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.     

Characterization of epididymal spermatozoa motility rate, morphology and longevity of springbok (Antidorcas marsupialis), impala (Aepyceros melampus) and blesbok (Damaliscus dorcus phillipsi): pre- and post-cryopreservation in South Africa

Cryopreservation of antelope epididymal spermatozoa could play a vital role in future breeding by developing a successful protocol for cryo-conserving them. The aim of this study was to characterize morphology, motility rates and longevity of epididymal spermatozoa from springbok, impala and blesbok. Cauda epididymal spermatozoa were collected post-mortem from both testicles of free-ranging springbok (n=18), impala (n=21) and blesbok (n=21), and divided into two groups (pre- and post-cryopreservation). Spermatozoa were cryopreserved in Biladyl supplemented with 20% egg yolk and 7% glycerol under field conditions. Pre-freeze and post-thaw sperm quality was evaluated. The longevity of thawed spermatozoa was evaluated under culture conditions that support domestic cattle in vitro fertilization. There was a significant difference between pre-freeze and post-thaw sperm motility index (SMI) (p<0.05), plasma membrane integrity (p<0.05) and acrosome integrity (p<0.05) for all species. Post-thaw SMI and plasma membrane integrity were comparable between species (p>0.05). The effects of cryopreservation on sperm cell morphology differed between species and between specific abnormal morphology. Blesbok had the least abnormalities in post-thaw spermatozoa. Cryopreservation substantially reduced the survivability and motility rates of antelope species. Blesbok spermatozoa tolerated cryopreservation and thawing process better than impala and springbok. The antelope cauda epididymal sperm maintained viability and acrosome integrity for at least 4h following incubation under conditions that support domestic cattle in vitro fertilization (IVF) with a decline in longevity over time across species however; species responded differently over time in terms of plasma membrane integrity and acrosome integrity. The antelope species may have different in vitro culture requirements, indicating differences in sperm physiology between the species. This research could contribute species-specific protocol development for IVF thus promoting ex-situ conservation strategies of African antelope species in South Africa.     

The effect of cryoprotectant on kangaroo sperm ultrastructure and mitochondrial function

This study examined the effect of cryoprotectants (20% DMSO, a 10% DMSO/10% glycerol mixture, 20% glycerol and 1 M sucrose solution) on kangaroo sperm structure and function, along with the effect of varying concentrations of glycerol on sperm mitochondrial function. Eastern grey kangaroo cauda epididymidal spermatozoa were incubated for 10 min at 35 °C in each cryoprotectant and the plasma membrane integrity (PMI) and motility assessed using light microscopy. The same samples were fixed for TEM and the ultrastructural integrity of the spermatozoa examined. To investigate the effect of glycerol on the kangaroo sperm mitochondrial function, epididymidal spermatozoa were incubated with JC-1 in Tris–citrate media at 35 °C for 20 min in a range of glycerol concentrations (0%, 5%, 10%, 15% and 20%) and the mitochondrial membrane potential (MMP) and plasma membrane integrity determined. As expected, incubation of spermatozoa in 20% glycerol for 10 min resulted in a significant reduction in motility, PMI and ultrastructural integrity. Interestingly, incubation in 20% DMSO resulted in no significant reduction in motility or PMI but a significant loss of structural integrity when compared to the control spermatozoa (0% cryoprotectant). However, 20% DMSO was overall less damaging to sperm ultrastructure than glycerol, a combination of 10% glycerol and 10% DMSO, and sucrose. While all glycerol concentrations had an adverse effect on mitochondrial function, the statistical models presented for the relationship between MMP and glycerol predicted that spermatozoa, when added to 20% glycerol, would lose half of their initial MMP immediately at 35 °C and MMP would halve after 19.4 min at 4 °C. Models for the relationship between PMI and glycerol predicted that spermatozoa would lose half of their initial PMI after 1.8 min at 35 °C and PMI would halve after 21.1 min at 4 °C. These results suggest that if glycerol is to be used as a cryoprotectant for kangaroo spermatozoa then it is best administered at 4 °C and that mitochondrial function is more sensitive to glycerol than PMI. Future research should be directed at investigating strategies that reduce exposure of spermatozoa to glycerol during processing and that test the cryoprotective properties of 20% DMSO for kangaroo spermatozoa.     

Effects of different cryoprotective agents on ram sperm morphology and DNAintegrity

This study investigates the effects of glycerol, 1,2 propanediol, sucrose, and trehalose on post-thaw motility, morphology, and genome integrity of Awassi ram semen. Ejaculates of thick consistency with rapid wave motion (>+++) and >70% initial motility were pooled. Sperm were diluted to a final concentration of 1/5 (semen/extender) in 0% cryoprotectant, 6% glycerol, 6% 1,2 propanediol, 62.5 mM sucrose or 62.5 mM trehalose using a two-step dilution method. The equilibrated semen was frozen in 0.25-ml straws. Semen samples were examined for sperm motility, defective acrosomes (FITC-Pisum sativum agglutinin (FITC PSA)), DNA integrity (acridine orange staining (AO)) and apoptotic activity (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Caspase-3 activity) at four time points: after dilution with extender A, after cooling to 5 °C, after equilibration and post-thaw. Freezing and thawing procedures (cooling at 5 °C, dilution, equilibration, and thawing) had negative effects on motility (P < 0.001), acrosome integrity (P < 0.001), and DNA integrity as determined by AO (P < 0.001) and TUNEL (P < 0.001) assays. There were positive correlations between sperm with defective acrosomes and apoptotic (AO- and TUNEL-positive) spermatozoa. In contrast, a significant negative correlation was found between sperm motility and defective acrosomes and AO- and TUNEL positivity (P < 0.01). The cryopreservation process acts as an apoptotic inducer in ram semen; all cryoprotectants used in the present study allowed apoptosis to some extent, with negative effects on sperm morphology and DNA integrity. The glycerol group performed better than the propanediol, sucrose, trehalose, and control groups in terms of post-thaw sperm motility but not DNA integrity.     

Incorporation of penetrating cryoprotectants in diluents for pellet-freezing ram spermatozoa

Glycerol may be toxic to frozen-thawed ram spermatozoa and reduce their fertilizing capacity. This study examined the cryoprotective effects of dimethyl sulphoxide (DMSO), ethylene glycol, glycerol and propanediol alone and in combinations with each other in Triscitrate-glucose diluents on the post-thaw motility and acrosome integrity of pellet-frozen ram spermatozoa. The 4 cryoprotectants were examined in diluents at 5 concentrations (0, 1.5, 3.0, 6.0, 12.0% v/v). Post-thaw motility of spermatozoa was higher in diluents containing ethylene glycol (1.5 to 6.0% v/v), glycerol (at all levels tested) and propanediol (1.5 and 3.0% v/v) than in diluents without cryoprotectant (P<0.001), but there was no effect of DMSO on post-thaw motility. Motility of spermatozoa was higher in diluents containing ethylene glycol or glycerol than DMSO or propanediol (P<0.001). In diluents containing the 4 cryoprotectants at 3 concentrations (1.5, 3.0, 6.0% v/v), better recovery of spermatozoa was found with the addition of 18.0 than 4.5% v/v egg yolk. Combinations of ethylene glycol and/or propanediol (0 to 6.0% v/v) with glycerol (0 to 6.0% v/v) in diluents were also examined. In the presence of glycerol at all levels tested, increasing levels of ethylene glycol and/or propanediol decreased motility and acrosome integrity of spermatozoa (P<0.001). We conclude that the compounds examined exert a cryoprotective effect on pellet-frozen ram spermatozoa, except for DMSO which had no effect. In this study, glycerol remained the single most effective cryoprotectant, and there was no enhancement of this cryoprotection by addition of the other compounds.     

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 antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.     

四种渗透性防冻剂在猕猴精子低温冷冻保存中对精子功能状态的影响

以冷冻精子的复苏运动度、荧光染料Hoechst 3 3 2 5 8检测的细胞膜完整率、异硫氰酸荧光素标记的花生凝集素 (FITC PNA)检测的顶体完整率作为精子功能状态的指标 ,对甘油、二甲亚砜、乙二醇和丙二醇 4种常用渗透性防冻剂在猕猴精子冷冻保存过程中的作用进行了比较。结果表明 :冷冻保存精子的复苏运动度 ,甘油 ( 4 7 3± 5 7% )和乙二醇 ( 4 4 8± 6 7% ) >二甲亚砜 ( 2 2 9± 0 9% ) >丙二醇 ( 0± 0 % ) ;细胞膜完整率 ,甘油 ( 5 4 8± 3 2 % )和乙二醇 ( 5 4 0± 6 7% ) >二甲亚砜 ( 3 7 5± 7 0 % ) >丙二醇 ( 2 8 3± 6 5 % ) ;顶体完整率 ,甘油 ( 82 2± 2 4 % )和乙二醇 ( 82 4± 2 4 % ) >二甲亚砜 ( 6 8 7± 5 7% )和丙二醇 ( 72 3±3 5 % ) (P <0 0 5 )。结果提示 :二甲亚砜和丙二醇 ,尤其是丙二醇并不适合猕猴精子的冷冻保存 ;而乙二醇具有和甘油相似的保护作用 ,是一种极具潜力的猕猴精子冷冻保存的渗透性防冻剂。     

Effect of glycerol concentration, Equex STM® supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (Vicugna pacos) sperm

Two experiments were conducted to determine the effects of glycerol concentration and Equex STM® paste on the post-thaw motility and acrosome integrity of epididymal alpaca sperm. In Experiment 1, epididymal sperm were harvested from male alpacas, diluted, and cooled to 4 °C in a Lactose cooling extender, and pellet-frozen in a Lactose cryodiluent containing final glycerol concentrations of 2, 3, or 4%. In Experiment 2, epididymal sperm were diluted in Biladyl®, cooled to 4 °C, stored at that temperature for 18-24 h, and further diluted with Biladyl® without or with Equex STM® paste (final concentration 1% v:v) before pellet freezing. In Experiment 1, sperm motility was not affected by glycerol concentration immediately (2%: 16.1 ± 4.6%; 3%: 20.5 ± 5.9% and 4%: 18.5 ± 6.6%; P > 0.05) or 3h post thaw (< 5% for all groups; P > 0.05). Post-thaw acrosome integrity was similar for sperm frozen in 2% (83.6 ± 1.6%), 3% (81.3 ± 2.0%) and 4% glycerol (84.8 ± 2.0%; P > 0.05) but was higher 3h post-thaw for sperm frozen in 3% (75.7 ± 3.8%) and 4% (77.2 ± 4.1%) than 2% glycerol (66.9 ± 2.7%; P < 0.05). In Experiment 2, sperm motility was higher immediately after thawing for sperm frozen in the presence of Equex STM® (Equex®: 21.5 ± 3.5%; control: 14.4 ± 2.1%; P < 0.05) but was similar at 3h post-thaw (P > 0.05). Acrosome integrity was similar for sperm frozen with or without Equex STM® paste immediately (control: 89.6 ± 1.2%; Equex®: 91.1 ± 1.4%; P > 0.05) and 3 h post-thaw (control: 69.3 ± 3.7%; Equex®: 59.9 ± 5.8%; P > 0.05). Sperm cryopreserved in medium containing 3-4% glycerol and 1% Equex STM® retained the best motility and acrosome integrity, even after liquid storage for 18-24 h at 4 °C prior to cryopreservation.     

The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa

In an effort to improve the number of functional spermatozoa following sex-sorting and cryopreservation, the effects on in vitro sperm characteristics of the additives: (i) catalase (pre-sorting); (ii) cholesterol-loaded cyclodextrins (CLCs; pre-sorting); and (iii) seminal plasma (post-thawing) were investigated. For all experiments, spermatozoa (three males, n=3 ejaculates/male) were processed using a high speed flow cytometer before cryopreservation, thawing and incubation for 6h. Catalase had no effect (P>0.05) on post-thaw motility characteristics (as measured by CASA) of sex-sorted ram spermatozoa, but pre-sort addition of CLCs reduced (P<0.05) sperm quality after post-thaw incubation for 0 h (motility), 3h (motility, average path velocity, viability and acrosome integrity) and 6h (motility, average path and curvilinear velocity, straightness, linearity, viability and acrosome integrity). Seminal plasma had a differential effect (P<0.001) on sex-sorted and non-sorted spermatozoa. Post-thaw supplementation of increasing levels of seminal plasma caused all motility characteristics of sex-sorted, frozen-thawed spermatozoa to decline (P<0.05); conversely, non-sorted, frozen-thawed spermatozoa exhibited improvements (P<0.05) in motility, viability, acrosome integrity and mitochondrial respiration. In summary, incorporation of catalase, CLCs and seminal plasma into the sorting protocol failed to improve post-thaw sperm quality and, consequently efficiency of sex-sorting of ram spermatozoa. The paradoxical effect of seminal plasma supplementation on the in vitro characteristics of ram spermatozoa provides further evidence that sex-sorting by flow cytometry produces a selected population of cells with different functions compared with non-sorted spermatozoa.     

Effects of the supplementation of trehalose extender containing egg yolk with sodium dodecyl sulfate on the freezability of goat spermatozoa

Two experiments were conducted to determine the effect of sodium dodecyl sulfate (SDS) added to a trehalose-egg yolk extender on the cryopreservation of goat spermatozoa. In Experiment 1, semen from four goats was frozen in trehalose extender (osmolality = 370, pH = 7) containing 4 and 20% (v/v) glycerol and egg yolk, respectively, and 0.035-0.2% SDS. After thawing, sperm motility and acrosome integrity were assessed using a computer-assisted sperm analysis (CASA) system and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Both motility and progressive motility were improved (P < 0.05) by increasing the concentration of SDS in the trehalose-egg yolk extender, with the best results obtained with SDS at 0.1% (80.0 +/- 1.5% and 65.0 +/- 1.7%, respectively). There were no significant differences in path velocity when spermatozoa were frozen in a diluent containing 0.035, 0.05, 0.075, or 0.1% SDS, but path velocity decreased significantly with 0.2% SDS. The percentage of acrosome-intact sperm were highest (P < 0.05) when 0.05% (74.0 +/- 1.1) and 0.075% (70.0 +/- 1.2) SDS were used. In Experiment 2, the effect of diluent storage time (6, 24, or 48 h) before freezing on the cryoprotective effect of SDS was investigated. Prolonged storage of the diluent had slight cryoprotective effects when 0.2% SDS is used, while motility and the acrosome integrity of the cryopreserved spermatozoa improved slightly when the extender was stored for 48 h at 5 degrees C before use. In conclusion, goat sperm freezability was significantly improved when sperm were frozen in a trehalose-egg yolk extender containing an adequate concentration of SDS.     

Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing-thawing

Whole ejaculate or sperm-rich fraction, collected from four sexually mature boars, was frozen in an extender containing lactose-hen egg yolk with glycerol (lactose-HEY-G) or extender containing lactose, lyophilized lipoprotein fractions isolated from ostrich egg yolk and glycerol (lactose-LPFo-G), and Orvus Es Paste, respectively. The sperm samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Sperm DNA integrity was assessed using a modified neutral comet assay. Sperm characteristics such as motility, plasma membrane integrity (SYBR-14/PI), mitochondrial function (rhodamine 123) and acrosome integrity were monitored. Freezing-thawing caused a significant increase (P<0.05) in sperm DNA fragmentation, irrespective of the procedures of ejaculate collection and extender type. Sperm DNA fragmentation was significantly lower (P<0.05) in the whole ejaculate compared with the sperm-rich fraction, indicating that spermatozoa maintained in the whole seminal plasma prior to its removal for freezing-thawing procedure were less vulnerable to cryo-induced DNA fragmentation. Furthermore, spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender exhibited lower (P<0.05) DNA fragmentation than those frozen in the absence of cryoprotective substances. The levels of sperm DNA damage, as expressed by comet tail length and tail moment values, were significantly higher (P<0.05) in sperm samples frozen in the absence of cryoprotective substances. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. It can be suggested that evaluation of DNA integrity, coupled with different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, may aid in determining the quality of frozen-thawed boar semen.     

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.     

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.     

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

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

Use of single-layer centrifugation with Androcoll-C to enhance sperm quality in frozen-thawed dog semen

The aim of this study was to investigate whether single-layer centrifugation (SLC) with Androcoll-C could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from five dogs was collected and cryopreserved following a standard protocol. After thawing, the semen samples were divided in two aliquots, one of which was used as a control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining), viability (dual staining with propidium iodine/acridine orange), and acrosome integrity (dual staining with propidium iodine/isothiocyanate-labeled peanut [Arachis hypogaea] agglutinin) were performed on aliquots of fresh semen, frozen-thawed control samples, and frozen-thawed SLC-treated preparations. A multivariate clustering procedure separated 57,577 motile spermatozoa into three subpopulations (sP): sP1 consisted of poorly active and nonprogressive spermatozoa (48.8%), sP2 consisted of moderately slow but progressive spermatozoa (13.3%), and sP3 consisted of highly active and/or progressive spermatozoa (37.8%). SLC with Androcoll-C yielded sperm suspensions with improved motility, viability, and acrosome integrity (P < 0.01). The frozen-thawed SLC-treated samples were enriched in sP3, representing 38.5% of the sperm population. Likewise, sP2 was more frequently observed after SLC, but not significantly so. From these results, we concluded that for dog semen samples selected by SLC with Androcoll-C after thawing, the sperm quality parameters, including motility patterns, are better than in frozen-thawed control samples.     

Cryopreservation of sperm from farmed Pacific abalone,Haliotis discus hannai

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me₂SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me₂SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me₂SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN₂) vapor for 10 min at 5 cm above the LN₂ surface and then submerging them in LN₂ for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me₂SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me₂SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me₂SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.     

Boar spermatozoa cryopreservation in low glycerol/trehalose enriched freezing media improves cellular integrity

The use of glycerol for boar semen cryopreservation results in low fertility, possibly due to toxicity. This has led to recommend the use of solutions with less than 4% glycerol. Trehalose is a disaccharide known to stabilize proteins and biologic membranes during processes such as cryopreservation. Thus, it was decided to evaluate the cryoprotective effect of glycerol/trehalose mixtures. Effects on motility (M), viability (Vb) and acrosomal integrity (nA) were evaluated. Sperm samples were frozen in three different extenders: G4 contained 4% glycerol; T1 contained 1% glycerol plus 250 mM trehalose and T0.5 was constituted by 0.5% glycerol plus 250 mM trehalose. All extenders yielded similar post-freezing/thawing motility rates. Viability was diminished in T0.5 as compared to the others. In regard to acrosome integrity, it was twice as high (P < 0.05) in the trehalose enriched media as in G4, the glycerol-only extender. Thus, T1 twice as many spermatozoa were alive, motile and intact, than in either T0.5 or G4, i.e. during freeze/thawing the use of T1 resulted in twice as many fertile cells as when using the other extenders. During our study, we noted that there were wide individual variations both in sperm viability and in motility.     

Fertility of spermatozoa cryopreserved with 2% acetamide or glycerol through artificial insemination in the Japanese white rabbit.

The rabbit is considered to be a valuable laboratory animal. We compared 2% acetamide and glycerol as cryoprotectants in egg-yolk diluent for ejaculated Japanese white rabbit spermatozoa to improve sperm cryopreservation methods. Fertility through artificial insemination, forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rates of forward progressive motility and plasma membrane integrity of the spermatozoa frozen with acetamide (27.1 +/- 8.3% and 24.5 +/- 6.5%) were significantly (P < 0.05) higher than those of the spermatozoa frozen with glycerol (16.3 +/- 10.9% and 14.3 +/- 7.6%). Though there was no significant difference in the kindling rates, the litter size of females inseminated with spermatozoa frozen with acetamide (6.0 +/- 1.1) were significantly (P < 0.05) higher than those of spermatozoa frozen with glycerol (3.0 +/- 0.4). The results indicate that 2% acetamide has a higher cryoprotective effect than 2% glycerol for sperm cryopreservation in the Japanese white rabbit.     

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe²⁺) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n = 13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe²⁺, EE-CAT plus Fe²⁺, EE-GPx plus Fe²⁺ and EE-SOD plus Fe²⁺). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6 h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6 h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2 h after thawing (P < 0.05). Catalase supplementation, however, improved DNA integrity at 4 h (P < 0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6 h, linear motility at 6 h, mitochondrial activity at 6 h, membrane integrity at 2 and 6 h, and DNA integrity at 4 h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2 h after thawing (P < 0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2 h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe²⁺ negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P < 0.05). After thawing, there were, however, no significant differences between the control plus Fe²⁺ and the antioxidative enzymes supplementation plus Fe²⁺ groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.