Influence of Thawing Diluents on Vitality,Acrosome Morphology,Ultrastructure and Enzyme Release of Deep Frozen Roar Spermatozoa*

The present investigation was performed to study the effect of freezing and thawing on boar spermatozoa. Thirty-one ejaculates from four boars were investigated after thawing in three different thawing diluents (seminal plasma, OLEP, isotonic glucose solution). From each ejaculate one sample of 1 × 109 spermatozoa was thawed in each of the thawing diluents. Each sample was examined in a thermoresistance test in which motility was stimulated with caffeine 30 min. and 3 hrs. after thawing. Furthermore, acrosome morphology and ASAT release from the spermatozoa were investigated for each sample. One ejaculate from the two most frequently used boars was examined by electron microscopy after thawing in each of the thawing diluents. Differences in the aspects studied appeared between isotonic glucose solution and the other two thawing diluents in the thermoresistance test, in the response to caffeine stimulation 3 hrs. after thawing and in the amount of ASAT released from the spermatozoa. The influence on the acrosome morphology varied between the thawing diluents, but the acrosomal alterations did not seem to be connected with the damage reflected by the thermoresistance test and by the measurement of extracellular ASAT activity. The ultrastructural investigation showed that all spermatozoa examined had some degree of ultrastructural alteration as compared with freshly ejaculated boar spermatozoa treated in the same way. This alteration could not be related to any of the thawing diluents. Of the various laboratory tests the thermoresistance test and the measurement of ASAT release are suggested to be sensitive indicators of sperm damage during freezing and thawing. These tests might be useful indicators of variations in sensitivity of spermatozoa to the freezing-thawing procedure.     

Effect of semen collection method (artificial vagina vs. electroejaculation), extender and centrifugation on post-thaw sperm quality of Blanca-Celtibérica buck ejaculates

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl(?), Andromed(?) and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1-/PI- (intact spermatozoa), and Mitotracker+/YO-PRO-1- (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl(?) and Andromed(?) resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl(?) or Andromed(?) are used as freezing extenders.     

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm® gradient between freeze–thaw cycles

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze–thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing–thawing cycles on sperm quality and to analyze three different elapsed times between freezing–thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing–thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen–thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.     

Cryopreservation effects on domestic cat epididymal versus electroejaculated spermatozoa

Frozen-thawed epididymal spermatozoa have already been successfully used in artificial insemination in the domestic cat, proving to be a valuable resource for the reproduction of felid species, which are threatened with extinction. The aim of this study was to compare the effects of freezing and thawing on domestic cat semen collected by electroejaculation (EL) and from the epididymides (EP) and vasa deferentia. Ten adult cats were anesthetized, electroejaculated and immediately thereafter, orchiectomized. Epididymal spermatozoa were collected through the compression of caudae epididymidis and vasa deferentia. Spermatozoa were frozen-thawed following a single protocol. Sperm motility, sperm progressive status (0-5), plasma membrane integrity and morphology (light and transmission electron microscope) were assessed on two occasions, immediately after collection and after freezing and thawing. There were no significant differences between the electroejaculated and epididymal fresh or frozen-thawed spermatozoa for any of the variables. However, the incidence of acrosome defects after freezing and thawing increased by 19% based on light microscopy, whereas ultrastructural images revealed acrosome damages in most sperm cells. Since these acrosomal changes are known to affect sperm fertilising capacity, further studies are needed to optimize cryopreservation techniques for epididymal as well as electroejaculated domestic cat spermatozoa.     

Membrane status of boar spermatozoa after cooling or cryopreservation

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.     

Progesterone treatment of boar spermatozoa improves male pronuclear formation after intracytoplasmic sperm injection into porcine oocytes

Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.     

Decrease in glutathione (GSH) content in bovine sperm after cryopreservation: comparison between two extenders

Growing evidence suggests that among the causes which deteriorate qualitative and functional characteristics of semen after freezing and thawing, there are those linked to oxidative stress and decrease detoxification of antioxidants present in the cells and seminal plasma. Overall, cryopreservation reduces the spermatozoal GSH content. In the present study we assessed the motility, capacitation status, ability to undergo acrosome reaction and levels of GSH in bovine spermatozoa after freezing and thawing, and compared the effects of the egg yolk tris-citrate and a commercial extenders (Bioxcell). Six semen samples were collected from each of five bulls and frozen and thawed in the two extenders. The level of total GSH of bovine spermatozoa detected in the raw semen ranged from 246.5 to 776.2 pmoles/mg of protein. The Bioxcell extender was superior in preserving GSH content (647.2+/-300.1 versus 223.6+/-154.0 pmoles/mL; P<0.05), motility levels (44.3+/-4.8 versus 41.8+/-4.0%; P<0.05), non-capacitated state (68.3+/-13.6 versus 61.3+/-17%; P<0.05) and a lower proportion of acrosome reacted spermatozoa post thaw (3.8+/-2.1 versus 6.3+/-3.1%; P<0.001) compared to egg yolk tris-citrate extender. These findings could be partially ascribed to the high GSH level contained in the commercial extender which seem able to alleviate oxidative damages to spermatozoa surviving freezing thawing procedures. Further researches are required in order to validate in vivo these results.     

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm® gradient between freeze–thaw cycles

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze–thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing–thawing cycles on sperm quality and to analyze three different elapsed times between freezing–thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing–thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen–thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.     

Effects of Equex STM Paste on the quality of frozen-thawed epididymal dog spermatozoa

This study was carried out to investigate the cryoprotective efficacy of Equex STM Paste on the quality of canine post-thaw epididymal spermatozoa. Following castration, spermatozoa were flushed from the cauda epididymides. Epididymal spermatozoa from 13 of 16 dogs with a sperm motility of >70% were frozen in an egg yolk-Tris extender, supplemented with Equex STM Paste (0.5%, v/v); the extender free of Equex STM Paste served as a control cryoprotective diluent. The quality of spermatozoa, judged by its motility, plasma membrane integrity and acrosome integrity, was evaluated on four occasions, immediately after collection, after equilibration and at 0 and 2h post-thaw. Reducing the temperature to 4 degrees C for 2h prior to freezing decreased sperm motility (P=0.001), but had no effects on membrane integrity or acrosome integrity. Immediately after thawing, the percentage of acrosome-intact spermatozoa significantly decreased in samples frozen without Equex STM Paste compared to freshly collected or Equex-treated samples. After incubation at 37 degrees C for 2h post-thaw, a greater percentage of motile spermatozoa (P=0.018) and spermatozoa with intact acrosomes (P=0.001) were observed in Equex-treated samples compared with the control. The percentage of membrane-intact spermatozoa did not differ significantly between Equex-treated and control samples at any time. Supplementation with Equex STM Paste in the semen extender was effective for freezing canine epididymal spermatozoa because it protected acrosome integrity against damage induced by cryopreservation and it prolonged post-thaw sperm motility during in vitro incubation at 37 degrees C.     

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

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

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

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

Effect of prostatic fluid on the quality of fresh and frozen-thawed canine epididymal spermatozoa

Canine epididymal spermatozoa have a low freeze-tolerance ability compared with ejaculated spermatozoa, which could arise from the absence of prostatic fluid (PF). Therefore, the purpose of this work was to elucidate the influence of PF on the quality of canine epididymal sperm before and after freezing. Caudae epididymides were retrieved from eight dogs after routine castration. Spermatozoa were released by slicing the tissue and were extended in either Tris solution or PF before freezing. Frozen sperm samples were thawed at 70 °C for 8 seconds in a waterbath. Sperm concentration, motility using computer-assisted sperm analysis, morphology, plasma membrane, acrosome and chromatin integrity were assessed in the fresh sperm samples (after 20 minutes incubation) and at 0 and 4 hours after thawing. Progressive motility, distance straight line, distance average path, average path velocity, curvilinear velocity, straight line velocity, straightness, linearity, wobble, and beat cross frequency were significantly increased after extraction into PF. There was a higher proportion of spermatozoa with DNA damage in the PF treatment group at 4 hours after thawing than in the Tris treatment group (15.8% vs. 6.7%, P < 0.05). These results suggest that the addition of PF to canine spermatozoa activates sperm motility in fresh spermatozoa but has a negative effect on chromatin integrity after freezing–thawing.     

Cryopreservation of Atlantic croaker spermatozoa: evaluation of morphological changes.

The spermatozoon of the Atlantic croaker (Micropogonias undulatus) is a primitive type in that it lacks an acrosome. The kidney-shaped head has a diameter of about 1.5 microns and is occupied by a granular and electron-dense nucleus. The short midpiece contains 3 spherical mitochondria and encircles the basal body of the flagellum but is separated from it. The flagellum consists of the typical 9 + 2 axoneme and surrounding plasma membrane but lacks a lateral ridge. Spermatozoa of Atlantic croaker diluted in either NaCl or sodium citrate solutions with or without DMSO were examined with the electron microscope before freezing in liquid nitrogen and after thawing. Damage following cryopreservation appeared to be greater to the mitochondria, plasma membrane, and 9 + 2 axoneme than to the nucleus. The incidence of postthaw damage in spermatozoa diluted with NaCl solutions containing DMSO was remarkably lower than that with either pure NaCl solutions, pure sodium citrate solutions, or sodium citrate solutions containing DMSO.     

Supplementation of the thawing medium with reduced glutathione improves function of frozen-thawed goat spermatozoa

Sperm cryopreservation represents a useful tool in the management of reproduction in goat production. However, freezing and thawing produce physical and chemical stress on the sperm membrane that reduces their viability and fertilizing ability. In this study, firstly we evaluated the effects of reduced glutathione (GSH, 1 and 5 mM) supplementation of the thawing extender on parameters of frozen-thawed goat spermatozoa. We used a set of functional sperm tests that included sperm motility assayed by computer-assisted semen analysis (CASA), membrane lipid packing disorder, spontaneous acrosome reaction, free radical production (ROS generation) and sperm chromatin condensation. The main findings from this study were that addition of GSH to the thawing medium resulted in: (1) a higher motility and progressive motility; (2) a higher number of non-capacitated viable spermatozoa; (3) higher number of viable spermatozoa with intact acrosome; (4) a reduction in ROS generation and (5) lower chromatin condensation. In a second study, the additions of reduced (GSH, 5 mM) or oxidized glutathione (GSSG, 2.5 mM) to the thawing media were evaluated. We confirmed the protective effect of GSH on the sperm functionality. The addition of GSSG to the thawing media was less protective to sperm functions compared to GSH. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen goat spermatozoa. The information derived from this study suggests the importance of oxidative stress as responsible for cryo-injury to spermatozoa and opens new windows to explore the practical application of antioxidants to improve the quality of post-thaw goat semen.     

Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing.

The fertilizing capacity, motility and ultrastructure of fowl and turkey spermatozoa were examined at various stages of the freezing process. For both species, fertility and motility were depressed after equilibration with dimethyl-sulphoxide at 5 degrees C. After freezing, motility was maintained at 55% for fowl spermatozoa and 40% for turkey spermatozoa; however, fertility was 55% for the fowl and 0% for the turkey. Qualitatively, the damage to the spermatozoa of both species was nearly identical, as revealed by scanning and transmission electron microscopy. The plasmalemma was the primary site of damage. 'Bent' spermatozoa, coiled tails and swollen mitochondria were also present. Damage to the acrosome was only observed in spermatozoa which had been frozen to -180 degrees or -196 degrees C. These changes were attributed to adverse osmotic conditions. Binding of cationic ferritin to the plasmalemma of spermatozoa from both species remained unaltered.     

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.     

Cryopreservation of bull spermatozoa alters the perinuclear theca

The perinuclear theca (PT) is involved in several important sperm functions leading to fertilization. The objective of this study was to investigate the effect of cryopreservation of bull spermatozoa on the integrity of the PT and the relationship between PT integrity and semen characteristics. Semen from seven bulls was evaluated before and after cryopreservation, comparing the integrity of the plasma membrane (hypo-osmotic test), percentage of live and dead spermatozoa (triple stain), acrosome integrity (triple stain) and the integrity of the PT (negative stain by electron microscopy). Cryopreservation of bull semen caused substantial damage to the PT; the proportion of spermatozoa with a damaged PT was 15.2% versus 52.5% (P<0.05) in fresh versus frozen-thawed spermatozoa, respectively. Furthermore, on average, 67.4% (range, 64-72%) of fresh spermatozoa were live, compared to 53.1% (range, 49-58%) for frozen-thawed spermatozoa; there was an inverse correlation between the percentage of live spermatozoa and the percentage with damage to the PT. Although 59.1% of frozen-thawed spermatozoa had an intact acrosome, only 43.7% of them still remained alive. In frozen-thawed semen, there was a high correlation (r=0.69) between live spermatozoa with an intact acrosome and spermatozoa that maintained an intact PT. In conclusion, freezing/thawing of bull spermatozoa altered the PT and maintaining PT integrity may be necessary to maintain acrosome integrity.     

Spermatozoa of the Atlantic bottlenosed dolphin, Tursiops truncatus

Semen from a male dolphin in captivity was collected by electro-ejaculation and frozen to -176 degrees C. Sperm motility was excellent after thawing 10 days later. Electron microscopy showed 14-16 parallel ridges in the post-acrosomal region and two types of mitochondria in the mid-piece. The spermatozoa were capable of fusing with zona-free hamster eggs only after preincubation for 2 h, suggesting the need for sperm capacitation and acrosome reaction before fertilization in this species.     

Assessment of the efficacy of Sephadex G-15 filtration of bovine spermatozoa for cryopreservation

Semen from five dairy AI bulls was split-filtered through a Sephadex G-15 filter and frozen in a Tris-citric acid buffer egg yolk-based extender. The effect of filtration was studied morphologically for individual sperm abnormalities. Computer-assisted sperm analysis (CASA) was used for motility and sperm motion assessment. Flow cytometry was used to disclose sperm viability (SYBR-14/PI), mitochondrial membrane potential (Mitotracker Deep Red/SYBR 14), acrosome integrity (SYBR 14/PE-PNA/PI), plasma membrane stability (Merocyanine 540/YO-PRO 1/Hoechst 333342), and chromatin stability (acridine orange staining). Filtration significantly reduced the concentration of recovered spermatozoa (P < 0.01), but improved semen quality, reducing the number of spermatozoa with various forms of morphological defects. Filtration also affected percentages of sperm motility after equilibration and after freezing/thawing. Sperm motion characteristics were, however, not significantly affected by filtration at any stage of the cryopreservation protocol, including post-extension, equilibration, or freezing/thawing. Filtration enhanced sperm viability after thawing (P < 0.05), but had no significant effect (P > 0.05) on recovery of spermatozoa with high mitochondrial potential, intact acrosomes, or preserved sperm chromatin structure. Sperm plasma membrane stability was also not affected by the filtration method used (P > 0.05). It can be concluded that filtration effectively separates weaken or abnormal spermatozoa in pre-freezing semen samples and therefore the procedure could be recommended to improve post-thaw sperm viability of selected, fertile sires.