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Artificial insemination (AI) is critical for breeding in the dairy industry. High-merit bulls can present low freezability, hampering genetic dissemination. Thawed semen can be improved using density gradient centrifugation (DGC) with colloids, but little information deals with the pre-freezing application. Thus, the BoviPure colloid (optimized for bull spermatozoa) was tested for pre-freezing application as the usual double-layer (DLC) versus single-layer (SLC, quick and economical). Semen from twelve Holstein-Friesian bulls was extended with OPTIXcell extender, frozen (Control), or processed by SLC or DLC and frozen. Sperm were assessed pre-freezing for motility and viability and post-thawing (directly and after 4 h 38 °C) for apoptosis, capacitation status, acrosomal damage, mitochondrial activity, cytoplasmic and mitochondrial reactive oxygen species (ROS), and chromatin status (SCSA for DNA fragmentation and chromatin compaction and monobromobimane, mBBr, for disulfide bridges evaluation). The DGC improved parameters post-thawing (e.g., 57.5%±10.1 motility vs. control 53.3% ± 11.2) at the cost of sperm loss (sperm recovery of DGC 14.4% ± 2.5 and SLC 17.4% ± 2.5). DNA fragmentation (%DFI) decreased (0.21% ± 0.53 vs. control 1.30% ± 0.10), and SLC reduced chromatin compaction. A clustering procedure separated lesser (LF) and greater freezability (GF) bulls. LF samples were especially benefited by DGC, with SLC providing better post-thawing results for this group. In conclusion, pre-freezing DGC improved sperm parameters post-thawing, potentially improving the cryopreservation of low-freezability semen from high-merit bulls. SLC, quicker and economical, would be preferable since it showed similar or higher performance than DLC.

The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals.

In present work it is pretended to estimate elastic parameters of beams through the combined use of precision geomatic techniques (laser scanning) and structural behaviour simulation tools. The study has two aims, on the one hand, to develop an algorithm able to interpret automatically point clouds acquired by laser scanning systems of beams subjected to different load situations on experimental tests; and on the other hand, to minimize differences between deformation values given by simulation tools and those measured by laser scanning. In this way we will proceed to identify elastic parameters and boundary conditions of structural element so that surface stresses can be estimated more easily., Optical and Laser Remote Sensing

Sex determination in bovine embryos is a useful tool in reproductive biotechnology. This work compares two techniques of embryo sexing by polymerase chain reaction (PCR). Embryos were produced in vitro with sex-sorted sperm and two techniques of DNA lysis were tested (proteinase K versus heat shock). Subsequently, halves of each lysed sample was amplified both by amelogenin and BRY4a/SAT1 primers. Male embryos treated by both digestion methods and amplified by BRY4a/SAT1 gave higher rates of false negatives. Amelogenin amplification failed with embryos previously digested by proteinase K. In contrast, the lysis method allowed obtaining the best accuracy in terms of sex verification when using amelogenin amplification.

Mammalian oocytes can undergo artificial parthenogenesis in vitro and develop to the blastocyst stage. In this study, using real-time PCR, we analyzed the expression of genes representative of essential events in development. In vitro matured oocytes were either fertilized or activated with ionomycin + 6-DMAP and cultured in simple medium. The pluripotency-related gene Oct3/4 was downregulated in parthenotes, while the de novo methylation DNMT3A gene was unchanged. Among the pregnancy recognition genes, IFN-t was upregulated, PGRMC1 was downregulated and PLAC8 was unchanged in parthenotes. Among the metabolism genes, SLC2A1 was downregulated, while AKR1B1, COX2, H6PD and TXN were upregulated in parthenotes; there was no difference in SLC2A5. Among the genes involved in compaction/blastulation, GJA1 expression increased in parthenotes, but no differences were detected within ATP1A1 and CDH1. Expression of p66shc and the Bax/Bcl2 ratio were higher in parthenotes, and there was no difference in p53. Parthenotes and embryos may differ in the way they stimulate apoptosis, with a preponderant role for p66shc within parthenotes. Differentially affected functions may also include pluripotency, de novo methylation and early embryonic signalling.

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66shc and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1.Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66shc and p53 respectively, and in their mechanisms to control pluripotency and de novo methylation. © 2009 Society for Reproduction and Fertility.

High follicular testosterone levels have been associated with a skew in the sex ratio in favor of males following in vitro fertilization, whereas egg incubation temperature has been found to influence sex ratio in some reptiles. The incubation temperature interferes with the aromatase activity, resulting in a sex determination mechanism thought to be lost in mammals. In this work we aimed to test the effects of testosterone on sex ratio of bovine embryos produced in vitro and to determine whether effects of sex and temperature are effectively decoupled in mammals. Bovine oocytes were in vitro matured for 22hr in TCM199, PVA, FSH and LH after a 22hr meiotic arrest in TCM199, PVA and roscovitine 25mM. Matured oocytes were in vitro fertilized and cultured up to Day 3, and embryos having three or more cells were sexed. In the first experiment, testosterone (0, 30, 300 and 1,500 nM), present both during meiotic inhibition and subsequent in vitro maturation (IVM), did not affect development rates or embryonic sex ratio. In the second experiment, increasing incubation temperatures (38, 39 or 40°C) during meiotic inhibition and subsequent IVM, reduced embryo development, but did not change the sex ratio. Under our experimental conditions, testosterone does not promote a preferential selection of Y-chromosome bearing spermatozoa by the oocyte, and temperature and sex ratio seems to be decoupled in mammals. J. Exp. Zool. 311A448-452, 2009. © 2009 Wiley-Liss, Inc.