Use of biochemical markers to evaluate the quality of fresh and cryopreserved semen from the arctic fox (Vulpes lagopus).

The aim of this study was to use biochemical markers to evaluate the quality of fresh and cryopreserved semen from the arctic fox (Vulpes lagopus). Twenty-three manually collected ejaculates were analysed for the main indicators of semen quality (sperm concentration and ejaculate volume). Sperm motility and percentage of morphologically normal and abnormal spermatozoa were determined according to the stage of cryopreservation (fresh--measurement A; equilibrated--measurement B; frozen/thawed--measurement C). Furthermore, the seminal plasma and supernatants were analysed after equilibration and freeze/thawing for the activity of the enzymes alkaline phosphatase (ALP), acid phosphatase (AcP), lactate dehydrogenase (LDH) and aspartate aminotransferase (AspAT), and for the activity of acrosin inhibitors (AP). The mean concentration of sperm was 625.1 million/cm3, and ejaculate volume averaged 1.6 cm3. Seminal plasma was characterized by the highest activity of alkaline phosphatase (3.43 x 10(3) U/l) and lowest activity of acrosin inhibitors (4.55 x 10(3) U/l). After equilibration, the supernatants showed the highest activity of acid phosphatase (94.9 U/l) and after freeze-thawing, they showed a high activity of lactate dehydrogenase (535.8 U/l) and aspartate aminotransferase (577.1 U/l), which indicates that these proteins had leaked from spermatozoa into the extracellular medium during the biotechnique of semen cryopreservation. In addition, several significant relationships were found between some indicators of semen quality and plasma and/or supernatant enzyme activity.

Inhibition of ß-N-acetylglucosaminidase by acetamide affects sperm motility and fertilization success of rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii).

ß-N-Acetylglucosaminidase (ß-NAGase) is an enzyme found in the sperm acrosome of numerous animal species including fish. Fish spermatozoa differ in their morphology including acrosome or acrosomeless aquasperm in chondrostean (e.g., sturgeon) and teleostean (e.g., rainbow trout). It has been shown that ß-NAGase exists with high activity in both eggs and sperm of these species. The present study shows the potency of ß-NAGase in fertilization. In rainbow trout, increase in sperm motility parameters (VAP and MOT) were observed in the presence of acetamide, an inhibitor for ß-NAGase. In contrast, sperm motility parameters (VCL, VSL, VAP, MOT, and PRG) were reduced on the Siberian sturgeon in the presence of acetamide. The inhibition of the activity of ß-NAGase in rainbow trout spermatozoa was led to a reduction in the number of fertilized eggs from 79% to 40%, whereas in sturgeon no change was observed in fertilization. Moreover, inhibition of ß-NAGase in both spermatozoa and eggs of trout and sturgeon resulted in significant decrease in fertilization rate from 79% to 1% in rainbow trout and from 84% to 12% in Siberian sturgeon. Our research proves that ß-NAGase can play a significant role in the fertilization process in teleosteans.

Determination of sperm acrosin activity in the arctic fox (Alopex lagopus L.)--using method developed for human spermatozoa.

The aim of the study was to adapt a method to determine acrosin activity of human spermatozoa to arctic fox (Alopex lagopus L.) spermatozoa. We modified this method by reducing sperm count per sample from 1 divided by 10 x 10(6) to 25 divided by 200 x 10(3), incubation time from 180 minutes to 60 minutes, and Triton X-100 concentration in the reaction mixture from 0.01% to 0.005% per 100 cm3. It has also confirmed that arctic fox seminal plasma is rich in proteinases and their inhibitors. To completely abolish the inhibitory effect of seminal plasma on acrosin activity it is recommended to wash the spermatozoa four times. Benzamidine served an inhibitor of acrosin activity.

Effect of organic and inorganic forms of selenium in diets on turkey semen quality.

The effects of Se supplementation and its organic or inorganic form on semen quantitative parameters (ejaculate volume, sperm concentration, and total number of sperm) and biochemical parameters of seminal plasma (protein concentration, acid phosphatase activity, superoxide dismutase activity, and total antioxidant capacity) were investigated over a 25-wk reproductive season. Additionally, DNA fragmentation and motility characteristics of turkey spermatozoa were measured. The parameters of turkey semen in relation to yellow semen syndrome were also determined. Twenty-four males (Big 6) were divided into 3 experimental groups differing in form of Se supplementation (no Se supplementation, 0.3 mg/kg of inorganic Se from sodium selenite and 0.3 mg/kg of organic Se from Sel-Plex, Alltech Inc., Nicholasville, KY). Dietary Se supplementation enhanced the sperm concentration and total number of sperm and did not influence the antioxidative properties of turkey seminal plasma and most biochemical parameters. Only seminal plasma acid phosphatase activity was increased in turkeys fed inorganic Se. The main sperm DNA fragmentation parameters were not affected by dietary Se. The highest percentage of motile spermatozoa (85%) was recorded for the semen of turkeys fed organic Se. Values of the biochemical parameters (acid phosphatase, superoxide dismutase, total antioxidant capacity) of seminal plasma increased during the reproductive season. Yellow semen was characterized by increased biochemical parameters and decreased spermatozoa motility characteristics. However, the percentage of motile spermatozoa did not differ between white and yellow semen. Organic Se seemed to be the preferred form of diet supplementation in comparison with inorganic Se. Biochemical parameters of semen and spermatozoa motility parameters appear to be useful for evaluating the effect of age on semen quality. Monitoring the DNA fragmentation of spermatozoa at the end of the reproductive season could be a useful tool for monitoring turkey semen quality. Increased superoxide dismutase activity can be used as an indicator of yellow semen. A decline in the quality of yellow semen can be related to a decrease in the spermatozoa motility parameters of turkeys.

Isolation and characterization of alpha1-proteinase inhibitor from common carp (Cyprinus carpio) seminal plasma.

Using a three-step procedure, we purified (79 and 51.6-fold to homogeneity) and characterized the two isoforms (a and b) of alpha1-proteinase inhibitor-like protein from carp seminal plasma. The isoforms have molecular masses of 55.5 and 54.0 kDa, respectively. These inhibitors formed SDS-stable complexes with cod and bovine trypsin, chymotrypsin and elastase. The thirty-three amino acids within the reactive loop SLPDTVILNRPFLVLIVEDTTKSILFMGKITNP were identified for isoform b. The same first ten amino acids were obtained for isoform a, and this sequence revealed 100% homology to carp alpha1-proteinase inhibitor (alpha1-PI) from perimeningeal fluid. Both isoforms of alpha1-PI are glycoproteins and their carbohydrate content was determined to be 12.6 and 12.1% for a and b, respectively. Our results indicated that alpha1-PI is one of the main proteins of carp seminal plasma. Using polyclonal anti-alpha1-PI antibodies, alpha1-PI was for the first time localized to the carp testis. The presence of alpha1-PI in testis lobules and in the area surrounding spermatides suggests that this inhibitor may be involved in the maintenance of testis connective tissue integrity, control of spermatogenesis or protection of tissue and spermatozoa against unwanted proteolysis. Since similar alpha1-PI has been identified in rainbow trout semen it can be suggested that the presence of alpha1-PI in seminal plasma is a common feature of cyprinid and salmonid fish.

The effect of increased ozone concentrations in the air on selected aspects of rat reproduction.

Five-month-old male rates were exposed to 0.5 ppm ozone for 50 days, 5 hours a day. A week before the completion of ozone exposure, a biological test was performed to determine the fertilization rate and the survival rate of newborns in both ozone-exposed and control animals. After 50 days, the rats were sacrificed with an overdose of halotane, and testes were collected to assess the morphology and motility of spermatozoa. Neither the morphology of spermatozoa nor motility parameters determined by the CASA (computer-assisted sperm analysis) system showed statistically significant differences between ozone-exposed and control males. The number of successful matings and the survival rate of newborns per litter within one year postpartum were also similar in both groups. However, sperm concentration was by 17% lower in ozone-exposed rats, compared with the control animals.

Gelatinases and serine proteinase inhibitors of seminal plasma and the reproductive tract of turkey (Meleagris gallopavo).

This study examined proteolytic enzymes and serine proteinase inhibitors in turkey seminal plasma with relation to their distribution within the reproductive tract and to yellow semen syndrome (YSS). Proteases of blood plasma, extracts from the reproductive tract, and seminal plasma were analyzed by gelatin zymography. We found a clear regional distribution of proteolytic enzymes in the turkey reproductive tract. Each part was characterized by a unique profile of serine proteolytic enzymes of molecular weights ranging from 29 to 88 kDa. The ductus deferens was found to be a site of very intense proteolytic activity. Two metalloproteases of 58 and 66 kDa were detected in all parts of the reproductive tract and seminal plasma. Using electrophoretic methods for detection of anti-trypsin activity, we found three serine proteinase inhibitors in turkey seminal plasma. Two inhibitors were found in the testis and epididymis and a third in the ductus deferens and seminal plasma. Blood plasma was characterized by the presence of two metalloproteinases and one serine proteinase inhibitor (of low migration rate) that were also detected in the reproductive tract. Amidase and anti-trypsin activities (expressed per gram of protein) differed for yellow and white seminal plasma. We concluded that turkey seminal plasma contains metalloproteases, serine proteinases, and serine proteinase inhibitors. The metalloproteases and one proteinase inhibitor are related to blood proteinases but the other two inhibitors and serine proteinases seem to be unique for the reproductive tract.

Biochemical characteristics and sperm production of turkey semen in relation to strain and age of the males.

The effect of turkey strain [BUT (Big-6), Hybrid Large White (HLW), and Nicholas (N-700)] on semen quantitative parameters (ejaculate volume, sperm concentration, and total number of sperm) and biochemical parameters (seminal plasma protein concentration, acid phosphatase activity in spermatozoa and seminal plasma, and antiproteinase activity of seminal plasma) was investigated over the reproductive period. The Big-6 strain had better quantitative parameters of semen compared with HLW and N-700 strains. Besides protein concentration, the Big-6 strain had the highest level of acid phosphatase activity in semen and antiproteinase activity in seminal plasma. The N-700 strain was characterized by reduced quantitative parameters and the lowest levels of all biochemical parameters in semen. While quantitative parameters of semen showed very little trend over the 21 wk of the reproductive season, the biochemical parameters increased several fold. Biochemical parameters of semen seemed to be more affected than quantitative parameters by age of the males. These changes may be related to decreasing semen quality with increasing age of male turkeys.

Blood cells in rainbow trout Oncorhynchus mykiss milt: relation to milt collection method and sampling period.

The presence of blood cells in milt of rainbow trout (Oncorhynchus mykiss) collected every week between the middle at the end of the spawning season, either by stripping or by catheterization was investigated. Basic sperm biological and biochemical characteristics were also evaluated. Because milt often becomes contaminated with blood during collection, we also studied the influence of experimental blood contamination on sperm motility and biochemical parameters of seminal plasma. We demonstrated the presence of blood cells (erythrocytes, lymphoid, and phagocytes) in rainbow trout milt collected by both methods. Both sampling period and collection method influenced sperm characteristics, however the relationship between these characteristics and blood cells are not clear at present. A high number of blood cells in milt was found in some samples, possibly due to inflammation, because at the same time we observed bacteria and elevated levels of protein and antiproteinase activity in contaminated samples. Experimental contamination of milt with blood did not influence sperm motility, protein concentration and LDH activity of the 5-day-stored semen. Our study demonstrated that blood cells were present in rainbow trout milt. Blood cells may also appear in milt as a result of bleeding and their elevated levels are present during inflammation.

Acrosin activity in turkey spermatozoa: assay by clinical method and effect of zinc and benzamidine on the activity.

We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 x 10(3) spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate, Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.

Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa.

The effects of extender composition and equilibration time on fertilizing ability of cryopreserved spermatozoa from rainbow trout, Oncorhynchus mykiss, were investigated. In addition, enzyme activity in supernatants from thawed sperm was assessed. The use of the two extenders: Erdahl & Graham's + 10% DMA (dimethyl acetamide) + 10% egg yolk and 0.3 M glucose + 10% DMA yielded the highest post-thaw fertilization rates. We observed interactions between extender constituents and the equilibration of diluted semen. This indicates a multifactorial effect of the extender constituents on spermatozoal resistance against injuries. The 10-min equilibration of spermatozoa in extender before freezing generally lowered the fertilization ability of spermatozoa, except for DMA-based extenders. The addition of egg yolk to the extender was generally beneficial, especially in DMA- and DMSO-based extenders. The use of low-density lipoprotein fraction showed no advantage to full-yolk or free-of-yolk extenders. Aspartate aminotransferase and lactate dehydrogenase leakage from damaged spermatozoa correlated negatively with the ability of cryopreserved spermatozoa to fertilize eggs. Each factor tested, when analyzed separately, did not give general information about its effect on the fertilization ability of cryopreserved sperm. The multifactorial analysis of the important factors in cryopreservation of trout spermatozoa showed their cumulative effect. This is the most likely reason for divergent information reported elsewhere on the effect of various factors in the cryopreservation of rainbow trout spermatozoa.

Effects of season and breed on sperm acrosin activity and semen quality of boars.

Acrosin activity and semen quality (sperm concentration, ejaculate volume and number of spermatozoa) were assessed from March 1997 to March 1998 in semen of Large White, Pietrain and Duroc x Pietrain boars. Semen quality varied with season, including high production of spermatozoa in autumn and winter and low production in summer. Semen quality also differed across breeds. Acrosin activity of boar spermatozoa was not affected by breed (range 3.16-3.32 mU/10(6) spermatozoa), but exhibited distinct seasonal changes. Monthly changes in acrosin activity were parallel to changes in number of sperm in the ejaculate from November to March. On the other hand, dramatic changes in acrosin activity between July and October (range 1.85-4.59 mU/10(6) spermatozoa) were not paralleled by similar changes in number of ejaculated sperm. These fluctuations in acrosin activity may reflect either changes in sperm acrosin production or disturbances to sperm membranes, probably related to effects of high summer temperatures during spermatogenesis. Results confirmed seasonal and breed-related differences in boar semen quality characteristics.

Characteristics of sperm acrosin-like activity of paddlefish (Polyodon spathula Walbaum).

Spermatozoa of paddlefish and sturgeon fishes (Acipenseriformes), unlike teleost fish, have an acrosome. The objectives of this study were to characterize acrosin-like activity of cryopreserved sperm of paddlefish (Polyodon spathula) and to test and compare stability of paddlefish acrosin-like activity with that of lake sturgeon and bull spermatozoa. Mean acrosin-like activity of cryopreserved paddlefish sperm was 0.372 +/- 0.067 microU/10(6) spermatozoa. This activity was 79% higher in the whole semen than in spermatozoa. Highest activity was recorded at pH 8.0 and 8.5. Triton X-100, zinc ions and 4'-acetamidophenyl 4-guanidinobenzoate (AGB) inhibited the activity. Amidase activity was also inhibited by N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). TLCK at concentrations of 0.1 and 1.0 mM gave a significant decrease in activity of 19 and 61%, respectively. However, TPCK significantly inhibited amidase activity (by 19%) only at concentration 1.0 mM. After acidification and 60 min incubation at 4 degrees C of sperm suspensions only 4% of the activity was retained. A similar phenomenon was observed in the case of lake sturgeon but not bull sperm. These results suggest that trypsin-like activity of Acipenserid fish resembles rather fish trypsin that mammalian one. In frozen-thawed paddlefish sperm a minute chymotrypsin-like activity was also indicated, when GPNA was used as substrate. This activity amounted to 0.0415 +/- 0.0138 microU/10(6) spermatozoa and was 18% of total amidase activity. This suggests that chymotrypsin-like activity may also be present in paddlefish spermatozoa.

The effect of egg yolk, low density lipoproteins, methylxanthines and fertilization diluent on cryopreservation efficiency of northern pike (Esox lucius) spermatozoa.

The effect of egg yolk, low density lipoproteins (LDL) as well as methylxanthines (caffeine and theophylline) and fertilization diluent on cryopreservation efficiency of northern pike, Esox lucius, spermatozoa was tested. Milt was cryopreserved in pellets on dry ice then stored in liquid nitrogen. The extender consisted of 0.6 M sucrose + 15% DMSO supplemented with egg yolk or LDL fractions. The most effective results (77.3% hatched larvae vs 74.1% in the control group) were obtained from extender that contained only 0.6 M sucrose + 15% DMSO and was used for freezing, while the fertilization diluent was used for thawing. Addition of egg yolk or LDL to the extender did not improve the results. The presence of caffeine in the thawing solution significantly lowered fertilization rate of cryopreserved spermatozoa, whereas theophylline did not significantly affect the results. The addition of fertilization diluent to the eggs prior to insemination was superior to the other treatments. The proposed procedure constitutes a complete method for the efficient cryopreservation of northern pike semen.

Determination of acrosin activity of boar spermatozoa by the clinical method: optimization of the assay and changes during short-term storage of semen.

A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen.

Studies on a long-term use of rapeseed products in diets for boars. Pathomorphological changes in the reproductive system, liver and thyroid gland.

Three feeding groups were used: the control (SOY) was fed diets without rapeseed products, and the two experimental groups were fed with either 10% rapeseed meal (RSM) or with 12% OO rape seeds (PFRS). Half of the boars from each group were slaughtered after 1 or 2 years. In RSM and PFRS boars steroid-3-beta-ol-dehydrogenase activity was high, whilst Leydig cells were not numerous after 1 year. Degeneration and necrosis of seminiferous epithelium resulting in atrophy of seminiferous tubules appeared in RSM boars after 2 years. In the PFRS group the lesions were stronger and proliferation of Leydig cells with high steroid-3-beta-ol-dehydrogenase activity was observed. In 1-year-old RSM and PFRS boars there were foci of necrosis in the epididymal epithelium. Thyroid weight in RSM boars and liver weight in PFRS boars were distinctly higher only during the first year. In these thyroid glands flattening of glandular epithelium and enlargement of colloid masses were observed, while in the livers, parenchymatic degeneration and structural transformation appeared. Testis weight increased after 2 years in RSM and PFRS boars; however, this had little effect on semen production.

Activity of aspartate aminotransferase and acid phosphatase in cryopreserved trout sperm.

Milt of brown, rainbow and brook trout was cryopreserved. Activity of aspartate aminotransferase (AspAT) and acid phosphatase was assayed both in supernatants and in spermatozoa obtained from thawed sperm samples; additionally, post-thaw motility was evaluated. Enzyme activities differed according to fish species and were strongly affected by the type of cryoprotectant used. The activity in supernatants was usually higher than that in spermatozoa because of protein leakage from injured cells. AspAT activity in cryopreserved spermatozoa correlated positively with fertilization success in all three species. There was a negative correlation between activity of extracellular (supernatant) AspAT and fertilization rates in variants with dimethyl sulfoxide and dimethylacetamide-based extenders. The motility of thawed sperm, determined microscopically, provided some information on the cryopreservation efficiency of trout milt.

Urinary markers for exposures to alkylating or nitrosating agents.

Investigation of urinary markers as indices of endogenous nitrosation and of gastric cancer etiology has been a major focus of our work. As part of this effort, studies have been carried out on a Colombian population at high risk for gastric cancer. In this group, nitrosoproline excretion was highly correlated with nitrate excretion in the subpopulation with advanced gastric pathology, but not in control subpopulations with more normal stomachs. Neither urinary 7-methylguanine nor 3-methyladenine was strongly related to gastric pathology or to urinary nitrate or nitrosoproline levels. More recently, as evidence has accumulated concerning the importance of nitric oxide as a cellular messenger, we have begun research toward developing markers for the presence of nitric oxide and for endogenous nitrosation via this compound. Nitric oxide is formed from arginine by activated endothelial cells as a messenger for vasodilation. We have shown that prolonged exercise leads to increased urinary nitrate and that when 15N-arginine is ingested by humans, 15N-nitrate levels increase in 24-hr urine collections. Nitrosohydroxyethylglycine and 3-nitrotyrosine were evaluated as indices for the formation of N-nitrosomorpholine and for the nitration of protein, respectively, under experimental conditions (e.g., immunostimulation) expected to enhance nitric oxide formation. Nitrotyrosine has not proved useful as a biomarker for nitration/nitrosation reactions in immunostimulated rats. Immunostimulation of rats following administration of morpholine led to increases in urinary nitrate and nitrosohydroxyethylglycine. This procedure, however, would not be appropriate for humans due to the toxicity of morpholine and the carcinogenicity of N-nitrosomorpholine.