Ultrastructural and cytochemical characterization of follicular cell types in bovine (Bos taurus) cumulus-oocyte complexes aspirated from small and medium antral follicles during the estrus cycle

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.     

Effects of water-soluble Laminaria japonica polysaccharide 3 (LJP-P3) on bull cryopreservation sperm

In this study, water-soluble Laminaria japonica polysaccharide3 (LJP-P3) was investigated for the cryoprotective effects on bull sperm. Five concentrations of LJP-P3 with 0.1, 1, 10, 50 and 100 mmol/L were added into the extenders of bull semen, respectively, and the effects on quality of sperm after freezing-thawing were assessed. The results showed that the kinematic parameters of bull sperm including linear motile sperm (LM), curvilinear line velocity (VCL) value, straight line velocity (VSL) and velocity of the average path (VAP) were greater in the extenders containing LJP-P3 (P<0.05). In comparison to those of other treatments and control group the extenders containing 1.0, 10.0 and 50.0 mmol/L of LJP-P3 led to higher percentage of mitochondrial activity and sperm membrane integrity(P<0.05), and the acrosome integrity of bull cryopreservation sperm were significantly improved in all treatment groups. Moreover, the higher GSH-Px, SOD and CAT levels in bull cryopreservation sperm were favored from the extenders of 10.0, 50.0 and 100.0 mmol/L LJP-P3 added (P<0.05) compared with other treatments and control group. In addition, the results of artificial insemination showed that both the pregnancy rate and the number of calving were higher in the group of semen containing 10 mmol/L of LJP-P3 than that of control group (P < 0.05). In summary, LJP-P3 exhibited a greater cryoprotective effect to bull sperm and the most suitable concentration of LJP-P3 is 10.0 mmol/L.     

Effect of relaxin on cryopreserved beef bull semen characteristics

This study aimed to improve the quality of cryopreserved beef bull (Piedmontese) semen by incorporation of relaxin in diluted semen before cryopreservation procedures. Semen samples were collected from 4 proven fertile bulls, using artificial vagina, once per week for 8 consecutive weeks and pooled together then diluted with Bullxcell® extender, and supplemented with different concentrations of relaxin (0 (control), 25, 50 and 100 ng/ml) before cooling, equilibration and freezing procedures. Frozen semen was thawed and assessed for motility by Computer-Assisted Sperm Analysis and vitality parameters such as acrosome, plasma membrane and DNA integrities, apoptosis, mitochondrial membrane potential, mucus penetration and SOD activity. The developmental potential of bovine embryos produced in vitro by using relaxin-treated was also investigated. In the present study, 50 and 100 ng/ml relaxin incorporation in extended bull semen before cryopreservation induced a reduction of sperm motility immediately after thawing (0h), whereas, during long incubation periods (1–2 h), relaxin showed a significant positive effect on sperm quality by improving the sperm motility and velocity parameters. Interestingly, sperm vitality was improved by 25 and 100 ng/ml relaxin and the blastocyst developmental rate was significantly increased in the 25 ng/ml relaxin group compared with controls (52/118, 44.0% vs. 32/116, 27.6%, respectively). These findings suggest a potential use of relaxin at the doses tested in the present study as an additive in the cryopreservation media of bull semen to improve sperm quality.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi

Low survival of cryopreserved sperm impedes the application of cryopreservation technique in spermcasting oyster species. This study developed a simple method of liquid nitrogen vapor freezing to improve post-thaw sperm survival in the spermcasting oyster Ostrea angasi. The results indicate that the permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were non-toxic to sperm up to 20% concentration and 90 min exposure whereas methanol at 10% or higher was toxic to sperm for any exposure over 30 min. Among the treatments with permeable cryoprotectants, 15% EG produced the highest post-thaw sperm motility. Sperm motility was further improved by the addition of non-permeable cryoprotectants (trehalose and glucose), with 15% EG + 0.2 M trehalose resulting in the highest post-thaw sperm motility among all the combinations evaluated. The durations of 20, 30 and 60 min equilibrations produced a higher post-thaw sperm motility and plasma membrane integrity (PMI) than 10 min. Higher post-thaw motility and PMI were achieved by freezing sperm at the 8 cm height from the liquid nitrogen surface than at the 2, 4, 6, 10 or 12 cm height. Holding sperm for 10 min in liquid nitrogen vapor produced higher post-thaw motility and PMI than for 2, 5 or 20 min. The cryopreservation protocol developed in this study improved both post-thaw motility and PMI of O. angasi sperm at least 15% higher than those cryopreserved using programmable freezing method. Liquid nitrogen vapor freezing might have greater applicability in improving post-thaw sperm quality of spermcasting oyster species.     

Comparative cryopreservation of avian spermatozoa: benefits of non-permeating osmoprotectants and ATP on turkey and crane sperm cryosurvival

A comparative approach was used to evaluate the cryosurvival of turkey and crane sperm frozen in a dimethylacetamide (DMA) cryodiluent supplemented with osmoprotectants and ATP. A range (6-26%) of DMA concentrations was used alone or in combination with ATP (30, 60 or 118mM) or one of the following osmoprotectants: (1) sucrose (turkey, 8.0%; crane, 5.0%); (2) 5.0% sucrose and 5.0% trehalose; or (3) betaine hydrochloride (0.1, 0.2 or 0.4mM). The viability of thawed sperm was assessed using the nigrosin-eosin stain and sperm motility was determined using the hanging-drop technique. For semen frozen only with DMA, post-thaw sperm motility was greatest (P<0.05) for the 6.0%, 10.0% and 18% concentrations, regardless of species. Turkey sperm frozen with the sucrose/trehalose combination had greater (P<0.05) post-thaw motility for all DMA treatments compared to DMA alone. The lowest concentration of the osmoprotectant betaine hydrochloride substantially improved turkey sperm viability post-thaw in all treatments compared to DMA alone (P<0.05). The post-thaw motility of crane sperm was improved (P<0.05) with a combination of 18.0%, 24.0% or 26.0% DMA and 30mM ATP. Moreover, in the presence of osmoprotectants, crane sperm motility decreased as the osmoprotectant concentration increased. The lowest concentration of ATP also improved crane sperm viability post-thaw, especially for DMA concentrations 18% or greater. The combination of sucrose and trehalose improved (P<0.05) crane sperm viability only with 6% and 10% DMA. These data affirm that there are avian-specific differences in sperm survival after cryopreservation and suggest that post-thaw survival can be enhanced by including species-based osmoprotectant/ATP combinations in a cryodiluent where DMA is the cryoprotectant.     

Cooling and freezing of epididymal sperm in the common hippopotamus (Hippopotamus amphibius)

Knowledge concerning reproduction in common hippopotamus is scarce and in particular very little is known about male reproductive physiology and sperm cryopreservation. Testes were obtained from nine castrated bulls and sperm extracted from the epididymides of eight of these individuals. Mean ± SEM values of reproductive parameters were: testicular weight (including epididymis and tunicas)—275.9 ± 54.1 g, total sperm motility—88.1 ± 4.2%, total cells extracted—11.0 ± 3.6 × 10⁹, intact acrosome—87.7 ± 1.8%, intact sperm morphology—51.6 ± 4.1%, and, for 3 individuals, hypoosmotic swelling test for membrane integrity—83.3 ± 1.8%. Chilled storage extenders tested were Berliner Cryomedium (BC), Biladyl^®, modification of Kenney modified Tyrode's medium (KMT), and Human Sperm Refrigeration Medium (HSRM). Extender had significant effect on post-dilution motility and motility and intact morphology after 4h and 24h at 4°C (P ≤ 0.007 for all). Berliner Cryomedium and HSRM were superior to Biladyl^® and KMT. Freezing extenders tested were BC with either 6% dimethyl sulfoxide (Me₂SO), or 5%, 7%, or 10% glycerol. Post-thaw motility was < 5% in 3/7 bulls in all extenders. When frozen in BC with 6% Me₂SO, one bull had 15% post-thaw motility and 3/7 had 20 to 60%. In glycerol, 3/7 had 15-30% post-thaw motility in 5%, 2/7 in 7%, and 1/7 in 10%. The extender had significant effect on post-chilling motility (P = 0.008), post-thaw morphology (P = 0.016), and motility 30 min after thawing (P = 0.015). Berliner Cryomedium with 6% Me₂SO or 7% glycerol were the freezing extenders of choice. Information obtained in this study allows initiation of cryobanking of sperm from the common hippopotamus which is of particular importance for genetically valuable individuals.     

Mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality

Human spermatozoa cryopreservation is an important means of assisted reproductive technology and male fertility preservation. Although this technique is particularly useful, sperm cryopreservation significantly reduces the quality of spermatozoa after freezing and thawing. The objective of the study is to examine the efficacy of mitochondria-targeted antioxidant MitoTEMPO in improving sperm quality during semen cryopreservation processes. Semen samples were collected and cryopreserved in extenders containing different concentrations (0.0, 0.5, 5, 50, and 500 μM) of MitoTEMPO. Sperm motility, viability, membrane integrity, mitochondrial membrane potential and antioxidant activities were measured and analyzed. The results showed that the addition of MitoTEMPO (5–50 μM) significantly improved post-thaw sperm motility, viability, membrane integrity and mitochondrial membrane potential (P < .05). Meanwhile, antioxidant enzymes activities were enhanced and MDA content were decreased in the group supplemented with MitoTEMPO. In conclusion, mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality and antioxidant enzymes profile.     

Elamipretide as a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation

As a technique widely used in assisted reproduction, human spermatozoa cryopreservation makes it possible to conserve functional sperm for a long time, but the impact of cryodamage on sperm during the process could not be ignored. The objective of the present study was to investigate the efficacy of Elamipretide, a novel small mitochondrial targeting short cytoprotective peptide, in attenuating cryodamage during spermatozoa cryopreservation. Semen samples were collected and cryopreserved in freeze solution containing different concentrations (0.0, 0.1, 1, and 10 μM) of Elamipretide. Sperm motility, viability, membrane integrity, mitochondrial membrane potential, DNA fragmentation, antioxidant profiles, and acrosome reaction were measured and analyzed. The results showed that supplementation of the freeze media with Elamipretide (1 and 10 μM) significantly improved post-thaw sperm parameters including motility and viability, stability of the plasma membrane, and mitochondria and chromosomes. In addition, by adding Elamipretide, excessive oxidation and acrosome dysfunction in sperm cells undergoing freeze-thaw were also significantly attenuated. Therefore, Elamipretide may be a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation.     

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: Effects of extender supplemented with different antioxidants on sperm motility,velocity and fertility

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), l-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), l-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), β-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, l-methionine, SOD, l-carnitine, α-tocopherol and l-reduced glutathione (p < 0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, l-methionine, SOD, α-tocopherol and l-reduced glutathione (p < 0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 μm s⁻¹ swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 °C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 °C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 × 10⁶/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 °C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.     

The effect of nocturnal sampling on semen quality and the efficiency of collection in bovine species

This study evaluated night and day semen collection regimes in Holstein and Brahman bulls (four bulls of each breed) that were collected weekly, each during a morning and a night collection. Ejaculates (n=64) were obtained via artificial vagina over 4 weeks. The first collection of each week alternated between night and day. Two collection teams were employed. Bull behavior parameters included reaction time to first mount, time to ejaculation, a refractory period test, and a thrust intensity test. The numbers of interruptions were counted as a managerial parameter. Pre-freeze semen parameters included total volume, initial motility and concentration. Post-freeze semen parameters measured were: 0- and 3-h post-thaw motility; percent intact acrosomes; and percent sperm abnormalities. Data were analyzed by least squares methods. The bull within breed effect differed (P<0.05) for behavior parameters. The bull within breed effect for total motile sperm harvested was not significant. The bull within breed response was mixed for post-freeze semen viability parameters. Bull within breed was not significant for sperm abnormalities. The night versus day treatment was significant for the managerial parameter (P=0.002). Although a different collection schedule for Bos indicus cattle was not warranted, the efficiency of the collection process was affected by extraneous environmental conditions.     

Evaluation of Spermatological Parameters Used to Predict the Fertility of Frozen Bull Semen

Post-thaw motility, velocity and acrosome integrity of frozen semen were determined in 18 bulls with varying fertility (average non-return rates: 71.3 (± 2.8) - range: 65.2-75.7). Five semen straws were investigated from each bull. The average values for sperm motility (percentage motile spermatozoa), sperm velocity (graded from 0-3) and acrosome integrity (proportion of spermatozoa with intact acrosome) were 67.5%, 2.5 and 79.3%, respectively. Significant correlations were found between sperm motility and velocity, but not between sperm motility and acrosome integrity. Both sperm motility and velocity were significantly related to bull fertility. It was concluded that of the post-thaw semen characteristics investigated in this study these 2 parameters provided a reliable basis for prediction of bull fertility.     

Six novel single-nucleotide polymorphisms in SPAG11 gene and their association with sperm quality traits in Chinese Holstein bulls

Sperm-associated antigen 11 (SPAG11) is predominant in the male reproductive tract. Similar to β-defensin, aside from its antibacterial activity, SPAG11 also has an important role in male reproductive function. In the present study, the association of bovine SPAG11 gene polymorphism with sperm quality traits was examined, including ejaculate volume, sperm concentration, fresh sperm motility, post-thaw cryopreserved sperm motility, and deformity rate of bull semen. Six novel single nucleotide polymorphisms (SNPs) of the SPGA11 gene were investigated in 426 normal mature Chinese Holstein bulls using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), PCR-restriction fragment length polymorphism (PCR-RFLP), created restriction site-PCR (CRS-PCR), and DNA sequencing methods. Linkage disequilibrium analysis showed that g.1306G>A and g.1454G>A (SNP-1), and g.16904G>T, g.16974C>T, and g.17000A>G (SNP-2) are completely linked, respectively. Correlation analysis showed the SNP-2 marker had a marked effect on fresh sperm motility and sperm concentration (P<0.05). SNP-3 g.22696T>C had a marked effect on post-thaw cryopreserved sperm motility (P<0.05) and deformity rate (P<0.01). However, the presence of SNP-1 was not correlated with the sperm production traits (P>0.05). Furthermore, association analyses of the 8 haplotypes constructed from the 17 combined haplotypes and reproductive traits showed that the bulls with the combined haplotype H5H6 (GGT/TTC) have the highest ejaculate volumes and the bulls with combined haplotypes H1H1 (AAT/TTT) and H1H6 (AGT/TTC) had the highest fresh and post-thaw sperm motilities, respectively. These results indicate that new molecular markers associated with sperm quality traits can be used in marker-assisted selection in bull breeding programs.     

Effect of dialysis or centrifugation on post-thaw motility and fertility of Santa Gertrudis bull semen collected by electroejaculation

Electroejaculated semen from Santa Gertrudis bulls was used to study the effect of centrifugation (600 x g for 5 min) or dialysis [molecular weight cutoff <14,000 Daltons (Da)] on post-thaw motility and on fertility in beef cattle. Analysis of post-thaw motility showed that the main effects (bulls and semen treatment) were significant (P<0.05). Dialysis significantly improved post-thaw motility in four of seven bulls. Initial volume of seminal plasma in the ejaculate was negatively correlated to post-thaw motility (r = -0.73). No significant improvement in post-thaw motility was observed for bulls with high volumes of ejaculate. In a fertility trial, calving rates of heifers synchronized with PGF(2)alpha and inseminated at 72 and 96 h after the second PGF(2)alpha injection with dialyzed or commercial semen were not statistically different (P>0.05; 54.4% vs 55.4%). These results show that dialysis could be used to improve post-thaw motility of electroejaculated bull semen without altering its fertilizing capacity. However, a high initial volume of seminal plasma seems to have a deleterious effect on sperm freezability that cannot be reversed by dialysis.     

Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa

Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen–thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2 ± 5.4%, motility following 30 min incubation at 37 °C: 58.5 ± 6.0% and following 3 h incubation: 21.7 ± 7.6%, intact acrosome: 57.1 ± 5.2%, normal morphology: 52.0 ± 5.8% and viability: 67.3 ± 6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed.     

Improvement of the post-thaw qualities of Okinawan native pig spermatozoa frozen in an extender supplemented with ascorbic acid 2-O-alpha-glucoside

The technical establishment of boar sperm cryopreservation is indispensable for effective breeding of the scarce Okinawan native pig Agu. The objective of the present study was to determine whether ascorbic acid 2-O-α-glucoside (AA-2G), a stable ascorbate derivative, is capable of improving the quality of cryopreserved Agu spermatozoa. Ejaculated Agu sperm frozen in an extender supplemented with 0, 100, 200, 400 or 800 μM AA-2G was thawed, and then evaluated the sperm motility and other qualities. Treatment with 200 μM AA-2G has the most beneficial effect on the sperm motility and the plasmalemma integrity after frozen-thawing among the concentrations tested (P < 0.05). In particular, the incidences of total motile sperm and rapid progressive motility at 1 and 3 h after incubation were markedly increased by treatment with AA-2G at 200 μM. The addition of AA-2G during cooling and freezing efficiently protected spermatozoa against the lipid peroxidation and the DNA damage. Spermatozoa frozen in the presence of AA-2G possessed significantly higher levels (P < 0.05) of ATP even after thawing than those frozen without AA-2G, implying that sperm viability was effectively conserved. Furthermore, higher sperm penetrability to matured oocytes in vitro was maintained in sperm treated with AA-2G during cryopreservation. These effects were observed for all sperm derived from three individuals. These findings demonstrate that the addition of AA-2G to the freezing extender efficiently improves the post-thaw qualities of fragile Agu sperm through the protection of spermatozoa against cell damage caused by oxidative stress during cryopreservation.     

Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution

The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs–Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm.     

Effect of alpha-lipoic acid on oxidative stress,viability and motility in the common carp (Cyprinus carpio) spermatozoa after short-term storage and cryopreservation

As known for different metabolic functions, α-lipoic acid (ALA) has been tested for spermatozoa preservation of animals as well as of human, but not for fish spermatozoa. The present study determined the effects of ALA on short and long-term (cryopreservation) preservation of common carp (Cyprinus carpio) spermatozoa, for the first time. For that, spermatozoa were diluted in extenders containing 0 (control), 0.025, 0.05, 0.1, 0.5, 1, 2, 5, and 10 mM of ALA concentrations in both short-term preservation and cryopreservation. Spermatozoa motility parameters by computer-assisted semen analysis, viability, lipid peroxidation and catalase activity in spermatozoa were conducted in both 2nd and 120th hours of short-term storage and post-thaw samples. Higher percentages of total spermatozoa motility (80 ± 3) and viability (87 ± 3) were observed in 0.5 mM ALA group after 120 h of incubation. In post-thaw samples, higher percentages of these parameters were in 1 mM ALA group (74 ± 3 and 83 ± 2, respectively). Moreover, the results have shown that the addition of ALA until concentrations of 2 mM improved especially spermatozoa curvilinear velocity, maintained viability, and suppressed excessive lipid peroxidation during the preservations. In conclusion, the additions of 0.5 mM ALA for short-term preservation and 1 mM ALA for cryopreservation were the optimal concentrations, and shown the protective effects on common carp spermatozoa, when considering all measured parameters together.