Viability Of Frozen-thawed Spermatozoa Research Articles

The Beneficial Effect of Resveratrol on the Quality of Frozen-Thawed Boar Sperm.

This study aimed to determine the effect of resveratrol and its optimal concentration on the quality of frozen-thawed (FT) boar sperm. Semen ejaculates were obtained from 13 Duroc boars aged between 1.5 and 3 years. The sperm sample was separated into 7 groups based on the concentrations of resveratrol in the freezing extender, which were 0 (control), 25, 50, 75, 100, 125, and 250 µM, respectively. The sperm was frozen using liquid nitrogen vapor and thawed at 50 °C for 12 s. After thawing, total motility, progressive motility, viability, intact acrosomes, mitochondrial membrane potential and level of MDA were assessed. The supplementation of 50-100 µM resveratrol improved the sperm motility and viability of FT sperm in comparison to the control group (p < 0.05). Furthermore, the 50 µM resveratrol group was significantly more protective than the control group in terms of intact acrosome, mitochondrial membrane potential, and level of MDA (p < 0.05). Nonetheless, the detrimental effect of resveratrol was found at a concentration of 250 µM. In conclusion, the addition of 50-100 µM resveratrol to a freezing extender is the optimal concentration for enhancing the quality of cryopreserved boar sperm.

Protein signatures of seminal plasma from bulls with contrasting frozen-thawed sperm viability

The present study investigated the seminal plasma proteome of Holstein bulls with low (LF; n = 6) and high (HF; n = 8) sperm freezability. The percentage of viable frozen-thawed sperm (%ViableSperm) determined by flow cytometry varied from -2.2 in LF to + 7.8 in HF bulls, as compared to the average %ViableSperm (54.7%) measured in an 860-sire population. Seminal proteins were analyzed by label free mass spectrometry, with the support of statistical and bioinformatics analyses. This approach identified 1,445 proteins, associated with protein folding, cell–cell adhesion, NADH dehydrogenase activity, ATP-binding, proteasome complex, among other processes. There were 338 seminal proteins differentially expressed (p < 0.05) in LF and HF bulls. Based on multivariate analysis, BSP5 and seminal ribonuclease defined the HF phenotype, while spermadhesin-1, gelsolin, tubulins, glyceraldehyde-3-phosphate dehydrogenase, calmodulin, ATP synthase, sperm equatorial segment protein 1, peroxiredoxin-5, secretoglobin family 1D and glucose-6-phosphate isomerase characterized the LF phenotype. Regression models indicated that %ViableSperm of bulls was related to seminal plasma peroxiredoxin-5, spermadhesin-1 and the spermadhesin-1 × BSP5 interaction (R2 = 0.84 and 0.79; p < 0.05). This report is the largest dataset of bovine seminal plasma proteins. Specific proteins of the non-cellular microenvironment of semen are potential markers of sperm cryotolerance.

Improvement of cryopreservation protocol in both purebred horses including Spanish horses

There is a widely held belief that the semen of Purebred Spanish Horses (PRE) is of generally poorer quality than that of other breeds, and survives cryopreservation less well. To determine whether this is the case, sperm concentration, viability and morphological abnormalities were examined in a total 610 fresh ejaculates from 64 healthy PRE (N=47) and non-PRE stallions (N=17). Sperm concentration and viability were then re-examined after pre-freezing centrifugation, and once again after freezing-thawing. No differences were observed between the PRE and non-PRE stallions in terms of any sperm quality variable at any observation point. When considering all PRE and non-PRE samples together, differences in sperm viability were observed between fresh and fresh-centrifuged sperm viability (70.1±12.5% compared to 76.3±10.9%; p&lt;0.01). After centrifugation the samples were also more homogeneous in terms of the total number of recovered sperm cells. Centrifugation also improved frozen-thawed sperm viability, reducing differences in sperm quality between individual stallions. For all centrifugations, a sperm:extender ratio of 1:5 was used. This would appear to provide better final results than those reported in the literature for the 1:1 ratio commonly used for PRE stallion sperm cryopreservation. In conclusion, obtained results show that the quality and frozen/thawed results of PRE stallion sperm are not lower than that of non-PRE breeds. In addition, using a 1:5 sperm:extender dilution ratio when selecting sperms by centrifugation prior to freezing, seems to provide better results than those usually reported when using a 1:1 ratio.

Factors affecting storage of Slovak native rabbit semen in the gene bank.

In this study, fresh and frozen-thawed semen of Nitra and Zobor rabbit breeds were evaluated for potential inter-breed or inter-male differences in sperm quality traits. Individual male semen from four rabbits of each breed were diluted (v:v; 1:1) in a freezing medium composed of a commercial diluent, 16% of dimethyl sulphoxide (DMSO), 4% of Ficoll 70 and 2% of sucrose and frozen in liquid nitrogen vapours before being plunged into liquid nitrogen. Different motility traits, viability and plasma membrane integrity of fresh and frozen-thawed semen were evaluated in vitro using computer-assisted sperm analysis and flow cytometry. To evaluate the sperm fertilization ability, artificial insemination of fresh and frozen-thawed sperm was performed. Our results showed the effect of breed (P ≤ 0.05) on frozen-thawed sperm viability and plasma membrane integrity. Moreover, individual variability in semen quality among the rabbits was revealed (0.31 to 0.71 among quality traits). Our results thereby confirmed that the cryopreservation procedure could not ensure comparable sperm post-thaw survival for different breeds or males. Nevertheless, correlations between numbers of fresh total motile and progressively moving sperm and several quality parameters measured post thawing were revealed. Therefore, we suggest that the objective assessment of fresh rabbit sperm motility may be an effective indicator of frozen-thawed semen quality. Consequently, regular semen assessment is required in order to preserve good-quality insemination doses from native breeds.

Cryopreservation of epididymal semen of domestic cat

Domestic cat (Felis silvestris catus) is used as a model species for developing effective methods of wild felids’ semen cryopreservation. The present study represents a comparison of domestic cat semen cryopreservation with two commercially available cryoprotectant agents (CPAs): CaniPlus Freeze (CPF) and SpermFreeze (SF). Semen was collected from the caudal epididymises of adult males and frozen with CPF and SF, correspondingly. The viability of frozen-thawed spermatozoa was evaluated by VitalScreen kit, staining with hematoxylin and eosin was performed for analysis of the spermatozoa morphology; both analyses were combined with the light microscopy. The viability rate of the frozenthawed semen cryopreserved with CPF and SF did not differ: 32.3±4.4 % for CPF and 43.3±4.0 % for SF. Total percentage of morphologically normal spermatozoa after freezing and thawing domestic cat semen was 26.0±2.3 % for CPF and 23.9±1.9 % for SF. In both cases, there were no differences from non-frozen semen, in the latter group the total percentage of spermatozoa with normal morphology was 29.0±4.1 %. The most frequent anomalies were the anomalies of tail, and the rarest anomalies were head defects. The percentages of spermatozoa with anomalies of the head, mid piece, tail and combined did not differ in these three groups. Taken together these results suggest that both CPAs are suitable for the purpose of domestic cat semen freezing and cryopreservation, although CPF was designed for Canidae semen cryopreservation and SF was developed for human semen freezing and so far was used exclusively in reproductive medicine. It might be concluded that these two commercially available cryoprotectant media are applicable for the purposes of domestic cat breeds’ semen cryopreservation.

Investigations of motility and fertilization potential in thawed cryopreserved mouse sperm from cold-stored epididymides

Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48h maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72h, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.

The in vitro quality of frozen-thawed Asian elephant (Elephas maximus) spermatozoa in semen supplemented with Equex STM paste and oxytocin during and after cryopreservation

The effects of Equex STM paste (Equex) and oxytocin (OT) on the in vitro quality of frozen-thawed Asian elephant sperm were investigated in the study. The viability of frozen-thawed sperm was significantly higher in the Equex-treated (1 and 2%) groups than in the control group. There were no differences in the examined sperm parameters among the control and OT-treated (0.05–5IU) groups.

Osmolarity of Coconut Water (Cocos nucifera) Based Diluents and their Effect Over Viability of Frozen Boar Semen

Problem statement: Boar sperm cells are sensitive to the freezing process, which compromises viability of frozen-thawed sperm. In a constant search for minimizing or suppressing sperm cell damage caused by the temperature and osmolarity changes during the freezing process, crioprotective and antioxidant substances have been added to the freezing media, such as coconut water, in order to increase the viability of frozen-thawed swine semen. The addition of any substance to the freezing diluent, directly affects osmolarity of the media, which can have positive or negative effects over the sperm cell. Approach: There are no published studies currently that indicate the effect of adding coconut water over the osmolarity of freezing media and their effect over viability of sperm cells, therefore, the objective of the present study was to evaluate the effect of coconut water based diluents osmolarity over the Motility (Mot), Acrosome Integrity (AI), Membrane Integrity (MI) and Mitochondrial Activity (MA) of thawed boar sperm cells. The treatments used were control T1 (LEY with bidistilled water + LEYGO) with an osmolarity range of 296-368 mOsmol Kg-1, T2 (LEY and deionized coconut water + LEYGO) between 381 and 480 mOsmol Kg-1 and T3 (LEY and in natura coconut water + LEYGO) between 519 and 1041 mOsmol Kg-1. The Westendorf modified method was the freezing method used. The obtained data were statistically analyzed by GLM, using the SAS software (SAS, 2000). Results: A significant difference was observed on T2 compared to T1 regarding Mot 41.9 Vs 36.9% and MI 58.0 Vs 50.2. T3 had a detrimental effect on all studied variables. Conclusion: Under our study conditions, the osmolarity range of T2, due to the non ionic solutes content, contributed to improve the viability of frozen-thawed sperm cells.

Effects of exposure of pre-pubertal boars to di(2-ethylhexyl) phthalate on their frozen-thawed sperm viability post-puberty

Late effects of pre-pubertal oral exposure to di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in, for example, polyvinyl chloride-products, on semen quality in young boars have not been clear-cut. The aim of this study was to determine whether stress imposed on spermatozoa would reveal such effects. Semen was collected from post-pubertal boars (8-9 months of age), which had been exposed to 300 mg kg(-1) body weight of DEHP per os three times a week from 3 to 7 weeks of age and from control siblings given placebo (water). The semen was cryopreserved and examined for plasma membrane integrity post-thaw using the short hypo-osmotic swelling test and flow cytometry (propidium iodide /SYBR-14). Sperm motility was assessed by computer-assisted sperm analysis. No significant difference in plasma membrane integrity could be found between the groups. The DEHP-exposed group had a significantly lower percentage of linearly motile spermatozoa at 30 min (P < 0.05) and 120 min (P < 0.001) after thawing, and a larger amplitude of lateral displacement of the head 120 min after thawing (P < 0.05), compared with controls. In summary, spermatozoa from boars pre-pubertally exposed to low doses of DEHP, showed kinematic deviations post-thaw that could be related to DEHP exposure.

Comparison of Semen Characteristics, Frozen-Thawed Sperm Viability, Testosterone Concentration and Embryo Development between Yorkshire Boar A and B

This study was carried out to compare the semen characteristics, frozen-thawed sperm viability and testosterone concentration and in vitro fertilization (IVF) and development of in vitro matured pig oocytes between two Yorkshire boars. Semen and blood samples were collected once per week from October to November 2002 from two adult Yorkshire boars at 18 months of age with 170 kg body weight. Sperm were deep frozen in 5 ml maxi-straws with lactose-egg yolk and N-acetyl-D-glucosamine (LEN) diluent and stored in liquid nitrogen. Blood samples were obtained at 10 a.m. by inserting a 21 gauge, hypodermic needle attached to 10 ml syringe into surface veins in the ear. The concentration of testosterone was determined by Competitive Enzyme Immunoassay. Ovaries were collected from prepubertal gilts at a local slaughter house. Cumulus oocyte complexes were aspirated from antral follicles (3 to 6 mm in diameter). The medium used for oocyte maturation was modified TCM 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5°C, 5% CO2 in air. For IVF, one frozen 5 ml straw was thawed at 52°C in 40 sec and was diluted with 20 ml Beltsville thawing solution at room temperature. Sperm were washed 2 times in mTLP-PVA and inseminated without preincubation after thawing. Oocytes were inseminated with 2×10 7 /ml sperm concentration. Oocytes were coincubated for 6 h in 500 µl mTBM fertilization medium. At 6 h after IVF, oocytes were transferred into 500 µl NCSU-23 culture medium for further culture of 48 and 144 h. There were no significant differences in the semen volume, motility, normal acrosome morphology and sperm concentration of raw semen between A and B of Yorkshire boar. However, motility and normal acrosome of boar A were higher than those of boar B at 0.5, 2, 3, 4, 5 and 6 h incubations of frozen-thawed sperm. Testosterone concentration (3.75 ng/ml) of boar A was higher than that (2.34 ng/ml) of boar B. The rate of blastocyst formation (15.1%) of boar A was higher than that (10.4%) of boar B. In conclusion, serum testosterone concentration of boar showed very important role for the frozen-thawed sperm viability and the blastocyst formation of pig oocytes matured in vitro. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 5 : 612-616)

Comparison of semen characteristics, sperm freezability and testosterone concentration between Duroc and Yorkshire boars during seasons

This study was carried out to investigate the effects of Duroc and Yorkshire boars and seasons influencing semen characteristics, frozen-thawed sperm viability and serum testosterone concentration. Yorkshire boars produced higher semen volume compared to Duroc boars among seasons. However, sperm concentration did not differ significantly between Duroc and Yorkshire boars among seasons. Sperm motility of frozen-thawed sperm in Yorkshire boars was higher than in Duroc boars in spring and summer. Normal acrosome of frozen-thawed sperm in Yorkshire boars was higher than in Duroc boars in winter. Serum testosterone concentrations in Duroc and Yorkshire boars were higher in spring than in summer, autumn and winter. Serum testosterone concentrations in spring and summer were higher in Yorkshire boars than in Duroc boars. In conclusion, when serum testosterone concentrations were higher in Duroc and Yorkshire boars among seasons, semen volume, sperm concentration and frozen-thawed sperm viability were higher.

Effect of Season Influencing Semen Characteristics, Frozen-Thawed Sperm Viability and Testosterone Concentration in Duroc Boars

This study was carried out to investigate the effects of season influencing semen characteristics, frozen-thawed sperm viability and testosterone concentration in Duroc boars. There were no significant differences in the semen volume and sperm concentration of Duroc boars among spring, summer, autumn and winter. However, the pH of sperm-rich and sperm-poor fractions in autumn and winter season was higher than in spring and summer season in Duroc boars. Sperm motility and normal acrosome of raw semen in Duroc boars did not differ significantly among spring, summer, autumn and winter. However, motility and normal acrosome of frozen-thawed sperm were higher in spring season than in summer, autumn and winter. Serum testosterone concentrations in Duroc were higher in spring than summer, autumn and winter. In conclusion, when serum testosterone concentrations were higher in seasons, frozen-thawed sperm viability in Duroc boars were higher. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 4 : 500-503)

Semen collection, characterization, and cryopreservation in a Magellanic penguin ( Spheniscus magellanicus )

A cooperative method was developed for collecting semen from a Magellanic penguin. Ejaculate parameters and semen production during a breeding season were characterized. Experiments were performed to study the effect on penguin spermatozoa of two temperatures (4°C and 21°C) for short-term storage, and two cryoprotectants (dimethylsulfoxide [DMSO] and ethylene glycol [EG]) for long-term storage (cryopreservation). All dilutions were made using modified Beltsville Poultry Semen Extender. Sperm quality was assessed by evaluating motility and forward progression (sperm motility index [SMI]), viability, and morphology. A total of 39 ejaculates was collected over the 40-day study period. Thirty-eight ejaculates contained spermatozoa, but semen quality decreased toward the end of the study period. Varying levels of urate contamination were present in all ejaculates. Sperm quality parameters were similar for diluted samples held at 4°C and 21°C, and samples maintained high numbers of viable (77.8 ± 5.4%) and morphologically normal (67.9 ± 2.5%) spermatozoa at 3 hr. SMI and percentage of viable sperm decreased (P 0.05). Both SMI and viability of frozen-thawed spermatozoa were higher (P < 0.05) for clean than for contaminated ejaculates. This is the first report on penguin ejaculate parameters, semen production, and preliminary methods for short- and long-term semen storage. Zoo Biol 18:199–214, 1999. © 1999 Wiley-Liss, Inc.