Sperm Protection Research Articles

CRYOPRESERVATION OF RED BROCKET DEER SEMEN (MAZAMA AMERICANA): COMPARISON BETWEEN THREE EXTENDERS

In recent years, genome banks have grown as a way of maintaining the genetic variability of populations. However, the quality of gamete cryopreservation will determine their efficiency. This study aimed to evaluate prefreeze and postthaw sperm motility, vigor, membrane integrity, and morphology of semen of red brocket deer (Mazama americana) using three extenders: E1, Tris-Yolk; E2, Tes-Tris-Yolk; and E3, Tes-Tris-Yolk-Equex. Six bucks were used, and three collections per buck were performed at 90-day intervals. Before freezing, semen volume, ejaculate concentration, motility, vigor, membrane integrity, and sperm morphology were evaluated. To compare the effect exerted by the extenders after sample thawing, further analyses of sperm motility, vigor, membrane integrity, and morphology were performed. Mean ejaculate volume and sperm concentration were 365.33 +/- 120.5 microL and 2,675.73 +/- 810.4 sperm/mL, respectively. Prefreeze motility for the extenders showed no significant differences (approximately 60%). Postthaw motility (E1 = 16.33 +/- 5.5, E2 = 5.44 +/- 5.2, E3 = 24.66 +/- 10.0) was significantly different between E2 and E3, whereas postthaw vigor (E1 =2.66 +/- 0.8, E2= 1.89 +/- 1.2, E3 = 3.83 +/- 0.4) was greater for E3 (P < or = 0.05). Analysis of postthaw membrane integrity revealed no significant differences between the extenders regarding counts of cells presenting intact membranes; however, E3 promoted the lowest number of cells with damaged membranes and higher cell counts for partially damaged membranes (P < or = 0.05). Analysis of sperm morphology revealed an increase in severe abnormalities when using E2 and E3 (P < 0.05). However, observation verified that counts of altered cells were lower using E3 than E2, suggesting a protective effect of Equex. These findings indicate that E3 promoted better semen quality postthaw. However, the performance of this extender in protecting sperm cells of M. americana during freezing was lower than that verified for other species. Thus, further studies are needed for characterization of red brocket deer semen and the optimization of extender for sperm cryopreservation.

In vitro antimicrobial sperm protection by an ejaculate‐like substance

Summary Ejaculate substances are hugely diverse among and within species. The functional significance of this diversity has been little studied except for a few accessory gland proteins. These studies suggest that ejaculate components evolve mainly by sexual selection, that is, sperm competition, male–female co‐evolution or by female choice for paternal investment. However, male benefits of protecting sperm from microbial harm, that is, antimicrobial ejaculate protection, has received little attention in the evolutionary literature, while it has been discussed in the human and livestock literature. Here, we examine in the common bedbug, Cimex lectularius, the two basic components of the antimicrobial ejaculate protection hypothesis: 1) microbes damage sperm cells and 2) antimicrobial ejaculate substances prevent such microbe‐induced sperm damage. We found that environmental microbes, which are sexually transmitted in bedbugs, induce sperm mortality. Sperm mortality was high (increased 40% compared with controls without bacteria) and very rapid (within 10 min) in vitro. Consistent with the antimicrobial sperm protection hypothesis, a lysozyme dose equivalent to the amount of antibacterial lysozyme‐like activity that is transferred in the seminal fluid during a single mating was sufficient to reduce sperm mortality to control levels. While this result is not necessarily predicted by the paternal investment hypothesis, it is plausible that female benefits of male immune substances may evolve under the male–female co‐evolution hypothesis. We found that weekly physiological lysozyme doses equivalent to the observed lysozyme‐like activity in seminal fluid benefit females to some extent, indicating that immune‐active ejaculate substances may have a secondary gift function and may release females from a trade‐off between reproduction and immunity. Lysozyme seems to stimulate early egg‐laying above the female optimum and later cause earlier reproductive senescence, which is not consistent with the idea of an overall direct benefit but is consistent with the idea that males transfer manipulative substances to the females. Our data are consistent with antimicrobial sperm protection. They support the idea of a naturally selected origin of ejaculate components and provide a mechanistically well‐defined pathway how sexual selection can affect ejaculate composition via antimicrobial sperm protection. As sperm and microbes frequently encounter each other in many species, antimicrobial sperm protection may be a common phenomenon and has the potential to inform models of male and female co‐evolution and biological diversification.

Proteomics of Tribolium castaneum seminal fluid proteins: Identification of an angiotensin-converting enzyme as a key player in regulation of reproduction

Seminal fluid proteins (SFPs) play important roles in regulation of reproduction and behavior. Proteomics approaches were used to identify 13 SFPs, including 3 new proteins in the red flour beetle, Tribolium castaneum. The 13 SFP genes identified code for Serpin, cysteine-rich protein, odorant binding protein-like (OBPL, G10064 and G10065), Kunitz-like protease inhibitor precursor, and WD 40 family protein and are predominantly expressed in the male accessory glands. The genes coding for 13 putative SFPs were knocked down in males; the RNAi males were mated with virgin females, and the number of eggs produced by the mated females was quantified. Knockdown in the expression of the gene coding for a protein similar to angiotensin-converting enzyme 9 (G15465, TcACE) in the males caused a decrease in egg production by the females when compared to the eggs produced by the females mated with control males. In addition, knockdown in the expression of the gene coding for heat shock cognate 70 led to a reduction in the amount of proteins produced by the male accessory glands by 55%. These data suggest that angiotensin-converting enzyme produced in the male seminal vesicles plays important roles in sperm protection during and after transfer to females.

Protective Effect of Gangliosides on DNA in Human Spermatozoa Exposed to Cryopreservation

Gangliosides, the sialic acid-containing glycosphyngolipids, are amphiphilic compounds which in micellar form affect the properties and functions of a cellular membrane. The aim of this study was to test whether exogenous gangliosides supplied to cryopreservation media before freezing could protect sperm cells from cryopreservation-induced DNA damage assessed by Comet assay. Additionally, to investigate whether gangliosides were also able to reduce membrane integrity damage, malonaldialdehyde as a measure of lipid peroxidation and sperm-specific lactate dehydrogenase-C4 activity as an enzyme marker of sperm membrane leakage were determined. The monosialogangliosides (GM1) and trisialogangliosides (GT1b) were examined at a concentration of 100 μM, which was above their respective critical micellar concentrations. Exogenous gangliosides were not found to protect sperm membrane from lipid peroxidation. However, a freezing-/thawing-induced increase in Comet parameters was equally significantly prevented by the presence of both GM1 and GT1b (P < .05), indicating that the ceramide moiety, rather than the polar groups, is involved in the protective ability of gangliosides. The observed phenomena suggest that ganglioside micelles could modulate hydrophobic properties of the sperm membrane responsible for better tolerance to DNA fragmentation, thus protecting DNA integrity from cryopreservation-induced damage.

Influence of Calcium on Milk Protein Aggregation and Binding Dynamics Between Binder of Sperm (BSP) Proteins and Milk Proteins.

Over the years, skim milk has been used as extender for cryopreservation of semen from domestic animals due to its protective role against cold shock. The protective effect is attributed to its protein fraction and approximately 80% of the total milk proteins are casein micelles. In skim milk, calcium ions are associated with casein micelles and also responsible for their internal stability. Calcium forms linkages between proteins as colloidal calcium phosphate or it directly binds to caseins. Recent studies have shown that major proteins of the bovine seminal plasma, named Binder of Sperm (BSP) proteins as well as their homologues from stallion, boar and ram interact with the constituents of milk-based extenders. This interaction is implicated in sperm protection during cooling and storage of semen. Thus, the aim of this study was to investigate if calcium ions modulate the interaction between milk proteins and BSP proteins from animals of agricultural importance. In order to evaluate such interaction, heated skim milk was incubated with seminal plasma proteins from bulls, stallions, boars and rams. Then, these solutions were loaded onto Sepharose CL-4B column previously equilibrated with buffer-C (20 mM Tris, 50 mM NaCl, 10 mM CaCl2, 0.02% sodium azide, pH 7.4). The proteins were eluted with buffer-C and fractions were analysed by immunobloting. In parallel, seminal plasma proteins alone was used as controls and subjected to gel filtration chromatography in buffer-C. In the presence of calcium, casein micelles and whey protein aggregated and co-eluted. Bull, stallion and boar BSP proteins also co-eluted with milk protein aggregates in the presence of calcium. These results suggest that, in these species, calcium modulates protein-protein interaction as a result of an increase in milk protein aggregation. In contrast, ram BSP proteins did not bind to milk protein aggregates, suggesting that ram BSP proteins may differ in structural organization as compared to the bull, boar and stallion BSP homologues. In conclusion, our results indicate that calcium influences the formation of larger casein aggregates and the binding of bull, stallion and boar BSP proteins to milk proteins. These findings are crucial to understand the mechanisms of sperm protection by extender components and for the development of novel extenders. (Supported by CAPES - Brazil and NSERC of Canada)

Study of adenosine-5'-triphosphate (ATP) in spermatozoa of boars

The data obtained suggest that the dialysis method of diluting the sperm of boars contributes to more effective protection of sperm during their cryopreservation than the traditional dilution of ejaculate. In this case, the preservation of adenosine-5'-triphosphate (ATP) and mobility was higher by 12 and 1 to 13.5% compared with control. Key words: Preservation, adenosine-5'-triphosphate (ATP), mobility, freezing, membrane, sperm, boars.

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction.

Effect of Rabbit Epididymal Antimicrobial Peptide, REHbβP, on LPS-Induced Proinflammatory Cytokine Responses in Human Vaginal Cells In Vitro.

Antimicrobial peptides (AMP's) protect epithelial surfaces including epididymis against pathogens and play a key role in orchestrating various defensive responses. Recently, we have identified one such AMP, rabbit epididymal hemoglobin-β subuit (REHbβP) from the epididymal fluid of rabbit, Oryctologus cuniculus. The demonstration of a protective role of REHbβP in epididymal epithelial cells (EPEC's) led us to investigate: (1) the identification of LPS interactive domain in REHbβP, and (2) whether the REHbβP of rabbit origin mediates vaginal cellular immune responses of another species (human). HeLa-S3, human vaginal epithelial cells (hVECs) were exposed to LPS or the LPS-stimulated cells treated with REHbβP or neutral peptide, nREHbβP. Effect of LPS and cytokines (IL-6 and IL-1α) and chemokines (IL-8, MCP-1) levels was determined in the culture supernatants. In response to the LPS, hVECs synthesized these mediators and the levels were significantly higher than controls. This enhancing effect was ameliorated when the LPS-induced hVECs were treated with REHbβP. Similar results were obtained on NF-κB protein and hBD-1 mRNA expression. Confocal microscopy studies revealed that REHbβP attenuated the LPS-induced internalization of E. coli by macrophages. The chemotaxis studies performed using Boyden chamber Transwell assay, which showed elevated migration of U937 cells when the supernatants of LPS-induced hVECs were used, and the effect was inhibited by REHbβP. REHbβP was found to be localized on the acrosome of rabbit spermatozoa, suggesting its role in sperm protection beside sperm function. In conclusion, REHbβP may have the potential to develop as a therapeutic agent for reproductive tract infections (RTI's).

The determination of ascorbic acid and uric acid in human seminal plasma using an HPLC with UV detection

Oxidative stress has been proposed as one of the potential causes for infertility in men. Ascorbic acid and uric acid play important role in protection of spermatozoa against free radicals. A method for the simultaneous determination of ascorbic acid and uric acid in human seminal plasma using HPLC with UV detection and investigation their clinical significance as antioxidants protecting male germ cells against oxidative damage are described. Semen samples were obtained from consecutive male partners of couples presenting for a fertility evaluation. After liquefaction, the samples were centrifuged and the supernatants were diluted with dithiothreitol solution and after a filtration injected onto an analytical column. For the separation, a reverse-phase column MAG 1, 250 mm × 4.6 mm, Labiospher PSI 100 C18, 5 μm, was used. The mixture of ethanol and 25 mmol/L sodium dihydrogenphosphate (2.5:97.5, v/v), pH 4.70 was used as a mobile phase. Analytical performance of this method is satisfactory for both ascorbic acid and uric acid: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked seminal plasma were between 92.1 and 102.1%. We have found no significant differences in both ascorbic acid and uric acid concentration between the smokers and non-smokers (351.0 ± 237.9 μmol/L and 323.7 ± 99.5 μmol/L vs. 444.8 ± 245.5 μmol/L and 316.6 ± 108.9 μmol/L, p > 0.05). This assay is a simple and reproducible HPLC method for the simultaneous measurement of ascorbic acid and uric acid in human seminal plasma.

The Epididymal Transcriptome and Proteome Provide Some Insights Into New Epididymal Regulations

Once shed from their fostering Sertoli cells, spermatozoa leave the testis and are transported passively by seminiferous fluid through the rete testis. Then, these immature cells enter the complex efferent duct system that is joined to the unique and convoluted epididymal duct. This epididymal duct, lined by a continuous layer of epithelial cells joined by tight junctions, is a tube several meters long (up to 60 m in domestic mammals) and forms an organ that is classically subdivided into 3 major anatomical regions: the head/caput, the corpus/body, and the tail/cauda. Spermatozoa travel throughout the duct for several days to weeks, depending on the species, and may be stored for even longer periods in the cauda part of the epididymis and vas deferens. During their journey the proportion of potentially "mature" spermatozoa increases, but it is only when they reach the cauda epididymidis that almost all spermatozoa have acquired their natural fertilizing ability, which involves progressive motility, the ability to undergo the postejaculatory events (capacitation and hyperactivation), and the capacity to recognize and to bind to the oocyte investments and egg plasma membrane. Recent secretomic, proteomic, and transcriptomic studies have provided new information on the functions and the regionalization of the epididymis and revealed some insights into the complexity of epididymal fluid. Among genes and proteins highly expressed by this tissue, many have roles related to sperm protection (such as oxidation), but a large number of new compounds related to innate immunity have also been discovered. This review will focus on possible new control mechanisms that these studies have suggested for this tissue.

The Major Proteins of Bovine Seminal Plasma Interact with Caseins and Whey Proteins of Milk Extender

Milk has been used routinely as an extender for sperm preservation. Caseins, the major proteins in milk, are proposed to be the protective constituents of milk during sperm preservation. It is unclear whether the whey proteins in milk are also implicated in the protection of sperm. Our previous studies have shown that the major proteins of bovine seminal plasma (recently named as binder of sperm or BSP, which comprises BSP1, BSP3, and BSP5 proteins) mediate a continuous phospholipid and cholesterol efflux from the sperm plasma membrane that is detrimental for sperm preservation. In this study, we investigated whether the protective effect of milk could be due to an interaction between BSP proteins and milk proteins. The binding of BSP proteins to milk proteins was demonstrated by gel filtration chromatography. Milk was fractionated into three fractions: the first containing whey protein aggregates and kappa-casein, the second containing all milk proteins, and the third containing small peptides, salts, and sugars. BSP1 has a higher affinity for the milk proteins in the milk fractions as compared to BSP3 and BSP5. The binding of BSP proteins to milk proteins was further characterized by isothermal titration calorimetry. We demonstrated that BSP1 binds to caseins and the titration could be simulated with a Scatchard approach, leading to an affinity constant (K(a)) of 350 mM(-1) and a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin was characterized by a K(a) of 240 mM(-1) and an n value of 0.8. These results indicate the existence of an interaction between BSP proteins and milk proteins that could be the origin of the protection of sperm during preservation in milk.

Identification of Increased Amounts of Eppin Protein Complex Components in Sperm Cells of Diabetic and Obese Individuals by Difference Gel Electrophoresis

Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ≤ 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, β-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress.

Thermodynamics of the interaction between bovine binder of sperm BSP1 and low-density lipoprotein from hen's egg yolk

Egg yolk is used in extender to protect sperm from cold shock and freezing during preservation. It is the low-density lipoprotein (LDL) fraction of egg yolk that protects sperm. Even though essential for sperm capacitation, the major proteins from bull seminal plasma, the Binder of SPerm (BSP) proteins, are detrimental for sperm preservation because they induce a continual phospholipids and cholesterol efflux from sperm membranes. The BSP proteins were proposed to bind to egg yolk LDL, preventing the sperm membrane damage. We characterized the binding between the BSP proteins and the LDL by isothermal titration calorimetry, providing the thermodynamics and quantitative description of this putative association. The association between BSP1 (major BSP proteins) and LDL is characterized by an affinity constant ( K a) of 3.4 ± 0.4 μM −1. A protein/LDL ratio of 104 ± 5 was determined indicating that 104 molecules of BSP1 would bind to one LDL particle. This stoichiometry leads to proposing that the association involves 1.6 ± 0.1 phosphatidylcholines (PC) per BSP protein. This finding is satisfactorily consistent with the fact that each BSP1 protein has 2 binding sites for choline group. In conclusion, the formation of a high affinity complex between BSP1 and LDL is proposed to be important for the protection of sperm by egg yolk extender.

Identification and isolation of gene differentially expressed on scrotal circumference in crossbreed bulls

Differential display reverse transcription polymerase chain reaction (DDRT-PCR), reverse northern blot analysis and real-time polymerase chain reaction (RT-PCR) were used to identify tissue-specific expression genes in scrotum of differential scrotal circumference bulls and the function of these specific genes on the development of the bull’s scrotum was analyzed in this study. The results indicate that 19 positive fragments were screened out by DDRT-PCR and reverse northern blot analysis. Results of BLAST with GenBank show that three genes or expressed sequence tag (ESTs) were unknown, and there were eight sequences highly identified to be Bos taurus mRNA for proline-rich protein P-B and other sequences were B. taurus ebd-P2 pseudogene, B. taurus similar to F-box only protein 21 isoform 2, B. taurus similar to Kinesin heavy chain isoform 5C, B. taurus similar to ankyrin repeat domain protein 15 isoform and B. taurus similar to galactosidase, beta 1-like. The result of real-time PCR analysis testified more that B. taurus was similar to galactosidase, beta 1-like, kinesin heavy chain isoform 5C and ankyrin repeat domain protein 15 isoform. B. taurus ebd-P2 pseudogene was highly expressed in bulls which had bigger scrotal circumference. They may be involved with sperm maturation in the epididymis, sperm protection and prevention of the ascent of microorganisms into the adjacent testes and also responsible for converting immature sperm into competent functional cells, as well as movement of spermatozoa. Key words: Bulls, scrotal circumference, differential display reverse transcription polymerase chain reaction (DDRT-PCR), real time polymerase chain reaction (RT-PCR).

Human prostasomes express CD48 and interfere with NK cell function

Prostasomes are small vesicles secreted by the prostate gland to the seminal fluid. In male fertility, the fusion of prostasomes to sperm has been associated with an enhancement of sperm function. Prostasomes also contribute to sperm protection within the female reproductive tract and immunologically relevant proteins have been identified on the prostasomal membrane that may prevent immune-mediated destruction of the sperm.The female genital tract is a potentially hostile environment for spermatozoa and a large number of NK cells have been found in the female reproductive tract. Since it has been suggested that NK cells may represent an important component of innate immunity in the female reproductive tract, we have analyzed the role of prostasomes in the regulation of NK cell activity.Flow cytometric analysis revealed that prostasomes expressed high levels of CD48, the ligand for the activating receptor CD244. The interactions between NK cells and purified prostasomes resulted in a decrease of CD244 expression. Moreover, the decreased NK cell activity observed in NK cells cultured in the presence of prostasomes suggests that prostasomes may immunomodulate the local environment within the female reproductive tract preventing immune-mediated sperm destruction and prolonging their survival rate.

In vitro antioxidant activity of the prostatic secretory granules in rabbit semen after exposure to organic peroxides

BackgroundThe prostate gland of rabbits produces numerous granules, which are specifically implicated in the inhibition of sperm capacitation during the first hours after mating. These granules are rich in vitamin E, but their role in the antioxidant protection of rabbit sperm has not been studied.Aim of studyThe objectives of this study were to investigate whether the prostatic secretory granules (PSGs) could prevent sperm induced-lipid peroxidation and to verify a potential involvement of tocopherols from the PSGs to the sperm.MethodsWashed sperm samples taken from eighteen White New Zealand rabbits were either incubated with tert-butyl-hydroperoxide (t-BHP, an oxidative stressor) or with buffered Tyrode's medium for 1 hour. The same number of sperm samples that contained PSGs were subjected to the previously mentioned treatments and thiobarbituric acid reactive substances (TBARS), vitamin E compounds and the acrosome status were assessed.ResultsThe incubation of the sperm with t-BHP resulted in a noticeable production of TBARS (0.38 vs. 0.22 nmol/10(7) cells) and an associated decrease of alpha-tocopherol (alpha-T, 72.3 vs. 103.2 nmol/10(8) cells) with respect to the sperm samples containing PSGs. The sperm incubated with the PSGs had a higher amount of alpha-T compared to the control (292.2 vs. 251.0 nmol/10(8) cells). The acrosome status was not affected by the occurrence of the organic peroxide in the medium and the amount of capacitated sperm was lower when the PSGs were also present.ConclusionsOverall, these results suggest that the PSGs may represent a source of protection for rabbit sperm against in vitro oxidative stress by supplying the sperm with endogenous alpha-T. This mechanism could be in part involved in the inhibition of sperm capacitation by the granules.

Are the Immunocompetence and the Presence of Metazoan Parasites in Cyprinid Fish Affected by Reproductive Efforts of Cyprinid Fish?

Each organism has the limited resources of energy that is distributed among important life traits. A trade-off between immune response and other physiological demands of organism especially costly reproduction is expected. Leuciscus cephalus, the cyprinid fish, was investigated during three periods varying in reproductive investment, that is, before-breeding, breeding, and after-breeding periods. We tested whether a potentially limited investment in immunity during the breeding is associated with higher susceptibility to the metazoan parasites. Following the immunocompetence handicap and sperm protection hypotheses, males expressing more elaborated sexual ornamentation should produce better quality sperm and be more parasitized. We found that reproductive investments in fish play an important role for energy allocation into somatic condition, immunity, and reproduction. The immune parameters including respiratory burst and leukocyte count were higher in breeding; however, parasite species richness and abundance appeared low. Males investing more in spawning tubercles reached high spermatocrite and were more parasitized by digeneans.

108 EFFECT OF DIMETHYLFORMAMIDE AND METHYLFORMAMIDE ON OXIDATIVE STATUS OF MANGALARGA STALLIONS CRYOPRESERVED SEMEN

Cryopreservation of equine semen has been widely studied by several research groups because of the large breed and individual variation in sperm freezability. A key factor in sperm cryopreservation is the high incidence of oxidative stress, an imbalance between reactive oxygen species (ROS) and antioxidant protection, which impairs sperm functionality by attacking plasma membrane, acrosome, mitochondria, and DNA. In order to study the resistance of equine spermatozoa to different reactive oxygen species (ROS), sperm samples from 4 Mangalarga stallions were collected using an artificial vagina. Samples were cryopreserved in extenders containing dimethylformamide (DMF) or methylformamide (MF). After thawing and washing, sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produced hidroxyl radical), and malondialdehyde (MDA, lipid peroxidation product). Samples were evaluated using the 3-3′ diamino benzidine (DAB) stain, as an indicator of mitochondrial activity; the eosin nigrosin staining, to evaluate plasma membrane integrity; the simple stain (fast green/Bengal rose), to assess acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS), a lipid peroxidation product. Statistical analysis was performed using the Student t-test and LSD test. Results showed that sperm mitochondrial potential of frozen-thawed samples in MF was highly susceptible to the attack of hydroxyl radical and hydrogen peroxide. No effect of ROS was observed on membrane and acrosome integrity. On the other hand, samples cryopreserved in DMF showed no differences in susceptibility to ROS. When evaluating the main effects of different extenders, results showed a higher protective effect of the MF extender on acrosome integrity and mitochondrial potential (MF: 12.1 ± 2.2 and 7.8 ± 2.3% v. DMF: 3.4 ± 0.7 and 1.1 ± 0.7%, respectively, P &lt; 0.05). However, a negative effect of MF extender was observed regarding the percentage of sperm showing intact membrane and TBARS content (MF: 2.0 ± 0.8% and 517 ± 115 ng/106 sperm v. DMF: 20.6 ± 1.7% and 118 ± 44 ng/106 sperm, respectively, P &lt; 0.05). A strong negative correlation was found between TBARS and plasma membrane integrity (r = -0.88; P = 0.004) for samples cryopreserved in DMF, whereas a positive correlation was found between TBARS and sperm with full mitochondrial potential (r = 0.73; P = 0.04). Results of the present study indicate that DMF may play a role in the protection of sperm against the attack of ROS. However, such action is apparently limited to the plasma membrane. On the other hand, the MF-supplemented extender exerts an intracellular protection. Therefore, the antioxidant therapy, especially hydrogen peroxide and hydroxyl radical scavengers, may be an alternative to improve the post-thaw quality of MF-supplemented cryopreserved semen in stallions, by increasing extracellular antioxidant capacity. The authors thank Nutricell for financial support and the media used in the present experiment.

Pro-inflammatory and oxidative stress pathways which compromise sperm motility and survival may be altered by L-carnitine.

The testis is an immunologically privileged organ. Sertoli cells can form a blood-testis barrier and protect sperm cells from self-immune system attacks. Spermatogenesis may be inhibited by severe illness, bacterial infections and chronic inflammatory diseases but the mechanism(s) is poorly understood. Our objective is to help in understanding such mechanism(s) to develop protective agents against temporary or permanent testicular dysfunction. Lipopolysaccaride (LPS) is used as a model of animal sepsis while L-carnitine (LCR) is used as a protective agent. A total of 60 male Swiss albino rats were divided into four groups (15/group). The control group received Saline; the 2(nd) group was given LCR (500 mg/kg i.p, once). The third group was treated with LPS (5 mg/kg i.p once) and the fourth group received LCR then LPS after three hours. From each group, five rats were used for histopathological examination. Biochemical parameters were assessed in the remaining ten rats. At the end of the experiment, animals were lightly anaesthetized with ether where blood samples were collected and testes were dissected on ice. Sperm count and motility were evaluated from cauda epididymis in each animal. Also, oxidative stress was evaluated by measuring testicular contents of reduced glutathione (GSH), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-HDG, the DNA adduct for oxidative damage) in testicular DNA. The pro-inflammatory mediator nitric oxide (NO) in addition to lactate dehydrogenase (LDHx) isoenzyme-x activity as an indicator for normal spermatozoal metabolism were assessed in testicular homogenate. Serum interlukin (IL)-2 level was also assessed as a marker for T-helper cell function. The obtained data revealed that LPS induced marked reductions in sperm's count and motility, obstruction in seminiferous tubules, hypospermia and dilated congested blood vessels in testicular sections concomitant with decreased testicular GSH content and LDHx activity. Moreover, the testicular levels of MDA, 8-HDG (in testicular DNA) and NO as well as serum IL-2 level were increased. Administration of LCR before LPS returned both sperm count and motility to normal levels. Also, contents of testicular GSH, MDA, 8-HDG and NO returned back to the corresponding control values. In addition, serum IL-2 level as well as histological abnormalities were markedly improved in LCR + LPS-treated rats. In conclusion, LPS increased proinflammatory and oxidative stress markers in the testis leading to a marked testicular dysfunction. L-carnitine administration ameliorates these effects by antioxidant and/or anti-inflammatory mechanisms suggesting a protective role against male infertility in severely infected or septic patients.

Sperm proteins in teleostean and chondrostean (sturgeon) fishes

Sperm proteins in the seminal plasma and spermatozoa of teleostean and chondrostean have evolved adaptations due to the changes in the reproductive environment. Analysis of the composition and functions of these proteins provides new insights into sperm motility and fertilising abilities, thereby creating possibilities for improving artificial reproduction and germplasm resource conservation technologies (e.g. cryopreservation). Seminal plasma proteins are involved in the protection of spermatozoa during storage in the reproductive system, whereas all spermatozoa proteins contribute to the swimming and fertilising abilities of sperm. Compared to mammalian species, little data are available on fish sperm proteins and their functions. We review here the current state of the art in this field and focus on relevant subjects that require attention. Future research should concentrate on protein functions and their mode of action in fish species, especially on the role of spermatozoa surface proteins during fertilisation and on a description of sturgeon sperm proteins.