Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa

The decline in boar semen quality after cryopreservation may be attributed to changes in intracellular proteins. Thus, the aim of the present study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually mature boars (mean age = 25.5+/-12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15 degrees C, after cooling to 5 degrees C, following thawing after freezing to -100 degrees C, and following thawing after 1 wk of cryopreservation at -196 degrees C. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5x10(6) spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was 90 kDa heat-shock protein (HSP90). Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5 degrees C. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5 degrees C. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100 degrees C. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The length of time between a decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa.     

Impact of storage prior to cryopreservation on plasma membrane function and fertility of boar sperm

Occasionally, boar semen must be shipped to another location for cryopreservation. We increased the initial holding time for the cooling of extended semen at 15 degrees C from 3 to 24 h to determine the effects on sperm characteristics and fertility. Thirty-one gilts and sows were inseminated once with subsequently cryopreserved and thawed semen. Increasing the holding time from 3 to 24 h had no significant effect on pregnancy rate 23 days after AI with frozen-thawed semen (64.5%) but decreased (P<0.05) embryo number from 15 to 9 and recovered embryos as fraction of CL from 73 to 47%. While the longer holding time at 15 degrees C did decrease potential litter size, the loss incurred was not too great to preclude the incorporation of a longer holding time into the cryopreservation protocol. An experiment was conducted to test the hypothesis that processing and freeze-thawing of boar semen would induce phospholipid scrambling in the plasma membrane similar to that evoked by incubation in bicarbonate-containing media. Merocyanine staining after incubation in the presence and absence of bicarbonate indicated that changes in plasma membrane phospholipid scrambling of processed and cryopreserved sperm differed from those in fresh semen undergoing bicarbonate-induced capacitation. The level of Annexin-V binding in boar spermatozoa increased from 1.6% in live spermatozoa in fresh semen to 18.7% in cryopreserved sperm. Apoptosis is unlikely to operate in mature spermatozoa. Apoptotic morphology in ejaculated spermatozoa is probably a result of incomplete deletion of apoptotic spermatocytes during spermatogenesis. Increased Annexin-V binding in thawed spermatozoa probably results from plasma membrane damage incurred during freezing and thawing.     

New aspects of boar semen freezing strategies

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2-3% in the freezing media, cooling rates of -30 to -50 degrees C/min, and thawing rates of 1200-1800 degrees C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1x10(9) spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.     

Chlortetracycline analysis of boar spermatozoa after incubation,flow cytometric sorting,cooling, or cryopreservation

In this study, the effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was evaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC-pisum sativum agglutenin and propidium iodide (PI). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR-14 and PI. Staining of spermatozoa with CTC alone and in combination with PI and/or Hoechst 33342 had no effect on the proportion of spermatozoa allocated to the F (uncapacitated), B (capacitated), or AR (acrosome-reacted) CTC fluorescent staining categories. The mean percentages of acrosome-intact and acrosome-reacted cells were 88.4 and 6.8 or 0.8 and 96.5 in semen treated with 0 or 100 μg/ml lysophosphatidylchloine (LPC), respectively (P < 0.001). Most spermatozoa were also in the AR CTC-stained category after treatment with LPC compared with a small proportion in the controls. Using flow cytometry to examine sperm suspensions stained with CTC, a gated population of spermatozoa with low fluorescence (population 1) comprised predominantly F-pattern cells (F-pattern: population 1 vs. population 2, 80.5 vs. 14.4%; P < 0.001), whereas population 2 (high fluorescence) comprised mainly B-pattern cells (B-pattern: population 1 vs. population 2, 8.5 vs. 62.3%; P < 0.001). Incubation (38°C, 4 hr), flow sorting, cooling (to 15 or 5°C) and freezing reduced the proportion of F-pattern and live spermatozoa, and increased the proportion of B-, AR-pattern, and dead spermatozoa, in comparison with fresh semen. There were more membrane changes in spermatozoa cooled to 5°C (30.4, 48.5, 21.1%) than in those cooled to 15°C (56.1, 32.6, 11.5% F-, B-, and AR-pattern spermatozoa, respectively). Mol. Reprod. Dev. 46:408–418, 1997. © 1997 Wiley-Liss, Inc.     

Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione.

Cryopreservation induces extensive biophysical and biochemical changes in the membrane of spermatozoa that ultimately decrease the fertility potential of the cells. Sulfhydryl groups of sperm proteins regulate a number of activities of the cells. Qualitative and quantitative analyses of sulfhydryl groups in the sperm membrane were performed by fluorescence microscopy, fluorimetry and electrophoresis. Fluorimetric analysis using 5-iodoacetamidofluoresceine indicated a two-fold increase in the content of sulfhydryl groups in sperm membrane after a freezing/thawing cycle. Electrophoresis of Triton-soluble sperm proteins after labeling with 3-(N-maleimidylpropionyl) biocytin indicated that proteins of 40-65 and 34 kDa range expose more sulfhydryl groups after cooling at 4 degrees C and freezing/thawing. Cryopreservation of spermatozoa changed the distribution pattern of sulfhydryl groups on sperm surface measured with fluorescence microscopy using 5-iodoacetamidofluoresceine. The percentage of spermatozoa labeled at the level of the mid-piece decreased by 50 and 90% after cooling and freezing/thawing, respectively. Spin labeling studies showed a 15% faster rotational diffusion (mobility) of sulfhydryl containing proteins in the membrane of frozen/thawed spermatozoa as compared to that of fresh spermatozoa. Addition of glutathione, reduced (GSH) or oxidized (GSSG), to the cryoprotectant partially prevented the effects of freezing/thawing, such as higher exposure of sulfhydryl groups, changes in the cellular distribution, and enhanced rotational diffusion of sulfhydryl containing proteins of sperm membrane. Addition of GSSG to the cryoprotectant reduced by 35% the loss of motility of spermatozoa undergoing a freezing/thawing cycle. We concluded that cryopreservation perturbs sperm membrane sulfhydryl containing proteins and that these modifications could be partially prevented by the addition of GSSG to the cryopreservation medium.     

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 +/- 0.21 nM GSH/10(8) 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 5 mM 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.     

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.     

The effects of rapid cooling (cold shock) of ram semen, photoperiod, and egg yolk in diluents on the survival of spermatozoa before and after freezing

The effects of rapid cooling of semen (cold shock) from 30 degrees C to various temperatures above 0 degrees C on survival of ram spermatozoa suspended in diluents with or without egg yolk were assessed before and after freezing. Rapid cooling of extended semen from 30 to 15 degrees C had little or no effect on spermatozoa survival before or after freezing. Rapid cooling of extended semen from 30 degrees C to 10, 5, or 0 degrees C was accompanied by a progressive decrease in percentage of motile spermatozoa and percentage of intact acrosomes before freezing and a decrease in percentage of motile spermatozoa and after freezing. The ability of spermatozoa motile after cold shock to survive freezing and thawing, evaluated as cryosurvival, was not significantly (P greater than 0.05) affected by the temperature to which semen was cooled. The addition of egg yolk to the initial extender had a beneficial effect on percentage of motile spermatozoa particularly after rapid cooling of semen to 10 and 5 degrees C. Although egg yolk had little effect before freezing on semen rapidly cooled to temperatures above 15 degrees C and therefore not actually cold shocked, it substantially improved the subsequent survival of spermatozoa after freezing and thawing. Percentage of motile spermatozoa after cooling and after freezing was generally higher when the semen was collected during a decreasing photoperiod than during an increasing photoperiod.     

Cold-induced ultrastructural changes in bull and boar sperm plasma membranes

The effect of low temperatures on the ultrastructure of the plasma membrane of bull and boar spermatozoa was investigated. Cold-induced changes in the organization of sperm plasma membrane components were demonstrated by the use of fast-freezing combined with freeze-fracture electron microscopy. This preparation technique ensures fixation without artifacts. At 38 degrees C bull and boar spermatozoa exhibited a random distribution of intramembranous particles over the plasma membrane of both head and tail. Exposure to 0 degree C resulted in redistribution of the intramembranous particles: on the head and principal piece of bull spermatozoa and on the principal piece of boar spermatozoa, particle-free areas were observed, whereas on the boar sperm head, particle aggregates were present. The original particle distribution was restored upon rewarming of bull and boar spermatozoa to 38 degrees C, as well as after freezing and thawing of bull spermatozoa. Dilution of bull and boar semen into Tris-dilution buffer and Beltsville Thaw Solution-dilution buffer, respectively, could not prevent cold-induced redistribution of intramembranous particles. The observed particle reorganization upon cooling was interpreted as the result of lateral phase separation in the plasma membrane. Species-dependent differences in cold-induced ultrastructural changes were considered to be determined by lipid composition and asymmetry of the plasma membrane, and might be related to differences in cold resistance between species.     

Detrimental effects of non-functional spermatozoa on the freezability of functional spermatozoa from boar ejaculate

In the present study, the impact of non-functional spermatozoa on the cryopreservation success of functional boar spermatozoa was evaluated. Fifteen sperm-rich ejaculate fractions collected from five fertile boars were frozen with different proportions of induced non-functional sperm (0--native semen sample-, 25, 50 and 75% non-functional spermatozoa). After thawing, the recovery of motile and viable spermatozoa was assessed, and the functional of the spermatozoa was evaluated from plasma membrane fluidity and intracellular reactive oxygen species (ROS) generation upon exposure to capacitation conditions. In addition, the lipid peroxidation of the plasma membrane was assessed by the indirect measurement of malondialdehyde (MDA) generation. The normalized (with respect to a native semen sample) sperm motility (assessed by CASA) and viability (cytometrically assessed after staining with Hoechst 33342, propidium iodide and fluorescein-conjugated peanut agglutinin) decreased (p<0.01) as the proportion of functional spermatozoa in the semen samples before freezing decreased, irrespective of the semen donor. However, the magnitude of the effect differed (p<0.01) among boars. Moreover, semen samples with the largest non-functional sperm subpopulation before freezing showed the highest (p<0.01) levels of MDA after thawing. The thawed viable spermatozoa of semen samples with a high proportion of non-functional spermatozoa before freezing were also functionally different from those of samples with a low proportion of non-functional spermatozoa. These differences consisted of higher (p<0.01) levels of intracellular ROS generation (assessed with 5-(and-6) chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester; CM-H(2)DCFDA) and increased (p<0.01) membrane fluidity (assessed with Merocyanine 540). These findings indicate that non-functional spermatozoa in the semen samples before freezing negatively influence the freezability of functional spermatozoa.     

The effects of cooling to 5 degrees C and freezing and thawing on the ultrastructure of bull spermatozoa.

Semen from 6 bulls was examined under the transmission electron microscope immediately after collection, after dilution and cooling to 5 degrees C and after freezing and thawing. Conception rates were determined following artificial insemination of the frozen and thawed semen. Dilution and cooling to 5 degrees C caused acrosomal swelling in about 50% of the spermatozoa. Subsequent freezing and thawing caused considerable ultrastructural changes to the acrosomes (disruption of the plasma and outer acrosomal membranes and dispersion of the acrosomal contents) and middle pieces (breakage of the plasma membrane and a reduction in the electron density of the mitochondrial matrix) of a high proportion of spermatozoa. The average non-return rate following insemination of semen from 5 of the bulls was 61.6% and higher (P greater than 0.001) than for the sixth bull (15%). Although this difference in semen viability was also demonstrated in the structural studies (acrosome, P greater than 0.05: middle piece, P greater than 0.001), more work is required to assess the relationship between structure and function of spermatozoa.     

Effect of freezing and thawing rates on the post-thaw viability of boar spermatozoa frozen in FlatPacks and Maxi-straws

The effects of different freezing and thawing rates on the post-thaw motility and membrane integrity of boar spermatozoa, processed as split samples in Maxi-straws or flat PET-plastic packages (FlatPack) were studied. A programmable freezing device was used to obtain freezing rates of either 20, 50 or 80 degrees C/min. Thawing of the samples was performed in a bath of circulating water; for 40s at 50 degrees C or 27s at 70 degrees C for Maxi-straws and 23s at 35 degrees C, 13s at 50 degrees C or 8s at 70 degrees C for the FlatPacks. Sperm motility was assessed both visually and with a computer assisted semen analysis (CASA) apparatus, while plasma membrane integrity was assessed using the fluorescent probes Calcein AM and ethidium homodimer-1. Temperature changes during freezing and thawing were monitored in both forms of packaging. Values for motile spermatozoa, sperm velocity and lateral head displacement variables were significantly (p<0.05) higher for samples frozen in FlatPacks than in Maxi-straws, with superior results at higher thawing rates. Freezing at 50 degrees C/min yielded better motility than 20 or 80 degrees C/min, although the effect was rather small. Neither freezing rate nor thawing rate had any effect on membrane integrity (p>0.05). A significant boar effect was seen for several parameters. The most striking difference in temperature courses between containers was a 4-5-fold lowering of the thawing rate, between -20 and 0 degrees C, in the center of the Maxi-straw, compared with the FlatPack. This is apparently due to the insulating effect of the thawed water in the periphery of the Maxi-straw. The improvement in sperm motility seen when using the FlatPack appears to be related to the rapid thawing throughout the sample, which decreases the risk of cell damage due to recrystallization during thawing. Since sperm motility patterns have been reported to be correlated with fertility both in vitro and in vivo it is speculated that the use of the FlatPack might improve the results when using frozen-thawed boar spermatozoa for artificial insemination.     

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.     

Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate freezers: a comparison

This study compared variation in the quality of cryopreserved boar spermatozoa and the control and accuracy of cooling rates between three semen freezers (CryoLogic Freeze Control CL3000, Planer Products Kryo Save Compact KS1.7/Kryo 10 Control module and a controlled rate 'Watson' freezing machine developed within our laboratory). Five ejaculates were collected from each of 15 boars (five boars from each of three breeds). Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% v/v glycerol) and placed into 0.5 ml straws. Three straws per treatment, from each ejaculate were cooled to -5 degrees C at 6 degrees C/min, held at -5 degrees C for 30s while ice crystal formation was induced, then further cooled from -5 to 80 degrees C at either 40 degrees C/min (Kryo Save Compact KS1.7 and Watson) or 6 degrees C/min (Freeze Control CL3000). Precise measurements of temperature fluctuations during the programmed cooling curves were made by inserting thermocouples into the semen filled straws. Semen was assessed for %motile cells, motility characteristics using computer-assisted semen analysis (CASA), plasma membrane integrity (%SYBR-14 positive stained spermatozoa) and acrosome integrity (%FITC-PNA positive stained spermatozoa). Spermatozoa cryopreserved using the Freeze Control CL3000 system (maximum rate of 6 degrees C/min) exhibited reduced post-thaw viability (14.2+/-2.8% mean plasma membrane intact spermatozoa) when compared to both the KS1.7 and Watson freezers (optimal rate of 40 degrees C/min) (18.4+/-3.2 and 25.7+/-3.7% mean plasma membrane intact spermatozoa, respectively). Differences in motility characteristics were observed between spermatozoa cryopreserved at 40 degrees C/min with the Watson apparatus preserving a larger proportion of sperm with progressive motility. Cooling curves in the CL3000 and KS1.7 were interrupted by a pronounced increase in temperature at -5 degrees C that corresponded with the latent heat of fusion released with ice crystal formation. This temperature change was significantly reduced in the cooling curves produced by the Watson freezer. These findings suggest that preserving spermatozoa using the Watson freezer improved post-thaw semen quality, with regard to sperm motility characteristics. Furthermore, that post-thaw semen viability was enhanced by minimising temperature fluctuations resulting from the release of the latent heat of fusion at ice crystal formation.     

The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa

Cryopreservation of epididymal spermatozoa is a potentially valuable tool for preserving genetic material from individuals of endangered species that die accidentally. Improvement of sperm-freezing protocols would increase the efficacy of gene banking from endangered felids, and the domestic cat can be used as a model for the wild felids. Addition of the detergent Equex STM paste to semen freezing extenders has been found to improve post-thaw survival and longevity of spermatozoa from various species but has never been tested for cat spermatozoa. Spermatozoa from cats with a high percentage of morphologically abnormal spermatozoa are more susceptible for cold injury and osmotic stress than spermatozoa from normozoospermic cats. Therefore, the aims of this study were to investigate: (a) if addition of Equex STM paste to a semen freezing extender would improve post-thaw sperm survival, and (b) if there is a relation between the percentage of morphologically normal spermatozoa and cryopreservation induced damage in cat epididymal spermatozoa. Spermatozoa harvested from epididymides of 10 male cats were frozen in a Tris egg yolk extender with or without the addition of Equex STM paste (0.5%, v/v). Sperm motility, membrane integrity and acrosomal status were evaluated immediately after harvesting, and at 0, 2, 4 and 6 h post-thaw. Sperm membrane integrity and acrosomal status were also evaluated after cooling to 4 degrees C, just before freezing. Cooling did not cause significant damage to the spermatozoa, whereas freezing damaged sperm membranes and acrosomes. Addition of Equex to the freezing extender had a significant positive effect on the percentage of intact acrosomes immediately after thawing (P > 0.05), but had a negative effect on the longevity of the spermatozoa; the percentages of membrane intact and motile spermatozoa being significantly lower in the presence of Equex than in the controls at 6h after thawing. The percentage of morphologically normal spermatozoa was not found to be correlated with either cryopreservation induced acrosome or plasma membrane damage, or with post-thaw motility (P > 0.05). The results clearly show that addition of Equex STM paste in the freezing extender protects the acrosomes of cat epididymal spermatozoa during the freezing--thawing process, but reduces the sperm longevity during in vitro incubation at 38 degrees C. Our results also indicate that the percentage of morphologically normal epididymal spermatozoa is not correlated with cryopreservation induced sperm damage using the described freezing protocol.     

Effect of glycerolization procedure and removal of seminal plasma on post-thaw survival and got-release from Boer goat spermatozoa

Forty ejaculates (20 for each of 2 experiments) were collected from 4 Boer goat bucks at weekly intervals to study the effect of glycerolization procedure and removal of seminal plasma on progressive motility, percent live spermatozoa and release of glutamic oxaloacetic transaminase (GOT) before and after the freezing of semen. Stepwise glycerolization at 37 degrees C gave higher progressive motility and percentage of live spermatozoa both before freezing and after thawing than onestep glyceroliza-tion at 37 degrees C or stepwise extension with glycerol being added after cooling to 5 degrees C. The GOT-release was reduced before freezing and after thawing of semen with stepwise glycerolization (P < 0.05). Progressive motility and the percentage of live spermatozoa were higher (P < 0.05) after the freezing of whole semen than in washed spermatozoa. The concentration of GOT in the extra-cellular fluid was lower in washed spermatozoa prior to freezing (P < 0,05); but after thawing, the washed spermatozoa released more GOT than spermatozoa in whole semen. Removal of seminal plasma prior to freezing spermatozoa in an extender containing egg yolk had an unfavorable effect on their post-thaw motility and integrity.     

Flow cytometric assessment of fresh and frozen-thawed Canada goose (Branta canadensis) semen

The present study was conducted to investigate spermatozoal membrane integrity, acrosome integrity, mitochondrial activity, and chromatin structure in fresh and frozen-thawed Canada goose (Branta canadensis) semen with the use of the flow cytometry. The experiment was carried out on ten, 2-year-old, Canada goose ganders. The semen was collected twice a week, by a dorso-abdominal massage method, then pooled and subjected to cryopreservation in straws, in a programmable freezing unit with the use of dimethyloformamide (DMF) as a cryoprotectant. Frozen samples were thawed in a water bath at 60 °C. The freezing procedure was performed ten times. For the cytometric analysis the fresh and the frozen-thawed semen was extended with EK extender to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with SYBR-14 and propidium iodide (PI), acrosomal damage was evaluated with the use of PNA-Alexa Fluor^®488 conjugate, mitochondrial activity was estimated with Rhodamine 123 (R123), and spermatozoal DNA integrity was measured by the sperm chromatin structure assay (SCSA). The cryopreservation of Canada goose semen significantly decreased the percentage of live cells, from 76.3 to 50.4% (P < 0.01). Moreover, we observed the significant decrease in the percentage of live spermatozoa with intact acrosomes (P < 0.01), but we did not detect significant changes in the percentage of live spermatozoa with ruptured acrosomes. However, after thawing 50% of Canada goose live spermatozoa retained intact acrosomes. Furthermore, the percentage of live spermatozoa with active mitochondria was significantly lower in the frozen-thawed semen than in the fresh semen (P < 0.05). Nevertheless, after thawing the mitochondria remained active in almost 50% of live cells. In the present study, we observed no changes in the percentage of sperm with fragmented DNA after freezing-thawing of Canada goose semen. In conclusion, the present study indicates that even the fresh Branta canadensis semen might have poor quality, the cryopreservation of its semen did not provoke spermatozoal DNA defragmentation and half of the spermatozoa retained intact acrosomes and active mitochondria after freezing-thawing.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.     

Acrosin activity is a good predictor of boar sperm freezability

The main aim of this study was to determine whether acrosin activity could predict boar sperm freezability. For this purpose, we characterized the changes in sperm quality and acrosin activity throughout the cryopreservation procedure of sperm samples from 30 Pietrain boars by analyzing four critical steps: step 1 (extended sperm at 15 °C), step 2 (cooled sperm at 5 °C), step 3 (30 minutes postthaw), and step 4 (240 minutes postthaw). Freezability ejaculate groups were set on the basis of sperm motility and membrane integrity after freeze–thawing. Results obtained highlighted the low predictive value in terms of freezability of sperm motility and kinematics and sperm membrane integrity, as no differences between good and poor freezability ejaculates were seen before cryopreservation. Significant differences (P < 0.05) between ejaculate groups were observed in the cooling step at 5 °C for sperm kinetic parameters, and after thawing for sperm motility and membrane integrity. In contrast, acrosin activity appeared as an indicator of boar sperm freezability because the differences (P < 0.05) between good and poor freezability ejaculates manifested yet in extended samples at 15 °C. On the other hand, we also found that variations in sperm kinematics, membrane lipid disorder, intracellular calcium content, acrosome integrity, and acrosin activity throughout the cryopreservation procedure were indicative of a significant damage in spermatozoa during the cooling step in both ejaculate groups. In conclusion, the main finding of our study is that acrosin activity can be used as a reliable predictor of boar sperm freezability because it differs significantly between good and poor freezability ejaculates yet before freeze–thawing procedures took place, i.e., in the refrigeration step at 15 °C.     

Membrane changes during different stages of a freeze-thaw protocol for equine semen cryopreservation

Many theories have been postulated concerning the possible effects of cryopreservation on spermatozoa, including suggestions the freeze-thawing process produces membranes that have greater fluidity and are more fusogenic, thus inducing changes similar to those of capacitation. The main objectives of this study were to determine at what stage of the freeze-thaw process membrane changes occur and whether evaluation with chlortetracycline (CTC) stain could predict the freezability of stallion sperm. Sperm viability and state of capacitation were simultaneously evaluated using CTC and Hoechst 33258 (H258) techniques. Membrane function was evaluated using the hypo-osmotic swelling test (HOS) and progressive motility (PM) was evaluated under light microscopy at each stage of a freeze-thaw protocol. Evaluated were raw semen; after dilution and centrifugation; after redilution and equilibration at room temperature; after cooling to 5 degrees C; after super cooling to -15 degrees C; and after thawing. The most pronounced functional damage to membranes and the greatest decrease in PM occurred in samples of all stallions after thawing (P<0.05). Cryopreservation, as evaluated by CTC/H258 staining, significantly (P<0.05) affected sperm membrane integrity after centrifugation, after redilution and equilibration at room temperature and after cooling to 5 degrees C. The HOS and H258 tests gave similar results (R values of approximately 0.75) and correlated inversely with the number of live noncapacitated sperm cells (R values of approximately -0.75). Remarkably, the subpopulation of capacitated live cells was unaffected in all freeze-thawing steps and the number of live acrosome reacted cells increased by a factor of 4. However, it was not possible to determine whether the changing CTC patterns reflect a true capacitation phenomenon or an intermediate destabilized state of the sperm cell membrane. This increase may indicate that the subpopulation of functional sperm cells capable of binding to the zona pellucida increases after freeze-thawing despite the deteriorative effect of this procedure for the entire live sperm population.