Influence of various permeating cryoprotectants on freezability of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of concentration and temperature of addition

With the aim of finding an ideal cryoprotectant (CPA) in a suitable concentration for red deer epididymal spermatozoa cryopreservation, we evaluated the effects of the 3 most commonly used CPAs, glycerol (GLY), ethylene glycol (EG), and propylene glycol (PG), on sperm cryoresistance. The aim of Experiment 1 was to evaluate the influence of 3 different final concentrations (3%, 6%, and 12%) of each CPA on sperm freezability. Sperm samples were diluted to a final sperm concentration of approximately 400 x 10(6) spermatozoa/mL with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Thawed samples were incubated at 37 degrees C for 2 hours in the freezing medium. At the end of this incubation period, sperm suspensions were again assessed. Our results showed that 12% of any CPA was toxic to red deer epididymal spermatozoa membrane integrity (P < .05). Moreover, regardless of the level of CPA, results indicated that the cryoprotective effects on red deer epididymal spermatozoa of the 3 CPAs after thawing are in the following sequence: GLY > EG > PG (higher symbols mean P < .001). Furthermore, our results also showed an improvement in sperm parameters when the TCF diluent contained 6% of GLY. In Experiment 2 extenders were prepared using GLY 6%. This experiment was designed to investigate the effect of 2 different temperatures of GLY addition -22 degrees C (ambient temperature) and 5 degrees C- on sperm freezability. Our results showed a differential response (P < .05) of motility (SMI) to temperature of GLY addition before freezing, the best being 22 degrees C (81.94 +/- 2.4% vs 72.38 +/- 2.4%). Although there were no statistically significant differences (P > .05) between the 2 temperatures of GLY addition after thawing in terms of sperm quality, after 2 hours of incubation, results tended to be better when CPAs were added at 22 degrees C. In conclusion, our work showed the efficacy of a TCF diluent with 6% of GLY and its addition at 22 degrees C, as an alternative to the more common 3%-4% of GLY and addition at 5 degrees C, in red deer semen freezing protocols.     

Effects of thawing procedure on postthawed in vitro viability and in vivo fertility of red deer epididymal spermatozoa cryopreserved at -196 degrees C

In this study, we have determined the effects of individual factor and thawing procedure on in vitro viability and in vivo fertility of frozen-thawed red deer epididymal spermatozoa. The spermatozoa that were collected from 13 Iberian deer stags were diluted at room temperature in a Triladyl-20% egg yolk medium and frozen in nitrogen vapors. In the first experimental series, sperm samples were collected from 10 mature stags. For thawing, the frozen straws were subjected to 3 different procedures: I (37 degrees C for 20 seconds), II (60 degrees C for 8 seconds) and III (70 degrees C for 5 seconds). Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SM) and of plasma membrane and acrosome (NAR) integrities. Statistically significant variations were found (P <.05) between stags for most of the seminal parameters evaluated. The thawing procedure did not have an effect on the seminal characteristics evaluated after this process, except for SM (P <.05), with the best overall recovery rates after freezing and thawing found with the use of protocol I. Our results also show a differential resistance to return to isosmotic conditions of spermatozoa thawed using the different thawing protocols. In the second experimental series (insemination artificial trial), with spermatozoa from 3 stags, results of fertility were statistically higher (69.7% vs 42.4%, P =.014) when spermatozoa were thawed at 37 degrees C for 20 seconds than were warmed at 60 degrees C for 8 seconds. Therefore, thawing protocol I, which provides slow thawing rates, was the most beneficial for epididymal spermatozoa thawing of the cervid subspecies analyzed in this study. In summary, high in vitro survival and in vivo fertility of frozen-thawed deer epididymal spermatozoa were dependent on warming rates, but each stag exhibited its own sensitivity to cryopreservation.     

Sperm characteristics and DNA integrity of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa frozen in the presence of enzymatic and nonenzymatic antioxidants

The main goal of this study was to investigate the potential protective effects of enzymatic and nonenzymatic antioxidants on cryopreservation injuries to red deer epididymal spermatozoa. In Experiment 1, the effects on sperm freezability of the enzymatic antioxidants catalase, superoxide dismutase, and a combination thereof were studied. In Experiment 2, sperm cryoresistance was evaluated when different nonenzymatic antioxidants, such as vitamin E, vitamin C, and butylated hydroxytoluene (BHT), were added to the freezing extender. Sperm quality was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and acrosome (ie, spermatozoa with normal apical ridges; % NAR) and membrane (by means of the HOS test) integrity. To address fully these topics, we incorporated a new set of functional sperm tests for mitochondrial function, membrane phospholipid disorder, and sperm chromatin stability. Samples were evaluated after freezing and thawing, and after a 2-hour period of incubation at 37 degrees C. The present study demonstrates that the addition of enzymatic antioxidants to freezing extenders improves sperm viability after cooling, and improves sperm motility, acrosome integrity, and mitochondrial status (P<.05) after thawing. After a 2-hour incubation period at 37 degrees C in the presence of enzymatic antioxidants, an improvement in membrane integrity (P<.05) was observed. However, when nonenzymatic antioxidants were present in the freezing diluents, no positive effects on thawed sperm parameters were noted. The chromatin stability test did not show significant differences between the treatments. We conclude that enzymatic antioxidants should be present in the early steps of cryopreservation of epididymal spermatozoa from red deer, so as to improve motility and acrosome integrity.     

Effect of Cooling, Freezing and Thawing Rates and Storage Conditions on Preservation of Human Spermatozoa

Human spermatozoa was relatively resistant to cooling shock. However, when diluted semen was cooled faster than 10 degrees C per minute from room temperature (RT) to 5 degrees C and rewarmed to RT, percentage motility and percentage alive of spermatozoa decreased when compared to the slower cooling rates (less than 5 degrees C/min). The optimum cooling rate from RT to 5 degrees C resulting in maximum survival of human spermatozoa was found to be 0.5 to 1 degree per minute when cooled from RT to 5 degrees C and subsequently frozen-thawed in liquid nitrogen (LN2). The optimal freezing rate of 10 degrees C per minute, from 5 degrees to -80 degrees C, resulted in higher survival of human spermatozoa than slower (1.1 degrees C/min) or faster (87.1 degrees C/min) freezing rates. Slow thawing in 20 or 35 degrees C air, on a dry bench, resulted in better survival than the other slower or faster thawing methods used. The temperature at which human semen samples were transferred to LN2 significantly influenced spermatozoa survival. Survival was higher when transferred at -30 degrees C or lower when compared with samples transferred at -15 degrees C or higher. However, maximal spermatozoa survival was obtained when the samples were transferred at -80 degrees C or lower. Transfer of human semen from LN2 to -25 to -30 degrees C and storage for 24 hours significantly reduced spermatozoa viability when compared with storage at 196 degrees C or -80 to -85 degrees C. No significant differences were found between storage temperatures of -80 to -85 degrees C and -196 degrees C in the maintenance of spermatozoa viability for up to 90 days.     

Antioxidant activity of Nigella sativa Seeds Aqueous Extract and its use for cryopreservation of buffalo spermatozoa

The free radical scavenging activity (RSA) of Nigella sativa extract and its efficiency for cryopreservation of buffalo spermatozoa was investigated. In experiment 1, Nigella sativa extract was prepared and evaluated for RSA using 2,2‐diphenyl‐1‐picrylhydrazyl assay. The results showed increased pattern of RSA at 1%–5% of Nigella sativa extract. In experiment 2, buffalo semen from three bulls (24 ejaculates) was incubated at 0%, 0.1%, 0.3%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5% and 6% extract to assess in vitro tolerability to Nigella sativa in terms of progressive motility (PM). Buffalo spermatozoa showed tolerance to all levels; rather, sperm PM was increased at 1%–4% extract. In experiment 3, semen from three bulls (24 ejaculates) was cryopreserved with 0%, 1%, 2%, 3%, 4% and 5% of Nigella sativa extract. Sperm PM and plasma membrane integrity (PMI) were evaluated after dilution and cooling, while PM, PMI, viability and DNA integrity were evaluated after thawing. Nigella sativa extract at 4% in extender improved (p < .05) post‐dilution, post‐cooling and post‐thaw sperm quality. In conclusion, Nigella sativa extract at all concentrations (1%–6%) showed antioxidant activity and its supplementation at 4% in extender improved buffalo sperm quality at all stages of cryopreservation.     

Conventional slow freezing cryopreserves mouflon spermatozoa better than vitrification

This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre‐freezing equilibration times (2–3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm ). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60–85°C min^?1), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm ) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified–warmed sperm variables were at their best when the spermatozoa was diluted in TCG–6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.     

Effects of methyl-beta-cyclodextrin on cryosurvival of boar spermatozoa

Methyl-beta-cyclodextrin (MBCD), which leads to the stimulation of cholesterol efflux from the cell membrane, was examined for its ability to increase the cryosurvival of spermatozoa from G?ttingen miniature pigs. The intactness of the acrosome and various motion parameters of spermatozoa after freezing and thawing were used to monitor cryosurvival. Spermatozoa were exposed to MBCD or a combination of MBCD and cholesterol-3-sulfate over a period of 3 hours while being cooled slowly from 25 degrees C to 5 degrees C, and were subsequently cryopreserved by the pellet method. After freezing and thawing, the acrosomal status was monitored by fluorescein isothiocyanate-labeled peanut agglutinin, and the motion parameters were assessed with a computer-assisted sperm motility analysis system. In post-thaw spermatozoa the values of the intact acrosome, motility, progressive motility, progressive velocity, straightness, and linearity of the cell path increased greatly with the concentration of MBCD (P < .05). In contrast, the lateral head displacement amplitude and the beat cross-frequency of post-thaw spermatozoa were not different among all treatments. On the contrary, the addition of cholesterol-3-sulfate to the diluent containing MBCD abolished the protective effect against freeze-thaw injury that MBCD provides to spermatozoa. These results indicate that cryosurvival of boar spermatozoa is enhanced by exposure to MBCD before freezing. This protective effect of MBCD may be due to active depletion of sperm membrane cholesterol.     

Survival and fertility of boar spermatozoa after freeze-thawing in extender supplemented with butylated hydroxytoluene

This study evaluated the protective effect of butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, against cryopreservation injuries to boar spermatozoa. In experiment 1, the lowest BHT concentrations able to reduce lipid peroxidation in boar spermatozoa were determined. Nine BHT concentrations (ranging from 0.025 to 3.2 mM) were evaluated, and the lowest (P <.05) production of malondialdehyde (MDA), as an indicator of lipid peroxidation, was obtained when BHT ranged from 0.2 to 1.6 mM. In experiment 2, sperm survivability was evaluated when BHT was added to a postthaw freezing extender by measuring the degree of sperm lipid peroxidation (using MDA production) and by measuring parameter such as motility, plasma membrane and acrosome integrity, and cell apoptosis. The ability of thawed spermatozoa to fertilize in vitro-matured oocytes and of embryos to develop to the blastocyst stage in vitro was also assessed. Pooled sperm-rich fractions collected from 3 mature Pietrain boars were frozen in 0.5-mL straws after dilution with lactose-egg yolk-glycerol-Orvus ES Paste extender supplemented with 0, 0.2, 0.4, 0.8, and 1.6 mM BHT. Postthaw sperm survival, evaluated 30 and 150 minutes after thawing, was higher in BHT-treated spermatozoa, being significant (P <.05) when the freezing extender was supplemented with 0.2, 0.4, and 0.8 mM BHT. The addition of BHT to the freezing extender resulted in a significant (P <.05) decrease in the MDA concentration in thawed spermatozoa, irrespective of the level of BHT used. BHT had no effect on oocyte cleavage rates, but the development to blastocyst was improved for embryos derived from spermatozoa frozen in extender supplemented with 0.4 mM BHT (16% vs 29% of blastocysts per total oocytes; P <.05). In conclusion, under the conditions tested in the present study, the addition of BHT to the freezing extender improved the overall efficiency of thawed boar spermatozoa.     

Detection of "apoptosis-like" changes during the cryopreservation process in equine sperm

The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure. Freezing and thawing caused an increase in membrane permeability and changes in the pattern of caspase activity; decreases in mitochondrial membrane potential were observed after centrifugation and cooling to 4 degrees C and after freezing and thawing. It is proposed that sperm mitochondria may be directly involved in the subtle damage that is present in most spermatozoa surviving freezing and thawing.     

Hyperactivated motility is coupled with interdependent modifications at axonemal and cytosolic levels in human spermatozoa.

Whether the motility characteristics of hyperactivated spermatozoa were determined by stable changes at the axonemal level and whether the presence of cytosolic factors was required for the expression of these changes was investigated. Different degrees of sperm hyperactivation were produced in Percoll-washed spermatozoa after incubation for 1 hour to 3 hours at 37 degrees C in Ham's F-10 supplemented with human blood plasma or fetal cord serum. Decomplemented fetal cord serum induced the highest percentage of hyperactivation (19 +/- 3%), followed by human plasma (13 +/- 2%). Fetal cord serum that was not decomplemented did not induce a level of hyperactivation (1.7 +/- 0.2%) significantly different from control levels (0.9 +/- 0.2%). Dialyzed fetal cord serum induced intermediate levels of hyperactivation (6 +/- 1%). The motility characteristics of demembranated sperm models of hyperactivated spermatozoa induced by decomplemented fetal cord serum and nonhyperactivated spermatozoa were compared by videomicroscopy and computer-assisted digital image analysis. After demembranation with Triton X-100 and reactivation of motility by Mg. adenosine triphosphate (Mg.ATP), hyperactivated and nonhyperactivated spermatozoa showed similar motility characteristics. However, hyperactivated spermatozoa that were demembranated and reactivated in cytosolic extracts from hyperactivated spermatozoa had significantly higher (P less than 0.05) linear velocity (33 +/- 4 mu/sec) and lower linearity (0.23 +/- 0.04) than control spermatozoa that were demembranated and reactivated in control cytosolic extracts (velocity = 24 +/- 1 mu/sec; linearity = 0.32 +/- 0.02). The data suggest that the expression of hyperactivated motility requires interdependent changes at the axonemal and cytosolic levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryopreservation—induced decrease in heat—shock protein 90 in human spermatozoa and its mechanism

Aim: To study the protein changes of spermatozoa associated with sperm motility during sperm cryopreservation and its mechanism. Methods: In 18 healthy men, the seminal sperm motility and HSP90 levels were studied before and after cryopreservation using SDS-PAGE, Western blotting and computerized image analysis. Results: The sperm motility declined significantly after cryopreservation (P<0.01). The average grey level and the integrated grey level of sperm HSP90 before cooling were 34.1±3.2 and 243.0±21.6, respectively, while those after thawing were 23.2±2.5 and 105.7±28.5, respectively. Both parameters were decreased significantly (P<0.01). No HSP90 was found in the seminal plasma before and after cryopreservation. Conclusion: HSP90 in human spermatozoa was decreased substantially after cryopreservation. This may result from protein degradation, rather than leakage into the seminal plasma.     

The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas

This experiment documented the incidence and variability of sperm characteristics found in freshly collected and ex vivo-manipulated semen samples from a population of disease-free captive koalas with a special emphasis on the dynamic aspects of DNA fragmentation. These changes were analyzed in light of the putative negative effect of iatrogenic damage after chilled storage and cryopreservation with respect to different semen extender compositions to maximize sperm longevity. Sperm DNA fragmentation (SDF) dynamics (SDF assessment after a varying period of time) was investigated with the sperm chromatin dispersion assay after either dilution in tris-citrate media and chilled preservation at 4°C for upward of 16 days or cryopreservation in either glycerol or dimethylacetamide (DMA) tris-citrate-based cryoprotectant media; corresponding data on progressive sperm motility, plasma membrane integrity, and the proportion of koala sperm with relaxed chromatin were also recorded. SDF analysis of the captive koala population revealed a low mean (±SEM) basal level of only 6.7% ± 0.9%. The percentage of progressive sperm motility, percentage of intact sperm plasma membranes, and the percentage of relaxed chromatin did not correlate significantly with that of basal SDF. Moreover, despite the absence of cysteine residues in marsupial protamines, koala spermatozoa showed remarkable stability in terms of their DNA integrity after the incubation of either fresh, chilled, or frozen-thawed semen samples; observations of progressive motility (P < .05) and plasma membrane integrity (P < .05) revealed that chilled koala spermatozoa declined after 4 days, whereas the incidence of relaxed chromatin increased after 8 days. Although koala SDF increased significantly (P < .05) with the period of chilled storage, these values remained less than 16% after 16 days storage and subsequent incubation at 35°C for a further 48 hours. Survivorship of prefreeze sperm DNA damage was not different when compared with sperm frozen in DMA or between sperm frozen in DMA or glycerol; however, spermatozoa frozen in glycerol showed a higher (P = .042) rate of DNA fragmentation than prefreeze spermatozoa. This result differed from that of observations of progressive motility, plasma membrane integrity, and relaxed chromatin, which were all adversely affected (P < .05) after cryopreservation in either glycerol or DMA; however, the postthaw characteristics of sperm cryopreserved in either glycerol or DMA were not different. After thawing, koala sperm chromatin tended to decondense; however, the incidence of sperm DNA fragmentation was not correlated with the incidence of sperm chromatin relaxation after glycerol (R = .2) or DMA (R = -.04) cryopreservation.     

Cryopreservation of rhesus monkey (Macaca mulatta) epididymal spermatozoa before and after refrigerated storage

Recently, there has been an increased interest in preservation of epididymal sperm as a potential source of material for genetic resource banking; however, cryopreservation of epididymal sperm from the rhesus monkey has not been explored. This study evaluated the effect of prolonged refrigerated storage of the intact cauda epididymides at various conditions on the postthaw motility of rhesus monkey epididymal spermatozoa, and also tested whether altering cryoprotectants and cooling methods could improve post-thaw motility for epididymal sperm after refrigerated storage. Motility before freezing decreased significantly after refrigerated storage (0 degrees C) for a period of 24 or 48 hours. Although postthaw motility was not significantly different after 24 hours of refrigerated storage, epididymides stored at a higher temperature (4 degrees C-10 degrees C) yielded better results, but postthaw motility still decreased significantly after 48 hours of refrigerated storage at 4 degrees C. Comparisons of glycerol and ethylene glycol at 3% and 6% revealed similar postthaw motility. However, consistently high postthaw motility was obtained with 3% glycerol throughout all freezing trials regardless of whether samples were collected fresh or after refrigerated storage for 24 or 48 hours. Cooling at a higher rate of 220 degrees C/min was found to yield better postthaw motility than the slower rate of 29 degrees C/min. Thawing time duration was evaluated, and a minimum of 30 seconds was required for thawing 0.25-mL straws containing 50-microL semen samples. An overall average of 42% postthaw motility was obtained for rhesus monkey epididymal sperm packed in 3% glycerol and cooled after 24 or 48 hours refrigerated storage. These postthaw motility results for epididymal sperm indicate that this method should be practical for use in preserving epididymal sperm, even if tissue must be shipped from sites remote from the cryopreservation laboratory.     

Controlled cooling during semen cryopreservation does not induce capacitation of spermatozoa from two portions of the boar ejaculate

Cryopreservation imposes dramatic changes in boar sperm survivability but it is as yet unclear which part of the process affects the spermatozoa the most. The present study monitored, along the entire process of cryopreservation, the stability (PMS) of the architecture of the lipid plasma membrane and its integrity (PMI), as well as the kinetics of the processed spermatozoa using two portions from the boar ejaculate (P1 = the first 10 mL of the sperm-rich fraction, SRF; P2 = the rest of the ejaculate), frozen in a recently developed package, the MiniFlatPack (MFPs, 0.5 x 10(9) sperm/dose). Evaluation was made at four specific stages, viz. S1 = after collection (suspended in Beltsville thawing solution, BTS); S2 = at 15 degrees C (suspended in lactose-egg yolk, LEY); S3 = at 5 degrees C (suspended in LEY plus glycerol); and S4 = post-thaw. Both sperm kinetics (using computer-assisted sperm analysis, CASA) and PMS [i.e. the degree of lipid disorder and of the exteriorization of phosphatidylserine (PS) in the plasma membrane, measured by flow cytometry using Merocyanine-540 (M-540), and Annexin-V (AV) respectively], as well as plasma membrane integrity [PMI, i.e. the degree of membrane damage, measured using Yo-Pro-1 or propidium iodide (PI)] were assessed after incubation in BTS at 38 degrees C. Moreover, spermatozoa were challenged by incubation in modified Brackett-Oliphant medium (mBO+) with 37 mm of bicarbonate at 38 degrees C for 30 min, and their PMS and PMI further explored. Total sperm motility was significantly higher in P1 than in P2 along the entire process (S1-S4; p < 0.01), decreasing significantly at S4 for both fractions (p < 0.0001). The proportion of spermatozoa showing linear motility (LinM) was similar between ejaculate portions (P1 and P2), with a significant increase post-thaw (S4; p < 0.0001). During cooling (S1-S3) but not post-thaw (S4), lateral head displacement (LHD) differed between portions and changed along the stages (p < 0.01). Sperm velocity differed between portions in S1 (p < 0.01), but remained similar, independently of the portion, thereafter (S2-S4). Both PMS and the total number of live spermatozoa remained similar between S1 and S3 while incubated in BTS for both ejaculate portions. Sperm mortality increased post-thaw (S4) in both portions but the degree of lipid disorder remained low in the live cells (1.28% for P1; 1.55% for P2). Exposure to mBO+, on the other hand, significantly increased membrane lipid disorder along cooling (S1-S3; p < 0.0001), increasing the percentages of dead spermatozoa, especially post-thaw (around 70%, both portions). PS-exteriorization (AV) was not evident along the cryopreservation process in control (BTS) samples and exposure to mBO+ only induced minor variations. The data showed that kinetics, PMS and PMI of boar spermatozoa suspended in BTS (S1), LEY (S2) or LEY plus glycerol (S3) were maintained during controlled cooling but were altered by thawing, showing more characteristics of cell injury than of sperm capacitation. The spermatozoa were able to capacitate but the bicarbonate challenge destabilized the plasma membrane during initial cooling and accelerated membrane changes post-thaw. We conclude that capacitation of boar spermatozoa does not occur during controlled cooling.     

Semen characteristics of the captive Indian leopard, Panthera pardus

Semen samples from 11 Indian leopards (Pantherapardus) from 3 different zoos in India were collected by electroejaculation. A computer-aided semen analyzer (CASA) was used for assessing the quality of the semen vis-à-vis sperm motility. The volume of the ejaculate, sperm density, and the number of motile and morphologically normal spermatozoa were found to be 1.57 +/- 1.26 mL, 55.78 million +/- 38.67 million per mL, 57.05% +/- 16.96% and 71.92% +/- 15.32%, respectively. Although the spermatology varied between individuals in the study, Box-Whisker-plot analysis suggested that the distribution was normal (P > .05). The ejaculated sperm were cryopreserved after diluting in test-yolk buffer. The post-thaw motility was 32.14% and did not differ at 30 or 60 days after cryopreservation. CASA indicated that the progressive velocity (VSL) of cryopreserved spermatozoa was decreased and, as a consequence, they moved more slowly than the neat (VSL 76.3 microm/sec in neat vs 53.8 microm/sec in cryopreserved spermatozoa) and the trajectories were less planar. However, both cryopreserved and neat spermatozoa penetrated the zona-free hamster oocyte with equal efficiency (79% neat vs 80% cryopreserved). The study also reports application of CASA for feline spermatozoa and provides information for the first time on the spermatology of the Indian leopard. This baseline data could be used in captive breeding programs. The results are compared and discussed with the available information on other felines.     

Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical-chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT--cooling rate of -0.55 °C min(-1) and freezing rate of -19.1 °C min(-1) and automated (AT--cooling rate of -0.23 °C min(-1) and freezing rate of -15 °C min(-1)), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein-conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fisher's test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 ± 1.41% and 30.50 ± 1.06%, with ethylene glycol was 21.17 ± 1.66% and 21.67 ± 1.13% and with dimethyl formamide was 8.33 ± 0.65% and 9.17 ± 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 ± 1.49% (CT) and 15.83 ± 1.26% (AT) to glycerol, 9.20 ± 1.31% (CT) and 9.92 ± 1.29% (AT) to ethylene glycol 4.65 ± 0.93% (CT) and 5.17 ± 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.     

Kinematic changes during the cryopreservation of boar spermatozoa

The present study evaluates the effect that various steps of a conventional cycle of cryopreservation have on the patterns of movement exhibited by boar spermatozoa. Sperm-rich ejaculate fractions collected from 24 mature fertile boars (1 ejaculate per boar) were cryopreserved following a standard freeze-thaw procedure with 0.5-mL plastic straws. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in 5 steps of the cryopreservation procedure. These steps were as follows: 1) at the time that the fresh semen was extended, 2) at 17 degrees C, after sperm concentration by centrifugation and re-extension of the pellet with lactose-egg yolk extender; 3) at 5 degrees C, after added freezing extender; 4) at the time that thawed semen was held in a water bath at 37 degrees C for 30 minutes; and 5) at the time that thawed semen was held in a water bath at 37 degrees C for 150 minutes. Data from individual motile spermatozoa, defined by 7 kinematic parameters (curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], straightness [STR], mean amplitude of lateral head displacement [ALH], and beat cross frequency [BCF]), were analyzed using a pattern analysis technique (PATN) to identify and quantify populations and subpopulations of motile sperm within the semen samples. After the first cluster analysis, 3 motile sperm populations (P) were identified (P1: progressive and/or vigorous cells [90.4%], P2: poorly progressive cells [8.3%], and P3: nonprogressive cells [1.3%]). These populations remained constant (P > .05) throughout the 5-step cryopreservation procedure. A second PATN was carried out within the P1 sperm population, which identified 3 sperm subpopulations (sP) (eg, sP1: cells with progressive and vigorous movement [58.7%], sP2: progressive cells only [24.6%], and sP3: vigorous cells only, hyperactive-like [16.7%]). Although the relative frequency of these 3 subpopulations varied among ejaculates (boars), there was no interaction with any cryopreservation step we examined. Whereas sP1 remained constant (P > .05), sP2 and sP3 varied significantly (P < .05) through the cryopreservation procedure, with the increase in sP3 after centrifugation at 17 degrees C and during cooling at 5 degrees C considered particularly relevant. In conclusion, the present study confirms the heterogeneity of sperm movement patterns in boar semen, patterns that vary through the cryopreservation procedure, especially after removal of the seminal plasma by centrifugation and subsequent extension at 17 degrees C and after the slow cooling at 5 degrees C, when obvious increases in hyperactivated movement appeared. The vast majority of spermatozoa, those exhibiting progressive and vigorous movement, remained constant during the cryopreservation procedure, although the proportion differed among boars.     

Effects on the quality of frozen-thawed alpaca （Lama pacos） semen using two different cryoprotectants and extenders

Aim： To evaluate two extenders and two cryoprotectant agents （CPA） for alpaca semen cryopreservation. Methods： Semen samples were obtained from four adult alpacas （Lama pacos） and frozen using extender Ⅰ （TRIS, citrate, egg yolk and glucose） or extender Ⅱ （skim milk, egg yolk and fructose）, each containing either glycerol （G） or ethylene glycol （EG） as CPA. Consequently, four groups were formed： 1） extender Ⅰ-G; 2） extender Ⅰ-EG; 3） extender Ⅱ-G; and 4） extender Ⅱ-EG. Semen was diluted in a two-step process： for cooling to 5 ℃ （extenders without CPA）, and for freezing （extenders with CPA）. Viability and acrosome integrity were assessed using trypan blue and Giemsa stains. Results： When compared, the motility after thawing was higher （P 〈 0.05） in groups Ⅱ-EG （20.0 %±6.7 %） and Ⅱ-G （15.3 %±4.1%） than that in groups Ⅰ-G （4.0 %±1.1%） and Ⅰ-EG （1.0 %±1.4 %）. Viable spermatozoa with intact acrosomes in groups Ⅱ-EG （18.7 %±2.9 %） and Ⅱ-G （12.7 %±5.9 %） were higher than that in groups Ⅰ-G （5.7 %±1.5 %） and Ⅰ-EG （4.0 %±1.0 %）. Conclusion： The skim milk- and egg yolk-based extenders containing ethylene glycol or glycerol to freeze alpaca semen seems to promote the survival of more sperm cells with intact acrosomes than the other extenders. （Asian J Androl 2005 Sep; 7： 303-309）     

Ultrastructural alterations of frozen-thawed Asian elephant (Elephas maximus) spermatozoa

Intact plasma and acrosome membranes and functional mitochondria following cryopreservation are important attributes for the fertilizing ability of spermatozoa. In the present study, functional and ultrastructural changes of Asian elephant spermatozoa after cryopreservation either in TEST + glycerol or HEPT + dimethyl sulphoxide (DMSO) were evaluated by fluorescent techniques and electron microscopy. Sperm frozen in TEST + glycerol had higher proportion of sperm with intact plasma (49.1 +/- 9.2% vs. 30.9 +/- 3.9%) and acrosomal (53.7 +/- 4.9% vs. 35.8 +/- 6.1%) membranes, as well as active mitochondria (57.0 +/- 7.2% vs. 42.0 +/- 5.0%) than those cryopreserved in HEPT + DMSO. The results obtained from electron microscopy were similar to those obtained by fluorescence microscopy. The percentage of normal spermatozoa was higher when spermatozoa were frozen in TEST + glycerol than those frozen in HEPT + DMSO (31.8 +/- 5.6 vs. 28.5 +/- 6.4). The ultrastructural alterations revealed by transmission electron microscopy could be classified as (i) distension of plasma membrane, while the acrosome was swollen; (ii) disruption or loss of plasma membrane, while acrosome was swollen with distended outer acrosomal membrane; (iii) disruption or loss of plasma and outer acrosomal membrane with leakage of acrosome content; (iv) extensive vesiculation of plasma and outer acrosomal membrane and leakage of acrosome content; (v) a complete loss of both plasma membrane and outer acrosomal membrane; and (vi) swelling of mitochondria. These findings suggest that the freezing and thawing procedure caused structural damage to elephant spermatozoa, especially in the plasma membrane, acrosome and mitochondria. Fluorescence and electron microscopic evaluations are potentially a powerful tool in the analysis of elephant spermatozoa after freezing and thawing.