Feasibility of using hard gelatin capsules for the packaging and deep-freezing of bull semen

Various packaging systems have been used for deep freezing of semen. In this study, feasibility of using hard gelatin capsules was established. Of the four types of capsules developed and tested, polymer-treated capsules were found to be suitable for the purpose, and were therefore used subsequently. French medium (0.5 ml) straws were used for control. Five semen samples from each of 12 bulls were processed and included for study. Semen was frozen by fast-freezing. Parameters studied after thawing of semen were comparable for the two methods. Upon analysis, the percentages of progressive motile spermatozoa, live spermatozoa and morphologically abnormal spermatozoa obtained for semen frozen in hard gelatin capsules and French medium straws were found to be nonsignificant. The percentage of intact acrosomes was found to be significantly higher (P < 0.05) for semen frozen - thawed in straws as compared to semen in capsules.     

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.     

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.     

A new pellet technique for cryopreserving ram and bull spermatozoa using the cold surface of cattle fat

A technique for freezing ram and bull spermatozoa in pellet form, using the cold surface of cattle fat was compared to other freezing procedures. Three freezing methods were compared to cryopreserve ram spermatozoa: 0.25 ml straws, pellets frozen on the cold surface of paraffin wax and pellets frozen on the cold surface of cattle fat. In addition, two cryoprotectants, glycerol or sucrose, in an egg yolk-Tris diluent were compared. Ram spermatozoa frozen as pellets on cattle fat exhibited higher percentages of motile cells after thawing (54%) than spermatozoa frozen in straws (49%) or as pellets on paraffin wax (42%, S.E.M. = 1; P < 0.05). However, the percentages of acrosome intact cells were similar for spermatozoa frozen as pellets (49%) and spermatozoa frozen in straws (48%; P > 0.05), but higher than for spermatozoa frozen as pellets on paraffin wax (39%, S.E.M. = 1; P > 0.05). Ram spermatozoa exhibited higher percentages of motile cells after thawing when the cryoprotectant was sucrose (51%) compared to glycerol (46%; P < 0.05). Similarly, acrosomal integrity was greater with sucrose (49%) than with glycerol (42%; P < 0.05). Bull spermatozoa exhibited higher percentages of motile cells after thawing, when cells were frozen in straws (47%) than in the pellet form, regardless of the surface on which the pellets were frozen (31-37%, S.E.M. = 3; P < 0.05). However, bull spermatozoa exhibited higher percentages of motile cells when frozen as pellets on the surface of cattle fat (66%) or dry ice (61%), than when frozen on paraffin wax (53%, S.E.M. = 4; P < 0.05). In conclusion, although bull spermatozoa survive cryopreservation more effectively in straws, ram spermatozoa can be cryopreserved as pellets on the cold surface of cattle fat using sucrose as the cryoprotectant. This technique is simple, requires little equipment, is less expensive than using straws and may prove useful for cryopreserving ram and possibly bull spermatozoa in developing countries.     

The effects of nonpenetrating cryoprotectants added to TEST-yolk-glycerol extender on the post-thaw motility of ram spermatozoa

The effects of adding five different concentrations of 17 polymeric compounds to TEST-yolk-glycerol extender on ram spermatozoa survival was studied. These were Aquacide (I, II, and III); dextran (0.8-1.6, 1.9, 15-20, 70, and 200-300 kDa); three types of Dri-Sweet; hydroxyethyl starch; methylcellulose, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and Supercol 912. All the compounds tested except the Dri-Sweet compounds and hydroxyethyl starch significantly (P less than 0.05) decreased percentages of motile cells in unfrozen samples. The use of dextran (0.8-1.6 kDa; hydrolyzed dextran separated by ethanol) and Aquacide II significantly (P less than 0.05) increased post-thaw motility of spermatozoa frozen in pellets. Dextran (15-20 kDa), dextran (0.8-1.6 kDa), Aquacide II, and hydroxyethyl starch significantly (P less than 0.05) increased the percentages of post-thaw motility of ram spermatozoa frozen in the presence of glycerol and egg yolk.     

The effects of rapid cooling (cold shock) of ram semen, photoperiod, and egg yolk in diluents on the survival of spermatozoa before and after freezing

The effects of rapid cooling of semen (cold shock) from 30 degrees C to various temperatures above 0 degrees C on survival of ram spermatozoa suspended in diluents with or without egg yolk were assessed before and after freezing. Rapid cooling of extended semen from 30 to 15 degrees C had little or no effect on spermatozoa survival before or after freezing. Rapid cooling of extended semen from 30 degrees C to 10, 5, or 0 degrees C was accompanied by a progressive decrease in percentage of motile spermatozoa and percentage of intact acrosomes before freezing and a decrease in percentage of motile spermatozoa and after freezing. The ability of spermatozoa motile after cold shock to survive freezing and thawing, evaluated as cryosurvival, was not significantly (P greater than 0.05) affected by the temperature to which semen was cooled. The addition of egg yolk to the initial extender had a beneficial effect on percentage of motile spermatozoa particularly after rapid cooling of semen to 10 and 5 degrees C. Although egg yolk had little effect before freezing on semen rapidly cooled to temperatures above 15 degrees C and therefore not actually cold shocked, it substantially improved the subsequent survival of spermatozoa after freezing and thawing. Percentage of motile spermatozoa after cooling and after freezing was generally higher when the semen was collected during a decreasing photoperiod than during an increasing photoperiod.     

A pilot study on post-thawing quality of Iberian red deer spermatozoa (epididymal and electroejaculated) depending on glycerol concentration and extender osmolality

The optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes-Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 degrees C. Then, they were diluted down to 100x10(6) sperm/mL and frozen at -20 degrees C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample.     

Isolation of a population of intact, highly-motile porcine spermatozoa using a discontinuous bovine serum albumin gradient

This study was conducted to determine the efficacy of isolating intact, highly-motile porcine spermatozoa using a discontinuous bovine serum albumin (BSA) gradient. Either 0.25, 0.5, 1, 2.5 or 5 x 10(9) spermatozoa extended to 26 ml with Kiev extender were layered on a discontinuous BSA gradient (4% BSA over 10% BSA) contained within a 500-ml separatory funnel. After 1 h of sperm migration at 23 degrees C, four 30-ml aliquots designated Fractions 1, 2, 3, 4 and Fraction T were collected from the bottom of the funnel. Fraction 4 was the bottom fraction and Fraction T was the remnant of the applied sample. For all concentrations of applied spermatozoa, the percentage of progressively motile spermatozoa was greater in Fraction 4 than in Fractions T or 1 (P<0.05). Fraction 4 contained a greater (P < 0.05) proportion of spermatozoa with normal apical ridge acrosomes than Fraction T. Regardless of the concentration of spermatozoa applied, Fraction 4 contained more than 90% of progressively motile spermatozoa and spermatozoa possessing normal apical ridge acrosomes. The percentage of applied cells recovered in Fraction 4 decreased as the concentration of spermatozoa applied to the gradient increased.     

Effect of freezing temperature,at which straws were plunged into liquid nitrogen,on the post-thaw motility and acrosomal status of ram spermatozoa

The present study was conducted to observe the effect of initial freezing temperature on subsequent survival and acrosomal integrity of Malpura and Bharat Merino ram spermatozoa during post-thawing incubation. Semen samples were diluted in TEST-yolk-glycerol extender, loaded in 0.25 ml straws and cooled down to -25, -75 or -125 degrees C freezing temperature using a programmable cell freezer. Computer assisted sperm analysis and acrosomal integrity of thawed samples were assessed after thawing and at hourly intervals during incubation at 37 degrees C for 4 h. The percentage of motile cells in samples frozen at -125 degrees C were 80.3 and 63.7 after post-thawing and -thawing incubation, compared to 75.9 and 39.7 at -25 degrees C or 73.9 and 51.8 at -75 degrees C temperatures, respectively. The spermatozoa with normal acrosome were also significantly, respectively, higher in samples frozen at -125 degrees C, compared to -25 and -75 degrees C temperatures. There were no significant breed variations on percentage of motile, percentage of rapidly motile cells, percentage of normal acrosomes, curvilinear velocity and lateral head displacement except straight line velocity and average path velocity of spermatozoa. The results indicated that -125 degrees C initial freezing temperature conferred the best cryopreserving ability to ram spermatozoa for post-thawing thermoresistance test compared to -25 or -75 degrees C freezing temperature.     

Comparison of different techniques to determine the percentage of intact acrosomes in frozen-thawed bull semen

Twenty-seven different batch dates of frozen bull semen from 26 bulls were used in this study. The semen was in 0.5-ml straws, and 23 of the batch dates were in whole milk extender while 4 were in egg yolk-tris extender. Straws from each batch of semen were incubated for 2 hours in a water bath at 37 degrees C. Following this, the percentage of progressive motility, the rate of motility, and the percentage of intact acrosomes were determined for each unfixed sample. Each batch of semen was fixed in 2 different solutions of 0.2% glutaraldehyde in phosphate-buffered saline (glutaraldehyde 1 and glutaraldehyde 2) and in 10% neutral buffered formol saline (formol saline). The percentage of intact acrosomes for each sample in these fixatives was determined at Day 0 and Day 7. There were no significant differences in the percentages of intact acrosomes among the unfixed samples and the samples in the 3 fixatives at Day 0. At Day 7, the samples in formol saline had a significantly higher percentage of intact acrosomes than those in glutaraldehyde 2. When the percentage of intact acrosomes for the unfixed samples at Day 0 was compared with the percentages of intact acrosomes for glutaraldehyde 1, glutaraldehyde 2, and formol saline at Day 7, only the percentage of intact acrosomes for formol saline was significantly higher than for the unfixed samples. Only one of the batches of semen in egg yolk-tris extender could be evaluated in formol saline because of a heavy precipitate that formed. There was a significant interaction between extender and storage. For the whole milk extender, the percentages of intact acrosomes at Day 7 were higher than for Day 0 for all the fixatives used. For the egg yolk-tris extender, the percentage of intact acrosomes decreased from Day 0 to Day 7. The correlations between the percentage of intact acrosomes for the unfixed samples and the post-incubation percentage of progressivé motility and rate of motility were 0.65 and 0.46, respectively.     

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.     

Effect of post-thaw incubation on sperm kinematics and acrosomal integrity of ram spermatozoa cryopreserved in medium-sized French straws

The objectives were to assess the effect of post-thaw in vitro incubation on motion characteristics and acrosomal integrity of ram spermatozoa of native Malpura and Bharat Merino breeds maintained under a semi-arid tropical environment. Good quality semen samples of both breeds were diluted, packaged in medium-sized straws, and frozen under controlled conditions. Straws were thawed at 60 degrees C for 10s and thawed samples were incubated at 37 degrees C for 4h. Post-thaw motion characteristics and acrosomal integrity of incubated spermatozoa were assessed (by computer-aided semen analysis and Giemsa staining, respectively) just prior to incubation and at hourly intervals thereafter. There was a significant effect of incubation time on motility characteristics and the proportion of spermatozoa with normal acrosomes; 81.4% (arcsin transformed value, 65.2) of spermatozoa were motile at the start of incubation, with 47.9% (arcsin transformed value, 44.4) motile after 4h. At the corresponding times, there were normal acrosomes in 65.8 (arcsin transformed value, 54.8) and 55.7% (arcsin transformed value, 48.9) of spermatozoa, respectively. The percentage straightness of spermatozoa varied during incubation (P < 0.01). However, there was no significant change in percentage linearity, curvilinear velocity, average path velocity, straight line velocity, lateral head displacement, and beat cross frequency of spermatozoa during incubation. There were no breed variations in any motility parameters during incubation, except percentage straightness (P < 0.05), lateral head displacement (P < 0.05) and beat cross frequency (P < 0.01). That sperm motility and acrosomal morphology were very acceptable immediately post-thaw and after 4h of incubation indicated the efficacy of cryopreserving ram spermatozoa under controlled conditions in medium-sized straws.     

A technique for the evaluation of sperm penetrating ability and quality of bovine semen processed in an extender made with Brackett-Oliphant medium and egg yolk

Egg yolk-sodium citrate (EYC) semen extender was compared with an extender made of Brackett-Oliphant medium and egg yolk (BOEY). Ejaculates were divided into equal portions, processed and frozen. Semen was thawed and evaluated for quality. Additional semen was thawed, stained with Hoechst 33342 and the spermatozoa capacitated, after which they were co-incubated with zona-free hamster oocytes to determine their penetrating ability. Sperm penetration of non-compressed, unfixed oocytes was evaluated using an optical sectioning technique on a standard research microscope. Sperm penetration was considered successful if a fluorescing sperm head was observed within the living oocyte in a hanging drop of fertilization medium. There were small differences in percentage of secondary abnormalities and percentage of progressive motility immediately after thawing between spermatozoa extended in EYC or BOEY diluent. There were no differences due to by extender composition in percentage of spermatozoa with intact acrosomes or percent of progressively motile after a 3 h incubation at 37 degrees C, nor the percentage of spermatozoa with head abnormalities. While there were significant correlations between all seminal quality characteristics, no quality measurements were correlated to percentage of oocyte penetration. The new penetration evaluation method allowed for examination of the fertilized oocytes using fluorescent microscopy initially and again after re-incubation for further development.     

Single layer colloid centrifugation technique improves motility,viability and chromatin integrity of ram spermatozoa after thawing

The cell membrane of ram spermatozoa is more sensitive to the freezing process than in other species due to its composition. As a result, the quality and viability of frozen thawed ram spermatozoa are often poor, which together with the specific structure of the ewe's cervix are the main reasons for lower fertility in ewes after intracervical insemination. In the present study we investigated the effects of semen centrifugation through a single layer of a species-specific colloid (Androcoll-O) on post-thaw quality of ram spermatozoa. Motility, viability and morphology were analysed 0, 6, 12 and 24 h after thawing. DNA fragmentation index (%DFI) of the samples was assessed 0 h after thawing, by SCSA™. Membrane and acrosome integrity of spermatozoa were analysed by Sybr-14/PI/PNA test 0 h after thawing.The proportion of motile spermatozoa was significantly higher in SLC – selected samples in comparison to control (not SLC – selected) samples at 0, 6, 12 (P < 0.001) and 24 h (P < 0.05). The proportion of viable spermatozoa was also significantly higher in SLC - selected samples in comparison to control samples at all times (P < 0.001). The proportion of abnormal acrosomes and morphologically abnormal spermatozoa (MAS) were significantly lower in SLC – selected samples compared to control samples at all times (P < 0.001). Analysis of chromatin stability revealed significantly lower %DFI values in SLC – selected samples compared to control samples (P < 0.001). The SYBR-14/PI/PNA test also revealed significantly better values in SLC – selected compared to control samples (P < 0.05). In conclusion, single layer colloid centrifugation significantly improved post-thaw quality and longevity of ram spermatozoa, making it suitable for artificial insemination initiatives.     

Effect of cooling of equine spermatozoa before freezing on post-thaw motility: preliminary results

The ability to ship cooled stallion semen to a facility that specializes in cryopreservation of spermatozoa would permit stallions to remain at home while their semen is cryopreserved at facilities having the equipment and expertise to freeze the semen properly. To accomplish this goal, methods must be developed to freeze cooled shipped semen. Three experiments were conducted to determine the most appropriate spermatozoal extender, package, time of centrifugation, spermatozoal concentration and length of time after collection that spermatozoa can be cooled before cryopreservation. In the first experiment, spermatozoa were centrifuged to remove seminal plasma, resuspended in either a skim milk extender, a skim milk-egg yolk-sugar extender or a skim milk-egg yolk-salt extender, cooled to 5 degreesC and frozen in 0.5- or 2.5-mL straws either 2.5 or 24 h after cooling. Samples frozen 2.5 h after cooling had higher percentages of progressively motile (PM) spermatozoa (27%) than samples frozen 24 h after cooling (10%; P < 0.05). Samples frozen 2.5 h after cooling in skim milk extenders containing egg yolk had higher percentages of PM spermatozoa (average 32%) than did spermatozoa frozen in extender containing skim milk alone (average 16%; P < 0.05). The percentages of PM spermatozoa frozen in 0.5- or 2.5-mL straws were similar (21 and 28%, respectively; P > 0.05). In the second experiment, spermatozoa were centrifuged to remove seminal plasma either before (25 degreesC) or after cooling (5 degreesC), and spermatozoa were frozen after being cooled to 5 degreesC for 2, 6, or 12 h. The percentages of PM spermatozoa were higher (P < 0.05) for spermatozoa centrifuged before cooling (30%) than for spermatozoa centrifuged after cooling (19%). Spermatozoa centrifuged at 25 degreesC then cooled for 12 h to 5 degreesC had higher (P < 0.05) post-thaw progressive motility (23%) compared to spermatozoa cooled for 12 h and centrifuged at 5 degreesC (13%). In the third experiment, spermatozoa were centrifuged for seminal plasma removal, resuspended at spermatozoal concentrations of 50,250 or 500 x 10(6)/mL, cooled to 5 degreesC for 12 h and then frozen. Samples with spermatozoa packaged at 50 or 250 x 10(6)/mL had higher (P < 0.05 percentages of PM spermatozoa (25 and 23%) after freezing than did samples packaged at 500 x 10(6) spermatozoa/mL (17%). We recommend that semen be centrifuged at 25 degreesC to remove seminal plasma, suspended to 250 x 10(6) spermatozoa/ml and held at 5 degreesC for 12 h prior to freezing.     

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.     

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.     

Effect of filtration on post-thaw quality of bull semen

Semen from 4 Holstein bulls was diluted in 4 different extenders, filtered with Sephadex ion-exchange column, and frozen in liquid nitrogen. Sperm motility, progressive motility, path velocity, progressive velocity and the percentage of normal acrosomes of filtered and nonfiltered semen were recorded before and after freezing. Semen characteristics were significantly influenced by extender, filtration and freezing. Before and after freezing, motility measurements and the percentage of normal acrosomes were higher (P < 0.001) in filtered than in nonfiltered spermatozoa. Post-thaw recovery rate of motile spermatozoa was higher in filtered semen than nonfiltered (68 vs 39%, P < 0.0001). The reduction in motility, progressive motility and the percentage of normal acrosomes during freezing and thawing processes were significantly lower (P < 0.0001) in filtered semen (34, 34 and 4%, respectively) than nonfiltered (59, 54 and 15%, respectively). Post-thaw viability of spermatozoa was significantly affected by extender, filtration and time (P < 0.0001). Immediate (0 h) post-thaw motility of nonfiltered semen (29%) was similar to 4-h post-thaw motility of filtered semen (25%; P > 0.05). In conclusion, bull spermatozoa recovered by Sephadex ion-exchange filtration showed better post-thaw viability.     

Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season

The study compared quality and freezability of stallion semen during breeding and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mixed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl and skim milk). Samples of 0.5ml were cryopreserved in straws using a programmed freezer. Parameters of sperm quality were evaluated before and after freezing/thawing. These included percentages of motile spermatozoa and of morphological intact sperm. Typical injuries were demonstrated by scanning electron microscopy (S.E.M.). The acrosomal status was visualised using FITC-conjugated peanut agglutinin, and the acrosome reaction was induced by calcium ionophore A 23187. The chromatin stability was estimated by acridine orange test.In winter, the average percentages of motile and morphologically normal sperm (67 and 74.3%, respectively) were higher than during the breeding season in May (59 and 65.9%; P < 0.05). After freezing/thawing the proportions of vital and intact sperm decreased significantly. The number of motile sperm declined to 15 and 18% in May and December (range 5-40%), and of morphologically intact sperm to 51% in both seasons. Results of S.E.M. showed typical membrane ruptures in the acrosomal region and some sperm with abnormal necks. The proportion of frozen sperm with spontaneous acrosome reaction was higher during winter (86.5 versus 77.0%), suggesting a higher degree of membrane reactivity. Percentages of spermatozoa with denaturated chromatin were minimal and showed minimal differences between fresh and frozen state, stallions or seasons. An additional decondensation treatment with papain and DTE revealed a slightly enhanced number of spermatozoa with denaturable DNA after cryopreservation, especially in December (5.4 +/- 1.3%). The influence of cryoextenders was not significant for most sperm parameters, but there was a high variability between the stallions. Altogether, the influence of factors on the quality of spermatozoa has the following rank order: cryopreservation > stallion > season. Different cellular structures seem to have different susceptibilities to physicochemical stress. The cryopreservation of sperm during December results in survival rates similar to those measured during the breeding season, even more important for successful preservation is the selection of suitable semen donors.     

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.