Production of sexed bovine embryos in vitro can be improved by selection of sperm treatment and co-culture system.

The study investigated the effects of sperm sorting, capacitation treatment and co-cultivation on sexed bovine in vitro embryo production. The effect of treatment and co-culture on production of embryos of the preferred sex from unsorted sperm was also studied. Sperm from five breeding bulls was used for fertilization of mature oocytes as follows: Experiment 1, sorted and unsorted sperm (bulls A-E) treated only with heparin in standard co-cultures; Experiment 2, sorted sperm (bulls A-E) treated with heparin-PHE (penicillamine, hypotaurine, and epinephrine) or heparin-caffeine in drop co-cultures; and Experiment 3, unsorted sperm (bull E) treated with either heparin-PHE or heparin-caffeine in both standard and drop co-cultures. In all bulls, treatment with heparin resulted in significantly (p < .05) reduced cleavage and blastocyst rates from sorted sperm, as compared with those from unsorted sperm. In bulls A, B, D and E, treatment of sorted sperm with heparin-PHE in drops significantly increased the blastocyst rate (p < .05). In unsorted sperm of bull E, heparin-PHE treatment in drops resulted in the XX/XY sex ratio inverse to that obtained by heparin-caffeine treatment in standard co-cultures (32.3%/67.7% and 66.7%/33.3%, respectively). In conclusion, the treatment of sorted sperm with heparin-PHE in modified drop co-cultures can be recommended for production of in vitro sexed embryos. The use of unsorted sperm for production of embryos of the preferred sex by selected capacitation treatment and co-culture can be the method of choice in bulls with low IVF yields from sorted sperm.

Aneuploidy Detection and mtDNA Quantification in Bovine Embryos with Different Cleavage Onset Using a Next-Generation Sequencing-Based Protocol.

Bovine embryos are now routinely used in agricultural systems as a means of disseminating superior genetics worldwide, ultimately with the aim of feeding an ever-growing population. Further investigations, common for human IVF embryos, thus have priority to improve cattle IVF, as has screening for aneuploidy (abnormal chromosome number). Although the incidence and consequences of aneuploidy are well documented in human preimplantation embryos, they are less well known for the embryos of other animals. To address this, we assessed aneuploidy levels in thirty-one 2-cell bovine embryos derived from early- and late-cleaving zygotes. Contemporary approaches ( Whole Genome Amplification and next-generation sequencing) allowed aneuploidy assessment for all chromosomes in oocytes from donors aged 4-7 years. We also quantified mitochondrial DNA (mtDNA) levels in all blastomeres assessed, thereby testing the hypothesis that they are related to levels of aneuploidy. The overall incidence of aneuploidy in this cohort of bovine embryos was 41.1% and correlated significantly with the timing of cleavage (77.8% in late-cleaving vs. 31.8% in early-cleaving embryos). Moreover, based on mtDNA sequence read counts, we observed that the median mtDNA quantity is significantly lower in late-cleaving embryos. These findings further reinforce the use of the bovine system as a model for human IVF studies.

Oxidative damage induced by carcinogenic polycyclic aromatic hydrocarbons and organic extracts from urban air particulate matter.

We investigated the role of oxidative damage in the mechanism of action of selected individual carcinogenic PAHs (c-PAHs: benzo[a]pyrene, B[a]P; dibenzo[a,l]pyrene, DB[a,l]P), an artificial mixture of c-PAHs (c-PAHs mix) and extractable organic matter (EOM) from urban air particulate matter (PM). Two cell lines (human hepatoma cells, HepG2; human diploid lung fibroblasts, HEL) were treated for 24 and 48h with various concentrations of compounds and mixtures. A panel of oxidative stress markers included 8-oxodeoxyguanosine (8-oxodG), 15-F(2t)-isoprostane (15-F(2t)-IsoP) and protein carbonyl groups. The response of the cell lines to the test compounds was substantially different. In HepG2 cells, oxidative damage to DNA was generally not induced by individual c-PAHs and the c-PAHs mix, but EOM increased 8-oxodG levels in these cells. In HEL cells, none of the compounds induced oxidative DNA damage. Lipid peroxidation, measured as the level of 15-F(2t)-IsoP, was induced by c-PAHs in HepG2 cells only after 48h of incubation, while the effect of EOM was detected already after 24h. In HEL cells, individual c-PAHs and the c-PAH mix generally decreased 15-F(2t)-IsoP levels. This effect was even stronger for EOM treatment. Protein oxidation, assessed as carbonyl levels in cell lysates, was not induced after 24h of treatment with any compound in either cell line. Individual c-PAHs and the c-PAH mix generally induced protein oxidation in both cell lines after 48h treatment, with the exception of DB[a,l]P in HepG2 cells. Oxidative damage to proteins caused by EOM was generally increased in HepG2 cells after 48h of incubation, while in HEL cells the effect was observed for only one dose of EOM. In summary, our results demonstrate the ability of EOM to induce oxidative damage to DNA and lipids after 24h of treatment, and to proteins after 48h, in HepG2 cells, while the effect of c-PAHs was substantially less. The induction of oxidative stress by c-PAHs and EOM in HEL cells was weak.

Limited nitrogen availability has cultivar-dependent effects on potato tuber yield and tuber quality traits.

An excess of nitrogen (N) is used in agriculture endangering the environment and food quality. One approach to circumvent this is to generate crops with a stable or even increased productivity under limited N. Here, we studied the effect of reduced N availability on potato (Solanum tuberosum) tuber yield and quality traits using five varieties: the wild Andigena and the commercial cultivars Désirée, Milva, Saturna and Alegria. Growth on limited N resulted in less tubers with a reduced weight except for Andigena. Tubers from low N-grown plants contained more starch, less sucrose and were delayed in sprouting. Some of the trait differences can be explained by changes in hormone levels between cultivars and N conditions. In general, Saturna and Alegria performed better under limited N making them excellent breeding candidates. Our results suggest that wild species more flexibly adapt to limited N, a trait lost in commercial potatoes.

Supplementation of l-carnitine during in vitro maturation improves embryo development from less competent bovine oocytes.

The present study was designed to define the impact of l-carnitine, supplemented during maturation, on bovine oocytes with different meiotic competence in terms of their IVF outcomes. Meiotically more competent (MMC) and less competent (MLC) oocytes were obtained separately from differently sized follicles at selected phases of folliculogenesis. The oocytes were matured with or without l-carnitine, fertilized and cultured to the blastocyst stage. The oocytes were examined for nuclear maturation, mitochondrial cluster formation, lipid consumption, fertilization and embryo development. The proportion of oocytes at metaphase II was significantly higher in the l-carnitine-treated MMC than that in the l-carnitine-treated MLC oocytes. However in comparison with the untreated controls, the proportion of MII oocytes with mitochondrial clusters was significantly higher only in the l-carnitine-treated MLC oocytes, which also showed a significantly lower mean lipid content. The l-carnitine-treated MLC oocytes showed significantly higher fertilization and syngamy rates than the untreated MLC oocytes. On the other hand, in the l-carnitine-treated MMC oocytes, the fertilization rate was similar to that of the untreated controls and the syngamy rate was significantly delayed. Although no significant differences in cleavage on Day 2 were found among all oocyte categories, l-carnitine treatment resulted in a significantly higher blastocyst yield in the MLC oocytes on Day 7 and Day 8 and a significantly higher proportion of expanded blastocysts in relation to the total number of blastocysts in MMC oocytes on Day 8. It can be concluded that l-carnitine supplementation during maturation improves the development of bovine embryos from meiotically less competent oocytes and accelerates blastocyst formation from more competent oocytes.

Gene expression in bovine embryos derived from oocytes with different developmental competence collected at the defined follicular developmental stage.

The aim of this study was to compare the expression of selected genes in bovine embryos developed from oocytes with different developmental competence. Four oocyte populations were collected, separately either from small (2-5 mm) or medium (6-10 mm) follicles, in the growth/stagnation (G/S) or dominance/regression (D/R) stage of the first follicular wave. They were matured, fertilized and cultured to D7 or D8 blastocysts by a standard protocol. Poly (A)+ mRNA was extracted from pooled blastocysts and the expression of bax-alpha (Bax), connexin 43 (Cx 43) and connexin 31 (Cx 31) was estimated using real-time RT-PCR. The cleavage rates were significantly higher in oocytes collected from both medium and small follicles, (p < or = 0.05 and p < or = 0.01, respectively) in the G/S than in the D/R stage. There were no significant differences in the D7 blastocyst rates between oocytes from both medium and small follicles in the G/S or D/R stage. But the D8 blastocyst rate was significantly higher in oocytes from small follicles in the G/S stage compared with those in the D/R stage. The relative abundance of Bax and Cx 31 made no significant difference in both D7 and D8 blastocysts developed from oocytes collected from medium or small follicles in the G/S or D/R stages. But the relative abundance of the Cx 43 transcript was significantly higher in D8 blastocysts developed from oocytes collected from both medium and small follicles in the G/S stage compared with those in the D/R stage. We conclude that the relative abundance of Cx 43 can be used as a marker of developmental potential for embryos derived from oocytes with different developmental competence because the level of Cx 43 transcript was greater in embryos derived from oocytes with greater developmental competence compared with those derived from oocytes with lesser developmental competence.

A potential relationship between the acrosome response characteristics of bovine spermatozoa and their in vitro fertilizing ability.

The aim of the work was to study a potential relationship between acrosome response characteristics of bovine spermatozoa and their ability to fertilize oocytes and produce in vitro embryos. Sperm of artificial insemination bulls with a high rate (22.0 +/- 4.1%, group A, n = 7) or a low rate (10.3 +/- 4.1%, group B, n = 8) of embryos were used. For acrosome assessment, motile spermatozoa from a Percoll gradient were incubated with or without heparin and examined by the fix-vital sperm assay (FVSA). The differences between the heparin-treated (H+) and the non-treated (H-) spermatozoa were significant (p < 0.01) in all bulls at all tested intervals. According to the kinetics of the heparin response, the bulls fell into three categories: fast (FR, n = 7), moderate (MR, n = 5) or slow (SR, n = 3) acrosome responses (p < 0.01). Five MR bulls were found in group A in comparison with two MR bulls in group B (57.1 vs 12.5%; p < 0.05). Intensity of the acrosome response (response index) was significantly higher in bull group A compared with bull group B (7.0 vs 4.6, p < 0.01). A positive correlation was recorded between response index and embryo rate (r = 0.668, p < 0.01). In conclusion (a) the kinetics of spermatozoa response to heparin may be important for in vitro fertilization, bulls with a moderate response appearing to be most suitable for embryo production; (b) greater spermatozoa response to heparin was related to more effective embryo production.

Embryos produced in vitro from bulls carrying 16;20 and 1;29 Robertsonian translocations: detection of translocations in embryos by fluorescence in situ hybridization.

Robertsonian translocation rob(16;20) in the heterozygous state was discovered in a subfertile bull of the Czech Siemmental breed. A chromosomal analysis of its family has shown that this dicentric fusion is formed de novo. The present experiments were designed to detect rob(16;20) and determine its incidence for in vitro produced embryos, using fluorescence in situ hybridization (FISH) and rob(1;29) as a detection control. To characterize semen of both bulls with the rob translocations, their sperm was examined for DNA integrity by the sperm chromatin structure assay (SCSA). For in vitro fertilization of oocytes, spermatozoa from a rob(16;20) bull carrier (Czech Siemmental breed) and those from a rob(1;29) bull carrier (Charolais breed) were used. Embryos at the 6- to 8-cell stage were cultured in a vinblastine-supplemented medium for 17 h, and embryos at the blastocyst stage were cultured in a colcemide-supplemented medium for 4 h. The embryos were fixed in methanol and acetic acid with Tween-20. Painting probes for chromosomes 16 (Spectrum Green) and 20 (Spectrum Orange) and chromosomes 1 (Spectrum Orange) and 29 (Spectrum Green) were simultaneously hybridized. In the embryos derived from the rob(16;20) bull, the presence of this translocation was not detected. On the other hand, 52.5% of the embryos derived from the rob(1;29) bull were translocation carriers. There was no significant difference in the frequency of this translocation between early and advanced embryos.