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Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 degrees C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.

Boar sperm viability post-thaw differs depending on the ejaculate fraction used, with spermatozoa present in the first 10 mL of the sperm-rich fraction (SRF) (portion 1, P1, sperm-peak portion) displaying the best cryosurvival in vitro compared with that of spermatozoa from the rest of the ejaculate (portion 2 of the SRF plus the post-spermatic fraction), even when using simplified freezing routines. This viability apparently relates to the specific profile of seminal plasma in P1 (i.e., glycoprotein and bicarbonate concentrations, and pH). However, spermatozoa from PI have not been compared with spermatozoa from the rest of the SRF (SRF P1, usually 30-40 mL of the SRF), which is routinely used for freezing. We compared P1 with SRF P I in terms of sperm kinematics (using the QualiSperm (TM) system), while membrane integrity (SYBR-14/PI), acrosome integrity (FITC PNA/PI), and sperm membrane stability (Annexin-V) were explored using flow cytomety. As well, total protein concentration and the proteomics of the seminal plasma (SP) of both portions of the SRF were studied using two-dimensional electrophoresis (2DE), mass fingerprinting (MALDI-TOF), and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) on selected peptides. The SRF portions were collected weekly from four mature boars (4-5 replicates per boar, sperm concentration: P1, 1.86 +/- 0.20; SRF P1, 1.25 +/- 0.14 x 10(9) spz/mL) and processed using a quick freezing method in MiniFlatPacks. Post-thaw sperm motility reached 50%, without differences between SRF portions, but with clear inter-boar variation. Neither plasma membrane nor acrosome integrity differed (ns) between fractions. These results indicate that there are no differences in cryosurvival after quick freezing of boar spermatozoa derived from either of the two SRF portions. While P1 and SRF-P1 clearly differed in relative total protein contents, as expected, they displayed very similar protein profiles as assessed using 2DE and mass sp, Original Publication: A P Siqueira, M Wallgren, M S Hossain, A Johannisson, L Sanz, J J Calvete and Heriberto Rodriguez-Martinez, Quality of boar spermatozoa from the sperm-peak portion of the ejaculate after simplified freezing in MiniFlatpacks compared to the remaining spermatozoa of the sperm-rich fraction, 2011, THERIOGENOLOGY, (75), 7, 1175-1184. http://dx.doi.org/10.1016/j.theriogenology.2010.11.024 Copyright: Elsevier Science B.V., Amsterdam. http://www.elsevier.com

Low sperm survival post-thaw and time-consuming procedures for conventional freezing (CF) hamper the commercial application of cryopreserved boar semen. We had previously proven that boar spermatozoa in the first 10 mL of the sperm-rich fraction, SRF (the so-called PI, the sperm-peak portion of the ejaculate) sustain best handling in vitro, since they probably bathe in an aliquot of seminal plasma (SP) with specific composition. Here, we performed three experiments to determine: Exp I: the concentration of bicarbonate among portions of the ejaculate; Exp II: the effects of bicarbonate doses on sperm motility and; Exp III: the outcome of a faster, simpler freezing method (SF), handling PI-spermatozoa packed in MiniFlatPacks(TM) (MFP) vs. CF and vs. SRF-spermatozoa (2 x 2 factorial design). The bicarbonate content in SP was, among portions/fractions of the ejaculate, lowest in P1 (13.71 mM/L, P less than 0.0001, Exp 1). Boar spermatozoa require bicarbonate in the extender (to the levels present in PI) to maintain acceptable motility over a 120-h period at 16-17 degrees C (Exp II). Sperm freezing was dramatically shortened (from 8 to 3.5 h) by the SF-procedure. P1 - and SRF-spermatozoa survived equally both CF- and SF-freezing (% total motility 30 min PT; P1-CF: 65.2 +/- 5.4% and P1-SF: 68.9 +/- 2.4%; SRF-CF: 64.4 +/- 2.7%; SRF-SF: 55.8 +/- 3.1%, ns). Interestingly. in contrast to SRF, there were no significant variations in 30-min PT-survival among either ejaculates or boars when the P1 was frozen, independent of the handling method (CF or SF). in conclusion, such a faster freezing protocol of semen packed in MFP could be advantageously applied to P1-spermatozoa (P1-SF), while the rest of the ejaculated spermatozoa could still be used for production of conventional artificial insemination (AI) doses, thus allowing for a maintained routine management of commercially relevant stud boars. (C) 2009 Elsevier B.V. All rights reserved.

Seminal plasma (SP) is a complex fluid which exerts biological actions in the female reproductive tract. In pigs, SP elicits endometrial inflammation and consequent immune changes after mating. This study tested whether heparin-binding spermadhesins (HBPs) and the heterodimer of porcine sperm adhesions I and II (PSP-I/PSP-II) in SP recruit different lymphocyte subsets (CD2(+), CD4(+) and CD8(+) T cells) or polymorphonuclear leukocytes (PMNs) to the superficial endometrium or luminal epithelium and lumen, respectively, of oestrous sows. In Experiment 1, endometrial biopsies were taken between 2 and 120 min after infusion of uterine horns with HBPs, PSP-I/PSP-II or saline and evaluated by immunohistochemistry or histology. In Experiment 2, the uterus of oestrous sows was infused with PSP-I/PSP-II or saline to assess PMN numbers in the uterine lumen 3 h later. PSP-I/PSP-II elicited CD2(+) T cell recruitment from 10 min, and CD8(+) T cells from 60 min after infusion, while HBPs increased CD4(+) T cell recruitment by 120 min. PSP-I/PSP-II but not HBPs induced PMN migration to the surface epithelium by 10 min. PMN numbers were elevated 5-fold by 30 min and 7-fold from 60 min. with PMNs detectable in the lumen from 30 min after infusion. Six-fold more PMNs were collected from the uterine lumen of PSP-I/PSP-II-infused sows compared to controls at 3 h after infusion. These data show that PSP-I/PSP-II heterodimer in seminal plasma has a predominant role in triggering the recruitment of uterine PMNs and T cells after mating, initiating a cascade of transient and long-lasting immunological events. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

The objective of this study was to determine the short and long term effects of a gonadotropin-releasing hormone (GnRH) vaccine (Improvac (TM) Pfizer Ltd.), on sexual maturity, development of the reproductive organs, and the morphology of caudal epididymal spermatozoa in non-castrated male pigs. The pigs were slaughtered 4, 16 or 22 weeks after the second Improvac (TM) vaccination. A total of 80 crossbred non-castrated male pigs were included in this study comprising two experiments, a short-effect (Experiment 1) and a long-effect (Experiment 2). The first experiment included 56 pigs, 24 of them were maintained as controls and 32 were vaccinated twice, and slaughtered 4 weeks after the second vaccination. The second experiment included 24 pigs, 12 controls and 12 vaccinated twice, and slaughtered either 16 weeks (n = 6) or 22 weeks (n = 6) after the second vaccination. None of the immunized pigs was sexually mature at slaughter, i.e. 4, 16 or 22 weeks after second vaccination. Corresponding results of the control pigs showed that 50% had reached sexual maturity at the age corresponding to 4 weeks after the second vaccination. and 100% at slaughter 16, respectively, 22 weeks after vaccination. At 4, 16 and 22 weeks after second vaccination both testes weight and bulbourethral length were significantly reduced (p less than 0.001). The percentages of proximal droplets and abnormal heads were significantly lower in the control pigs than in the immunized pigs at slaughter 4 weeks after vaccination, whereas distal droplets were higher. For the other morphological parameters no significant differences were seen, but all mean values except for acrosome defects were numerically lower in the control pigs compared with the immunized pigs. For pigs slaughtered 16 or 22 weeks after vaccination, the vaccination effect was significant for percentages of proximal droplets, distal droplets, acrosome defects, acrosome abnormality and abnormal heads (p = 0.017-0.001). The immunization

Seminal plasma (SP) is a complex fluid which exerts biological actions in the female reproductive tract. In pigs, SP elicits endometrial inflammation and consequent immune changes after mating. This study tested whether heparin-binding spermadhesins (HBPs) and the heterodimer of porcine sperm adhesions I and II (PSP-I/PSP-II) in SP recruit different lymphocyte subsets (CD2(+), CD4(+) and CD8(+) T cells) or polymorphonuclear leukocytes (PMNs) to the superficial endometrium or luminal epithelium and lumen, respectively, of oestrous sows. In Experiment 1, endometrial biopsies were taken between 2 and 120 min after infusion of uterine horns with HBPs, PSP-I/PSP-II or saline and evaluated by immunohistochemistry or histology. In Experiment 2, the uterus of oestrous sows was infused with PSP-I/PSP-II or saline to assess PMN numbers in the uterine lumen 3h later. PSP-I/PSP-II elicited CD2+ T cell recruitment from 10 min, and CD8(+) T cells from 60 min after infusion, while HBPs increased CD4(+) T cell recruitment by 120 min. PSP-I/PSP-II but not HBPs induced PMN migration to the surface epithelium by 10 min. PMN numbers were elevated 5-fold by 30 min and 7-fold from 60 min, with PMNs detectable in the lumen from 30 min after infusion. Six-fold more PMNs were collected from the uterine lumen of PSP-I/PSP-II-infused sows compared to controls at 3h after infusion. These data show that PSP-I/PSP-II heterodimer in seminal plasma has a predominant role in triggering the recruitment of uterine PMNs and T cells after mating, initiating a cascade of transient and long-lasting immunological events.

Sperm motolity is, for its relation to cell viability and fertility, a central component of the spermiogram, where consideration of motion patterns allows discrimination of sub-populations among boar spermatozoa. Extension and cryo-preservation imposes changes in these patterns in connection to handling, additives, temperature changes and the removal of boar seminal plasma (BSP) which apparently makes spermatozao susceptible to xodative stress, thus affecting survival and motility post-thaw. Detailed kinematic analyses during sperm cooling are sparse, particularly when considering the instrumentation and settings used for analyses, the effect of extenders, and of the BSP the processed spermatozoa are exposed to. Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction of the ejaculate (portion 1. P1-BSP), have shown an increased ability to sustain motility during and after cryo-preservation than spermatozoa immersed in the rest of the ejaculate (portion 2, P2). When P2-spermatozoa were cleansed from their BSP and exposed for 60 min to pooled P1-BSP, their motility post-thaw increased to similar levels as P1-spermatozoa. This BSP-influence is sire-dependent, presumably related to the protein concentration in the different ejaculate poriton,s andapparently unrealted to changes in membrane integrity or membrane stability through conventiona, controlled cooling. (C) 2008 Elsevier Inc. All rights reserved.

A recently developed triple staining (SNARF-1/YO-PRO-1/ethidium homodimer) was used to assess early changes in boar sperm membrane integrity (MI) with the results of cryopreservation procedures and to seek for correlations among MI-spermatozoa in pre-freeze semen and its freezeability. Ejaculates from five boars were evaluated in the fresh and frozen-thawed (FT) state, and its freezeability defined as % of membrane intactness, MI% (MI% = % of FT-spermatozoa with intact membranes x 100 divided by the % of prefreeze spermatozoa with intact membranes) estimated. Significant differences were found among boars for freezeability (MI%) and motility post-thaw (%). Interestingly, significant correlations were found between the percentage of YO-PRO-1-positive spermatozoa and freezeability (R = 0.440, p less than 0.01), indicating this new triple staining can be used to safely disclose among ejaculates prior to freezing. (c) 2006 Elsevier B.V. All rights reserved.

Boar semen can be successfully frozen - highly packed - in small containers (medium-straw, MS or MiniFlatPack, MFP). The use of deep intra-uterine artificial insemination (DIU-Al) can make possible the deposition of small volumes of this thawed, non re-extended semen deeply intra-Uterine, close to the sperm reservoir. The present experiments studied the fertility achieved after single or double DIU-Al per oestrus, with special attention to the interval between AI and spontaneous ovulation. Semen from two boars of proven fertility was frozen in MS or MFP holding 1 x 109 total spermatozoa. Multiparous (2-5 parity, n = 42) crossbred sows were checked for oestrous behaviour after weaning and the occurrence of spontaneous ovulation was checked with transrectal ultrasonography (TUS) to establish the mean interval between onset of oestrus (OO) and ovulation which was found to be when approximately 2/3 of the oestrus period has passed. The sows were, in the following standing oestrus, Subjected to DIU-Al using thawed semen from either MS (n = 20) or MFP (n = 22), inseminated Without further re-extension. The sows were randomly allotted to one of three groups: (1) single DIU-Al 8 h before expected ovulation (control group, n = 19); (2) single DIU-AI 4 h before expected ovulation (treatment group S, n = 15); and (3) double DIU-AI 12 and 4 h before expected ovulation (treatment group D, n 8). Occurrence of spontaneous ovulation was confirmed by TUS, performed as during the first oestrous period and used to determine the real interval of DIU-AI and ovulation. Pregnancy was also confirmed by TUS 28 days after 00 in those sows not returning to oestrus. These sows were slaughtered (30-45 days of pregnancy), and the appearance of the reproductive tract and ovaries, the number of live and dead foetuses, of implantation sites and of corpora lutea (CL) were recorded. Sows (n = 9) returning to oestrus ("open") were re-inseminated (either once [n = 4] or twice [n = 51) the following oes

The use of deep-frozen boar semen for artificial insemination (AI) is constrained by the need for high sperm numbers per dose, yielding few doses per ejaculate. With the advancement of new, intrauterine insemination strategies, there is an opportunity for freezing small volumes containing high sperm numbers, provided the spermatozoa properly sustain cryopreservation. The present study aimed to concentrate (2 x 10(9) spz/mL) and freeze boar spermatozoa packed in a 0.5 mL volume plastic medium straw (MS) or a multiple FlatPack (MFP) (four 0.7 mL volume segments of a single FlatPack [SFP]) intended as AI doses for intra-uterine AI. A single freezing protocol was used, with a conventional FlatPack (SFP, 5 x 10(9) spz/5 mL volume) as control. Sperm viability post-thaw was monitored as sperm motility (measured by computer-assisted sperm analysis, CASA), as plasma membrane integrity (PMI, assessed either by SYBR-14/PI, combined with flow cytometry, or a rapid hypo-osmotic swelling test [sHOST]). Sperm motility did not differ statistically (NS) between test-packages and control, neither in terms of overall sperm motility (range of means: 37-46%) nor sperm velocity. The percentages of linearly motile spermatozoa were, however, significantly higher in controls (SFP) than in the test packages. Spermatozoa frozen in the SFP (control) and MFP depicted the highest PMI (54 and 49%, respectively) compared to MS (38%, P less than 0.05) when assessed with flow cytometry. In absolute numbers, more viable spermatozoa post-thaw were present in the MFP dose than in the MS (P less than 0.05). Inter-boar variation was present, albeit only significant for MS (sperm motility) and SFP (PMI). In conclusion, the results indicate that boar spermatozoa can be successfully frozen when concentrated in a small volume. (C) 2004 Elsevier Inc. All rights reserved.

In the pig, a functional tubal sperm reservoir (SR) is established before ovulation to ensure availability of suitable numbers of viable spermatozoa for fertilization. The boars large ejaculate is split: most spermatozoa are delivered in a sperm-rich fraction (SRF) followed by a post-SRF fraction containing increasing amounts of the spermadhesin PSP-I/PSP-II-rich seminal vesicle secretion. This heterodimer acts as leukocyte chemoattractant both in vitro and in vivo, contributing to the phagocytosis of those spermatozoa not reaching the SR. Sequential ejaculate deposition of marked spermatozoa and SR screening showed that most spermatozoa in the SR arose from the fortuitous PSP-poor, first portion of the SRF fraction, escaping phagocytosis and replenishing the SR within 23 h. The SR-sperm numbers diminish gradually in relation to ovulation, spermatozoa. being continuously redistributed toward the upper isthmus. In vitro, only uncapacitated spermatozoa bind to epithelial explants, suggesting that the SR influences sperm capacitation. In vivo, most viable spermatozoa - usually harbored in the deep furrows in the pre- or peri-ovulatory SR during spontaneous standing estrus - are uncapacitated, but capacitation significantly increases after ovulation. Pre-/peri-ovulatory SR spermatozoa promptly capacitate in vitro when exposed to the effector bicarbonate, an influence that can be reversed by co-incubation with SR fluid or its component hyaluronan. Fluid collected from the ampullar segment (rich in bicarbonate) induces capacitation in vitro. In conclusion, the lack of massive sperm capacitation in the SR and the diverse individual response to capacitation shown by tubal spermatozoa would relate both to the insurance of full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation in the upper oviduct, thus maximizing the chances of normal fertilization. (C) 2004 Elsevier Inc. All rights reserved.

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 computer-assisted. 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 hew information on the biological characteristics of boar ejaculates that is not given by conventional sperm analysis.

The Kidd smelter and refinery, Ontario, produces 135 000 tonnes/a blister Cu, 117 000 t/a cathode Cu, 132 000 t/a Zn, 820 t/a Cd and 24 t/a In. A furnace integrity programme was initiated in 1993 to improve the reliability and safety of the smelting-cleaning-converting furnace line. The converting furnace is one of three vessels forming the Mitsubishi continuous smelter line, but its campaign life has been limited by break-outs through the hearth and walls, as well as overloading of Cu cooling blocks in the roof. The converting furnace is undergoing a series of upgrades to overcome severe refractory wear to the roof, walls and hearth. Wall modifications have included a new upper shell, wall coolers and an increase in the number of cooling blocks. Improvements to the roof include increased cooling, a novel brick/cooler suspension system and roof compression for gas tightness., The Kidd smelter and refinery, Ontario, produces 135 000 tonnes/a blister Cu, 117 000 t/a cathode Cu, 132 000 t/a Zn, 820 t/a Cd and 24 t/a In. A furnace integrity programme was initiated in 1993 to improve the reliability and safety of the smelting-cleaning-converting furnace line. The converting furnace is one of three vessels forming the Mitsubishi continuous smelter line, but its campaign life has been limited by break-outs through the hearth and walls, as well as overloading of Cu cooling blocks in the roof. The converting furnace is undergoing a series of upgrades to overcome severe refractory wear to the roof, walls and hearth. Wall modifications have included a new upper shell, wall coolers and an increase in the number of cooling blocks. Improvements to the roof include increased cooling, a novel brick/cooler suspension system and roof compression for gas tightness.