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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.

One of the main sources of repeat breeding in dairy cattle, caused by fertilization failure or early embryonic death, is metabolic stress during lactation. Nutrition seems also to play a role when the condition is seen in heifers, where oocyte cytoplasmic maturation is impaired. To determine whether over conditioning affects oocyte morphology, immature oocytes were collected by ovum pick-up (OPU) twice weekly during 5 weeks from three over-conditioned repeat breeder dairy heifers (RBH) and two normal virgin heifers (VH, controls) of the Swedish Red breed, monitored by body weight and condition. Oocyte quality was assessed under stereomicroscope and further examined by transmission electron microscope for accumulation of cytoplasmic lipid deposits. After OPU, the RBH yielded more low quality oocytes (60% vs 52% for VH, p = 0.14). The relative occupancy of osmophilic lipid droplets in the cytoplasm was higher in oocytes of bad quality compared with good ones, especially in RBH (p = 0.08) but also in VH (p = 0.11). Moreover, the oocytes from over-conditioned RBH showed higher amounts of cytoplasmic lipid deposits both in good (p = 0.14) and, even more prominent, in bad quality oocytes (p = 0.06). Such accumulation of lipid droplets may imply increased sensitivity to oxidative stress, hinder cytoplasmic maturation and lead to subfertility, as accounted in over-conditioned repeat breeders of the Swedish Red breed.

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, PI I have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2). probably in relation to different features of the surrounding seminal plasma (SP). In the present study. We investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing Moreover, sperm plasma membrane intactness (investigated using, SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of PI spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP. sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it Was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions. (C) 2009 Elsevier Inc. All rights reserved.

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, PI I have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2). probably in relation to different features of the surrounding seminal plasma (SP). In the present study. We investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing Moreover, sperm plasma membrane intactness (investigated using, SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of PI spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP. sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it Was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions. (C) 2009 Elsevier Inc. 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 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.

Visual-motility assessment is a tool used to determine the quality of boar ejaculates. This method is subjective by nature, and consequently, computer-assisted sperm analysis (CASA), with different software designs, has been developed for more objective assessment using conventional image analysis or particle counting. In the present study, we compared the results of sperm analysis using a conventional CASA system (Cell Motion Analyzer, SM-CMA (TM)), with those using a novel software (QualiSperm (TM)) and those of visual assessment performed by two operators. Ejaculates were collected weekly from four Swedish Landrace boars for 4 weeks. Each ejaculate was divided into three aliquots of different sperm concentration (300, 125, and 40 million spermatozoa/mL) and stored at similar to 17 degrees C for 96 h. Only samples at 40 million spermatozoa/mL concentration were analyzed using both automated systems; for the remaining concentrations, the SM-CMA (TM) was not used due to its inability to examine higher sperm concentrations. The number of spermatozoa analyzed was highest for the QualiSperm (TM) (similar to 300-5000 spermatozoa), followed by the SM-CMA (TM) (similar to 200 spermatozoa), and lastly, by subjective motility evaluation (similar to 100 spermatozoa). There was a time-course decrease in motility of the liquid-stored semen, detectable by either computerized method. Although the percentage of motile spermatozoa measured by the two automated systems correlated well (r greater than= 0.75), there was disagreement between operators. In conclusion, because of the lower degree of variation, the numbers of spermatozoa analyzed, and the speed of analysis (similar to 1 min per sample), QualiSperm (TM) appears to be a suitable instrument for routine work, provided it maintains stability and is available at an affordable price. (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.

Use of AI-doses containing low-sperm numbers are increasingly been used to optimise use of elite bulls as well as to accommodate an eventual wider application of sex-sorted semen. Since spermatozoa might, however, suffer from high extension rates, thus compromising fertility, this study evaluated the post-thaw sperm quality of semen from commercial progeny-tested, high-ranked AI-sires whose semen was within acceptable limits of normality, frozen in a split-design to 15 (control, 15M) or 2 x 106 total spermatozoa (treatment, 2M) per straw. Assessment post-thaw included computer-evaluated sperm motility (CASA), membrane integrity (SYBR-14/PI), membrane stability (Annexin-V/Pl), acrosome integrity (Carboxy-SNARF-1/PI/ FITC-PSA), and chromatin integrity (AO of in situ acid-induced DNA denaturation). High extension did not affect the proportions of linearly motile spermatozoa, of membrane integrity or stability nor chromatin integrity, immediately post-thaw. However, high extension clearly affected linear sperm motility following incubation at 38 degrees C for 30 min, sperm viability when assessed by SNARF and, particularly, acrosome integrity of the otherwise viable spermatozoa. Individual sire variation was evident. Fertility was preliminarily evaluated for one of the less affected bulls in a blind field trial. A total of 109 dairy cows were randomly inseminated with 15M or 2M-straws without differences in pregnancy rate between them (47% versus 43%). This similarity in fertility rates, confirmed the in vitro methods used were appropriate for identifying cryosurvival and further suggested the site of sperm deposition was not crucial for the fertility of low-sperm AI-numbers for this particular sire. However, the inter-bull variation seen calls for caution when cryopreserving low concentrations of bull spermatozoa with conventional freezing protocols. (C) 2007 Elsevier Inc. All rights reserved.

This study evaluated the effects of cooling, freezing and thawing on the plasma membrane integrity, kinetics and expression of two sugar transporters glucose transporter-3 and -5 (GLUT-3 and GLUT-5) in spermatozoa from Iberian boars. Semen samples were collected twice weekly from eight young, fertile Iberian boars of the Entrepelado and Lampino breeds. The samples were suspended in a commercial extender and refrigerated to 17 degrees C for transport to the laboratory (step A), where they were further extended with a lactose-egg yolk-based extender and chilled to 5 degrees C (step B) prior to freezing in the presence of glycerol (3%). Spermatozoa were assessed for plasma membrane integrity and sperm motility at each of the steps, including post-thaw (step C). Aliquots were also prepared for immunocytochemical localisation of the sugar transporters (fixed and thin smears for transmission and scanning electron microscopy levels respectively) and for SDS-PAGE electrophoresis and subsequent western blotting, using the same antibodies (rabbit anti-GLUT-3 and anti-GLUT-5 polyclonal antibodies). The results showed lower percentages of progressively motile spermatozoa at step C in both breeds, while the percentage of live spermatozoa was significantly lower only in the Entrepelado breed. The results obtained from electron microscopy clearly showed that Iberian boar spermatozoa expressed the hexose transporters, GLUT-3 and GLUT-5. The pattern of expression, in terms of location and concentration, was characteristic in each case but, in the case of isoform GLUT-5, it remained constant during the different steps of freezing-thawing protocol. These results indicate that cryopreservation affects the status of sperm cells of Iberian boars by altering the distribution of some membrane receptors and decreasing the percentage values of parameters linked to sperm quality.

Although sperm head shape and relative dimensions are considered reliable indicators of sperm quality, their quantification is most often operator-driven, e.g., subjective. Artificial insemination semen doses from 35 mature stud boars of known fertility and belonging to three breeds and two hybrid breeds (Duroc, Large White, Landrace, respectively, Yorker and Risco) were used in this study. Sperm samples were extended to 100 x 10(6) Cells per mL and 10 mu L of the sperm suspension used to made smears which, stained, were examined using phase contrast microscopy interfaced with an automated sperm morphology analyzer (ASMA, 2 ISAS (R)). Each sperm head was measured for four primary parameters [area (A) mu m(2), perimeter (P) mu m, length (L) mu m, width (M mu m], and four derived parameters of head shape [(L/W, (4 pi A/P-2), ((L - W/(L + M), (pi LW/4A)]. Definition of head size was statistically performed. The threshold for each class was established on the basis of the area values, considering the 25th percentile as small and the 75th percentile as large spermatozoa. In a second step, sperm head shape was determined as normal, elliptic, abnormal (rugose) contour, long or irregular and percentiles set as above to define spermatozoa with normal values for each shape parameter. Significant differences were found among breeds in the size of morphologically normal spermatozoa, which were significantly larger and more elliptic (P less than 0.001) in the Duroc breed. Sperm chromatin integrity was studied using the SCSA-assay, with significant differences observed in the degree of fragmentation intensity (DFI) although this value was consistently low in all animals studied. The hereby-validated ASMA was able to determine significant differences in sperm shape and dimensions among breeds, which were not accompanied by deviations in chromatin structure neither within nor between fertile AI-boars. (c) 2007 Elsevier Inc. All rights reserved.

Cryopreservation imposes dramatic changes in boar sperm survivability but it is as yet unclear which part of the process affects the spermatozoa the most. The present study monitored, along the entire process of cryopreservation, the stability (PMS) of the architecture of the lipid plasma membrane and its integrity (PMI), as well as the kinetics of the processed spermatozoa using two portions from the boar ejaculate (P1 = the first 10 mL of the sperm-rich fraction, SRF; P2 = the rest of the ejaculate), frozen in a recently developed package, the MiniFlatPack (MFPs, 0.5 x 10(9) sperm/dose). Evaluation was made at four specific stages, viz. S1 = after collection (suspended in Beltsville thawing solution, BTS); S2 = at 15 degrees C (suspended in lactose-egg yolk, LEY); S3 = at 5 degrees C (suspended in LEY plus glycerol); and S4 = post-thaw. Both sperm kinetics (using computer-assisted sperm analysis, CASA) and PMS [i.e. the degree of lipid disorder and of the exteriorization of phosphatidylserine (PS) in the plasma membrane, measured by flow cytometry using Merocyanine-540 (M-540), and Annexin-V (AV) respectively], as well as plasma membrane integrity [PMI, i.e. the degree of membrane damage, measured using Yo-Pro-1 or propidium iodide (PI)] were assessed after incubation in BTS at 38 degrees C. Moreover, spermatozoa were challenged by incubation in modified Brackett-Oliphant medium (mBO+) with 37 mM of bicarbonate at 38 degrees C for 30 min, and their PMS and PMI further explored. Total sperm motility was significantly higher in P1 than in P2 along the entire process (S1-S4; p less than 0.01), decreasing significantly at S4 for both fractions (p less than 0.0001). The proportion of spermatozoa showing linear motility (LinM) was similar between ejaculate portions (P1 and P2), with a significant increase post-thaw (S4; p less than 0.0001). During cooling (S1-S3) but not post-thaw (S4), lateral head displacement (LHD) differed between portions and changed along the stag

Late effects of pre-pubertal oral exposure to di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in, for example, polyvinyl chloride-products, on semen quality in young boars have not been clear-cut. The aim of this study was to determine whether stress imposed on spermatozoa would reveal such effects. Semen was collected from post-pubertal boars (8-9 months of age), which had been exposed to 300 mg kg(-1) body weight of DEHP per os three times a week from 3 to 7 weeks of age and from control siblings given placebo (water). The semen was cryopreserved and examined for plasma membrane integrity post-thaw using the short hypo-osmotic swelling test and flow cytometry (propidium iodide /SYBR-14). Sperm motility was assessed by computer-assisted sperm analysis. No significant difference in plasma membrane integrity could be found between the groups. The DEHP-exposed group had a significantly lower percentage of linearly motile spermatozoa at 30 min (P less than 0.05) and 120 min (P less than 0.001) after thawing, and a larger amplitude of lateral displacement of the head 120 min after thawing (P less than 0.05), compared with controls. In summary, spermatozoa from boars pre-pubertally exposed to low doses of DEHP, showed kinematic deviations post-thaw that could be related to DEHP exposure.

Late effects of pre-pubertal oral exposure to di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in, for example, polyvinyl chloride-products, on semen quality in young boars have not been clear-cut. The aim of this study was to determine whether stress imposed on spermatozoa would reveal such effects. Semen was collected from post-pubertal boars (8-9 months of age), which had been exposed to 300 mg kg(-1) body weight of DEHP per os three times a week from 3 to 7 weeks of age and from control siblings given placebo (water). The semen was cryopreserved and examined for plasma membrane integrity post-thaw using the short hypo-osmotic swelling test and flow cytometry (propidium iodide /SYBR-14). Sperm motility was assessed by computer-assisted sperm analysis. No significant difference in plasma membrane integrity could be found between the groups. The DEHP-exposed group had a significantly lower percentage of linearly motile spermatozoa at 30 min (P less than 0.05) and 120 min (P less than 0.001) after thawing, and a larger amplitude of lateral displacement of the head 120 min after thawing (P less than 0.05), compared with controls. In summary, spermatozoa from boars pre-pubertally exposed to low doses of DEHP, showed kinematic deviations post-thaw that could be related to DEHP exposure.

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

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.

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.

Hormonal asynchronies during oestrus, related to the presence of suprabasal plasma-progesterone (p4) concentrations and a delayed ovulation, interfere with the fertility of repeat-breeder heifers (RBH). Since tubal dysfunction can occur in connection with hormonal asynchronies and constrained availability of fertile spermatozoa at the time of ovulation, the present study tested the hypothesis that frequent sperm deposition from onset of oestrus to ovulation may improve pregnancy rates in RBH. Five RBH and five virgin heifers (VH; controls) were repeatedly artificially inseminated (AI) at 6 h intervals from onset of oestrus to spontaneous ovulation. Hormone analyses revealed suprabasal P-4 concentrations and a delay in the occurrence of the luteinising hormone (LH) surge, but a normal cortisol profile in RBH. Compared with controls, RBH presented longer interval from onset of oestrus to ovulation, and therefore, received more AIs. Pregnancy rates in RBH reached control levels (60%; NS), indicating that the hypothesis might be correct. Pregnancy rates in VH were below the expected range, presumably attributed to a deleterious influence of the frequent handling. The study suggests that pregnancy rates can be improved in RBH by frequent AI in relation to spontaneous ovulation. However, this practice of repeated manipulations, while seeming not to show adverse effects, lacks practicality for routine use.

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.