New assays for detection and localization of endogenous lipid peroxidation products in living boar sperm after BTS dilution or after freeze-thawing

Reactive oxygen species have been implicated in sperm aberrations causing multiple pathologies including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries for semen storage prior to artificial insemination but unusual in porcine breeding industries as semen dilution and storage at 17 degrees C is sufficient for artificial insemination within 2-3 days. However, longer semen storage requires cryopreservation of boar semen. Freeze/thawing procedures induce sperm damage and induce reactive oxygen species in mammalian sperm and boar sperm seems to be more vulnerable for this than bull sperm. We developed a new method to detect reactive oxygen species induced damage at the level of the sperm plasma membrane in bull sperm. Lipid peroxidation in freshly stored and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous lipid classes, phosphatidylcholine and cholesterol and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY(581/591). Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in the living sperm cells. Cytoplasmatic droplets in incompletely matured sperm cells were intensely peroxidized. Furthermore, lipid peroxidation was particularly strong in the mid-piece and tail of frozen/thawed spermatozoa and significantly less intense in the sperm head. Induction of peroxidation in fresh sperm cells with the lipid soluble reactive oxygen species tert-butylhydroperoxide gave an even more pronounced effect, demonstrating antioxidant activity in the head of fresh sperm cells. Furthermore, we were able to show using the flow cytometer that spontaneous peroxidation was not a result of cell death, as only a pronounced subpopulation of living cells showed peroxidation after freeze-thawing. Although the method was established on bovine sperm, we discuss the importance of these assays for detecting lipid peroxidation in boar sperm cells.     

Lipid peroxide formation in relation to membrane stability of fresh and frozen thawed stallion spermatozoa

In this study we used a new method to detect reactive oxygen species (ROS) induced damage at the level of the sperm plasma membrane in fresh and frozen-thawed stallion sperm. Lipid peroxidation (LPO) in sperm cells was assessed by a fluorescent assay involving the labeling of stallion sperm with the LPO reporter probe C11-BODIPY(581/591). The peroxidation dependent spectral emission shift of this membrane probe could be localized using inverted spectral confocal microscopy and quantified on living and deteriorated sperm cells using flow cytometry. Mass spectrometric analysis of the main endogenous lipid class, phosphatidylcholine (PC), was carried out to determine the formation of hydroxy- and hydroperoxyphosphatidylcholine in fresh sperm cells. Peroxidation as reported by the fluorescent probe corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of LPO. This allowed us to correlate endogenous LPO with localization of this process in the living sperm cells. In absence of peroxidation inducers, only relatively little peroxidation was noted in fresh sperm cells whereas some mid-piece specific probe oxidation was noted for frozen-thawed sperm cells. After induction of peroxidation in fresh and frozen-thawed sperm cells with the 0.1 mM of lipid soluble ROS tert-butylhydrogen peroxide (t-BUT) intense probe oxidation was produced in the mid-piece, whereas the probe remained intact in the sperm head, demonstrating antioxidant activity in the head of fresh sperm cells. At higher levels of t-BUT, probe peroxidation was also noted for the sperm head followed by a loss of membranes there. Frozen-thawed sperm were more vulnerable to t-BUT than fresh sperm. The potential importance of the new assays for sperm assessments is discussed.     

Determination of glutation peroxidase and superoxide dismutase activities in canine seminal plasma and its relation with sperm quality and lipid peroxidation post thaw

Lipid peroxidation (LPO) of dog spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezeability. Innate levels of LPO were low in fresh spermatozoa but increased after thawing in one of the dogs included in our study. The level of lipid peroxidation in fresh spermatozoa was not correlated with that of thawed spermatozoa. Negative correlations were detected between the activity in seminal plasma of GPx and sperm velocities post thaw (P < 0.01), however SOD activity was positively correlated with the percentage of linear motile sperm post thaw (P < 0.05).     

Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing

In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.     

The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability

The Sperm Class Analyzer was used to investigate the effect of freeze-thawing procedure on Florida buck sperm head morphometry, and to relate possible changes in sperm head dimensions to cryopreservation success. Semen samples (n=76) were frozen with tris and milk-based extenders and thawed. Sperm quality samples (motility, morphology, acrosome), and sperm head morphometric values (length, width, area, perimeter, ellipticity) were compared between fresh and frozen-thawed samples. Sperm freezability was judged according to the sperm quality parameters assessed. Fertility data was obtained after artificial insemination with cryopreserved semen. Cryopreservation success was different between freezing methods. Sperm head dimensions were significantly (p<0.05) smaller in cryopreserved tris and milk spermatozoa respectively than in those of the fresh samples. The sperm head morphometric parameters that had changed after cryopreservation were lower in suitable semen samples after thawing and with successful pregnancies after artificial insemination. These data suggest that changes in sperm head morphometry might reflect spermatozoa injury occurred during cryopreservation.     

Iron-induced luminescence as a method for assessing lipid peroxidation of frozen-thawed goat spermatozoa

Freezing/thawing procedures induce enhanced reactive oxygen species (ROS) formation in mammalian sperm and these ROS may be a cause for the decrease in sperm function following cryopreservation. In the present study, we used a chemiluminescence method to detect ROS-induced damage in goat spermatozoa. Iron-induced luminescence of fresh and frozen/thawed sperm cells was assessed using a luminometer. It was shown that the freezing/thawing procedure had a significant effect on some luminescence parameters. Semen freezing significantly increased the values of integral, peak max, T.half (rise) and T.max (peak) parameters. A significant correlation was observed between the percentage of motile spermatozoa and integral, peak max and T.half (rise) parameters. In conclusion, the results of the present study indicate that measurement of induced luminescence can be an alternative, sensitive and relatively simple method for assessing the effect of cryopreservation on oxidative damage to spermatozoa.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.     

Positive effect of butylated hydroxytoluene (BHT) on the quality of cryopreserved cat spermatozoa

The semen cryopreservation processes are associated with state of oxidative stress induced by high levels of reactive oxygen species (ROS), causing damage to functional spermatozoa. Whereby, antioxidants have been utilized to scavenge or neutralize the elevated levels of ROS. The aim of at the present study was to evaluate the effect of adding BHT to the freezing extenders on post-thaw characteristics of domestic cat spermatozoa. Semen samples were frozen in Tris-fructose-citric acid-based extender, supplemented with different concentrations of BHT (0.5 mM, 1.0 mM and 2.0 mM) and a control sample without antioxidant. After thawing, sperm samples were assessed for motility by computer‐assisted sperm analysis and viability, acrosome integrity, superoxide anion production and membrane lipid peroxidation status by flow cytometry. In the study, the parameters of sperm motility and acrosome integrity were significantly higher in 2.0 mM BHT compared to sperm frozen in the extender with other concentrations and control (P < 0.05), in addition, this concentration reduced significantly the superoxide anion production and lipid peroxidation of the sperm. The results demonstrated that the supplementation of BHT to the freezing extender could protect the function and cellular structure of domestic cat sperm from cryoinjuries.     

Detection of lipid peroxidation in frozen-thawed avian spermatozoa using C(11)-BODIPY(581/591)

The aim of this study was to perform flow cytometric analysis of C11-BODIPY581/591 oxidation in fowl and geese sperm as a marker for membrane lipid peroxidation (LPO) and to establish if the cryopreservation process would make sperm membranes more susceptible to oxidative stress. The experiment was carried out on 10 meat type line Flex roosters and 10 White Koluda® geese. The semen was collected two times a week, by dorso-abdominal massage method and pooled from 10 individuals of each species. Fowl semen samples were subjected to cryopreservation using the “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. Geese semen samples were cryopreserved in plastic straws in a programmable freezing unit with Dimethyloformamide (DMF) as the cryoprotectant. A fluorescent lipid probe C11-BODIPY581/591 provided with two double bonds that are oxidized during their contact with ROS, was used for the purpose of the assessment of the LPO in freshly diluted semen samples and frozen-thawed semen samples. This probe changes its color according to its state (non peroxidized: red; peroxidized: green). Flow cytometric analysis was used to monitor these changes. The White Koluda® geese fresh semen had a higher level of LPO than the Flex fresh semen (P > 0.01). The cryopreservation of fowl semen significantly (P > 0.01) increased the percentage of live and dead spermatozoa with lipid peroxidation. In frozen-thawed semen of White Koluda® geese the percentage of live spermatozoa with LPO significantly decreased (P > 0.05) whereas significantly (P > 0.01) higher level of dead cells with LPO was observed. There were significant differences between the two studied species. After thawing, the percentage of live and dead spermatozoa with lipid peroxidation was higher in fowl semen than in geese semen (P > 0.01). In conclusion, our data clearly indicate the existence of species specific differences in susceptibility of spermatozoa to the oxidation of PUFAs in the cell membranes, where such oxidation is caused by cryopreservation. This study shows that avian spermatozoa are vulnerable to radicals and frozenthawed sperm have higher level of LPO than fresh sperm. According to our observation, fowl semen is more susceptible to LPO than geese semen.     

Cryopreservation disrupts lipid rafts and heat shock proteins in yellow catfish sperm

Lipid rafts and associated membrane proteins (flotillin, caveolin) play important roles in cell signaling and sperm fertilization while heat shock proteins (Hsp) ensure properly protein folding to fulfill their physiological functions. The markedly reduced fertility in thawed sperm after cryopreservation could result from disrupted membrane lipid rafts and these proteins. To explore the effect of sperm cryopreservation on lipid rafts and heat shock proteins, we compared lipid raft integrity, and the expression levels of lipid raft associated proteins (Flot-1, Flot-2, Cav-1) as well as heat shock proteins (Hsp90, Hsp70) in fresh and thawed sperm cryopreserved under different scenarios in yellow catfish. We found higher lipid raft integrity, higher protein expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 in fresh sperm samples than in thawed sperm samples, in thawed sperm samples cryopreserved with optimal cooling rate than those cryopreserved with sub-optimal cooling rate, and in thawed sperm samples cryopreserved with extenders supplemented with cholesterol than those supplemented with methyl-β-cyclodextrin (for cholesterol removal). Our findings indicate that lipid raft integrity, and expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 are clearly associated with sperm quality, and together they may play a cumulative role in reduced fertility associated with thawed sperm in aquatic species.     

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.     

Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen

The sensitivity of dog sperm cells for extracellular Ca(2+)/Ca(2+)-ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm-rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrode's medium (HBT containing 0.1 mM Ca(2+)), without (control sample) and with 2.5 microM Ca(2+)-ionophore (induced sample) and incubated for 60 min at 38 degrees C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5-ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer-driven three-step freeze protocol and subsequently thawed for 50 sec in a 37 degrees C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein-conjugated peanut agglutinin (PNA-FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD-1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca(2+)-ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca(2+)-ionophore treatment correlated very negatively (r = -0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca(2+)-ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca(2+)-ionophore treatment followed by simultaneous determination PNA-FITC and EthD-1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors.     

The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen Microscopic and oxidative stress parameters after freeze-thawing process

There is a lack of information regarding lipid peroxidation and antioxidant capacity in cryopreserved ram semen, and cryopreservation is associated with the production of reactive oxygen species (ROS) which lead to lipid peroxidation (LPO) of sperm membranes, resulting in a loss of motility, viability and fertility of sperm. The aim of this study was to determine the influence of certain additives and their different doses on standard semen parameters, lipid peroxidation and antioxidant activities after the cryopreservation/thawing of ram semen. Ejaculates collected from four Akkaraman rams, a native breed of sheep, were evaluated and pooled at 33 degrees C. Semen samples which were diluted with a Tris-based extender containing additives including trehalose (50, 100mM), taurine (25, 50mM), cysteamine (5, 10mM), and hyaluronan (0.5, 1mg/ml), and an extender containing no additives (control) were cooled to 5 degrees C and frozen in 0.25ml French straws, being stored in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. The use of a Tris-based extender supplemented with 50mM trehalose, 25mM taurine, and 5 and 10mM cysteamine led to higher percentages of post-thaw motility, in comparison to the control group (P<0.01). No significant differences were observed in the percentages of acrosome and total abnormalities, and the hypoosmotic swelling test upon the supplementation of the freezing extender with antioxidants after the thawing of semen. In biochemical assays, the addition of antioxidants did not cause significant differences in levels of malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-Px), after thawing, when compared to groups with no additives. In this study, catalase (CAT) activities were higher in the group that was applied 25mM taurine as an antioxidant, than in all of the other groups (P<0.001). Compared to the controls, antioxidant treatment with 100mM trehalose, 50mM taurine, 5mM cysteamine and 0.5mg/ml hyaluronan, significantly elevated vitamin E (vit E) levels in samples (P<0.001).     

Evaluation of gilthead sea bream, Sparus aurata, sperm quality after cryopreservation in 5 ml macrotubes

Cryopreservation produces several types of damage in spermatozoa, leading to fertility impairment. The reduction arises both from a lower viability post-thaw and from sublethal dysfunctions in some of the surviving cells. In the present study, we have analysed the effect of cryopreservation in 5 ml macrotubes on the quality of post-thawed gilthead sea bream sperm. Several standard sperm quality parameters were determined: pH and osmolarity of seminal plasma, sperm concentration, and motility. An exhaustive determination of sperm quality before and after cryopreservation was investigated. Several parameters related with spermatozoal status were determined: ATP content, plasma membrane integrity and functionality, mitochondrial functionality, and sperm fertility. Our results demonstrated that gilthead sea bream spermatozoa suffer several types of damage after freezing/thawing. The percentage of viable cells slightly decreased after cryopreservation, however plasma membrane was affected by cryopreservation, since cells could not resist the hyperosmotic shock. Mitochondrial status was affected by cryopreservation since there was a decrease in the parameters of sperm motility, ATP content (3.17 nmol ATP/10(5) spermatozoa to 1.7 nmol ATP/10(5) spermatozoa in 1:20 frozen samples) and an increase of the percentage of cells with mitochondrial depolarized membranes (11% for fresh and 27% for 1:20 frozen samples). Fertility rate was similar either using fresh or frozen/thawed sperm (77 and 75% hatched larvae, respectively).     

Protein degradation and phosphorylation after freeze thawing result in spermatozoon dysfunction

Cryopreservation is widely used in many assisted conception units. Semen cryopreservation is the only proven method that offers many couples the chance to have children. However, spermatozoa are exposed to physical and chemical stressors during freezing and thawing that result in adverse changes in membrane lipid composition, sperm motility, viability, and acrosome status. Wang et al. (Proteomics 2014, 14, 298–310) evaluate the protein content of freeze‐thawed sperm samples relative to that of fresh sperm samples from the same normozoospermic donors. Four proteins are verified via Western blot and immunofluorescent staining, which are putatively involved in spermatozoon dysfunction. These marked differences demonstrated by Wang et al. suggest that dysfunctional spermatozoon after cryopreservation may be due to protein degradation and protein phosphorylation.     

Mass spectrometric detection of cholesterol oxidation in bovine sperm

We report on the presence and formation of cholesterol oxidation products (oxysterols) in bovine sperm. Although cholesterol is the most abundant molecule in the membrane of mammalian cells and is easily oxidized, this is the first report on cholesterol oxidation in sperm membranes as investigated by state-of-the-art liquid chromatographic and mass spectrometric methods. First, oxysterols are already present in fresh semen samples, showing that lipid peroxidation is part of normal sperm physiology. After chromatographic separation (by high-performance liquid chromatography), the detected oxysterol species were identified with atmospheric pressure chemical ionization mass spectrometry in multiple-reaction-monitoring mode that enabled detection in a broad and linear concentration range (0.05-100 pmol for each oxysterol species detected). Second, exposure of living sperm cells to oxidative stress does not result in the same level and composition of oxysterol species compared with oxidative stress imposed on reconstituted vesicles from protein-free sperm lipid extracts. This suggests that living sperm cells protect themselves against elevated oxysterol formation. Third, sperm capacitation induces the formation of oxysterols, and these formed oxysterols are almost completely depleted from the sperm surface by albumin. Fourth, and most importantly, capacitation after freezing/thawing of sperm fails to induce both the formation of oxysterols and the subsequent albumin-dependent depletion of oxysterols from the sperm surface. The possible physiological relevance of capacitation-dependent oxysterol formation and depletion at the sperm surface as well as the omission of this after freezing/thawing semen is discussed.     

Cryopreservation of semen from endangered pheasants: the first step towards a cryobank for endangered avian species

In order to improve the genetic management of bird species within the European Endangered Programs (EEP), a research project on artificial insemination and cryopreservation of Galliformes semen has been developed. The aim of the program is to create a sperm cryobank for threatened bird species. During this study, semen was collected from 17 pheasant species and specific characteristics of ejaculates were analyzed (volume, sperm concentration, motility, pH). Artificial insemination with fresh semen was performed in nine species and with frozen semen in eight species. Inseminations with frozen and thawed semen were made in 17 species. Viability of fresh and frozen semen was assessed in vitro using double stains, eosin and nigrosin. The effect of pH (7-8.5) on viability of fresh and frozen/thawed spermatozoa was also studied. Chicks hatched in eight and three species after insemination with fresh and frozen/thawed semen, respectively. Species varied widely in semen viability: 1-30% of spermatozoa survived freezing and thawing. There was a negative correlation between the viability of frozen spermatozoa and semen pH. In our experimental conditions, the pH of diluents had no effect on semen viability. However, semen with the highest pH had the lowest quality after freezing and thawing. These experiments demonstrated the feasibility of using a very simple and inexpensive method to achieve artificial insemination and cryopreservation of semen in endangered pheasant species.     

Detrimental effects of non-functional spermatozoa on the freezability of functional spermatozoa from boar ejaculate

In the present study, the impact of non-functional spermatozoa on the cryopreservation success of functional boar spermatozoa was evaluated. Fifteen sperm-rich ejaculate fractions collected from five fertile boars were frozen with different proportions of induced non-functional sperm (0--native semen sample-, 25, 50 and 75% non-functional spermatozoa). After thawing, the recovery of motile and viable spermatozoa was assessed, and the functional of the spermatozoa was evaluated from plasma membrane fluidity and intracellular reactive oxygen species (ROS) generation upon exposure to capacitation conditions. In addition, the lipid peroxidation of the plasma membrane was assessed by the indirect measurement of malondialdehyde (MDA) generation. The normalized (with respect to a native semen sample) sperm motility (assessed by CASA) and viability (cytometrically assessed after staining with Hoechst 33342, propidium iodide and fluorescein-conjugated peanut agglutinin) decreased (p<0.01) as the proportion of functional spermatozoa in the semen samples before freezing decreased, irrespective of the semen donor. However, the magnitude of the effect differed (p<0.01) among boars. Moreover, semen samples with the largest non-functional sperm subpopulation before freezing showed the highest (p<0.01) levels of MDA after thawing. The thawed viable spermatozoa of semen samples with a high proportion of non-functional spermatozoa before freezing were also functionally different from those of samples with a low proportion of non-functional spermatozoa. These differences consisted of higher (p<0.01) levels of intracellular ROS generation (assessed with 5-(and-6) chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester; CM-H(2)DCFDA) and increased (p<0.01) membrane fluidity (assessed with Merocyanine 540). These findings indicate that non-functional spermatozoa in the semen samples before freezing negatively influence the freezability of functional spermatozoa.     

Acrosin release and acrosin activity during incubation in capacitating media using fresh and frozen-thawed dog sperm

We evaluated the effect of time and temperature on acrosin release from the acrosomal cap and the activity of this enzyme during in vitro capacitation in fresh and frozen/thawed dog sperm. Sperm-rich fractions of six ejaculates from three dogs were processed as fresh and frozen samples. Each sperm sample was incubated in canine capacitation medium (CCM) for 0, 1, 2 and 3 h at 20°C and at 37°C. After incubation, the samples were assessed by the indirect immunofluorescent staining technique. The probability of having unlabeled sperm (PUS), indicating acrosin loss, was modelled by a binomial distribution using logistic regression. There was a linear relationship between PUS and time at both temperatures (p<0.001); however, a major percentage of unlabeled sperm was observed in frozen/thawed samples soon after incubation, indicating that the release of acrosin was affected by capacitation time, mainly in frozen samples. Temperature influenced acrosin release only in cryopreserved sperm (p<0.05). Acrosin activity was measured by digestion halos on slides coated with gelatin-substrate film during each time period; a significant increase in the number of large halos was observed in fresh samples throughout the experiment, whereas frozen/thawed sperm showed a decreased rate of halo diameters during culture. Thus, there appears to differences between fresh and frozen dog sperm in terms of acrosin release and the level of acrosin activity in the course of in vitro capacitation.     

Effects of reactive oxygen species on sperm function

Reactive oxygen species (ROS) formation and membrane lipid peroxidation have been recognized as problems for sperm survival and fertility. The precise roles and detection of superoxide (SO), hydrogen peroxide (HP), and membrane lipid peroxidation have been problematic, because of the low specificity and sensitivity of the established chemiluminescence assay technologies. We developed flow cytometric assays to measure SO, HP, membrane lipid peroxidation, and inner mitochondrial transmembrane potential in boar sperm. These methods were sufficiently sensitive to permit detection of early changes in ROS formation in sperm cells that were still viable. Basal ROS formation and membrane lipid peroxidation in the absence of ROS generators were low in viable sperm of both fresh and frozen-thawed boar semen, affecting less than 4% of the sperm cells on average. However, this is not the case in other species, as human, bovine, and poultry sperm have large increases in sperm ROS formation, lipid peroxidation, loss of motility, and death in vitro. Closer study of the effects of ROS formation on the relationship between sperm motility and ATP content in boar sperm was conducted using menadione (mitochondrial SO generator) and HP treatment. Menadione or HP caused an immediate disruption of motility with delayed or no decrease in sperm ATP content, respectively. Overall, the inhibitory effects of ROS on motility point to a mitochondrial-independent mechanism. The reduction in motility may have been due to a ROS-induced lesion in ATP utilization or in the contractile apparatus of the flagellum.