Cryopreservation Of Boar Semen Research Articles

Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars

A major limiting factor for commercial cryopreservation of boar semen for artificial insemination (AI) is the large individual variation to cooling, where the degree of cell dehydration during ice (re)shaping seems to play a major role. This study investigated, in the frozen state, the degree of dehydration and ice crystal distribution in boar semen doses whose spermatozoa displayed different viability after thawing. Cross-sectioned medium-straws (0.5 mL, n = 10 ) from a total of 10 stud boars classified as “good” ( n = 5 ) or sub-standard (e.g., “bad” freezers, n = 5 ) by conventional analyses (computer assisted motility and sperm viability) were examined by Cryo-scanning electron microscopy (Cryo-SEM) to determine whether differences between groups could be already distinguishable prior to thawing. The degree of hydration was monitored in relation to the areas of ice crystal formed extracellularly (lakes), the areas of frozen, concentrated extender (veins) where spermatozoa were located and the degree of compartmentalization (number of lakes) present. Irrespectively of the region studied, the gradient of main dehydration (as lakes) observed along the cross-section area of the straws was very irregular. Most spermatozoa were enclosed in the freezing extender matrix and no obvious signs of external membrane damage were observed. None of the Cryo-SEM variables significantly correlated with post-thaw sperm parameters ( p > 0.05 ) . However, we identified significant differences ( p < 0.0001 ) among boars for all ultrastructure variables studied, including the size of the veins, where differences in solute concentration is expected. We concluded that despite the large variability in ice crystal formation during the conventional freezing process among boars, this is unrelated to inter-boar post-thaw sperm differences.

Evaluation of a cushioned method for centrifugation and processing for freezing boar semen

The purpose of this investigation was to evaluate the use of an iodixanol cushion during centrifugation on sperm recovery and yield after centrifugation (sperm recovery, sperm motility, viability, membrane lipid disorder, acrosome reaction and ROS generation); and to investigate how this procedure affects sperm function after freezing–thawing (sperm motility, membrane lipid disorder, acrosomal status and homologous in vitro penetration test). The sperm-rich fractions from fertile boars were centrifuged under two centrifugation régimes: 800 × g for 10 min (standard method) and 1000 × g for 20 min with an iodixanol (60% w/v) cushion at the bottom of the centrifuge tubes (Cushion method). The highest recovery was achieved using the cushion method (sperm loss for cushion method was 0.50% ± 0.18 versus 2.97% ± 0.43 for standard method, P < 0.01) and sperm quality was not significantly affected by the centrifugation régime. The motion parameters (% progressive motility, % motility, VCL, VSL, VAP, ALH, BCF, P < 0.05) of frozen–thawed samples showed higher values using the standard method. However, a higher number of viable spermatozoa with lower lipid disorders were found in spermatozoa processed with the cushion method. The in vitro penetration assay showed that the individual boar influenced the parameters studied but there were no differences between the two centrifugation régimes used. Our results support the hypothesis that the proportion of sperm loss in frozen–thawed semen was significantly influenced by the centrifugation régime. Therefore, the iodixanol cushion method is a suitable tool for cryopreservation of boar semen in order to reduce sperm loss without affecting sperm quality.

Effects of platelet-activating factor and platelet-activating factor: acetylhydrolase on in vitro post-thaw boar sperm parameters

Cryopreservation of boar sperm compromises fertility after thawing by reducing sperm longevity and inducing acrosome reaction-like changes. In an attempt to improve the post-thaw motility and acrosome integrity of boar sperm, semen was frozen using a modified Westendorf method in which the medium was supplemented with either platelet-activating factor (PAF) or a recombinant platelet-activating factor:acetylhydrolase (PAF:AH; Pafase) before or after freezing. Platelet-activating factor is a phospholipid that is present in boar semen and PAF:AH is the naturally occurring enzyme that converts PAF to biologically inactive Lyso-PAF. Addition of PAF to the cryopreservation medium improved post-thaw motility immediately after thawing and after 3 h incubation at 37 °C (60.0 ± 0.0% and 25.0 ± 2.9%; mean ± S.E.M.) compared to the control sperm (41.7 ± 1.7% and 10.0 ± 2.9%; P < 0.05). Acrosome integrity was higher immediately after thawing and after 3 and 6 h incubation at 37 °C when sperm were frozen in the presence of Pafase (55.7 ± 3.2%, 45.7 ± 3.7% and 23.0 ± 3.1%), compared to the control sperm (42.7 ± 1.5%, 25.7 ± 5.7% and 12.3 ± 2.7%) and sperm frozen in the presence of PAF (33.0 ± 3.7%, 26.3 ± 2.2% and 11.7 ± 0.3%; P < 0.05). Addition of PAF to sperm after thawing improved motility immediately post-thaw (41.6 ± 2.6%), compared with addition of Pafase (23.3 ± 2.2%) or the control sperm with no supplementation of the medium (26.7 ± 2.2%; P < 0.05). However, this beneficial effect was lost by 3 h post-thaw. Supplementation of boar semen cryopreservation medium with PAF and Pafase appeared to have beneficial effects on the in vitro quality of the sperm post-thaw.

Studies on the Effect of Supplementing Boar Semen Cryopreservation Media with Different Avian Egg Yolk Types on in Vitro Post‐thaw Sperm Quality

Fertility after insemination of cryopreserved boar semen is currently below that of fresh semen. In an attempt to improve the post-thaw motility and acrosome integrity of boar sperm, semen was frozen using an adapted Westendorf method in which the chicken egg yolk was replaced by either duck or quail egg yolk. The different composition of the yolk types, particularly the amount of cholesterol, fatty acids and phospholipids, were thought to potentially afford a greater level of protection to sperm against damage during freezing and thawing. Sperm frozen in medium containing chicken egg yolk displayed higher motility immediately after thawing, but there was no difference in the motility of sperm frozen with different types of egg yolk 3 or 6 h after thawing and maintenance at 37 degrees C. Sperm frozen in media containing chicken or duck egg yolk had a higher proportion of intact acrosomes immediately after thawing than sperm frozen in medium containing quail egg yolk, but 6 h after thawing and maintenance at 37 degrees C the sperm that had been frozen in medium containing chicken egg yolk had a higher proportion of intact acrosomes than the sperm frozen in media containing duck or quail egg yolk. Analysis of the composition of the different yolk types showed that the basic components of the yolks were similar, but the ratios of fatty acids and phospholipid classes differed. Duck egg yolk had more monounsaturated fatty acids (MUFA) than chicken egg yolk, which had more MUFA than quail egg yolk. Duck egg yolk contained more phosphotidylinositol (PI) than chicken or quail egg yolks and quail egg yolk contained more phosphotidylserine than either chicken or duck egg yolks. The differences in post-thaw motility and acrosome integrity of boar sperm when frozen in media containing the different types of egg yolk may be due to the variation in composition.

New assays for detection and localization of endogenous lipid peroxidation products in living boar sperm after BTS dilution or after freeze–thawing

Reactive oxygen species have been implicated in sperm aberrations causing multiple pathologies including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries for semen storage prior to artificial insemination but unusual in porcine breeding industries as semen dilution and storage at 17 ̊C is sufficient for artificial insemination within 2–3 days. However, longer semen storage requires cryopreservation of boar semen. Freeze/thawing procedures induce sperm damage and induce reactive oxygen species in mammalian sperm and boar sperm seems to be more vulnerable for this than bull sperm. We developed a new method to detect reactive oxygen species induced damage at the level of the sperm plasma membrane in bull sperm. Lipid peroxidation in freshly stored and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous lipid classes, phosphatidylcholine and cholesterol and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY 581/591. Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in the living sperm cells. Cytoplasmatic droplets in incompletely matured sperm cells were intensely peroxidized. Furthermore, lipid peroxidation was particularly strong in the mid-piece and tail of frozen/thawed spermatozoa and significantly less intense in the sperm head. Induction of peroxidation in fresh sperm cells with the lipid soluble reactive oxygen species tert-butylhydroperoxide gave an even more pronounced effect, demonstrating antioxidant activity in the head of fresh sperm cells. Furthermore, we were able to show using the flow cytometer that spontaneous peroxidation was not a result of cell death, as only a pronounced subpopulation of living cells showed peroxidation after freeze–thawing. Although the method was established on bovine sperm, we discuss the importance of these assays for detecting lipid peroxidation in boar sperm cells.

Cryopreservation of boar semen: equilibrium freezing in the cryomicroscope and in straws

Supercooling causes very abrupt temperature and osmotic changes and can thus lead to freezing damage. Supercooling can be prevented by seeding, using a sample volume and geometry that allows rapid spreading of the ice throughout the sample. In a split-sample comparison of such samples on the cooling stage of a cryomicroscope and seeded at −5 and −15 °C, respectively, the percentages of membrane-intact sperm and sperm with acrosomes with a ‘normal apical ridge’ (NAR) were 72.5 ± 3.8 and 75.8 ± 2.0 versus 46.3 ± 4.8 and 36.0 ± 3.7 (means ± S.E.M., n = 4). In ejaculates of 15 unselected AI boars, after seeding at −5 °C, the post-thaw % live and % NAR were 66.3 ± 10.4 and 74.8 ± 7.5, respectively. Our present research is aimed at translating these findings to freezing in straws and at a high sperm concentration. We have designed a novel type of freezing apparatus for controlled-rate freezing of straws, in which supercooling can be effectively prevented in the entire straw. In a split-sample comparison of semen frozen in straws at a sperm concentration of 1.5 × 10 9 cells/ml with nine ejaculates from eight unselected AI boars, we found 54.8 ± 1.9% versus 40.7 ± 1.7% (means ± S.E.M.) membrane-intact sperm for the new apparatus and a conventional freezing apparatus, respectively. With bull semen (eight ejaculates from six bulls), we obtained 67.3 ± 3.0% versus 59.3 ± 2.9% (means ± S.E.M.) membrane-intact sperm for the new apparatus and conventional freezing, respectively. Additionally, the temperature curve after ice nucleation is of great importance. We have developed a model that allows us to predict that optimal cryopreservation requires a non-linear cooling curve in which the cooling rate varies as a function of subzero temperature.

Changes in sperm quality and lipid composition during cryopreservation of boar semen

Egg yolks are commonly used in diluents in order to improve the freezability of semen. Two aspects of the role of lipids in boar semen freezability are reported in this article. The first one concerns the eventual exchanges of lipid components between the spermatozoa and the yolk-based diluent during cryopreservation. Two types of yolk have been considered as ingredients in diluents for cryopreservation: yolks with a standard fatty acid composition and yolks enriched in docosahexaenoic acid (DHA). The relation between lipid exchanges and the quality of fresh semen is considered. The other aspect concerns the possibility to enhance the freezability of boar spermatozoa by altering the plasma membranes under the influence of dietary fatty acids. Spermatozoa were damaged significantly by the cryopreservation cycle in all experiments. Spermatozoa with the best fresh quality had accumulated the largest quantity of lipids upon thawing. A general decrease in the proportion of polyunsaturated fatty acids was observed after thawing. The yolks enriched in n-3 fatty acids failed to improve the quality of sperm following cryopreservation. The proportion of DHA was significantly higher in spermatozoan phospholipids from thawed cells that had been in contact with n-3 yolks. A significant reduction in cholesterol was observed in spermatozoa after the cryopreservation cycle, which correlated with an increased number of acrosome-reacted cells and changes in the parameters of motility. The addition of 3% fish oil to the daily boar ration significantly increased the content of DHA (from 33 to 45% of the total fatty acids) in the spermatozoa. Ejaculate concentrations were significantly increased in the experimental group. DHA-enriched semen did not show improved freezability, at least not as assessed by in vitro parameters.

Decrease in glutathione content in boar sperm after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen–thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84±0.21nM GSH/108 sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P<0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P=0.0102). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.

Changes in sperm quality and lipid composition during cryopreservation of boar semen

The effect of cryopreservation on boar sperm viability, motility, lipid content and antioxidant enzymatic activities was studied. Three classes of semen were determined according to a cluster analysis on the basis of the proportion of live and dead cells after freezing and thawing. The classes identified were: high (H, n = 4), average (A, n = 12) and low (L, n = 3) viability. The concentration of sperm cells decreased from class H to A to L. Fresh semen samples with higher viability and a higher proportion of motile cells also maintained better quality after the freezing and thawing procedure. Sperm viability and motility in both fresh and thawed samples were similar in classes H and A, while significantly lower values were measured in class L. The relative decrease in sperm viability and motility after cryopreservation increased from class H to A to L. The lipid content of spermatozoa (micrograms per 10(9) cells) increased significantly after freezing and thawing in classes H and A but not in class L. This result indicated that active sperm lipid metabolism might be responsible for the increase in lipid content. Phospholipid and triacylglycerol contents increased whereas free cholesterol content decreased after thawing. The fatty acid composition of fresh spermatozoa was similar in all three classes. The proportion of polyunsaturated fatty acids decreased significantly after freezing and thawing, indicating contamination from the diluent or peroxidation. After freezing and thawing, superoxide dismutase activity in spermatozoa was significantly higher in class L than in classes H and A, which did not differ from each other.

Changes in sperm quality and lipid composition during cryopreservation of boar semen

The effect of cryopreservation on boar sperm viability, motility, lipid content and antioxidant enzymatic activities was studied. Three classes of semen were determined according to a cluster analysis on the basis of the proportion of live and dead cells after freezing and thawing. The classes identified were: high (H, n = 4), average (A, n = 12) and low (L, n = 3) viability. The concentration of sperm cells decreased from class H to A to L. Fresh semen samples with higher viability and a higher proportion of motile cells also maintained better quality after the freezing and thawing procedure. Sperm viability and motility in both fresh and thawed samples were similar in classes H and A, while significantly lower values were measured in class L. The relative decrease in sperm viability and motility after cryopreservation increased from class H to A to L. The lipid content of spermatozoa (micrograms per 10(9) cells) increased significantly after freezing and thawing in classes H and A but not in class L. This result indicated that active sperm lipid metabolism might be responsible for the increase in lipid content. Phospholipid and triacylglycerol contents increased whereas free cholesterol content decreased after thawing. The fatty acid composition of fresh spermatozoa was similar in all three classes. The proportion of polyunsaturated fatty acids decreased significantly after freezing and thawing, indicating contamination from the diluent or peroxidation. After freezing and thawing, superoxide dismutase activity in spermatozoa was significantly higher in class L than in classes H and A, which did not differ from each other.

Fundamentals and recent development in cryopreservation of bull and boar semen

Summary This paper describes some of the effects of cryopreservation on living sperm, and some recent results for bull and boar semen. During freezing and thawing of semen, the sperm can be damaged by the rapid and dramatic changes in the physicochemical conditions during cooling and ice formation. The rate and extent of these changes, and their impact on the cells, are strongly affected by the cooling rate and the medium composition. For many species, including humans and several farm animals, the optimal cooling rate has been determined, sometimes in combination with the cryoprotectant concentration. Recent results indicate that the cryopreservation of bull semen at the AI station could be improved by using controlled freezing at a cooling rate of about 100 °C/min, and by changing the composition of the freezing medium. An interaction between glycerol concentration and cooling rate has been described for boar semen. Current cryopreservation methods for boar semen based on an optimal combinations of these two factors allow consistent sperm survival in the frozen semen of AI boars, with acceptable variation among boars. The fertility results for semen processed in this fashion seem to be adequate for special applications, such as export, or long‐term storage.

An indirect immunofluorescence assay to assess sperm cryodamage using anti-bull sperm monoclonal antibodies

This study was undertaken to examine the effect of cholesterol-loaded-cyclodextrin (CLC) on boar sperm viability and spermatozoa cryosurvival during boar semen cryopreservation, and methyl-β-cyclodextrin (MBCD) was treated for comparing with CLC. Boar semen treated with CLC and MBCD before freezing process to monitor the effect on survival and capacitation status by flow cytometry with appropriate fluorescent probes. Sperm viability was higher in 1.5 mg CLC-treated sperm (76.9 ± 1.01%, P < 0.05) than un-treated and MBCD-treated sperm before cryopreservation (58.7 ± 1.31% and 60.3 ± 0.31%, respectively). For CTC patterns, F-pattern was higher in CLC treated sperm than MBCD-treated sperm, for B-pattern was higher in CLC-treated sperm than fresh sperm (P < 0.05). For AR pattern (an acrosome-reacted sperm) was lower in CLC-treated sperm than MBCD-treated sperm (P < 0.05). Moreover, we examined in vitro development of porcine oocytes after in vitro fertilization using CLC-treated frozen–thawed semen, in which CLC treatment prior to freezing and thawing increased the development of oocytes to blastocyst stage in vitro. In conclusion, CLC could protect the viability of spermatozoa from cryodamage prior to cryopreservation in boar semen.

Phospholipase A2 activity in fresh and cryopreserved spermatozoa from boars

The enzyme phospholipase A2 (PLA2) is considered to play an important role in the fertilizing ability of mammalian spermatozoa, but PLA2 activity has not been identified in boar spermatozoa. Fertility rates obtained using cryopreserved boar semen are considerably less than those with fresh boar semen, which might be attributed to altered PLA2 activity. Phospholipase A2 activity, determined by the formation of radiolabelled fatty acid from a radiolabeled phospholipid substrate, was successfully detected in the sonicates and acid extracts of boar spermatozoa. The PLA2 in acid extracts was calcium-dependent, active at neutral and alkaline pH and was maximized with increasing incubation temperatures, time and spermatozoal protein. Assaying acid extracts under conditions of 35 °C, 24 h, pH of 7.5 and 2 mM calcium, the PLA2 activity of freshly collected boar spermatozoa was found to be 8.22 ± 1.47 pmoles palmitic acid h−1 mg−1 protein (mean ± SE). Matched samples of spermatozoa exposed to cryopreservation had a significantly lower (P &lt; 0.05) activity of 4.26 ± 0.59 pmoles palmitic acid h−1 mg−1 protein. This study has identified PLA2 in boar spermatozoa for the first time and determined that its activity is reduced by procedures known to reduce fertility. Key words: Phospholipase, boar, semen, cryopreservation

Cryopreservation of Boar Semen

The present review summarizes information concerning the methods available to cryopreserve boar semen, covering the historical background, cryobiology and cryoprotecting considerations, technological developments and recent advances in cryopreservation methodologies. Successful methods for cryopreservation of boar semen have not been achieved despite numerous efforts world wide. Improvements in semen preservation technologies have been deterred by lack of in vitro methods that can accurately predict in vivo fertilizing capacity of frozen boar semen. The cell membrane is of crucial importance with regard to freeze-thaw survival of spermatozoa. It is important to optimize the survival of the plasma membrane as this is a non homogenous entity both in structure and function. The boar sperm membrane exhibits extreme sensitivity to freezing treatment. Freezing and thawing results in considerable changes in electrolyte dynamics and damages have mainly been associated with alterations in the head membranes especially at thawing. To date fruitless efforts have been carried out to find a cryoprotectant for the spermatozoa membranes and glycerol still continues to be used despite its harmful effects to the membranes.

Biotechnological methods score efficacy the cryopreservation of boar semen

Aim. Estimate boar semen for suitability to cryopreservation using informative biotechnological methods (thermoresistant probe, heat resistance test) to replenish genetic resources of Animal Gene bank Institute of Animal Breeding and Genetics n. M.V. Zubets, NAAS. Methods. In carrying out this work used biotechnology, cryobiological, morphological methods. Results. Mirgorod boars breed investigation we noted feature individual semen quality, which affect its suitability for cryopreservation. All boar genealogy analysis of experiment in Mirgorod breed descended from a line of boar named Lovchyk 1205, which was characterized by negative performance stability to semen cryopreservation. For morphometric and functional parameters of sperm, potentially of better quality sperm for cryopreservation was boar Mirgorod purebred No. 1159, for which thermoresistant probe, heat resistance test indices was respectively 40.0 % and 35.0 %. Most suitable for cryopreservation in terms of heat resistance test boar semen was number 1159 and No. 79, and amounted to 35.0 % and 30 %. Nevertheless, after thawing average sperm activity index fell to 83.7 %. Conclusions. Established a comprehensive method for assessing the suitability of native breeds boar semen cryopreservation involving of morphological and biotechnological methods Keywords: cryobank, spermatozoa, cryopreservation, gene pool.