Effects of glutamine on post-thaw motility of stallion spermatozoa: an approach of the mechanism of action at spermatozoa level

The cryoprotective effect of l-glutamine and an approach of its mechanism of action, in preserving motility of stallion spermatozoa during the freezing-thawing process, were studied. In Experiment 1, thirty-six ejaculates were collected from six stallions (two good, two middle, and two of poor sperm freezability) and were diluted with 10 different freezing media derived from INRA 82 medium supplemented with 20 mM HEPES and 2% (v/v) centrifuged egg yolk (BM). After thawing, sperm motility was evaluated by a computer-assisted semen motility analyser. The effects of glutamine and glycerol at different concentrations on post-thaw sperm motility were studied. A possible interaction between medium and semen freezability was investigated. Only the 50 mM glutamine + 2.5% glycerol medium significantly improved sperm motility compared to classical freezing medium (2.5% glycerol). The presence of glutamine at 50 mM was not sufficient to offset the need to use glycerol in the freezing extender. The use of glutamine at a higher concentration >100 mM in the presence of 2.5% of glycerol was toxic. Reducing the glycerol proportion from 2.5% to 2 or 1.5% in the presence of glutamine at 50, 75, and 100 mM had no influence on post-thaw motility of semen of middle and good freezability. Moreover, the substitution of 2.5% glycerol by 50 mM glutamine in BM, did not significantly change the post-thaw motility of semen of good freezability. In Experiment 2, 3H-glutamine and 3H-glycerol were used to study the kinetics of penetration of glutamine and glycerol in sperm cells. The radioactivity of each radio-labelled semen pellet was measured at different times (0, 15, 30, 60, 90, 120 min), by using a Packard tri-carb 4530 apparatus. The percentages of incorporated radioactivity (%IRA) in semen pellets were calculated at different times. The %IRA of 3H-glycerol in semen pellets were significantly higher than the %IRA of 3H-glutamine. The %IRA of 3H-glycerol in semen pellets increased greatly from time 0 to 60 min, and then it is stabilized from 60 to 120 min. In contrast, the %IRA of 3H-glutamine in semen pellets increased slightly from 0 to 60 min, then it stabilized until 120 min. These experiments demonstrate that glutamine has a synergistic cryoprotective effect with glycerol on cryopreservation of stallion spermatozoa, and suggest that glutamine acts at the extra-cellular level, independently of glycerol.     

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.     

Cryosurvival of Trichomonas vaginalis during cryopreservation of human semen

Despite a 90% cryosurvival of Trichomonas vaginalis in their growth medium trypticase yeast maltose (TYM) with DMSO, none of these parasites have previously been observed to survive during cryopreservation of infected human semen with glycerol (Andrologia 18, 323 (1986)). This could have been due to the failure of the culture method used to detect low numbers of survivors. The prospects of possible transmission of T. vaginalis by artificial insemination with cryobanked (-196 degrees C) semen prompted an investigation of the cryosurvival of this parasite in the presence of semen with the cryoprotectant glycerol, using a more sensitive culture method for viability evaluation. Semen and seminal fluid from the same 23 ejaculates, as well as culture medium, were inoculated with small clinical numbers of T. vaginalis and evaluated as to their survival before and after cryopreservation. Results indicated: (i) The highest cryosurvival of T. vaginalis (4.5%) was in cryobanked (glycerolated) semen, (ii) semen, as well as glycerol, shows cryoprotective action, and (iii) glycerol reduced survival of parasites in semen, seminal fluid, and TYM medium during exposure prior to freezing. Clinical information on infectivity of small numbers of T. vaginalis and the data presented here suggests that these organisms could be transmitted by artificial insemination with infected cryobanked human semen.     

Improved methods for freeze preservation of chimpanzee sperm

Freeze preservation of human and nonhuman semen has been used effectively for a number of years; however, the application of freezing to preserve nonhuman primate sperm has been less successful. This study compares five freeze methods and various concentrations of the cryoprotectants glycerol and dimethylsulfoxide (DMSO) for cryopreservation of chimpanzee (Pan troglodytes) sperm. The different methods were compared using quantitative analysis of sperm function and, by an indirect measure of fertilizing capacity, the denuded hamster oocyte penetration assay (SPA). The best results were obtained using an extender comprising Ham's F10 medium containing 15% heat inactivated human cord serum and 15.6% glycerol. Semen extended 1 to 1 by this method, for a final glycerol concentration of 7.8%, and frozen using a computer programmed freezer maintained approximately 65% of its original viability and up to 70% of its initial motility. The extended semen was used to initiate pregnancy in two female chimpanzees.     

Differences in ability of jennies and mares to conceive with cooled and frozen semen containing glycerol or not

A suitable method for the cryopreservation of donkey semen would be very valuable for the ex situ management of genetic diversity in this species. This report uses a variety of observation and trials to evaluate the effect of cryoprotectants in per-cycle pregnancy rates (PC) in equids females (jennies (donkey) and mares (horse)). This was explored by (1) comparing the results of insemination of jennies and mares with cooled or frozen donkey semen, (2) examining the possible toxic effect of the cryoprotectant (CPA) glycerol in these two species and (3) studying alternative solutions. Donkey and horse semen was either used immediately, or cooled according to some steps of the pre-freezing procedure or frozen and thawed. The pre-freezing procedure included semen dilution, centrifugation, resuspension in milk or in INRA82+2% egg yolk+various % CPA (expressed as final concentrations in extended semen (v/v)) and then cooling to 4 degrees C. PC was similar in mares and jennies inseminated with donkey semen cooled to 4 degrees C in milk. However, the PC was significantly higher in mares than in jennies when donkey semen was frozen with 2.2% glycerol (36%, n=50 cycles vs. 11%, n=38 cycles; P<0.01). Increasing the concentrations of glycerol (0, 2.2, 3.5, 4.8%) before cooling stallion semen resulted in a progressive decrease in mare PC (87, 53, 53, 13% (n=15 cycles for each concentration); P<0.0001). The addition of 2.2% glycerol before cooling donkey semen decreased the PC measured in jennies to 0. The replacement of glycerol by 2% dimethylformamide increased the fertility obtained in jennies with cooled donkey semen (PC: 67%, n=12 cycles) but did not increase the fertility obtained with frozen-thawed donkey semen (PC: 11%, n=28 cycles with dimethylformamide vs. 0%, n=16 cycles with glycerol). In conclusion, this study clearly shows that the ability of jennies to conceive after AI with donkey frozen semen is lower than that of mares. Glycerol affects the fertility of donkey and stallion spermatozoa as early as during the pre-freezing procedure. In consequence, the glycerol level must be low in frozen equine semen to provide good fertility. The toxic dose of glycerol for donkey spermatozoa seems to be almost half that for stallion spermatozoa. Whether this greater sensitivity of donkey spermatozoa to glycerol is responsible for the low success of semen cryopreservation in jennies is not so obvious because replacement of glycerol by dimethylformamide was not much more effective in terms of fertility.     

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.     

Nonbeneficial effects of glycerol on the oocyte penetrating capacity of cryopreserved and incubated human spermatozoa

The effect of cryopreservation on human spermatozoa in the presence or absence of glycerol was assessed by using sperm motility, functional integrity of sperm membrane, and denuded hamster oocyte penetration tests. Glycerol treated cryopreserved spermatozoa yielded a significantly higher (P less than 0.01) percentage of motile sperm and percentage of sperm with functionally intact membrane immediately after thawing than the spermatozoa not treated with glycerol but cryopreserved. However, no significant difference was observed between these cryopreserved spermatozoa (either treated or untreated with glycerol) on the percentage of motile sperm and the rate of oocyte penetration when the sperm were washed and incubated for 2 hr in a medium containing no glycerol. Thus, it appears glycerol may not be beneficial, since cryopreservation of spermatozoa either treated or untreated with glycerol essentially yields similar oocyte-penetrating capacity of sperm.     

Effect of glycerol level in Tris-basek extender and equilibration period on quality of frozen goat semen

Twenty ejaculates, 4 from each of 5 native goats, were collected using an artificial vagina, and the effects of glycerol level (4, 6.4 and 9 %) and the equilibration period (1, 3 and 5 h) were studied by split-sample technique. The extender used was Tris egg yolk citric acid fructose glycerol extender. The semen was frozen in 0.5-ml French straws by exposure for 10 min to liquid nitrogen vapor, 5 cm above the liquid nitrogen level. After 14 h of storage in liquid nitrogen, the straws were thawed in water at 37 degrees C for 12 - 15 sec. The percentage of progressively motile sperm (PPM) and the percentage of damaged acrosomes (PDA) were studied after equilibration and after thawing. The mean PPM after thawing was found to be 64.0 +/- 0.90, 66.92 +/- 0.54 and 63.65 +/- 1.07 when semen was frozen with 4, 6.4 and 9 % glycerol and 61.48 +/- 0.81, 65.05 +/- 0.78 and 68.03 +/- 0.87 in 1-, 3- and 5-h equilibrated semen, respectively. The mean PDA after thawing was 7.12 +/- 0.88, 8.23 +/- 0.76 and 10.58 +/- 0.84 when semen was frozen with 4, 6.4 and 9 % glycerol and 7.0 +/- 0.74, 9.0 +/- 0.95 and 9.93 +/- 0.81 in 1-, 3- and 5-h equilibrated semen, respectively. Both PPM and PDA differed significantly (P<0.01) between glycerol levels, between equilibration periods and between stages (after equilibration and after thawing). The PPM also differed significantly due to equilibration period x stage interaction (P<0.01) and glycerol level x stage interaction (P<0.05). The PDA did not differ significantly due to interactions. When the differences between pairs of means were tested by least significant difference, it was found that after equilibration PPM was not significantly affected by either glycerol level or equilibration period, while after thawing, it was significantly higher (P<0.05) for 6.4 % glycerol and 5-h equilibrated semen than for 4 or 9 % glycerol and 1- or 3-h equilibrated semen, respectively. The PDA was lower with 4 % glycerol and 1-h equilibrated semen.     

Quality of frozen-thawed semen in brown bear is not affected by timing of glycerol addition

We have tested several freezing protocols for brown bear semen, modifying the time when glycerol was added (before and after cooling to 5 °C). No differences were found among protocols, indicating a good tolerance of brown bear semen to glycerol. This finding indicates that freezing protocols for brown bear semen could be modified to fit practical solutions which would facilitate preparation of the seminal samples in the field with the addition of glycerol at ambient temperature.     

The uptake of glycerol, alpha-aminoisobutyric acid and 2-deoxy-D-glucose by spermatozoa of a line of chickens selected for fertility of frozen-thawed semen and a control line

The uptake of glycerol, alpha-aminoisobutyric acid and 2-deoxy-D-glucose by fowl spermatozoa was each measured in two trials using seventh generation males of a line selected for duration of fertility of frozen-thawed semen and those of a randomly selected control line. The selected line had significantly (P< 0.01) higher fertility of frozen-thawed and fresh semen than the control line. The association between glycerol level in spermatozoa before freezing and the fertility of frozen-thawed semen was examined. Significantly greater amounts (CPM/10(9) cells) of glycerol, alpha-aminoisobutyric acid and 2-deoxy-D-glucose were taken up by the spermatozoa of the selected line than those of the control line at 15, 30 and 60 min of incubation. Rank correlations between the fertility of frozen-thawed semen and glycerol levels were generally positive but low in magnitudes.     

Freezing rabbit semen by the use of BF5 diluent

Three experiments were carried out to find the optimal concentration of DMSO and glycerol in BF5 diluent for freezing rabbit spermatozoa. Semen was diluted 1:1 with diluent A (BF5 + DMSO) at 25 degrees C and diluted further 1:1 with diluent B (diluent A + glycerol) after cooling down to 5 degrees C. Diluted semen was frozen immediately and stored in liquid nitrogen. Maximum percentages for motility and normal acrosomes were obtained in the presence of 12% DMSO (as expressed in diluent A) and 3% glycerol (final concentration) after thawing.     

Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen.

The influence oftemperature, addition of glycerol, initial freezing temperature, method of dilution, level of glycerol in the diluted semen, equilibration time and type of diluent on the survival and fertilizing capacity of deep-frozen according to the best conditions was compared with that of "fresh" semen. The addition of glycerol at plus30 degrees C resulted in a highly significant decrease in the mean proportion of motile spermatozoa immediately after thawing compared with the effect of addition at plus 4 degrees C. The immersion of the straws at minus55 degrees C significantly reduced the revival of the spermatozoa compared with initial freezing at lower temperatures. The exposure time to glycerol had no significant effect on the survival of spermatozoa after thawing and incubation, but fertility was significantly higher with 4% than with 2% glycerol. The I. N. R. A. diluent provided better sperm survival and a significantly higher conception rate than did lactose-egg yolk extender. The semen frozen according to the best conditions (about 50% of the samples) had a fertilizing ability similar to that of "fresh" semen when the proportion of motile spermatozoa before, and after 1 or 3 hr of incubation was equal to or above 45, 40 and 30% respectively.     

Examination of some processing methods for freezing boar semen.

Five experiments were conducted to examine the effect of processing methods and diluents on survival and morphology of boar spermatozoa after freezing. Post-thawing survival of spermatozoa was better for Beltsville-F3 (BF3) than for tris-fructose-EDTA freezing diluent when the seminal plasma and glycerol were removed prior to freezing (method A). Both freezing diluents yielded similar viability results when the spermatozoa were frozen in the presence of siminal plasma and glycerol (method B). Viability of spermatozoa after thawing was better when glycerol concentration in the prefreezing diluent (method A) or in the freezing medium (method B) was 2-5 and 5-0 rather than 7-5%. Cooling of diluted semen to 5 degrees C beyond 4 h decreased the post-thawing survival of spermatozoa. The proportion of spermatozoa with undamaged acrosomes after processing and thawing by different methods was indistinguishable and relatively low. When the semen was frozen at cell concentrations ranging from 0-25 to 2-0 X 10(9)/ml, the viability of spermatozoa declined with increasing concentration following freezing in BF3, and S-1 diluents. Viability results were very similar for all cell concentrations examined when tris-fructose-EDTA diluent was used, indicating the possibility of freezing boar semen in a concentrated state.     

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws

The interaction of glycerol concentrations of 0-10% and cooling rates from 1 to 1,500 degrees C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30 degrees C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0-1% glycerol after cooling at 30 degrees C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30 degrees C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1 degrees C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500 degrees C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30 degrees C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.     

Freezing dog semen in presence of the antioxidant butylated hydroxytoluene improves postthaw sperm membrane integrity

In an attempt to evaluate the protective effect of a lipid-soluble antioxidant (butylated hydroxytoluene; BHT), semen from four dogs (Canis familiaris) was frozen in two different extenders (Uppsala or INRA-96 plus glycerol) with or without 1 mM BHT. Sperm membrane integrity using flow cytometry and motility using a computerized system were evaluated in each experimental group. The Uppsala extender was superior in all aspects of sperm function. The percentage of sperm membranes was significantly higher in semen samples frozen in presence of BHT. Our results suggest that the Uppsala extender can be improved with the addition of BHT.     

Post-thaw plasma membrane integrity of bull spermatozoa separated with a Sephadex ion-exchange column

Previous experiments have established that filtration of bovine semen through a Sephadex ion-exchange column improves its quality before and after freezing. The present study was conducted to determine the post-thaw membrane integrity of bull spermatozoa separated with a Sephadex ion-exchange column and to determine the kind of protection to spermatozoa is provided by glycerol during freezing and thawing. Semen from Holstein bulls diluted in TEST-yolk extender (with and without glycerol) was filtered through a Sephadex ion-exchange column and frozen in liquid nitrogen (-196 degrees C). After thawing, there were more normal acrosomes in filtered spermatozoa than nonfiltered (P < 0.01). Post-thaw plasma membrane integrity and swelling ability in a hypoosmotic solution revealed that the filtered spermatozoa had a stronger (P < 0.005) plasma membranes than the nonfiltered. Filtered spermatozoa demonstrated higher zona-free hamster oocyte penetration than the nonfiltered (30.5 vs 11.5%; P < 0.0005). Spermatozoa extended in TEST-yolk without glycerol had the lowest (P < 0.001) normal acrosomes, intact plasma membranes and swelling ability. Plasma membrane over the post-acrosomal region of the head and post-midpiece region of the tail was more sensitive to damages caused by freezing and thawing than acrosomal and midpiece regions of spermatozoa. Glycerol in the extender provided significant (P < 0.05) protection to the sensitive regions of filtered and nonfiltered spermatozoa during freezing and thawing. Filtered plus glycerolated spermatozoa had the highest (P < 0.01) normal acrosomes, intact plasma membranes and swelling ability. In conclusion, the pre-freezing filtration of bovine semen harvested the spermatozoa possessing stronger plasma membranes which enabled them to endure freezing and thawing stresses. The addition of glycerol to the extender protected the post-acrosomal region of the head and post-midpiece region of the tail of spermatozoa from freezing and thawing shocks.     

Cryopreservation of canine semen using a coconut water extender with egg yolk and three different glycerol concentrations

Semen was collected by digital manipulation from six adult dogs. The second fraction of the ejaculate was used in this study. The semen was assessed by macroscopic and microscopic criteria 1 min after collection, first dilution, cooling, glycerol addition and thawing. Experiments were conducted to compare the effect of three different concentrations of glycerol in coconut water extender. The freezing method employed was that one described for caprine semen with slight changes. Semen was thawed at 37 degrees C for 1 min. Spermatozoal motility after thawing was 49.2+/-26, 44.2+/-18.3 and 35.8+/-26.8% for groups with 4, 6 and 8% glycerol, respectively. The vigor after thawing was 2.6+/-1.1, 2.7+/-1.0 and 2.1+/-1.2 for these groups, respectively. There was no difference among groups in motility and vigor. However, a smaller percentage of total and secondary abnormalities was observed using 6% glycerol in coconut water extender. In conclusion, the three glycerol concentrations (4, 6 or 8%) can be used successfully in cryopreservation of canine semen using a coconut water extender.     

Artificial insemination of sheep: Effects on fertility of number of spermatozoa inseminated and of storage of diluted semen for up to 18 hours at 5°C

Ram semen was prepared in a buffered glucose-saline solution containing 3% (v/v) egg yolk so that insemination doses of 25 or 100 million spermatozoa in volumes of 50 or 250 μl could be given per ewe at artificial insemination (AI). Fertility was significantly reduced by dilution and, within the treatments of diluted semen, significantly higher lambing rates followed the use of doses of 100 million spermatozoa. The volume of the AI dose had no significant effect on fertility.Of 945 inseminations performed using diluted semen, 388 were with samples that had been cooled to 5°C and stored chilled for 5 or 18 hr. The mean lambing result of 40% for freshly diluted semen was significantly higher than 31.6% and 30.2% for samples stored chilled for 5 and 18 hr respectively. Ewes inseminated with doses of chilled semen containing 25 million spermatozoa had a low lambing rate of 21.3%. The presence of 7.5% glycerol (v/v) in the diluent did not significantly affect the fertility of chilled semen.     

Effect of some permeating cryoprotectants on CASA motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility because they provide objective results for thousands of mammalian spermatozoa. Mammalian spermatozoa experience osmotic stress when the glycerol is added to the cells prior to freezing and removal from the cells after thawing. In order to minimize osmotic damage, cryoprotectants having lower molecular weights and greater membrane permeability than glycerol, were evaluated to determine their effectiveness for cryopreserving bull spermatozoa. The aim of this study was to compare the cryopreservation effects of low molecular weight cryoprotectants (ethylene glycol and methanol) to glycerol, on post-thaw CASA sperm parameters. Bull semen was diluted with tris-egg yolk extender containing 3% glycerol, 3, 2 and 1% ethylene glycol or 3, 2 and 1% methanol. Bull semen was frozen in 0.5 straws. Bull spermatozoa exhibited higher percentages (p<0.01) for total (Mot, 72.4%) and progressively (Prog, 29.5%) motilities when frozen in extender containing 3% glycerol compared to 3, 2 and 1% ethylene glycol or 3, 2 and 1% methanol. In conclusion, no advantages were found in using ethylene glycol or methanol to replace glycerol in bull semen freezing. Glycerol provided the best sperm characteristics for bull spermatozoa after freezing and thawing. The possibility of using ethylene glycol or methanol as permeating cryoprotectants for bull semen deserves further investigation, and these cryoprotectants should also be evaluated in extenders that contain disaccharides or cholesterol.     

Effect of differential addition of glycerol and pyruvate to extender on cryopreservation of Mediterranean buffalo (B.bubalis) spermatozoa

The composition of the extender in which semen is diluted before freezing plays a major role in successful cryopreservation of spermatozoa. Substances of high osmolarity, like glycerol, protect sperm cells during the freezing process and energy-rich compounds, like pyruvate provide extra energy during capacitation and fertilization. Since cryopreservation procedures for Buffalo spermatozoa have not been adequately defined, the aim of the study was to improve the survival rate of buffalo (Bubalus bubalis) spermatozoa after cryopreservation by optimizing the timing for adding glycerol and by enriching the cryoprotectant extender with an energy source substrate. Semen was collected with an artificial vagina from 5 bulls and the ejaculates were immediately evaluated for motility, forward progressive motility and for viability, pooled and held at room temperature (28 degrees C) for 1 h. Then aliquots of pooled semen were subjected to dilution and equilibration in triplicate as follows: Experiment 1. Glycerol (3%) in a commercial extender was added to the semen at 28 degrees C and cooled to 5 degrees C for 1 h; then extender with 11% glycerol was added before further equilibration (initial glycerol addition; IGA) and the samples held at 5 degrees C for 1, 3 or 5 additional hours (IGA 1, n = 24; IGA 3, n = 24; IGA 5, n = 24) before freezing. Experiment 2. Glycerol (3%) was added and the mixture brought to 5 degrees C as described above. Then extender with 11% glycerol was added (late glycerol addition; LGA) and after equilibration for 1, 3 and 5 h (LGA 1, n= 24; LGA 3, n = 24; LGA 5, n = 24) the samples were frozen. In Experiments 3 and 4 Na pyruvate (1.25 mM) was added to the extender as described for IGA and LGA above (IPA and LPA samples). The effect of addition time (initial vs late) of glycerol and pyruvate was evaluated by measuring sperm motility, progressively forward motility and viability. After freezing-thawing the percentage of motile spermatozoa was significantly higher (0.001相似文献