Biomedical subjects

Szabolcs Nagy

Publications and source records attributed to Szabolcs Nagy.

8 recordsLinked to original sources

PubMed2005

Usefulness of a triple fluorochrome combination Merocyanine 540/Yo-Pro 1/Hoechst 33342 in assessing membrane stability of viable frozen-thawed spermatozoa from Estonian Holstein AI bulls.

In a situation where technology allows for the simultaneous measurement of numerous parameters of a single sperm cell, it becomes crucial to choose those parameters which may be useful in estimating in vivo fertility. Sperm membrane destabilization is believed to occur during chilling of semen, although its effect on the post-thaw (PT) fertility of the spermatozoa has not yet been fully assessed. For this reason, we tested a new combination of fluorophores, Merocyanine 540 (M540)/Yo-Pro 1/Hoechst 33342 (H33342), to detect sperm plasma membrane destabilization in bull spermatozoa conventionally processed for artificial insemination (AI). The samples were tested by flow cytometry (FC), both immediately PT and following an in vitro swimup (SU) technique, and results were thereafter compared with conventional sperm quality measurements (of concentration, motility, morphology, and membrane integrity), including in vivo fertility. Semen samples from six Estonian Holstein (EHF) AI bulls, frozen when the sires were aged 3, 5, and 7 years, allowed us to test the effect of bull age on quality of semen. Plasma membrane stability correlated to motility, normal head morphology (p<0.05), and membrane integrity (p<0.01). Following the SU selection, motility, membrane integrity (p<0.001), and membrane instability increased (p<0.01), as did stability (p<0.05). Bull age did not influence the degree of sperm membrane destabilization, except for the 3-year sample versus 7-year sample, in which the proportion of spermatozoa with destabilized plasma lemma increased PT (p<0.05) without affecting membrane integrity. Only parameters measured after SU, such as proportion of total motile and linearly motile spermatozoa, assessed with computer-assisted sperm analysis (CASA) (p<0.01), average path velocity (VAP) (p<0.001), and percentage of spermatozoa with unstable plasma lemma (p<0.05), had a significant relationship with non-return rate (NRR). The results indicate that a triple combination of the fluorophores M540/Yo-Pro 1/H33342 is suitable for monitoring the status of membrane stability in frozen-thawed (FT) bull spermatozoa. As well, a SU preselection method seems helpful in distinguishing relationships between sperm quality and fertility among bulls in a homogenous sire population.

Aging↗

PubMed2005

Sperm chromatin stability in frozen-thawed semen is maintained over age in AI bulls.

The aim of the present study was to investigate the effect of age of the sire on the in vitro quality of frozen-thawed (FT) bull spermatozoa, both when tested immediately postthaw (PT) and when assessed after cleansing and selection through a swim-up (SU) procedure. Semen samples from six Swedish Red and White Breed (SRB) artificial insemination (AI) bulls at age 1 and again, at 4 years were collected and frozen in 0.25 ml plastic straws. Also, semen was collected from six Estonian Holstein (EHF) bulls at the ages of 3, 5, and 7 years and likewise processed. The FT semen was tested for the susceptibility of sperm nuclear deoxyribonucleic acid (DNA) to undergo acid-induced denaturation in situ, as quantified by flow cytometry (FCM). The DNA denaturability was expressed as function alpha t, i.e., as the ratio of red (denaturated DNA) to red + green (total cellular DNA) fluorescence intensity. The results were expressed as the percentage of cells with high alpha t values, i.e., cells outside the main population (% COMP alpha t). Morphological evaluation of the same samples was performed to detect general and sperm head abnormalities and differences between ages. Fertility results were available as non-return rates (NRRs) for the semen of the sires when they were 1 year (SRB) and 3 years (EHF) old, varying from 62.2 to 70.7% in SRB and from 52.2 to 76.0% in EHF animals. The COMP alpha t values ranged from 0.5-3.6% (PT) to 0.2-1.7% (SU) for SRB bulls and from 0.4-1.8% (PT) to 0.2-1.5% (SU) for EHF bulls. Both breeds lacked differences between ages, either PT or after SU. However, the SU procedure yielded a significantly higher population of spermatozoa with stable DNA following acid-induced denaturation, than PT samples (p < 0.001). No correlation was detected between field fertility and chromatin stability. The results indicate that for these bull populations, the SU procedure was able to select spermatozoa with stable chromatin from the bulk samples. However, the use of DNA denaturation as a challenge to assess sperm chromatin stability did not offer a more accurate tool to evaluate sperm quality than the conventional, light microscopical evaluation of morphology.

Aging↗

PubMed2005

Deep freezing of concentrated boar semen for intra-uterine insemination: effects on sperm viability.

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, intra-uterine 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 < 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 < 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.

Animals↗

PubMed2005

Assessment of the efficacy of Sephadex G-15 filtration of bovine spermatozoa for cryopreservation.

Semen from five dairy AI bulls was split-filtered through a Sephadex G-15 filter and frozen in a Tris-citric acid buffer egg yolk-based extender. The effect of filtration was studied morphologically for individual sperm abnormalities. Computer-assisted sperm analysis (CASA) was used for motility and sperm motion assessment. Flow cytometry was used to disclose sperm viability (SYBR-14/PI), mitochondrial membrane potential (Mitotracker Deep Red/SYBR 14), acrosome integrity (SYBR 14/PE-PNA/PI), plasma membrane stability (Merocyanine 540/YO-PRO 1/Hoechst 333342), and chromatin stability (acridine orange staining). Filtration significantly reduced the concentration of recovered spermatozoa (P < 0.01), but improved semen quality, reducing the number of spermatozoa with various forms of morphological defects. Filtration also affected percentages of sperm motility after equilibration and after freezing/thawing. Sperm motion characteristics were, however, not significantly affected by filtration at any stage of the cryopreservation protocol, including post-extension, equilibration, or freezing/thawing. Filtration enhanced sperm viability after thawing (P < 0.05), but had no significant effect (P > 0.05) on recovery of spermatozoa with high mitochondrial potential, intact acrosomes, or preserved sperm chromatin structure. Sperm plasma membrane stability was also not affected by the filtration method used (P > 0.05). It can be concluded that filtration effectively separates weaken or abnormal spermatozoa in pre-freezing semen samples and therefore the procedure could be recommended to improve post-thaw sperm viability of selected, fertile sires.

Acrosome↗

PubMed2005

Experiences in deer sperm cryopreservation under practical conditions--a pilot study.

The genetic potential of the red and fallow deer populations in Hungary is well known. Conserving the variability in this excellent genetic material for game preservation is one of our most important task. The aim of the present pilot study was to test the logistical steps of a sperm processing and storing system in which deer sperm can be stored at a level that meets quality standards accepted for domestic animals. Moreover, two different semen extenders, commercially used for freezing bull semen, were compared from the viewpoint of applicability to freeze fallow deer sperm. Sperm was collected from epididymes of eight red stags (Cervus elaphus hippelaphus) and six fallow bucks (Dama dama) during the rutting season. Red deer samples were washed in Triladyl extender, while fallow deer samples were split and processed in Triladyl or Bioxcell extender. In the samples, which had a shorter time interval between the death of the animal and the sperm collection, the percentage of viable spermatozoa with intact acrosome was typically higher.

Animals↗

PubMed2005

Mitochondrial activity of frozen-thawed spermatozoa assessed by MitoTracker Deep Red 633.

The present study was conducted to find a more objective method of evaluating sperm quality than the current subjective motility evaluations by testing the applicability of a novel fluorescent probe, Mitotracker Deep Red 633 (M-22426), for measuring the mitochondrial activity of spermatozoa both after freezing/thawing (PT) and after swim-up selection (SU), using flow cytometry (FC). The results from FC were compared to those of conventional microscopic motility evaluations and of computer-assisted sperm analysis (CASA) as well as to the fertility obtained after AI in the field. Semen from six Estonian Holstein bulls, processed when the sires were aged 3, 5, and 7 years, was included in the experiment. Sperm motility (measured either subjectively or by means of CASA) was always significantly (p<0.01-0.001) higher in the spermatozoa recovered by SU, for any of the age groups considered, or even when combining the age groups. Motility (measured subjectively) after SU was significantly (p<0.05) higher when bulls reached 7 years of age, compared to earlier collection ages, but no differences were registered between ages for CASA-assessed motility, either after SU or immediately PT. The numbers of spermatozoa with high red fluorescence also increased after SU: p<0.05 (for semen from bulls aged 3 years), p<0.001 (5 years), p<0.001 (7 years), and p<0.001 when all age groups were combined. The proportion of spermatozoa with high mitochondrial activity as determined by Mitotracker Deep Red 633 correlated with sperm motility (p<0.01) both PT and after SU, but not with the fertility results. In conclusion, MitoTracker Deep Red 633 seems to be a reliable marker for frozen-thawed bovine semen viability both PT and after SU.

Aging↗

PubMed2004

Changes in plasma membrane and acrosome integrity of frozen-thawed bovine spermatozoa during a 4 h incubation as measured by multicolor flow cytometry.

Cryopreservation of bull semen is sub-optimal, causing cell death of a majority of spermatozoa. Even the surviving cells are affected post-thaw, either structurally or functionally. The aim of this study was to investigate the sequence of events that take place when sperm plasma membrane and acrosome deteriorate during a 4 h incubation period post-thaw, with special attention paid to the acrosome status of dying cells. Frozen-thawed semen of six AI dairy bulls was used. Three straws per batch were pooled and incubated at 37 degrees C. Sub-samples were taken at 30 min intervals and stained with SYBR 14, propidium iodide (PI) and phycoerythrin-conjugated peanut agglutinin (PE-PNA). Plasma membrane and acrosome integrity were measured by flow cytometry. The experiment was repeated three times. Immediately after thawing, only 3.45% of the dying cells showed acrosomal exocytosis. This number increased dramatically during incubation, reaching 67% after 4 h. Within the intact cell population, the overall decrease in viability and acrosome integrity was kept at five percentage points. Flow cytometry and the triple fluorochrome combination presented a detailed picture of the time course in plasma membrane and acrosome deterioration of frozen-thawed bull semen. The results are expected to be useful for monitoring new cryopreservation protocols.

Acrosome↗

PubMed2002

A triple-stain flow cytometric method to assess plasma- and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles.

Simultaneously evaluating postthaw viability and acrosome integrity of spermatozoa by flow cytometry would provide a valuable testing tool in both research and routine work. In the present study, a new triple-stain combination was developed for the simultaneous evaluation of viability and acrosome integrity of bovine sperm processed in egg yolk-based extender by flow cytometer. SYBR-14 and propidium iodide (PI) enabled the discrimination of sperm cells from egg yolk and debris particles, which was instrumental for the flow cytometric analyses of frozen-thawed bovine sperm, because it implied that washing steps to remove egg yolk were no longer required. In addition, phycoerythrin-conjugated peanut agglutinin (PE-PNA) was used to discriminate acrosome-damaged/reacted sperm cells from acrosome-intact cells. Repeatability was calculated using two processed ejaculates of 10 bulls. Three straws per batch were analyzed in duplicate measurements. Method-agreement analysis between the SYBR-14/PE-PNA/PI and fluorescein isothiocyanate (FITC)-conjugated PNA was performed, with FITC-PNA/PI staining being carried out on 14 frozen-thawed semen samples immediately after thawing and after a 3-h incubation at 37 degrees C. The British Standards Institution repeatability index of the SYBR-14/PE-PNA/PI combination was 2.6%. On average, the FITC-PNA/PI method showed a 6.3% overestimation of the live and acrosome-intact sperm cell subpopulation. In conclusion, the new triple-stain combination is highly repeatable and easy to use in routine application, and it provides a more precise estimate for the rate of sperm cells with intact head membrane and acrosome compared to the generally used and validated FITC-PNA/PI staining.

Acrosome↗