A statistical approach using sequentially principal component analysis (PCA), clustering, and discriminant analyses was developed to identify sperm morphometric subpopulations in well-defined portions of the fresh boar ejaculate. Semen was obtained as 2 portions (the first 10 mL of the sperm-rich fraction and the rest of the ejaculate, respectively) and frozen using a conventional protocol. Before freezing, an aliquot was used for computerassisted sperm morphometry analysis (ASMA). Postthaw quality was evaluated using computer-assisted sperm analysis (CASA), and an annexin-V/PI assay evaluated sperm membranes. The PCA revealed that 3 variables represented more than 78% of the cumulative variance in sperm subpopulations. The clustering and discriminant analyses, based on 5780 individual spermatozoa, revealed the existence of 4 sperm subpopulations. The relative percentage of these subpopulations varied between boar and ejaculate portions. Linear regression models based on measured morphometric characteristics could account for up to 36% of the percentage of intact sperm membranes postthaw. The ASMA protocol used in our study was useful to detect subtle morphometric differences between spermatozoa, and the combination of this analysis with a multivariate statistical procedure gave new information on the biological characteristics of boar ejaculates that is not given by conventional sperm analysis.

No abstract

Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols and has previously been studied in the pig species using annexin-V detection of phosphatidylserine translocation. In the present study we designed a new assay to detect these changes in boar spermatozoa, based on the slight increase of sperm membrane permeability occurring during the early stages of cryoinjury, using the combination of three fluorescent probes, SNARF-1, YO-PRO-1 and ethidium homodimer. Four ejaculates from five different boars were frozen-thawed and flow cytometrically (FC) evaluated as paired samples. One of the samples was assayed using the annexin-V/propidium iodide staining and the other sample was evaluated using the new triple staining. Using this combination of probes, four sperm subpopulations were easily detected: viable, with stable membranes (SNARF-1 positive cells), and three with compromised membranes, one of YO-PRO-1+/Eth- cells, one ethidium homodimer+ spermatozoa and, finally spermatozoa stained both with YO-PRO-1 and ethidium homodimer (YO-PRO-1+/Eth+). The latter three categories corresponded to dead spermatozoa, but with different degree of membrane damage, being YO-PRO+/Eth- an earlier stage of membrane destabilization, (manifested by an increase in membrane permeability, while still maintaining membrane integrity) than YO-PRO+/Eth+. A method agreement analysis between both methods was performed revealing good agreement, although the percentage of live cells was 9.44% larger for the triple stain than the annexin-V assay. The new assay stained all sperm sub-populations present in the sample, making it especially suitable for both fluorescence microscopy and flow cytometry, facilitating the exclusion of debris and egg-yolk particles when using FC.

In this work, we study the chiral symmetry breaking in pseudo-quantum electrodynamics in (2 þ 1) dimensions, which is designed to reproduce a Coulomb potential for charged particles on a plane interacting via photons propagating in (3 þ 1) space-time dimensions and would be relevant for applications to condensed-matter systems. Using an ultraviolet cutoff in the momentum integrals, we show that there is a critical dimensionless coupling c ¼ =4 above which there is chiral symmetry breaking. In the case of the theory with N massless fermions, we obtain a critical value of the number of fermion flavors, N c , below which the chiral symmetry breaking occurs. Finally, we discuss the relevance of our results to graphene in the ultimate deep infrared regime where the Fermi velocity of the Dirac fermions approaches the velocity of light.

No abstract