Effect of storage method and extender osmolality in the quality of cryopreserved epididymal ram spermatozoa

Post-mortem sperm recovery and cryopreservation could be a complement to germplasm banking in sheep, especially for endangered breeds. This study is an attempt to identify factors for improving the success of cryopreserving ram epididymal spermatozoa, considering the decrease of sperm quality with post-mortem time. Epididymal spermatozoa from 9 rams were kept at 5°C using three storage methods: within the epididymes, undiluted sperm mass, and diluted in extenders of different osmolality (TES-Tris-fructose at 320, 370 or 420 mOsm/kg, 20% egg yolk, 8% glycerol). At 0, 24, 48 and 72h, spermatozoa were cryopreserved using each extender. Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability and acrosomal status). Post-mortem time decreased pre-freezing and post-thawing sperm quality. Some storage x extender combinations improved the effect of post-mortem time on sperm quality. Both epididymis storage combined with the 420 extender, and storing the spermatozoa diluted in the 320 extender improved post-thawing quality, especially at long post-mortem times. Storing the spermatozoa diluted in the 370 extender was detrimental for the acrosomal status. These findings have practical applications. The simplest storage method (within the epididymes) seems to be adequate if hyperosmotic extenders were used for freezing. An alternative method could be storing the spermatozoa diluted in a hypoosmotic extender. These recommendations are limited to the osmolalities tested in this study (420 mOsm/kg and 320 mOsm/kg); other osmolalities should be tested.     

Cryopreservation of epididymal stallion sperm

Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion’s breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen–thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose–egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters.     

Equine sperm post-thaw evaluation after the addition of different cryoprotectants added to INRA 96® extender

The rise of assisted reproduction techniques in equine medicine has fostered investigations that seek to optimize methods to increase fertility rates. Since cryopreservation continues to give low values of viability in stallions, the handling and preservation of the sperm is of vital importance. This reduction of fertility makes it essential for farmers to find new options that ensure reliability in the use of these techniques. The main objective of this study was to assess the effect of INRA 96® (manufactured commercial extender for cooling of Equine semen) as an extender for cryopreservation in combination with different cryoprotectants: Acetal (5%), Dimethylformamide (5%) and Glycerol (5%), alone and combined (2.5% each) on ejaculated and epididymal spermatozoa. Ejaculates collected from mature stallion and epididymal sperm samples were cryopreserved in INRA® varying content of cryoprotectant and cryopreserved. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. We conclude that INRA 96® is suited as extender for freezing when it is used in combination with Dimethylformamide (5%) or Dimethylformamide (2.5%) + Glycerol (2.5%) for samples of ejaculate. The combination of Dimethylformamide (2.5%) + Glycerol (2.5%) showed the best results on epididymal spermatozoa. In conclusion, the combination of Dimethylformamide and Glycerol as cryoprotectants in INRA® medium enhanced equine epididymal and ejaculated spermatozoa quality after cryopreservation.     

Effect of basic factors of extender composition on post-thawing quality of brown bear electroejaculated spermatozoa

The improvement of freezing extenders is critical when defining sperm cryopreservation protocols for wild species, in order to create germplasm banks. The aim of this study was to evaluate the effect of additives (Equex Paste and EDTA) supplementation, egg-yolk (10 and 20%) and glycerol (4 and 8%) concentrations and extender osmolality (300 and 320 mOsm/kg) on the post-thawing quality of brown bear semen. Semen was obtained from 20 adult males by electroejaculation, and centrifugated individually (600 × g for 6 min). The pellets were diluted 1:1 in the corresponding extender TTF (TES-Tris-Fructose with the aforementioned variants) and cooled to 5 °C. Then, it was diluted down to 100 × 10⁶ spz/mL, loaded in 0.25 mL straws and frozen at −20°C/min. After thawing (in water at 65 °C for 6s), the semen samples were assessed for motility (CASA), viability (SYBR-14 with propidium iodide), acrosomal status (PNA-FITC with propidium iodide) and mitochondrial activity (JC-1). Extender supplementation with additives rendered significantly higher results for these sperm parameters. Comparing the two percentages of egg yolk, 20% egg yolk showed the highest motility results, percentages of viable spermatozoa and viable spermatozoa with intact acrosome. No differences were detected among samples frozen using 4 or 8% glycerol. For extender osmolality, 300 mOsm/kg showed higher values of VAP, VCL, VSL, and ALH than 320 mOsm/kg. Based on the best performance of sperm motility, viability and acrosome status, we conclude that the most suitable extender to cryopreserve brown bear spermatozoa was TTF adjusted to 300 mOsm/kg, supplemented with 20% egg yolk, 4-8% glycerol, and the additives 1% Equex paste and 2% EDTA.     

Improved cryopreservation protocol for Blanca-Celtibérica buck semen collected by electroejaculation

The collection of sperm samples by electroejaculation (EE) leads to an increase of the production of seminal plasma which could modify the tolerance of spermatozoa to the cryopreservation procedure. This study aims to compare a standard sperm cryopreservation protocol for samples collected by artificial vagina (AV) with the same protocol and modifications to this for samples obtained by EE. Semen from six males of Blanca-Celtibérica goat breed was collected by AV (control) and EE, and three experiments were conducted. In Experiment 1, it was examined the effects of egg yolk concentration contained in freezing extender (0%, 1.5%, 10% and 20% of egg yolk); in Experiment 2, it was evaluated the cooling rate from 30 to 5 °C (fast: 10 min and slow: 90 min) and the temperature of glycerol addition (30 and 5 °C); and in Experiment 3, it was examined the time of equilibration at 5 °C (0, 1, 2 or 3 h). A heterologous in vitro fertilization test was carried out in order to compare the fertility of control samples with that resulting from the EE protocol which showed the highest sperm quality. Results showed greater sperm motility parameters after thawing for control samples cryopreserved in standard conditions in the three experiments. For samples collected by EE, extender with 20% egg yolk, a slow cooling rate and a longer equilibration time (3 h) provided higher sperm quality, and no differences were observed between temperatures of glycerol addition. Samples collected by EE and cryopreserved with the protocol which yielded the best sperm quality after thawing showed higher fertility compared to AV.     

Male reproductive traits, semen cryopreservation, and heterologous in vitro fertilization in the bobcat (Lynx rufus)

There is limited information on bobcat ejaculate traits and sperm cryopreservation and fertilizing ability. Bobcats were electroejaculated under general anesthesia in November (autumn) and April (spring), and endocrine and sperm traits were characterized. Testosterone (mean ± SEM: 0.90 ± 0.15 ng/mL) was not different between sampling times, but cortisol (average: 13.95 ± 1.73 μg/dL) was significantly higher in April. Average number of spermatozoa was 10.0 ± 3.4 × 10⁶ sperm/ejaculate, with values being significantly higher in April. Sperm motility (average 55.7 ± 5.8% motile sperm) was not different between sampling times. The proportion of normal spermatozoa in the ejaculate (average: 14.7 ± 2.1%) was significantly higher in April, but the percentage of spermatozoa with intact acrosomes (average: 43.7 ± 3.8%) was significantly higher in autumn. Spermatozoa were cryopreserved in a Tes-Tris-based diluent (TEST) or Biladyl, both containing 20% egg yolk and 4% glycerol. Diluted sperm were loaded into straws, refrigerated using a programmable thermoblock with a dry chamber, frozen in nitrogen vapors, thawed, and incubated in F-10 medium with 5% fetal bovine serum for up to 3 h. After cryopreservation in TEST, there were about 50% motile sperm upon thawing, and survival was high during incubation post-thaw. Cryopreservation in Biladyl led to similar results, but motility decreased substantially during incubation post-thaw. Bobcat spermatozoa fertilized domestic cat oocytes matured in vitro. Fertilization rates were higher for sperm collected in April and cryopreserved in TEST (46%) than for those cryopreserved using Biladyl (<3%). Fertilized oocytes cleaved in culture, and some (27%) reached the morula stage. This study has allowed us to gain further baseline information on bobcat reproduction, explore sperm cryopreservation conditions, and show that fertilizing capacity can be tested using in vitro-matured cat oocytes. These results will be important for future conservation efforts.     

Spermatozoa recovery and post-thawing quality of brown bear ejaculates is affected for centrifugation regimes

Sperm cryopreservation protocols for brown bear (Ursus arctos) require the centrifugation of semen samples to increase sperm concentration and to clean urine in contaminated samples. We evaluated the effect of centrifugation regimes (time and relative centrifugal force—RCF) on the quantity of sperm recovered and the quality of post-thawed sperm. Thirteen brown bears were electroejaculated. The ejaculates were diluted 1:1 in Tris–citric acid–glucose (TCG) extender and centrifuged with different RCF/time combinations: 600×g, 1,200×g and 2,400×g, for 3, 6 or 12 min. After centrifugation, spermatozoa were diluted in TES–Tris–fructose extender with egg yolk and glycerol (final glycerol concentration of 8%) and frozen in 0.25-mL straws. In the post-thawed semen, motility was assessed by CASA, and acrosomal status (PNA-FITC), viability (SYBR-14 with propidium iodide) and chromatin status (SCSA) were determined by flow cytometry. The longest centrifugation time (12 min) significantly decreased some motility parameters. Sperm recovery significantly decreased in brown bear at 600×g. Our results suggest that brown bear spermatozoa are more sensitive to long centrifugation times than to high RCF. Centrifugation regimes showed no effects on the post-thawing chromatin status. We recommend preparing the brown bear semen for freezing by centrifugation 1,200×g or 2,400×g for 6 min, after electroejaculation and dilution 1:1 in TCG extender, since these procedures increase the spermatozoa recovery without harmful effects on the post-thawed quality of brown bear spermatozoa.     

Fertilizing potential of mouse spermatozoa cryopreserved in a medium containing whole eggs

Experiments were conducted to improve survival of mouse spermatozoa through the cryopreservation process. In the first experiment, percentages of motile spermatozoa and fertilizing capacities of spermatozoa were evaluated when mouse spermatozoa were cryopreserved using three previously reported cryopreservation media: (1) 18% raffinose in 3% skim milk; (2) Tes/Tris medium containing 25% egg yolk and 1.25% glycerol; and (3) PBS containing 18% raffinose and 1.75% glycerol, each at three different cooling rates (-3, -10, and -50 degrees C/min). Spermatozoa frozen in the skim milk/raffinose medium exhibited the highest percentage of motile spermatozoa (39%) when cells were frozen at -10 degrees C/min (P<0.05). The second experiment evaluated the effects of modifying the Tes/Tris/egg yolk medium, comparing different concentrations of egg yolk, BSA, and sodium dodecyl sulfate. Reducing egg yolk from 25% of the medium volume to 5%, increased percentages of motile spermatozoa after cryopreservation from 29 to 36% (P<0.05). Addition of 1% BSA and sodium dodecyl sulfate to medium containing 5% egg yolk further improved percentages of motile spermatozoa after freezing. In the final experiment, 20% whole egg was substituted for 5% egg yolk and 1% BSA used in previous experiments and resulted in percentages of motile spermatozoa (51%) equal to that of the skim milk-raffinose medium. However, fertility rates were higher (68%) than for spermatozoa frozen in the skim milk-raffinose medium (P < 0.05) and were comparable to the fertility rates of fresh spermatozoa (77%; P>0.05). In conclusion, freezing mouse spermatozoa in a medium containing 20% whole egg, 0.035% sodium dodecyl sulfate, and 1.25% glycerol using a cooling rate of -10 degrees C/min preserves the motility and fertilization capacity of mouse spermatozoa.     

Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant

Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility >/= 60%), and were used for cryopreservation studies. Semen were frozen in TEST + glycerol, TEST + DMSO, HEPT + glycerol, or HEPT + DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42.0 +/- 4.3%; EM2: 26.0 +/- 17.3%) and the percentage of membrane and acrosome intact spermatozoa (EM1: 55.5 +/- 8.1%; EM2: 46.3 +/- 6.4%) cryopreserved in TEST + glycerol were significantly higher than (P < 0.05) those frozen in the other medium investigated choices for cryopreservation of Asian elephant spermatozoa. The data support the use of TEST + glycerol as an acceptable cryopreservation media of Asian elephant semen for the establishment of sperm banks.     

不同甘油浓度与平衡时间对食蟹猴精液冷冻效果的影响

探讨了不同甘油浓度(3%、5%、7%、11%)和不同平衡时间(30、60、90、120min)对食蟹猴(Macaca fascicularis)精液冷冻效果的影响,以建立和优化食蟹猴精液冷冻的程序。参照TTE稀释液成分组成改良型TTE,冷冻前和解冻后均检测精子的活力、畸形率、质膜完整性、顶体完整率。结果显示,平衡时间为30min时精子的冷冻解冻后活力、复苏率均高于平衡时间90min和120min组,差异显著(P<0.05),比60min组稍好;甘油浓度为3%、5%组的精子冷冻解冻后活力及复苏率均高于甘油浓度11%组,差异显著(P<0.05),比7%组好;不同甘油浓度各组间以及不同平衡时间各组间畸形率、质膜完整性、顶体完整率差异不显著(P<0.05)。由此得出如下结论,在食蟹猴精液冷冻中,在改良TTE中加入3%~5%的甘油且平衡30min可以获得较好效果,精子冻后活率和复苏率达到45%和62%。     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.     

Effect of egg yolk, cryoprotectant, and various sugars on semen cryopreservation in endangered Cuvier's gazelle (Gazella cuvieri)

Cryopreservation of spermatozoa from endangered species is a valuable tool for genetic management. Previous studies showed the feasibility of cryopreservation of spermatozoa from various endangered gazelles but have also revealed difficulties with available protocols for semen freezing in Cuvier's gazelle (Gazella cuvieri). Experiments were carried out to investigate the effect of (a) 5% or 20% egg yolk or 4% or 6% glycerol, and (b) addition of sugars (glucose, fructose, lactose and raffinose) on cryopreservation using a Tes-Tris-based diluent (TEST). A diluent containing 13.5% raffinose, 5% or 20% egg yolk, and 6% glycerol (REYG) was also evaluated. Semen was obtained by electroejaculation from 22 G. cuvieri males. Diluted samples were loaded into 0.25 ml straws, cooled to 5 °C over 1.5 h (−0.16 °C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapours for 10 min and plunged into liquid nitrogen. Subsamples were assessed for motility and acrosome integrity upon collection, after refrigeration–equilibration, after freezing and thawing, and 2 h after thawing. Use of TEST with 20% egg yolk or with 4% glycerol led to worse motility preservation, whereas TEST with 5% egg yolk and 6% glycerol led to better results. Addition of fructose, lactose or raffinose to TEST resulted in similar or worse preservation of motility than inclusion of glucose. On the other hand, use of a raffinose-based medium with 20% egg yolk and 6% glycerol (REYG) afforded better preservation of motility than use of TEST. With REYG, 20% egg yolk was better than 5% egg yolk for motility preservation. Differences were noted between males in their responses to cryopreservation when using different egg yolk or glycerol concentrations. Moreover, spermatozoa from most males exhibited better cryopreservation with REYG although some were better cryopreserved in TEST. The raffinose-based diluent thus represents an improvement over previous results but more work is needed to better characterize cryopreservation conditions for future routine banking of Cuvier's gazelle spermatozoa.     

An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm's response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 microM), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n = 4) and wombat (n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast-3 cm above liquid N2 interface; slow-6 degrees C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris-citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410 mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility.     

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008–2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24 h at 17 °C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P < 0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season’s influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.     

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 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.     

Improved cryopreservation protocol for Blanca-Celtibérica buck semen collected by electroejaculation

The collection of sperm samples by electroejaculation (EE) leads to an increase of the production of seminal plasma which could modify the tolerance of spermatozoa to the cryopreservation procedure. This study aims to compare a standard sperm cryopreservation protocol for samples collected by artificial vagina (AV) with the same protocol and modifications to this for samples obtained by EE. Semen from six males of Blanca-Celtibérica goat breed was collected by AV (control) and EE, and three experiments were conducted. In Experiment 1, it was examined the effects of egg yolk concentration contained in freezing extender (0%, 1.5%, 10% and 20% of egg yolk); in Experiment 2, it was evaluated the cooling rate from 30 to 5 °C (fast: 10 min and slow: 90 min) and the temperature of glycerol addition (30 and 5 °C); and in Experiment 3, it was examined the time of equilibration at 5 °C (0, 1, 2 or 3 h). A heterologous in vitro fertilization test was carried out in order to compare the fertility of control samples with that resulting from the EE protocol which showed the highest sperm quality. Results showed greater sperm motility parameters after thawing for control samples cryopreserved in standard conditions in the three experiments. For samples collected by EE, extender with 20% egg yolk, a slow cooling rate and a longer equilibration time (3 h) provided higher sperm quality, and no differences were observed between temperatures of glycerol addition. Samples collected by EE and cryopreserved with the protocol which yielded the best sperm quality after thawing showed higher fertility compared to AV.     

Effects of cryopreservation on Ca2+ signals induced by membrane depolarization, caffeine, thapsigargin and progesterone in boar spermatozoa

Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the Ca2+ handling of boar spermatozoa using several sperm activators. Intracellular Ca2+ signals from single spermatozoa were measured using confocal Ca2+ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external K+ concentration elicited a three times larger Ca2+ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited Ca2+ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the Ca2+ store with thapsigargin induced a rapid rise in Ca2+ in the control but generated a smaller increase of Ca2+ after thawing. Exposure to progesterone induced a biphasic rise of the Ca2+ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting Ca2+ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the Ca2+ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal Ca2+ store and progesterone in terms of the Ca2+ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.     

Evaluation of alternative cryoprotectants for preserving stallion spermatozoa

Although use of cryopreserved stallion spermatozoa is currently accepted by many breed registries, utilization of this technique remains limited due to poor fertility for some stallions. One reason for these results is osmotic stress that spermatozoa experiences when the cryoprotectant (glycerol) is added to the cells prior to freezing and removal from the cells after thawing. In an effort to minimize osmotic damage, alternative cryoprotectants, having lower molecular weights and greater membrane permeability than glycerol, were evaluated to determine their effectiveness for cryopreserving stallion spermatozoa. In the first experiment, equal molar concentrations of several amides were compared to determine if they could preserve the motility of sperm as well as glycerol. At 0.55 M concentration, addition of glycerol to a skim milk-egg yolk (SMEY) diluent resulted in higher percentages of motile sperm (61%) than methyl formamide (40%) or dimethyl formamide (38%, P<0.05), while formamide, acetamide, and methyl acetamide resulted in recovery of less than 20% motile cells (P<0.05). When methyl formamide or dimethyl formamide were increased to 0.6 or 0.9 M they resulted in percentages of motile cells (48-54%) similar to that achieved with glycerol (52%). Similarly, 0.9 M ethylene glycol also resulted in similar percentages of motile cells (43%). Replacing the glucose and fructose in the SMEY diluent with either raffinose or trehalose did not result in higher percentages of motile sperm (65 and 66%, respectively) than the control SMEY (63%). Similarly, addition of methyl cellulose also did not increase the percentages of motile spermatozoa in the samples, after cryopreservation (P>0.05). In conclusion, both methyl formamide and dimethyl formamide protected stallion spermatozoa from cryodamage as effectively as glycerol. Since these compounds permeate the plasma membrane more effectively than glycerol, they should cause less osmotic damage to stallion spermatozoa than glycerol. Therefore, these compounds may prove very effective in the cryopreservation of stallion spermatozoa, and may be particularly useful for spermatozoa from stallions that produce spermatozoa that have poor post-thaw characteristics when glycerol is used as the cryoprotectant.     

Cryopreservation of yellowfin seabream (Acanthopagrus latus) spermatozoa (Teleost, Perciformes, Sparidae)

The effects of both osmolality and cation in the initiation of sperm motility were examined in yellowfin seabream, Acanthopagrus latus. Various factors involved in the cryopreservation of yellowfin seabream spermatozoa on motility are discussed. Extender containing only glucose proved to be a suitable medium for freezing yellowfin seabream spermatozoa to -196 degrees C. Glycerol seems to have a direct osmotic effect on yellowfin seabream sperm cells, and it induced sperm motility before freezing and during thawing. However, this exhausted the energy needed for sperm motility for fertilization. Dimethyl sulfoxide (DMSO) proved superior to ethylene glycerol, propylene glycerol, glycerol and methanol as a cryoprotectant. Prolonged equilibration time had a detrimental effect on both prefreezing and post-thawing sperm motility. The estimated optimum freezing rate was in the range of -20 to -154 degrees C/min. More frozen-thawed than fresh spermatozoa are required to achieve comparable fertilization rates.