In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca)

Research was conducted to characterize seminal traits and to develop a sperm cryopreservation method using directional freezing (DF) for the killer whale (Orcinus orca). Experiments evaluated effects of: (i) freezing rate (SLOW, MED, FAST) by diluent (BF5F, Biladyl®, EYC) in 0.5 mL straws; and (ii) freezing method (straw or DF) by glycerol (3, 6, or 9% final concentration, v:v) on in vitro sperm quality. Fresh ejaculates (n = 161) were (mean ± SD) 7.8 ± 7.4 mL at 740 × 10⁶ sperm/mL with 92.2 ± 6.3% total motility (TM), 85.4 ± 6.9% progressive motility (PM), 89.6 ± 9.0% viability and 89.8 ± 9.2% acrosome integrity. Samples frozen using straws by the MED or SLOW method were improved (P < 0.05) over FAST across all diluents. At 3 h post thaw (PT), TM, PM, Rapid motility (RM), VAP, VCL, ALH and viability for 3% and 6% glycerol were improved (P < 0.05) over 9% glycerol. Directional freezing samples at 0 h and 3 h PT, at all glycerol concentrations, displayed higher (P < 0.001) TM, PM, RM, VAP, VSL, VCL and viability /intact acrosomes (PI/FITC-PNA) than straw. These data provided the first information on ejaculate characteristics and the development of a semen cryopreservation method using DF in the killer whale.     

Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell extender

This study was designed to compare commercially available extender Bioxcell^® with tris-citric egg yolk extender for post thaw quality and in vivo fertility of buffalo semen. For comparison of post thaw semen quality: semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina (at 42 °C) for three weeks (replicates). Qualifying ejaculates having motility >60% from each buffalo bull were divided in two aliquots and diluted (at 37 °C having 50 × 10⁶ spermatozoa/ml) in tris-citric egg yolk or Bioxcell^® extender. Diluted semen was cooled to 4 °C in 2 hours, equilibrated for 4 hours and filled in 0.5 ml straws. Semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. Straws were then plunged and stored in liquid nitrogen (−196 °C). After 24 hours of storage, semen straws were thawed at 37 °C for 30 seconds to assess sperm motility, viability, plasma membrane integrity, normal apical ridge, and abnormalities (head, mid piece, and tail). For comparison of in vivo fertility: semen from two buffalo bulls of known fertility was cryopreserved in tris-citric egg yolk and Bioxcell^® as described earlier, and used for inseminations under field conditions. Post-thaw percentage of sperm motility (45.3 ± 1.1, 45.0 ± 1.4), viability (66.2 ± 1.1, 64.4 ± 1.3) plasma membrane integrity (60.4 ± 1.2, 59.2 ± 1.4) and normal apical ridge (82.9 ± 0.5, 80.7 ± 0.5) did not differ (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. Similarly, sperm abnormalities of head (1.20 ± 0.1, 1.20 ± 0.1), mid piece (0.67 ± 0.1, 0.87 ± 0.1) and tail (11.7 ± 0.2, 11.6 ± 0.3) remained similar (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. In vivo fertility rates of buffalo semen cryopreserved in tris-citric egg yolk and Bioxcell^® also remained similar (44% vs. 47%). It is concluded that commercially available Bioxcell^® may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg yolk extender.     

The effect of supplementation of cryopreservation diluents with sugars on the post-thawing fertility of ram semen

This study was conducted to elucidate the effect of increasing the osmolality of a basic Tris, extender supplemented with sucrose, trehalose or raffinose on post-thawing ram semen quality (sperm motility, viability, acrosome integrity, total sperm abnormalities and membrane integrity). After primary evaluation of the collected ejaculates, only semen samples with more than 70% motile sperm, and a sperm concentration of higher than 3 × 10⁹ sperm/ml were used for cryopreservation. The semen samples were pooled and diluted (1:4) with a Tris-citric acid-fructose-yolk extender, supplemented with different concentrations (50, 70 or 100 mM) of sucrose, trehalose or raffinose. As control, semen was diluted and frozen in the base diluent, without additional sugars. Pooled semen samples were aspirated into 0.25 ml straws, cooled to 5 °C within 90 min and frozen by exposure to liquid nitrogen vapor (4-5 cm above the liquid nitrogen surface) for 10 min - before plunging into liquid nitrogen, for storage. After 24 h, straws were thawed in a water bath (37 °C) for 30 s. The frozen-thawed sperm characteristics were improved significantly (P < 0.05) by increasing the level of the sugars. Optimal results being obtained with 70 and 100 mM trehalose or raffinose. All extenders containing supplemental sugars were superior in terms of sperm quality to the control (P < 0.01) group. The highest sperm motility (60.6 ± 1.9%), viability (60.6 ± 2.5%) and membrane integrity (58.2 ± 2.1%) were recorded using 100 mM trehalose and the lowest with 50 mM sucrose (48.6 ± 1.9%, 51.4 ± 2.5% and 47.9 ± 2.1%, respectively). All sugar concentrations decreased the percentage of acrosomal and total sperm abnormalities (P < 0.05). The extenders containing 100 mM trehalose or raffinose significantly (P < 0.05) decreased the occurrence of sperm abnormalities, compared to the other treatments. The fertility rates obtained after cervical insemination of the frozen-thawed sperm were 46.8%, 44.1% and 16.7% for 100 mM trehalose, 100 mM raffinose and the control with supplementation of the diluents, respectively. The study showed that ram sperm can tolerate hyperosmotic diluents, and that a range of sugar concentrations (50-100 mM) may successfully be incorporated in the ram semen cryopreservation diluents, although further research is warranted.     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves,straw sizes,and thawing rates

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (−10 °C/min) or a fast (−40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8 s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P < 0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P > 0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P < 0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P > 0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples thawed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P < 0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.     

Successful artificial insemination using semen frozen and stored by an ultrafreezer in the Majorera goat breed

The semen of five Majorera breed bucks was collected and processed to reach a final concentration of 200 × 10⁶ spermatozoa/straw in the extender containing 4% of glycerol and 12% of egg yolk. Two freezing techniques were assessed: (LN) straws were frozen and stored in liquid nitrogen, and (ULF) straws were frozen and stored in the ultra-low freezer at −152 °C. Semen quality (sperm motility, acrosome integrity and abnormal sperm cells percentages) was determined for different storage times (1, 30, 90 and 365 days of cryopreservation). Thereafter, 150 Majorera goats were assigned to four experimental groups: for groups LN-1 (n = 40) and LN-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in liquid nitrogen, respectively, while for groups ULF-1 (n = 40) and ULF-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in an ultra-low freezer at −152 °C, respectively. The pregnancy rate was determined by transabdominal ultrasound scanning; in addition, the kidding rate and prolificacy were recorded at parturition. In vitro results showed that the freezing protocol did not affect sperm quality with similar values for up to 1 year of cryopreservation. The kidding rates were not significantly different between experimental groups (43.6%, 38.5%, 42.8% and 40.0% for groups LN-1, ULF-1, LN-6 and ULF-6, respectively). In all experimental groups, the kidding rate and prolificacy were significantly higher (p < 0.01) in multiparous than in nulliparous goats. Therefore, the in vitro results and fertility trials confirmed the efficiency of the ULF technique for freezing and storage of goat semen.     

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm

Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10⁵ spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p < 0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p < 0.05).     

Apoptosis in fresh and cryopreserved buffalo sperm

The objectives of present study were (a) validation of annexin V/PI assay for estimation of sperm apoptosis in buffalo (Experiment 1) and (b) determining the effect of stages of cryopreservation on sperm apoptosis and its correlation with sperm motility and plasma membrane integrity (Experiment 2). In Experiment 1, different levels of apoptosis were artificially induced in buffalo semen (100 × 10⁶ sperm/aliquot) through graded doses of camptothecin (5, 10 and 20 μM/aliquot). Higher concentrations of camptothecin (10 and 20 μM) successfully (P < 0.05) induced apoptosis as compared to the lower (5 μM) dose and/or control. In Experiment 2, semen samples (n = 9, three pooled semen samples from each of the three buffalo bulls separately) were cryopreserved using vapor freezing. The mean percentage of apoptotic, necrotic and viable sperm did not differ between fresh and before freezing stages. However, freezing and thawing increased (P < 0.05) the percentage of apoptotic sperm (25.4 ± 0.6 vs. 36.5 ± 1.9) while decreased (P < 0.05) the necrotic (35.1 ± 1.2 vs. 29.7 ± 0.7) and viable sperm (37.2 ± 1.3 vs. 32.8 ± 1.9, (P < 0.07). Likewise, the mean percent motility and plasma membrane integrity decreased (P < 0.05) (64 ± 2.1 vs. 49.4 ± 1.3) and (79.6 ± 0.5 vs. 38.7 ± 0.3) respectively, at post thaw compared to other stages. Coefficient of correlation, combined at all stages for each variable revealed that sperm apoptosis was inversely correlated with sperm motility and plasma membrane integrity. It is concluded that (a) the annexin V/PI assay can be used as a tool to determine the buffalo semen apoptosis and (b) freezing and thawing induces apoptosis in buffalo sperm.     

Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis)

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen-thawed semen quality of Nili-Ravi buffalo bulls. Ejaculated bull semen was extended in a Tris-citrate egg yolk extender containing various concentrations of BHT (0.5, 1.0, 2.0 and 3.0 mM). Semen was frozen at −196 °C using 50 × 10⁶ spermatozoa per 0.5 mL straws. Five straws from each treatment were thawed to assess the semen quality in terms of sperm motility, viability, plasma membrane integrity and acrosomal integrity. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability, plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining, hypo-osmotic swelling test and normal acrosomal reaction, respectively. The highest (P < 0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa was achieved by addition of 1.0 and 2.0 mM BHT to semen extender. However, highest (P < 0.05) viability of spermatozoa was achieved by inclusion of 2.0 mM BHT. The higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of the spermatozoa compared to other concentration used. In conclusion, BHT when added in the semen extender can improve the semen quality of buffalo bulls.     

Comparison of two different cryopreservation protocols for freezing goat semen

In this study, two different semen cryopreservation protocols were compared to freeze goat semen. The ejaculates (n = 12) were collected by using electro-ejaculator from six mature bucks (two ejaculates per each buck). Each ejaculate was divided into two groups as Protocol 1 (P1) and Protocol 2 (P2). In P1, semen was diluted directly in an extender containing 15% egg yolk, 300 mM Tris, 28 mM glucose, 95 mM citric acid 5% glycerol to a concentration of 200 × 10⁶ sperm/mL. In P2, after the removal of seminal plasma by centrifugation, the semen sample was diluted with the first portion of milk extender consist of 100 mg/mL skimmed milk powder and 27.75 mM glucose (without glycerol) to a concentration of 400 × 10⁶ sperm/mL. The second portion of the milk extender containing 14% glycerol was added to semen gradually in order to achieve sperm concentration 200 × 10⁶ sperm/mL and 7% glycerol level in the final volume. Extended semen was loaded in 0.25 mL straws, held for 2 h at 4 °C, frozen in nitrogen vapor and stored in liquid nitrogen. Post-thaw motility and live sperm rate (mean ± SEM) were significantly lower (P < 0.05) in P1 as compared to P2 (47.50 ± 1.23% vs. 55.63 ± 1.72%; 80.04 ± 1.29% vs. 84.04 ± 1.08%, respectively). However, live intact, total intact, abnormal, reacted acrosome and DNA damaged sperm rates were similar (P > 0.05) in both protocols. It was concluded that both protocols used in this study provided reasonable post-thaw parameters; however, P2 yielded better motility and live sperm rate compared to P1.     

A comparative study of freezing single cells and spheroids: Towards a new model system for optimizing freezing protocols for cryobanking of human tumours

Cryopreservation of human tumour cells and tissue is a valuable tool for retrospective analysis and for the transport and handling of biopsy material. Tumour tissue consists of different cell types, which have different optimal freezing conditions, and extracellular matrix. A well-defined and authentic model system is required for developing new freezing protocols and media. This work describes the use of L929 and PC-3 spheroids as new model systems for freezing human tumours. Cell suspension and spheroids were frozen in different vessels (1 ml cryovials and a special, cryo-compatible 30 × 25 μl multi well plate) at slow rate (1 °C/min). Freezing media were combinations of culture or tumour transport medium (Liforlab^®) with the cryoprotective agents, Me₂SO, trehalose and modified starch. We also present a new method of evaluating the viability of three dimensional multicellular systems to compare thawed spheroids objectively. Best viability (70%) of L929 spheroids occurred with a combination of Liforlab^® and starch hydrolysis product. The best cryopreservation results for spheroids were found with extracellular cryoprotectants, while optimum viability of single cells was achieved with Me₂SO.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios

This is the first study where the systematic application of theories and techniques used in mammalian sperm cryopreservation have been applied to honey bee (Apis mellifera L.) semen as a means to improve postthaw viability of cryopreserved sperm. Six newly designed diluents, three cryoprotectants (dimethyl sulfoxide, DMA, glycerol), and five diluent:semen ratios (1:1, 3:1, 6:1, 9:1, and 12:1) were tested. In addition, the sperm freezing tolerance of three honey bee strains was evaluated. Specific protocols were designed to control semen freezing and thawing rates. Sperm motility was assessed visually, whereas sperm viability was assessed using SYBR-14 and propidium iodide fluorescent stains. Diluent treatments did not affect fresh (nonfrozen) sperm viability yet affected fresh sperm motility (P < 0.05). Based on these assessments, two diluents were chosen and used in all successive cryopreservation experiments. Using the selected diluents, semen was collected at various diluent:semen ratios, along with one of the three cryoprotectants. Semen collected at high dilution ratios, using a hypotonic antioxidant diluent containing catalase, in combination with dimethyl sulfoxide, provided higher postthaw sperm viability than that of all other combinations tested (68.3 ± 5.4%; P < 0.05). Using this combination of dilution ratio, diluent, and cryoprotectant, there were no differences among honey bee strains for postthaw sperm viability (P = 0.805). Nevertheless, these new semen dilution and freezing methods improved postthaw viability of sperm to levels that could theoretically sustain worker populations in colonies, thus providing potential for further optimization of cryopreservation techniques for the genetic preservation and improvement of honey bee genotypes.     

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.     

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me₂SO), N,N-dimethylacetamide (DMA), N,N-dimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 °C; (2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me₂SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 °C/min) and were compared to Me₂SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ? 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ?10% which was significantly lower than that of methanol and ME. With Me₂SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ? 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.     

Quality evaluation of umbilical cord blood progenitor cells cryopreserved with a small-scale automated liquid nitrogen system

The performance of a small-scale automated cryopreservation and storage system (Mini-BioArchive system) used in the banking of umbilical cord blood (UCB) units was evaluated. After thawing the units, the viability and recovery of cells, as well as the recovery rate of hematopoietic progenitor cells (HPCs) such as CD34⁺ cells, colony-forming unit-granulocyte-macrophage (CFU-GM), and total CFU were analyzed. Twenty UCB units cryopreserved using the automated system and stored for a median of 34 days were analyzed. Mean CD34⁺ cell viabilities before freezing were 99.8 ± 0.5% and after thawing were 99.8 ± 0.4% in the large bag compartments and 99.7 ± 0.5% in the small compartments. The mean recovery values for total nucleated cells, CD34⁺ cells, CFU-GM, and total CFU were 94.8 ± 16.0%, 99.3 ± 18.6%, 103.9 ± 20.6%, and 94.3 ± 12.5%, respectively in the large compartments, and 95.8 ± 25.9%, 106.8 ± 23.9%, 101.3 ± 23.3%, and 93.8 ± 19.2%, respectively in the small compartments. A small-scale automated cryopreservation and storage system did not impair the clonogenic capacity of UCB HPCs. This cryopreservation system could provide cellular products adequate for UCB banking and HPC transplantation.     

Effect of oviductal proteins on sperm functions and lipid peroxidation levels during cryopreservation in buffaloes

A study was undertaken to find out the effect of addition of oviductal proteins on sperm functions and lipid peroxidation (LPO) levels in buffaloes. Oviductal flushings were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle), centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal and luteal oviductal fluid were precipitated overnight using ammonium sulphate, centrifuged (10,000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored frozen at -20 degrees C. Six pooled good quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was split into three parts and extended in Tris-Egg yolk-Citrate extender (20% egg yolk: 7% glycerol), so that final dilution yielded approximately 60 million sperm cells/ml and cryopreserved in 0.5 ml French straws (30 million sperm cells per straw) in LN2 (-196 degrees C). Before freezing, the nonluteal and luteal oviductal proteins (NLOP &LOP) were incorporated at the concentration of 1mg/ml of extended semen. The equilibrated and frozen thawed (37 degrees C for 30s) semen was evaluated for motility, viability and acrosomal integrity, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides these tests, LPO level was assessed in sperm and seminal plasma in equilibrated and frozen thawed semen. Results revealed that addition of oviductal proteins to semen before freezing convey beneficial effect in terms of spermatozoan motility, viability and acrosomal integrity. Nonluteal oviductal proteins favored significantly (P < 0.05) higher sperm penetration distance in cervical mucus (23.00+/-1.15 mm) than the control group (15.00+/-3.46 mm) in frozen thawed semen. Similarly, swollen sperm percentage was also significantly (P < 0.05) higher in NLOP treated group than the LOP included and control groups. In frozen thawed spermatozoa, the LPO level was significantly (P < 0.05) lower in NLOP added group than the LOP added and control group. It was inferred that incorporation of oviductal proteins in extender before freezing reduced the lipid peroxidation levels in buffalo spermatozoa during cryopreservation and thereby improved the post-thaw semen quality.     

Biophysics of zebrafish (Danio rerio) sperm

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (V_o) of 12.1 ± 0.2 μm³ (SEM), a water permeability (L_p) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) μm/min/atm, and a cryoprotectant permeability (P_s) of 0.10 ± 0.01 (SEM) × 10⁻³ cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 °C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.     

Cryopreservation of Acropora digitifera sperm with use of sucrose and methanol based solution

Coral biodiversity has recently been considered an important topic in environmental studies. Biodiversity could be preserved with successful cryopreservation of endangered species gametes or embryos. Herein, we developed cryopreservation protocols for Acropora digitifera sperm with use of sucrose and methanol based extender. We studied cryopreservation of A. digitifera sperm with floating frames, allowing the placement of 250 μl French straws 4 cm above the liquid nitrogen surface, resulting in a 40 °C/min freezing rate. This method enabled the successful cryopreservation of sperm in 0.9 M sucrose supplemented with 20% methanol. In this protocol, we used a 1:3 (sperm:extender) dilution ratio. The fertilization ratios of freezing:thawed sperm were similar to the control and reached 63%. This method might be a valuable option in the formation of A. digitifera gene banking. Further studies are needed to explore possibilities of using this method in cryopreservation of other coral’s sperm.