Effect of lactose and glycerol on the motility, normal apical ridge, chromatin condensation and chromatin stability of frozen boar spermatozoa

The effect of lactose and glycerol concentration, as well as the equilibration time with glycerol was studied on motility, normal apical ridge (NAR), and chromatin state of boar spermatozoa after the freezing and thawing process. In the first experiment, samples were frozen in first and second extenders containing different concentrations of lactose (11, 12 and 14%). In the second experiment samples were frozen using second extenders with different concentrations of glycerol (4, 6, 8 and 10%) and were incubated at 5 degrees C for 0 and 30 min. Motility, motility after caffeine treatment, NAR, chromatin condensation and stability (susceptibility to de-condense after heparin treatment) were evaluated. The results indicated that freezing spermatozoa in extenders with increasing concentrations of lactose adversely affected motility but provided a protective effect on acrosomes. Increased lactose concentration induced higher chromatin condensation but maintained the same stability. Increasing the glycerol concentration in the second extender from 4-6 to 8% led to higher motility and NAR as well as lower chromatin condensation and stability. When 30 min equilibration time was allowed after dilution with the same extenders, spermatozoa showed higher NAR and lower chromatin condensation and stability. The longer equilibration time was detrimental for motility when freezing in the 8% glycerol extender but favourable when using the 4% glycerol extender. Compared to the 8% glycerol, spermatozoa frozen in the 10% glycerol extender showed similar motility and increased chromatin condensation and stability, as well as low values of NAR that did not improve by longer incubation time.     

Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa

Four experiments were conducted to investigate the effects of egg yolk during the freezing step of cryopreservation (namely, the process except for the cooling step), on the viability of goat spermatozoa. The effects of egg yolk on sperm motility and acrosome integrity during the freezing step were investigated in Experiment 1. Spermatozoa diluted with Tris-citric acid-glucose (TCG) solution containing 20% (v/v) egg yolk were cooled to 5 degrees C, washed, and then frozen in TCG with egg yolk (TCG-Y), TCG without egg yolk (TGG-NY), 0.370 M trehalose with egg yolk (TH-Y), or trehalose without egg yolk (TH-NY). All extenders contained glycerol. In frozen-thawed spermatozoa, the inclusion of egg yolk in the freezing extenders increased (P<0.05) percentages of motile sperm, progressively motile sperm, and the recovery rate (ratio of post-thaw to pre-freeze values), but decreased (P<0.05) acrosomal integrity. Moreover, extenders with trehalose had better (P<0.05) post-thaw sperm viability. In Experiment 2, the effects of egg yolk on acrosome status before and after freezing were studied. Egg yolk significantly decreased the proportion of intact acrosomes before freezing, leading to fewer (P<0.05) intact acrosomes post-thaw and lower (P<0.05) recovery rates for intact acrosomes. In Experiment 3, including sodium dodecyl sulfate (SDS) in a diluent containing egg yolk tended to preserve the acrosome compared with the egg yolk containing diluent free of SDS, however, spermatozoa had a lower (P<0.05) proportion of intact acrosomes than those in a yolk-free diluent. However, after cooling, spermatozoa were diluted with a glycerolated extender containing egg yolk. Therefore, the objective of Experiment 4 was to explore whether the egg yolk or glycerol was responsible for the reduced intact acrosome percentage. In this experiment, after cooling and washing the spermatozoa were diluted in TCG with glycerol and/or egg yolk. The combination of glycerol and egg yolk in the extender reduced (P<0.05) the proportion of intact acrosomes compared with egg yolk or glycerol alone. In conclusion, the inclusion of egg yolk significantly improved sperm motility, indicating its beneficial effects during the freezing step of cryopreservation; trehalose appeared to synergistically increase its cryoprotective effects. Furthermore, although neither glycerol nor egg yolk per se affected the proportion of intact acrosomes, the combination of the two significantly reduced the proportion of acrosome-intact spermatozoa.     

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.     

Lactose-egg yolk diluent supplemented with N-acetyl-D-glucosamine affect acrosome morphology and motility of frozen-thawed boar sperm

These experiments were carried out to investigate the effect of N-acetyl-D-glucosamine, and to obtain additional information about the effect of orvus es paste (OEP) and egg yolk concentration in the freezing of boar sperm in the maxi-straw. The highest post-thaw acrosomes of normal apical ridge (NAR) and motility were obtained with 0.025 or 0.05% N-acetyl-D-glucosamine concentration in the first diluent. However, there were no effects of N-acetyl-D-glucosamine among the diluents with or without N-acetyl-D-glucosamine at the second dilution. The N-acetyl-D-glucosamine in the first and second diluents was added at room temperatures (20-23 degrees C) and 5 degrees C, respectively. It is suggested that the temperature of N-acetyl-D-glucosamine addition is important for the effect of boar sperm protection during freezing and thawing. When the 0.05% N-acetyl-D-glucosamine was supplemented in the first diluent, the optimum final OEP content was 0.5%. The optimum content of egg yolk in the diluent with 0.05% N-acetyl-D-glucosamine concentration was 20% and egg yolk was one of the main cryoprotective agents. In conclusion, we found out that the diluent with 0.025 or 0.05% soluble N-acetyl-D-glucosamine in the first diluent, 0.5% final orvus es paste concentration and 20% egg yolk concentration significantly enhanced NAR acrosomes and motility of boar sperm after freezing and thawing.     

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. 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 the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing–thawing caused a significant decrease (P < 0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P < 0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P < 0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.     

Extenders for preservation of canine and equine spermatozoa at 5 degrees C

Two experiments were conducted to evaluate the effects of six extenders and three glycerol levels on the motility of sperm stored at 5 degrees C. Using a split-ejaculated design, semen from 10 dogs and 12 stallions was extended with egg-yolk-tris (EYT), egg-yolk-bicarbonate (EGB), Beltsville F-3 (BF-3), Cornell University (CUE), caprogen (CAP) and heated skim milk (SM) extenders. After cooling to 5 degrees C, additional extender containing 0% to 12% glycerol was added to provide a final concentration of 0%, 3% or 6% glycerol. Regardless of glycerol level, a higher (P<0.05) percentage of canine sperm retained their potential for progressive motility in CAP extender than in EYT, SM, CUE, EGB or BF-3 extenders. The SM extender was the best (P<0.05) for maintaining motility of equine sperm. The inclusion of 6% glycerol depressed (P<0.05) motility of canine sperm, but there was no effect (P>0.05) of glycerol concentration on the percentage of motile equine sperm. For both species, the interaction of glycerol level and extender was nonsignificant. CAP may be useful for storage of canine sperm at 5 degrees C and SM may be satisfactory for storage of equine sperm.     

Evaluation of l-glutamine for cryopreservation of boar spermatozoa

The aim of the present study was to evaluate the protective effect of l-glutamine (l-Gln) against cryopreservation injuries on boar sperm. In Experiment 1, l-Gln from 20 to 80 mM was evaluated as a supplement for a standard freezing extender (egg yolk – EY – 20%, and glycerol 3%). No significant improvement (P > 0.05) was obtained for any post-thaw sperm parameter assessed (objective sperm motility – CASA system – and flow cytometric analysis of plasma and acrosomal membrane integrity −SYBR14/PI/PE-PNA− and plasma membrane stability −M540/YoPro1−). In Experiment 2, l-Gln was evaluated as a partial glycerol substitute in the freezing extender. Significant (P < 0.05) enhancement of post-thaw sperm motion parameters was achieved in sperm frozen in the presence of 2% glycerol and 80 mM l-Gln compared to control (3% glycerol). In Experiment 3, l-Gln was evaluated as an EY substitute in the freezing extender, and no functional sperm were recovered after thawing sperm frozen in the presence of l-Gln and the absence of EY. In conclusion, l-Gln has the ability to cryoprotect boar sperm when it is used as a partial glycerol substitute in the freezing extender.     

Effect of egg yolk lipids on the freezing of goat semen

Jamunapari goat buck semen contained both phospholipase and lysophospholipase activities which remained active during dilution (Step I) with diluents containing egg yolk, cooling to 5 degrees C (Step II), glycerolization and equilibration (Step III) and freezing and thawing (Step IV). A quantitative estimate of the phosphatidyl choline and phosphatidyl ethanolamine before and after freezing revealed that the lipids in egg yolk added to dilute goat semen were not hydrolysed to lysophospholipids and free fatty acids. Seminal plasma was, therefore, not removed and goat semen was frozen in egg yolk citrate-glucose, egg yolk-tris and skim milk-egg yolk. Dilution of goat semen 20 times with the three extenders containing 7% glycerol and an equilibration time of 3 h yielded optimum results. A comparative evaluation of freezing in the three diluents based on the assessment of sperm motility, live sperm count and acrosomal damage showed egg yolk-tris to be best extender for the successful freezing of goat semen. Insemination trials conducted with frozen semen and the number of actual kiddings yielded a fertility rate of approximately 81% in our study.     

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   

Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at ?196°C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.     

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

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

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) spermatozoa obtained by electroejaculation

We tested extenders and freezing protocols for Iberian red deer semen. Samples were obtained by electroejaculation (10 stags), and analyzed for motility (CASA), viability (propidium ioide), acrosomal (PNA-FITC) and mitochondrial status (JC-1). Samples were diluted 1+1 in extender, cooled and adjusted for glycerol (extender with higher glycerol concentration), brought to 160×10⁶ mL⁻¹ and frozen. Four experiments were carried out, repeating sperm analysis after thawing to compare treatments. In a first experiment, seven samples were frozen using Triladyl^® (20% egg yolk) and UL extender (Tes-Tris-fructose, 15% egg yolk, 4% glycerol). Triladyl^® yielded higher motility after thawing. In a second trial, 17 samples were frozen using Triladyl^®, Andromed^®, Bioxcell^®, and UL with 8% LDL (low-density lipoproteins). Triladyl^® and Andromed^® performed better than Bioxcell^® on motility, and than UL-LDL on viability and acrosomal status. In a third experiment, the performance of freezing the sperm-rich ejaculate fraction versus the whole ejaculate was tested on nine samples. The sperm-rich ejaculate fraction not only rendered more motile and viable spermatozoa but also showed higher freezability (higher motile spermatozoa recovery). In a fourth experiment, we tried three modifications of the freezing protocol, for improving the freezability of low concentration samples: prior removal of seminal plasma; replacing extender (second fraction) for pure glycerol to reduce dilution; and performing only the 1+1 dilution, not the second dilution. No differences were found, although only three samples could be used. Both Triladyl^® and Andromed^® were deemed appropriate for freezing Iberian red deer semen, and the rich fraction should be selected for freezing.     

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.     

Duration of thawing on post thaw acrosome morphology and motility of boar spermatozoa frozen in 5-ml maxi-straws

Ejaculates from six boars were frozen in 5-ml maxi-straws and stored in liquid nitrogen. Maxi-straws were thawed by submersion in a 52 degrees C circulating waterbath for 28, 34, 40, 46, 52, or 58 sec. The post thaw percentages of motile sperm and acrosomes with a normal apical ridge (NAR) at the initial microscopic examination were significantly higher (P < 0.05) for maxi-straws submerged for 40 sec than for any other duration of submersion. The mean core temperature of maxi-straws after 40 sec of submersion was 4.8 degrees C. Based on regression equations for post thaw motility and NAR acrosomes, a 37 sec thawing time would be expected to provide the maximal post thaw survival for boar sperm frozen in maxi-straws.     

A two-step dilution tris-egg yolk extender containing Equex STM significantly improves sperm cryopreservation in the African wild dog (Lycaon pictus)

Conservation management of endangered African wild dogs (AWD; Lycaon pictus) can benefit greatly from development of sperm freezing and artificial insemination. Previous freezing attempts yielded nearly 0% motile sperm within 2 h of thawing. In this study, two canine freezing protocols were tested: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution. Semen was collected by electroejaculation from n = 24 AWDs, of which eight ejaculates of sufficient quality (four good quality with initial sperm motility of 75.0 ± 4.4% and four poor quality; showing rapid decrease in sperm motility to 3.3 ± 3.3% prior to freezing) were frozen. For good quality samples, motility and sperm motility index persisted for up to 8 h for Protocol 2, and was higher between 2 and 6 h after thawing with a decrease from 4 h of incubation. Motility dropped to nearly 0% after 2 h incubation for Protocol 1. Viability was higher for Protocol 2 throughout the 8 h of incubation, with a decrease after 6 h, compared to 4 h for Protocol 1. Acrosome integrity was higher for Protocol 2 throughout post-thaw incubation, with a decrease after 2 h for both protocols. Protocols did not differ in normal sperm morphology or DNA integrity. Poor quality samples yielded similar results, except for acrosome integrity, which declined for Protocol 2. In conclusion, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it suitable for sperm banking and artificial insemination initiatives.     

Glutaraldehyde fixation of boar spermatozoa for acrosome evaluation

The acrosome morphology, as assessed by phase-contrast microscopy, was not altered by fixing boar spermatozoa with 0.1 to 2% glutaraldehyde in comparison to spermatozoa inhibited with NaF. The acrosome morphology of glutaraldehyde-fixed samples remained unchanged for 14 days.Boar semen diluted and stored in Beltsville L1 (BL1), egg-yolk-glucose-bicarbonate (EGB), and Beltsville F5 (BF5) could be fixed with glutaraldehyde for acrosome evaluation if the supernatant solution containing egg yolk was removed immediately after glutaraldehyde addition. Spermatozoa extended in Beltsville F3 (BF3) extender could not be microscopically evaluated after glutaraldehyde fixation because of casein precipitation. Acrosome morphology of glutaraldehyde-fixed boar spermatozoa remained unchanged for at least 19 days holding at room temperature or shipping by parcel post.     

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 centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility of equine sperm

Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm motility between semen cooled slowly in extender with or without glycerol to 5 degrees C prior to freezing to -120 degrees C and semen cooled continuously from 20 degrees C to -120 degrees C. From these experiments, a new procedure for processing, freezing and thawing semen evolved. The new procedure involved dilution of semen to 50 x 10(6) sperm/ml in centrifugation medium and centrifugation at 400 x g for 15 min, resuspension of sperm in lactose-EDTA-egg yolk extender containing 4% glycerol, packaging in 0.5-ml polyvinyl chloride straws, freezing at 10 degrees C/min from 20 degrees C to -15 degrees C and 25 degrees C/min from -15 degrees C to -120 degrees C, storage at -196 degrees C, and thawing at 75 degrees C for 7 sec. Post-thaw motility of sperm averaged 34% for the new method as compared to 22% for the old method (P<0.01).     

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.     

Dialysis of boar semen prior to freezing-thawing: its effects on post-thaw sperm characteristics

Low-molecular weight components of the seminal plasma have a detrimental effect on sperm function. The present study was undertaken to evaluate the effect of the removal of low-molecular weight components by dialysis on sperm characteristics prior to and after freezing. Semen, collected from 5 boars, was extended in Kortowo-3 extender (K-3, Poland) and cooled for 3h (control non-dialysis) or dialyzed for 5h in semi-permeable dialysis bags of 12-14kDa molecular weight cut-off prior to freezing. The semen samples were diluted in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or lactose-lyophilized lipoprotein fractions-glycerol extender (lactose-LPFo-G), packaged into aluminum tubes and frozen in a controlled programmable freezer. Pre-frozen and frozen-thawed spermatozoa were evaluated for motility, plasma membrane (SYBR-14 and propidium iodide) and acrosome integrity, mitochondrial function (Rhodamine 123) and ATP content. The results of the study showed that dialysis significantly improved the sperm characteristics prior to freezing. Dialysis enhanced (P<0.05) post-thaw sperm motility, plasma membrane integrity and mitochondrial function, but had no significant effect (P>0.05) on recovery of spermatozoa with intact acrosomes. Furthermore, dialyzed spermatozoa exhibited higher (P<0.05) ATP content compared with the control after freezing-thawing. Consistent inter-boar variability was detected mainly in dialyzed semen following freezing-thawing. These results indicated that the improvement in sperm quality characteristics prior to freezing and the post-thaw sperm recovery were due to the removal of low-molecular weight components from the seminal plasma. It can be suggested that dialysis is effective in improving the post-thaw quality of boar spermatozoa and has also great practical importance in improving the protocols for cryopreservation of semen. Dialysis may also contribute to a better understanding of different mechanisms underlying cryo-induced damage to boar spermatozoa.