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 thawing velocity on survival and acrosomal integrity of ram spermatozoa frozen at optimal and suboptimal rates in straws

The effect of various thawing velocities on the motility and acrosomal maintenance of ram spermatozoa frozen at 20 degrees C/min (optimal) or 2 degrees C/min (suboptimal) was studied. The freeze-thaw motility and the percentage of intact acrosomes of spermatozoa frozen at 20 degrees C/min increased progressively with the thawing velocity. In semen frozen at 2 degrees C/min, motility of spermatozoa and the percentage of intact acrosomes declined drastically when the thawing velocity obtained in air at 20 degrees C was increased by thawing in water at 20 degrees C. Thawing at higher temperatures markedly increased both motility and acrosomal preservation, but the best results with semen frozen at 2 degrees C/min were lower than those obtained with semen frozen at 20 degrees C/min. The optimal freeze-thaw conditions for semen protected by 4% glycerol were freezing at 20 degrees C/min and thawing in water at 60 or 80 degrees C for 8 or 5 sec, respectively. Semen collected from rams exposed to a decreasing photoperiod exhibited higher motility after freezing and thawing than those exposed to an increasing photoperiod. However, there was no effect on acrosomal preservation after freezing at 20 degrees C/min.     

Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa

A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.     

Comparative semen cryopreservation in ferrets (Mustela putorius furo) and pregnancies after laparoscopic intrauterine insemination with frozen-thawed spermatozoa

A study was conducted to determine an optimum technique for semen cryopreservation and the biological competence of frozen-thawed ferret spermatozoa. Fifty-two fresh electroejaculates from 4 males were evaluated for sperm percentage motility, forward progressive motility, motility index (SMI) and acrosomal integrity. To determine the optimum temperature for maintaining sperm motility in vitro and the influence of glycerol on sperm motility, seminal aliquants were diluted in TEST diluent (containing either 0 or 4% glycerol) and maintained at 25 degrees or 37 degrees C. For cryopreservation, semen was diluted in each of 3 cryodiluents (TEST, PDV, BF5F), cooled for 30 min at 5 degrees C and pelleted on solid CO2 or frozen in 0.25 ml straws (20 degrees C/min to -100 degrees C). Following thawing, SMI and acrosomal integrity were determined. Ten females with maximum vulval swelling were given 90 i.u. human chorionic gonadotrophin and laparoscopically inseminated in utero with spermatozoa previously frozen using the optimum diluent and freeze-thaw method. The maintenance temperature of 25 degrees C was superior (P less than 0.05) to 37 degrees C for sustaining sperm motility, and glycerol did not influence (P greater than 0.05) motility for up to 11 h of culture. After thawing, motile spermatozoa were recovered in all treatment groups, but sperm motility and normal acrosomal ratings were highest using the PDV diluent, the pelleting method and thawing at 37 degrees C (P less than 0.05). Seven of the 10 ferrets (70%) inseminated with spermatozoa frozen by this approach became pregnant and produced 31 kits (mean litter size 4.4; range 1-9 kits).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level

The effect of thawing velocities ranging from 10°C/min to 1.800°C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1°C/min (suboptimal), 5°C/min, and 30°C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1°C/min, the percentage of motile sperm (FMP) initially fell to 3.5–4.0% when the thawing rose to 200°C/ min, but, with further increases in thawing rate, increased and reached peak values (10.3–11.0% FMP) after thawing at 1,800°C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1°C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800°C/min. In semen frozen at the optimal rate of 30°C/min, the increases in thawing rates above 200°C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200°C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration. The peak value 53% NAR was recorded in semen protected by 2% glycerol, frozen at 30°C/min, and thawed at 1,200°C/min. In view of the inverse relationship between FMP and NAR, selection of optimal conditions from among the interacting variables, freezing rate, glycerol concentration, and thawing rate requires compromising between maximal FMP and maximal NAR. Accordingly, we have adopted as optimal a protocol with a thawing rate of 1,200°C/min, a freezing rate of 30°C/min and concentrations of 3% glycerol. © 1993 Wiley-Liss, Inc.     

Cryopreservation of spermatozoa from wild-born Namibian cheetahs (Acinonyx jubatus) and influence of glycerol on cryosurvival

Sperm cryopreservation is a valuable tool for the genetic management of ex situ populations. This study was conducted to assess: (1) semen characteristics of wild-born cheetahs; and (2) the impact of three types of glycerol influence (duration of exposure, temperature, and method of addition) on sperm cryosensitivity. To evaluate the impact of duration of glycerol exposure, spermatozoa were incubated in Test Yolk Buffer (TYB) with 4% glycerol at ambient temperature (approximately 22 degrees C) for 15 vs. 60 min before cryopreservation. To evaluate the influence of temperature and method of glycerol addition, spermatozoa were resuspended at ambient temperature either in TYB with 0% glycerol followed by addition of 8% glycerol (1:1 v/v; at ambient temperature vs. 5 degrees C) or directly in TYB with 4% glycerol. All samples were cryopreserved in straws over liquid nitrogen vapor and evaluated for sperm motility and acrosomal integrity after thawing. Semen samples (n = 23; n = 13 males) contained a high proportion (78%) of pleiomorphic spermatozoa. Ejaculates also contained a high proportion of acrosome-intact (86%) and motile spermatozoa (78%). Immediately after thawing, a significant proportion of spermatozoa retained intact acrosomes (range, 48-67%) and motility (range, 40-49%). After thawing, incubation in glycerol for 60 min at ambient temperature before freezing decreased (p < 0.05) sperm motility and acrosomal integrity at one time-point each (pre-centrifugation and post-centrifugation, respectively). However, method or temperature of glycerol addition had no (p > 0.05) impact on sperm cryosurvival. In summary, (1) wild-born cheetahs produce high proportions of pleiomorphic spermatozoa but with a high proportion of intact acrosomes; and (2) resuspension in 4% glycerol, followed by exposure for up to 60 min at ambient temperature, had minimal effect on sperm motility and acrosomal integrity after cryopreservation. Results indicate the feasibility of cryopreserving cheetah spermatozoa under field conditions, providing a user-friendly method to capture and store gametes to enhance genetic management.     

The effect of cooling rate on the survival of cryopreserved bull,ram, and boar spermatozoa: a comparison of two controlled-rate cooling machines

Spermatozoa from three species, bovine, ovine, and porcine, were frozen using standard techniques in two controlled-rate cooling machines, a commercial instrument and a custom-built device. Ice crystallisation was induced mechanically by touching the straws with a pre-cooled rod. The sperm samples were stored 24h, and then thawed rapidly and evaluated for motility, viability, and acrosomal integrity in the membrane-intact population. The custom-built controlled-rate cooling machine proved significantly better at all cooling rates for all species. This was particularly evident for the ram and the boar spermatozoa. In general, -30 or -50 degrees C/min were better than -1 degrees C/min, with a slight advantage being evident for -30 degrees C/min. However, this became very apparent for boar spermatozoa. It is clear that the higher cooling rates are necessary for successful freezing of spermatozoa from these species, and that careful control of the cooling rate is essential for maximal recovery of viable and functional cells. This is best achieved when the cooling profile is controlled from within a dummy sample.     

Effect of different glycerol treatments on frozen-thawed dog sperm longevity and acrosomal integrity

Four different concentrations of glycerol in a Tris-fructose-citric acid extender for frozen dog semen and the effects of adding glycerol at 37 degrees C or 4 degrees C to the extender were studied by monitoring the post-thaw sperm longevity and acrosomal integrity during incubation at 39 degrees C. In the first part of this study, ejaculates from 13 dogs were pooled and divided into 4 aliquots, which were centrifuged and the sperm pellets rediluted with a Tris-fructose-citric acid extender containing 2, 4, 6 and 8% (v/v) glycerol, respectively. Progressive motility by subjective estimation, live:dead spermatozoa ratio using eosin-nigrosin staining, and acrosomal integrity using phase contrast microscopy were evaluated before processing and at 0, 0.5, 1, 2 and 4 hours post-thawing incubating the semen samples in the dark at 39 degrees C. The experiment was performed using seven replicates and it was found that sperm motility and acrosomal integrity were superior following the use of 8% glycerol in the extender. In Experiment 2, 13 ejaculates from the same dogs used in the first experiment were pooled and divided into 3 aliquots, and an 8% glycerol diluent was added at 37 degrees C and 4 degrees C after 1 h of cooling or at 4 degrees C after 2 h of cooling, respectively. After freezing and thawing the same parameters as studied in the first experiment were assessed. The experiment was performed in 7 replicates, and no difference was found between treatments.     

Effects of pre- and post-thaw thermal insults on viability characteristics of cryopreserved bovine semen

The magnitude of damage to the viability of cryopreserved bovine spermatozoa by pre- and post-thaw thermal insults was compared. Semen collected by artificial vagina from 5 Holstein bulls was diluted in egg yolk-citrate-7% glycerol extender (EYCG) and cryopreserved in 0.5 mL French straws at a sperm concentration of 40 to 60 x 10(6) cells/mL. In Experiment 1, straws were subjected to 22, 5 or -18 degrees C static air temperature for a duration of 1, 2, 3, 4 or 5 min before or after thawing in a 37 degrees C water bath for 1 min. Control straws were thawed in a 37 degrees C water bath for 1 min without further thermal insult. In Experiment 2, straws were thawed for 1 min in a 37 (control), 20 or 5 degrees C water bath, or were loaded into an insemination gun and plunged into a 37 degrees C water bath for 3 min. In both experiments, straws were returned to a 37 degrees C water bath for incubation prior to viability analysis. Viability evaluations, conducted in triplicate, included the percentage of motile spermatozoa at 1 min and at 3 h post thermal insult and the percentage of intact acrosomal membranes at 3 h post thermal insult. In both experiments, acrosomal integrity was more sensitive than motility to thermal insult. In Experiment 1, a significant interaction was observed between timing of thermal insult (pre- or post-thaw), static air temperature and duration of straw exposure. At 22 and 5 degrees C, thermal insults applied before thawing significantly (P<0.05) reduced acrosomal integrity at > or = 2 and > or = 4 min of exposure, respectively. However, post-thaw exposure to 22 and 5 degrees C for up to 5 min had no effect on any of the sperm viability parameters evaluated. In contrast, at -18 degrees C static air temperature, post-thaw exposure for > or = 3 min decreased acrosomal integrity (P<0.05), while 5 min of pre-thaw exposure was required for alteration of acrosomal integrity. In Experiment 2, each alternative thawing method resulted in significantly (P<0.05) lower incubated acrosomal integrity relative to the controls. These findings suggest that bovine spermatozoa cryopreserved in EYCG extender are more sensitive to pre-thaw than post-thaw thermal insults and that acrosomal integrity following 3-h incubation at 37 degrees C is superior to motility evaluations for detection of damage to sperm viability due to thermal insult.     

Cryopreservation of seabream (Sparus aurata) spermatozoa

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.     

New thermal stress test to assess the viability of cryopreserved boar sperm.

A new, rapid, thermal stress test for assessing the viability of boar semen, requiring only 45 min of incubation at 42.5 degrees C, was developed and compared with a widely used stress test of 180 min incubation at 37 degrees C. The shorter procedure was found to have the same discriminatory ability as the standard test in assessing the effects of freezing conditions on the percentage of spermatozoa remaining motile. Neither test was able to show differences in the kinetic rating of motile sperm after freezing in relation to the glycerol concentration present during freezing. However, the new test had a greater ability to distinguish the effects of different concentrations of glycerol, over the range of 0 to 6%, and to reveal different degrees of acrosomal damage sustained during freezing. The longer procedure was unable to distinguish among glycerol concentrations from 0 to 4% with respect to acrosomal damage and produced an overall lower proportion of sperm having a normal apical ridge. The new thermal stress test thus has the advantages of greater sensitivity and more rapid execution over the test hitherto in widespread use.     

Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws

Experiments were conducted to evaluate two extenders (egg-yolk Tris and egg-yolk lactose), varying concentrations of two cryopreservatives (glycerol and dimethyl sulfoxide), and rates for cooling to 5 degrees C, cooling from 5 to -100 degrees C, and warming for canine spermatozoa packaged in 0.5-ml French straws. At optimal concentrations of glycerol, egg-yolk Tris extender was superior to egg-yolk lactose in preserving spermatozoal motility. Addition of dimethyl sulfoxide, alone or in combination with glycerol in either extender, was not beneficial to spermatozoal survival after thawing. Canine spermatozoa withstood a range of cooling and equilibration times with no detrimental effect on spermatozoal motility prior to freezing. However, there were differences in spermatozoal motility immediately after thawing; these differences were variable, resulting in a cooling time by equilibration time interaction. Spermatozoal motility after thawing was best preserved by freezing in egg-yolk Tris extender containing 2-4% glycerol, using a moderate rate of cooling from 5 to -100 degrees C (-5 degrees C/min from 5 to -15 degrees C, then -20 degrees C/min from -15 to -100 degrees C). Three of 12 bitches inseminated intravaginally with semen frozen using this protocol became pregnant.     

The effect of glycerol-related osmotic changes on post-thaw motility and acrosomal integrity of ram spermatozoa

Ram semen, collected by artificial vagina, was diluted and processed for long-term storage as described by P. S. Fiser, L. Ainsworth, and R. W. Fairfull (Canad. J. Anim. Sci. 62, 425-428, 1982). The concentration of the cryoprotectant, glycerol, was adjusted to 4% in the diluted semen prior to freezing by a one-step addition at 30 degrees C (Method 1), by cooling the semen to 5 degrees C and addition of the glycerol gradually over 30 min (Method 2), by one-step addition of glycerol prior to equilibration for 2 hr (Method 3), or by cooling to 5 degrees C, followed by a holding period of 2 hr at 5 degrees C, and the one-step addition of glycerol just prior to freezing (Method 4). After thawing, the glycerol concentration of the semen was reduced by stepwise dilution from 4 to 0.4% over 15 or 30 min or by a one-step ten-fold dilution. The average post-thaw percentage of motile spermatozoa was significantly lower after addition of glycerol by Method 1 (39.9%) than when the glycerol was added by the other three methods (range, 44.0-46.4% averaged over the glycerol dilution). The average post-thaw percentage of intact acrosomes (61.2%), highest in semen in which the glycerol was added by Method 2, was not significantly different from those in which glycerol was added to semen by Methods 3 and 4, but it was significantly higher than that found in semen in which the glycerol was added by Method 1 (54.4%). However, when averaged over the method of glycerolation, the post-thaw percentage of motile spermatozoa (range, 43.7-44.2%) and the percentage of intact acrosomes (range, 56.8-59.5%) did not differ significantly in semen subjected to gradual decrease in glycerol concentration and diluent osmolality (over 15 and 30 min) or by a one-step, 10-fold dilution. These data indicate that post-thaw survival of spermatozoa can be influenced by the way in which glycerol is added prior to freezing. However, post-thaw spermatozoa motility and acrosomal integrity can be maintained even after a rapid decrease in glycerol concentration such as that which accompanies insemination or dilution of semen for assessment of motility.     

Effects of spermatozoal concentration and post-thaw dilution rate on survival after thawing of dog spermatozoa

The objectives of this study were to evaluate the effects and interactions of freezing dog semen using 4 different sperm concentrations (50 x 10(6), 100 x 10(6), 200 x 10(6) and 400 x 10(6) spermatozoa/mL) in 0.5-mL straws and diluting the thawed semen at 4 different rates (1:0, 1:1, 1:2 and 1:4) on post-thaw survival and longevity of dog spermatozoa during incubation at 38 degrees C. Fifteen ejaculates were collected from 12 dogs and pooled. The semen pool was divided into 4 aliquots containing respectively 4,200 x 10(6), 2,100 x 10(6), 1,050 x 10(6) and 525 x 10(6) spermatozoa, which were centrifuged. Sperm pellets were rediluted with TRIS-glucose-egg yolk extender containing 5% glycerol and 0.5% of Equex STM Paste to obtain the designated sperm concentrations. The semen was frozen in 0.5-mL straws 4 cm above liquid nitrogen (LN2). The straws were thawed at 70 degrees C for 8 sec and the contents of each straw were divided into 4 aliquots and diluted with TRIS buffer at 38 degrees C at rates of 1:0, 1:1, 1:2 and 1:4 (semen:buffer), respectively, making a total of 16 treatments. Sperm motility was subjectively evaluated after thawing and at 1-h intervals during 8 h of incubation at 38 degrees C. Plasma membrane integrity and acrosomal status were evaluated at 1, 3, 6, 12 and 18 h post-thaw using a triple-staining procedure and flow cytometry. For data pooled across the post-thaw dilution rate, motility was higher (P< 0.001) in samples frozen with 200 x 10(6) spermatozoa/mu. The integrity of sperm plasma membranes after 18 h incubation was higher (P<0.05) in samples frozen with 200 x 10(6) and 400 x 10(6) spermatozoa/mL. For data pooled across sperm concentration, samples diluted at a rate of 1:2 or 1:4 had better (P<0.001) motilities after 8 h of incubation than undiluted samples or those diluted at 1:1. The integrity of the sperm plasma membranes was higher (P<0.001) at increasing dilution rates. When the 16 treatments were compared, the best longevity was obtained when semen packaged at a concentration of 200 x 10(6) spermatozoa/mL was diluted immediately after thawing at 1:4 dilution rate.     

Morphological change of the acrosome on motile bovine spermatozoa due to storage at 4 degrees C

Swelling of the apical ridge and anterior acrosome of motile bovine spermatozoa was observed during in-vitro storage using differential interference-contrast optics. This morphological alteration is different from that described as the false acrosome reaction on immotile spermatozoa, apparent in ageing semen samples and which has been associated with cell death. In this study, transmission electron microscopy revealed that the apical ridge acrosomal matrix was extended into complex folds and/or projections. Acrosomal and plasma membrane integrity was retained. Storing spermatozoa (1500 X 10(6)/ml) in seminal plasma at 4 degrees C for 1 day was most conducive to the swelling of the apical ridge. Replacing seminal plasma with egg yolk-citrate inhibited swelling. However, incubating semen at 37 degrees C in egg yolk-Tris-fructose extender (25 X 10(6) spermatozoa/ml) after storage in egg yolk-citrate at 4 degrees C for greater than or equal to 3 days restored the swelling characteristic.     

The use of ejaculated boar semen after freezing in 2 or 6% glucerol for in vitro fertilization of porcine oocytes matured in vitro

Two concentrations of glycerol in a freezing diluent were tested with respect to the in vitro fertilizing capacity of frozen-thawed boar spermatozoa which, before exposure to oocytes, were subjected to 3 methods of fractionation. These were 1) the upper fraction, 2) the swim-up and 3) percoll gradinet-centrifugation. The highest proportions of motile spermatozoa were obtained by the swim-up procedure, while acrosomal integrity was best preserved by the upper fraction procedure. Raising the glycerol concentration from 2 to 6% (v/v) during freezing decreased the proportion of spermatozoa with a normal apical ridge. Spermatozoa separated by the upper fraction method showed the greatest penetration of oocytes and produced the highest indidence of polyspermy. The glycerol level affected penetration and polyspermy only with spermatozoa separated in a percoll gradient, where the higher level of glycerol increased oocytes penetration and polyspermy. Pronuclei formation was influenced by the separation procedure and by the glycerol concentration in the freezing diluent. The results indicate that frozen boar semen can be used for in vitro fertilization more successfully than fresh semen since penetration by frozen upper fraction spermatozoa was similar to, the degree of polyspermy was lower, and the formation of two pronuclei was greater (P<0.01) than in oocytes exposed to fresh semen.     

Semen cryopreservation in Bactrian camel (Camelus bactrianus) using SHOTOR diluent: effects of cooling rates and glycerol concentrations

Experiments were conducted with a final goal of providing a suitable protocol for cryopreservation of Bactrian camel semen. In Experiment I, the effect of average cooling rate (slow cooling: 0.14 versus fast cooling: 0.55 degrees C/min) on the viability of chilled semen was evaluated. In Experiment II, the effect of different concentrations of glycerol (4, 6 and 8%) on the post-thaw viability of frozen sperm was investigated. In Experiment III, the efficiency of SHOTOR diluent was compared with IMV buffers for the cryopreservation of camel semen. Viability parameters including progressive forward motility (PFM), plasma membrane integrity and percentage of live spermatozoa were assessed. Progressive forward motility of sperm cooled at the faster rate was superior after incubating for 24h at 4 degrees C compared to that cooled at the slower rate (P<0.05). Post-thaw viability of Bactrian camel sperm was better using a final glycerol concentration of 6% compared to 4 and 8% (P<0.05). Progressive forward motility of frozen-thawed sperm was greater using SHOTOR diluent (29.9%) compared to IMV buffers (4.2%, P<0.05). In conclusion, semen cryopreservation in Bactrian camel is feasible when it is extended in SHOTOR diluent, cooled within 1h (average cooling rate: 0.55 degrees C/min) to 4 degrees C, and then exposed to glycerol, at the final concentration of 6%.     

The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws

The effect of varying the concentration of glycerol from 0 to 16% on the survival of ram spermatozoa frozen at increasing rates of cooling (1–100 °C/min) or by direct plunging of spermatozoa in 0.5-ml straws in liquid nitrogen was studied after thawing at a constant rate (in water at 39 °C for 30 sec). For each glycerol concentration, the ram spermatozoa tolerated a range of cooling velocities and the best survival rates (percentage motility and rating) were obtained when the glycerol concentration was 4 or 6% and when the rate of freezing ranged from 10 to 100 °C/min. No spermatozoa survived in any glycerol concentration following freezing in straws plunged into liquid nitrogen. In general, the range of cooling rates shifts to lower values as the glycerol concentration increases for optimum cryosurvival. However, the toxic effect of increasing the concentration of glycerol over 8% contributes greatly to the gradual decrease in cryosurvival of spermatozoa at these particular concentrations.     

Effects of holding time during cooling and of type of package on plasma membrane integrity, motility and in vitro oocyte penetration ability of frozen-thawed boar spermatozoa

The effect of a prolonged holding time (HT) during cooling on plasma membrane integrity (PMI), motility and in vitro oocyte penetration ability of boar spermatozoa frozen-thawed in different types of package was investigated. Boar semen was frozen in a split-sample design using 3 different HTs (3, 10 and 20 h) during cooling and three different types of freezing package: Maxi-straws, Medium-straws and FlatPacks. Assessment of PMI (SYBR-14 and propidium iodide, fluorescence microscopy) and sperm motility (visually and with CASA) was done during cooling (at 32 degrees C, 15 degrees C, 5 degrees C) and post-thaw (PT). The in vitro oocyte penetration ability of the spermatozoa was tested only PT, using a homologous in vitro penetration assay (hIVP). During cooling the HTs used had no significant (p<0.05) effect on either PMI or percentage of motile spermatozoa Post-thaw PMI was significantly higher (p<0.05) for 10 h and 20 h HT compared with 3 h, and the percentage of motile spermatozoa decreased significantly with 20 h HT as opposed to 3 h and 10 h. Regarding the freezing packages, the FlatPacks and Maxi-straws yielded significantly more PMI than did the Medium-straws (p<0.05). Post-thaw motility was significantly higher for FlatPacks than for straws, in terms of both percentage motile spermatozoa, and sperm velocity and lateral head displacement (LHD). The hIVP did not show any significant differences among the HTs, although FlatPacks yielded a significantly higher penetration rate and more spermatozoa per penetrated oocyte (p<0.05) than did the straws. Changes in motility patterns, toward a more circular motility during cooling and PT, could be noticed where individual spermatozoa showed a capacitation-like motility pattern. The changes were more obvious with 10-h and 20-h HTs than with 3-h HT.