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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.

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This is an author produced version of a paper published in Journal of Reproductive Immunology. This paper has been peer-reviewed and is proof-corrected, but does not include the journal pagination.Citation for the published paper: Rodriguez-Martinez, H., et al. AbstractThe heterogeneous seminal plasma (SP) is involved in endometrial inflammation and following immune changes that occur post-mating in pigs. This study tested whether SP spermadhesins (binding heparin [HBPs] or not [PSP-I/PSP-II]) recruit different lymphocyte subsets (CD2 + , CD4 + and CD8 + ) and polymorphonuclear leukocytes (PMNs) to the superficial endometrium/lining epithelium and lumen, respectively, of oestrous sows, in vivo. In Experiment 1, endometrial biopsies were taken under narcosis from six sows whose uterine horns were separated and their lumen infused with 3 mg/mL spermadhesins (HBP resp PSP-I/PSP-II) in 100 mL saline (treatment horn) or 100 mL saline (control horn). Endometrial samples removed between 2 and 120 min post-infusion were studied by immunohistochemistry (IHC, using mABs) or histology, to assess the degree of T-cell respectively PMN entry over time. In Experiment 2, eight conscious oestrous sows were singly infused intra-utero with 3 mg/mL of PSP-I/PSP-II in 100 mL saline (n = 4) or 100 mL of saline (n = 4), to assess relative PMN numbers in the uterine lumen, 3 h post-infusion.Compared to controls, PSP-I/PSP-II infusion significantly recruited uterine lymphocytes from 10 min (CD2 + ) or from 60 min (CD8 + ) onwards, while HBPs had only increased CD4 + cells by 120 min. As well, PSP-I/PSP-II, but not HBPs, significantly (P < 0.05) induced an early (10 min) PMN migration to the surface epithelium, 5-fold by 30 min and 7-fold from 60 min onwards (P < 0.001), with PMNs visible in the lumen from 30 min of infusion. Six-fold more PMNs were collected from the uterine lumen of PSP-I/PSP-II-infused sows compared to controls, 3 h after infusion (P < 0.001). Seminal plasma PSP-I/PSP-II heterodimer triggers the entry of cleansing uterine PMNs, initiating a cascade of transient and long-lasting immunological events in oestrous sows. IntroductionIn the pig, as in many other species, mating causes a transient endometrial inflammation (reviewed by Robertson et al., 2006) which, at first sight, is a logical step by the female genital tract to combat the entry of foreign cells (e.g. the spermatozoa), foreign proteins (in the seminal plasma, SP) and, eventually, of pathogens. Some spermatozoa escape this inflammation by reaching the oviductal sperm reservoir (SR) and participating in fertilization.Spermatozoa, and later the early embryo, the foetus and the placenta, are immuno-tolerated by HRM et al-2009-BSP as uterine immunoattractants the female, despite all being allogeneic (they contain transcripts of paternal origin) and thus liable to immune rejection. How is this accomplished?The boar ejaculate is large and fractionated; most spermatozoa are delivered in a sperm-rich fraction (SRF) followed by a post-SRF containing increasing am...