Margareta Wallgren

Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate

Antioxidant supplementation during cooling was assayed to improve the motility of frozen-thawed (FT) boar spermatozoa from two different fractions of the ejaculate, the first component of the sperm-rich fraction (Fraction I) and the rest of the bulk ejaculate (Fraction II). Using a split-sample design, addition of two different concentrations (100 and 200 microMl(-1)) of the water-soluble Vitamin E analogue Trolox (6-hydroxy -2,5,7,8-tetramethylchroman -2-carboxylic acid) was evaluated for an effect on sperm motility (measured both subjectively and by means of a computer assisted motility assessment (CASA)), and on mitochondrial membrane potential using flow cytometry after cell-loading with JC-1. The effect of the Vitamin E analogue was clearly dose-dependent and varied with the fraction of the ejaculate considered. Motility was significantly higher in Trolox-treated spermatozoa (200 microm), from either ejaculate fraction, albeit the effect was more evident in spermatozoa from Fraction II (P<0.05) for any Trolox-concentration. Antioxidant supplementation resulted, also dose-dependent, in a higher number of spermatozoa showing high mitochondrial activity as assessed by the JC-1 staining, in both ejaculate fractions. In the present trial, exogenous Trolox positively affected post-thaw sperm viability (as motility and mitochondrial membrane potential) in both fractions of the ejaculate. The magnitude of the effect appeared, however, to be dependent of the fraction of the ejaculate considered.

Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: a new method of evaluating sperm membrane integrity

Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols. The present study evaluated the ability of an Annexin-V binding assay to detect early changes in sperm membrane integrity using flow cytometry (FC) in two different portions of the boar ejaculate, in cryopreserved semen. Using a split sample design, sperm motility was evaluated in fresh (controls) and frozen-thawed (FT) samples, both subjectively and by means of a computer-assisted motility assessment (CASA) system, while membrane integrity was assessed using Annexin-V (A) and propidium iodide (PI) staining in spermatozoa derived from the first sperm-rich fraction (Portion I) or the remaining ejaculate (Portion II). The A/PI technique revealed four sperm subpopulations, two PI negative (either A- (alive) or A+ (apoptotic)); and two PI positive (dead cells), either A+ (dead, late apoptotic or early necrotic cells) or A- (dead, late necrotic cells). Significant differences were found between the two portions of the ejaculate in the fresh (control) and FT samples. In the fresh controls, significantly more live, nonapoptotic spermatozoa (A-/PI-) were present in Portion I than in Portion II (P<0.001). Although apoptotic spermatozoa were detected in both semen portions, the frequency of live, early apoptotic (A+/PI-) cells was significantly lower in Portion I than in Portion II (P<0.001). Irrespective of the ejaculate portion considered, freezing and thawing significantly decreased the mean percentages of live spermatozoa (P<0.01), and dramatically increased the percentages of apoptotic or early necrotic cells (P<0.01), but not of early apoptotic cells (N.S.). The latter finding might suggest that apoptotic changes due to cryopreservation using the procedures applied in this trial are transient and lead to cell death. In conclusion, the Annexin-V binding assay was able to detect deleterious changes in the sperm plasma membrane at an earlier point than PI staining, thus representing a novel approach to investigating membrane integrity in this species. The finding that fewer spermatozoa in Portion I of the ejaculate showed early apoptosis post-freezing, suggests boar spermatozoa in this portion of the seminal plasma are less sensitive to the stress induced by cryopreservation.

Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture

Previous studies have shown sperm quality after cryopreservation differs depending on the fraction of seminal plasma the boar spermatozoa are contained in. Thus, spermatozoa contained in the first 10 ml of the sperm-rich fraction (portion I) withstand handling procedures (extension, handling and freezing/thawing) better than those contained in the latter part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction; portion II). The present study evaluated whether an exogenous antioxidant, the water-soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), could, when added to the freezing extender in a split-sample design trial, improve the post-thaw viability and membrane quality of this particular portion of the ejaculate, with particular attention to the status of the plasma membrane. Using a split-sample design, the initial changes in the fluidity status of the sperm plasmalemma after thawing were measured by flow cytometry (FC) after loading with Merocyanine-540 and YO-PRO-1. The FC-derived data revealed a clear ejaculate portion-dependent effect of the antioxidant supplementation. While no beneficial effect of the antioxidant supplementation was visible in spermatozoa from portion I, more spermatozoa with intact membranes were observed in the supplemented samples of portion II, suggesting the protective effect of vitamin E is dependent of the portion of the boar ejaculate considered.

Photoperiodic effects on pubertal maturation of spermatogenesis, pituitary responsiveness to exogenous GnRH, and expression of boar taint in crossbred boars

Forty-eight weaned, winter-born crossbred males (average age of 42 days) were exposed to either a natural photoperiod (January-June at 60 degrees N, Control) or one of two artificial photoperiods (1400 lx) in light-sealed rooms. The Spring/Summer group was exposed to an artificial photoperiod simulating conditions from the vernal equinox (mid-March) to August at 60 degrees N and the Autumn/Winter group to a photoperiod, simulating conditions from the autumnal equinox (mid-September) to February at 60 degrees N. Plasma samples were collected biweekly until the pigs were slaughtered, after reaching 115 kg, and analysed for testosterone, estrone sulfate, thyroxine and prolactin. Additionally, three animals per treatment (n = 9) were injected with gonadotropin-releasing hormone (GnRH) and plasma samples were collected every 15 min and analysed for luteinizing hormone and testosterone. Boar taint, carcass composition and reproductive traits were measured at slaughter. Live-weight gain from start to slaughter was lower among the Control animals compared with the Autumn/Winter and Spring/Summer animals. There was a peak in plasma testosterone in both the Spring/Summer and Autumn/Winter groups at 71 days of age, whereas plasma testosterone in the Control group remained at prepubertal levels. At 113 and 127 days of age, the Control group had somewhat higher testosterone levels than the Spring/Summer group, but at 141 days of age and on the day before slaughter, the Autumn/Winter group had a higher mean plasma testosterone concentration. There were no differences between treatments in the endocrine response to the GnRH challenge. Bulbourethral gland weight at slaughter was lower in the Spring/Summer group than in the Autumn/Winter group. The percentage of proximal cytoplasmic droplets was higher in the Spring/Summer group than in both the Control and Autumn/Winter groups. Spermatogenesis at the time of slaughter was clearly more mature in animals in the Autumn/Winter group than in those in the Spring/Summer and Control groups. Fat androstenone was lower in the Spring/Summer group than in the Control group. In the sensory evaluation, the Spring/Summer group had less boar taint than the Autumn/Winter group. Artificial short days with moderate initial changes in photoperiod, stimulated spermatogenesis compared with long days, in accordance with the pattern seen in European Wild Boars (Sus scrofa). Boar taint was also affected with higher scores in the Autumn/Winter group than in the Spring/Summer group, although this was not clearly indicated by the traditional measurements of boar taint-fat contents of androstenone and skatole.

Influence of seminal plasma on the kinematics of boar spermatozoa during freezing

Sperm motility 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 spermatozoa susceptible to oxidative 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 10mL 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 60min 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 portions, and apparently unrelated to changes in membrane integrity or membrane stability through conventional, controlled cooling.

Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, P1) 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 P1 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.

Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation

We investigated the effect of supplementing extended boar semen with different amounts of hyaluronan (HA) prior to freezing on post-thaw sperm characteristics. Using a split sample design, the effect of HA at a final concentration of 500 or 1000 microg/ml semen on post-thaw motility parameters, and membrane lipid architecture status assessed by merocyanine-540/YOPRO-1 and flow cytometry were evaluated. HA-supplementation improved motility parameters (P < 0.05 to P < 0.001) and decreased the percentage of hyperactivated spermatozoa (P < 0.05). HA-supplemented samples had more spermatozoa showing high lipid membrane stability as assessed with merocyanine-540. In conclusion, HA appeared to preserve post-thaw spermatozoa viability in vitro and maintained membrane stability after cryopreservation.

Advances in Boar Semen Cryopreservation

The present paper highlights aspects of the cryopreservation of boar semen, a species with particular large, fractionated ejaculates, and a cumbersome cryotechnology that had prevented its commercial application. With the dramatic increase of use of liquid pig semen for artificial breeding over the past decade, developments on cryopreservation alongside the routine use of stud boar semen for AI had been promoted. Recent advances in our laboratory, accommodating the best use of portions of the sperm-rich fraction of the ejaculate for cryopreservation of the sperm-peak portion (P1) and parallel use of the rest of the collected ejaculated spermatozoa, appears as a suitable commercial alternative.

Short- and long-term effects of immunization against gonadotropin-releasing hormone, using Improvac™, on sexual maturity, reproductive organs and sperm morphology in male pigs

The objective of this study was to determine the short and long term effects of a gonadotropin-releasing hormone (GnRH) vaccine (Improvac 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 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<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 clearly disrupted the number and morphology of the interstitial Leydig cells, lasting throughout the study period (4-22 weeks after vaccination). Spermatogenesis was also clearly affected in the immunized pigs, to various degrees, from mild disruption (spermatocyte loss, decrease of the normal number of layers of germ cells) to severe loss of germ cells including tubuli with Sertoli cells-only (complete disappearance of germ cells), also covering the entire study period. The results indicated that the effect of immunization persisted for at least 22 weeks after the second vaccination.

Spermadhesin PSP-I/PSP-II heterodimer induces migration of polymorphonuclear neutrophils into the uterine cavity of the sow

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.