among the three Piwi genes analysed. In prepubertal and pubertal oocytes Piwil1 transcript was the most abundant whereas the expression of Piwil4 was undetectable. We also demonstrated that expression of piRNA occurs preferentially in the gonads of adult male and female pigs. Moreover, a piRNA subset isolated from ovaries was 2-3 nucleotides longer than the piRNA from testes.

Preliminary in vitro study on the effect of xanthohumol on rumen methanogenesis.

There is a great interest in reducing the methane emission from ruminants as one possible cause of global warming. The aim of the presented study was to determine the effects of xanthohumol, one of the hop (Humulus lupulus) phytofactors, on methane production, microbial population and basic parameters of ruminal fermentation. The experiment was carried out in a batch culture system. The basic substrate (400 mg) consisting of meadow hay and barley meal (60:40) was supplemented with 0 (Control), 0.1, 0.2 or 1.0 mg of xanthohumol. The basic parameters of rumen fermentation and composition of microbial population were measured after 24 h of incubation. For the first time, the results of this in vitro study have demonstrated that xanthohumol is capable to reduce the methane production, even at the lowest dose applied (0.1 mg/400 mg). The observed reduction in methane production by 12-13% was not accompanied by altering the basic rumen fermentation parameters. However, the practical utility of this supplement needs further investigation under long-term in vivo conditions.

Low incidence of chromosome aberrations in spermatozoa of fertile boars.

Chromosomal imbalance in gametes and embryos is one of the factors contributing to early embryonic mortality. Although the rate of chromosomally abnormal sperm cells is low and usually does not exceed 1%, there is no clear indication of fertilizing potential of such gametes. The aim of the experiment was to investigate the type and incidence of numerical chromosomal aberrations in spermatozoa produced by fertile boars used in artificial insemination (AI). We used the protocol of fluorescent in situ hybridization (FISH) on sperm interphase nuclei with molecular probes for porcine chromosome pairs 1 and 10. Altogether 12 348 sperm cells were examined. Disomy was observed in spermatozoa of all seven AI boars whereas only one diploid cell was identified in all screened sperm cells. The average rate of chromosomally unbalanced sperm was 0.105% (13/12 348) with an inter-individual variation from 0.048% to 0.194%. Among abnormal sperm cells, both disomy (0.097%) and diploidy (0.008%) were detected. Nullisomy was not included into calculations. The estimated aneuploidy rate calculated by doubling the number of disomic cells was 0.194%. Chromosome pair 10 was significantly more often involved in non-disjunction (75%, 9/12 aneuploid sperm cells) than chromosome pair 1 (25%, 3/12). We have shown for the pig that the rate of disomic cells falls into a range presented by other authors, whereas that of diploid spermatozoa appeared to be lower in the present study. In conclusion, numerical chromosome aberrations were present in spermatozoa of all AI boars analyzed in this study. Therefore, it can be assumed that the presence of unbalanced spermatozoa at the level observed in fertile males does not significantly affect their reproductive potential.

The quality of porcine oocytes is affected by sexual maturity of the donor gilt.

Although differences in the quality of oocytes derived from young gilts and adult sows are well documented, evidence concerning gametes of pre-pubertal and cycling gilts is scarce and inconsistent. The aim of this work was to establish whether sexual maturity of gilts affects the quality of their oocytes with the use of the brilliant cresyl blue (BCB) test, oocyte diameter and apoptosis. Ovarian morphology was evaluated, and gonads with corpus luteum or albicans were recognized as originating form cycling gilts (C) and those with follicles as originating form pre-pubertal females (P). Altogether 952 cumulus-oocyte complexes (COCs; group P: 554; group C: 398) were examined, whereas 149 COCs, not subjected to BCB test, served as a control for TUNEL. COCs of proper morphology were evaluated by the BCB test which differentiated two categories of gametes: more competent, BCB+, and less competent BCB- oocytes. The control group comprised oocytes of proper morphology aspirated from ovaries of P and C gilts not subjected to BCB test. Finally five groups of COCs were matured in vitro: 1/P-BCB+, 2/P-BCB-, 3/C-BCB+, 4/ C-BCB- and 5/ control. Significantly more large oocytes (≥ 120 µm), more BCB+ oocytes and more high quality (both BCB+ and ≥ 120 µm) oocytes originated from ovaries of cycling gilts than pre-pubertal gilts (p<0.001). The rate of mature oocytes at the MII stage differed significantly between C-BCB+ (68.5%) and P-BCB+ (32.9%) oocytes. The incidence of apoptosis among BCB-treated oocytes after in vitro maturation was 21.4% and was similar to that observed in control oocytes (17.4%). BCB+ oocytes from cycling gilts showed significantly higher (28.7%) incidence of apoptosis than that of the group P (16.2%). Interestingly, high quality oocytes displayed a similar level of apoptosis regardless of the donor puberty. We demonstrated that C gilts provided more BCB+ oocytes as well as more large oocytes than P gilts, although C-BCB+ oocytes showed higher apoptotic rate. In conclusion, high incidence of apoptosis and a big variation in the diameter of more competent BCB+ oocytes make the BCB test a less effective selection tool than previously reported.

Timing of the first zygotic cleavage as a marker of developmental potential of mammalian embryos.

Embryo quality related to its developmental potential is now one of the most important issues in modern embryology. It has been demonstrated that some in vitro produced blastocysts fail to hatch and implant after transfer despite a normal morphology. Although embryos are able to adjust to sub-optimal culture conditions, significant changes in expression profiles of developmentally important genes have been noticed. Timing of the first zygotic cleavage is considered a non-invasive marker of embryo developmental potential and has been successfully used in human IVF programs for identifying embryos of superior quality. Early-cleaving zygotes are more likely to develop to the blastocyst stage than their late-cleaving counterparts. The timing of the first zygotic cleavage has been associated with several parameters that may affect developmental potential of the resulting embryos. The mechanism causing variation in the timing of the first zygotic cleavage has not been identified. It may be related to culture environment or to some intrinsic factors within the oocyte, the sperm or both. In this paper we discuss some of the important aspects related to the timing of the first zygotic cleavage and its influence on the developmental competence of resulting embryos.