Mariola Słowińska

Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic cod, Gadus morhua L.

There is a lack of biomarkers or indices that can be used to predict the quality of fish semen samples following the freezing and thawing cycle. In the present study, a series of semen indices were tested to assess if they could accurately forecast the cryopreservation potential of Atlantic cod (Gadus morhua) semen. Fresh and frozen-thawed sperm activity variables were compared, and relationships between frozen-thawed sperm activity and fertilization success were examined. In comparison with fresh sperm, activity variables of frozen-thawed spermatozoa were reduced. Of the 18 males examined, mean (± SEM) spermatocrit of fresh sperm was 40.72 ± 4.23%, osmolality of the seminal plasma 366.32 ± 4.95 mOsmol/kg, pH 8.32 ± 0.04, protein concentration 1.05 ± 0.08 mg/mL, anti-trypsin activity 153.83 ± 19.25 U/L, and total antioxidant capacity 0.15 ± 0.03 μmol Trolox equivalents/mL. Frozen-thawed fertilization success was highly variable among males with values ranging from 18.5 to 90.2%. Regressions yielded significant positive relationships between frozen-thawed motility, velocity, track crossing frequency, and subsequent fertilization success. Sequential multiple regressions explained up to 95% of the variation in frozen-thawed sperm activity. Spermatocrit and pH of fresh semen were negatively related, whereas osmolality and antioxidant capacity were positively related to frozen-thawed motility and velocity. Each of these indices can be measured within minutes of collecting a fresh sample of semen and are thus early indicators of the capacity of semen samples to withstand cryopreservation. These results have many benefits for conservation of wild stocks, aquaculture production, and for understanding semen biology and cryobiology of fishes.

Isolation and characterization of transferrin from common carp (Cyprinus carpio L) seminal plasma.

Transferrin (Tf) in fish is recognized as a component of non-specific humoral defense mechanisms against bacteria. It is a major protein of common carp seminal plasma but its structure and localization in carp testis is unknown. In this study we developed a simple and efficient three-step purification procedure consisting of affinity chromatography (Con A-Sepharose), hydrophobic interaction chromatography (Phenyl Sepharose) and gel filtration (Superdex 200). The molecular mass of Tf has been determined to be 73.6 kDa and isoelectric point 5.1. The peculiar characteristics of carp transferrin were the lack of carbohydrate component and binding of iron ions by only one functional iron-binding site. Western blot analysis revealed a strong similarity of carp seminal plasma Tf to carp blood Tf and Tf from seminal plasma of other cyprinids but a lower similarity to salmonid and percid fishes. Tf was localized to the blood vessels of the carp testis which strongly suggest that most Tf of carp seminal plasma originates from blood. In conclusion, seminal plasma Tf has a unique structure and is similar or identical to blood Tf.

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.

Seminal plasma biochemistry and spermatozoa characteristics of Atlantic cod (Gadus morhua L.) of wild and cultivated origin.

Our objectives were to compare spermatozoa activity, morphology, and seminal plasma (SP) biochemistry between wild and cultivated Atlantic cod (Gadus morhua). Swimming velocities of wild cod spermatozoa were significantly faster than those of cultivated males. Wild males had a significantly larger spermatozoa head area, perimeter, and length, while cultivated males had more circular heads. Total monounsaturated fatty acids and the ratio of n-3/n-6 were significantly higher in sperm from wild males, while total n-6 from cultivated males was significantly higher than the wild males. Significantly higher concentrations of the fatty acids C14:0, C16:1n-7, C18:4n-3, C20:1n-11, C20:1n-9, C20:4n-3, C22:1n-11, and C22:6n-3 were observed in wild males, while significantly higher concentrations of C18:2n-6, C20:2n-6, and C22:5n-3 occurred in cultivated males. Osmolality, protein concentration, lactate dehydrogenase and superoxide dismutase activity of SP of wild males were significantly higher than the cultivated males. Antioxidant capacity of SP was significantly higher in cultivated males, while pH and anti-trypsin did not differ between fish origins. Four bands of anti-trypsin activity and nine protein bands were detected in SP. Performing a discriminant function analysis, on morphology and fatty acid data showed significant discrimination between wild and cultivated fish. Results are relevant to breeding programs and aquaculture development.

Comparison of three staining techniques for the morphometric study of rainbow trout (Oncorhynchus mykiss) spermatozoa.

This study was designed to compare the performance of the kits Diff-Quick, Hemacolor and Spermac for staining the spermatozoa of rainbow trout. Automated sperm morphology analysis (ASMA) was performed using two image analysis programs to determine the sperm measurements: head size (length, width, area and perimeter), shape (ellipticity, rugosity, elongation and regularity) and tail length. Diff-Quick was found to be the best procedure for staining the trout spermatozoa. The use of this method rendered the highest number of cells correctly analyzed, and provided good colour intensity and contrast of the sperm head. No differences among the methods were detected in terms of tail length measurements. Mean values established using Diff-Quick for the main morphometric variables were: head length 2.93+/-0.13 microm; head width 2.33+/-0.15 microm and tail length 34.16+/-1.66 microm. Based on these findings, we recommend the Diff-Quick staining kit for its accurate and reproducible morphometric results. Notwithstanding, when analyzing the sperm tail of the rainbow trout, the Spermac method offers improved contrast.

Isolation and identification of fetuin-B-like protein from rainbow trout seminal plasma and its localization in the reproductive system

Seminal plasma of rainbow trout (Oncorhynchus mykiss, Salmonidae) contains an inhibitory system consisting of three fractions (I-III) characterized by different electrophoretic migration rates. Using a two-step isolation procedure we purified (20- and 43-fold to homogeneity) and characterized the two subforms of inhibitor I (Ia and Ib). On the basis of the homology alignment of the amino acid sequences, inhibitor I was classified to the family of cysteine proteinase inhibitors - fetuins. The molecular masses were determined to be 61,146.5Da and 63,096.0Da, and the isoelectric points were estimated to be 6.04 and 6.22 for inhibitor Ia and Ib. Both inhibitors were glycoproteins with a carbohydrate content about 13% for inhibitor Ia and 19% for inhibitor Ib. The equilibrium association constant of inhibitor Ib with cod trypsin was determined to be 7.1×10(8)M(-1). Except for the cod trypsin inhibition, the inhibitor Ib effectively inhibited papain belonging to the cysteine proteainases. Comparative studies of the distribution of inhibitor I and the previously described inhibitor II were performed. The presence of inhibitor I in the seminal plasma was a common feature of several Salmoniformes, which was contrary to inhibitor II detected in seminal plasma of other fish families. Inhibitors I and II showed different expression patterns in the testes and spermatic duct of the rainbow trout.

Isolation, characterization and cDNA sequencing of acrosin from turkey spermatozoa.

Acrosin (EC 3.4.21.10) is serine proteinase localized in the sperm acrosome and considered to play an essential role in fertilization. In contrast to mammalian, there are only limited data concerning avian acrosin, mostly focused on the characterization of mature enzyme. In the present study, acrosin was isolated from turkey spermatozoa using gel filtration in the presence of 4 M urea at acidic pH. N-terminal Edman sequencing allowed the identification of the first 26 N-terminal amino acids: VVGGTEALHG SWPWIVSIQNPRFAGT. This sequence was used to construct primers and obtain a cDNA sequence from the testis. The amino acid sequence deduced from the cDNA shows that turkey acrosin is initially synthesized as prepro-protein with 19-residue signal peptide. This signal sequence is followed by a 327-residue sequence corresponding to the acrosin zymogen. Turkey proacrosin does not contain a proline-rich segment at the C-terminal portion. Mature turkey acrosin is a two-chain molecule consisting of light and heavy chains and was found to be glycoprotein. The proacrosin/acrosin system exists in turkey spermatozoa and this system can be activated similarly to that of mammals. The high value of association constant strongly suggests that acrosin activity in turkeys can be controlled by a seminal plasma Kazal inhibitor under physiological conditions.

Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo)

The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.

Isolation and Characterization of an Ovoinhibitor, a Multidomain Kazal-Like Inhibitor from Turkey (Meleagris gallopavo) Seminal Plasma

ABSTRACT Turkey seminal plasma contains three serine proteinase inhibitors. Two of them, with low molecular masses (6 kDa), were identified as single-domain Kazal-type inhibitors responsible for regulating acrosin activity. Our experimental objective was to isolate and characterize the inhibitor with the high molecular weight from turkey seminal plasma. The inhibitor was purified using hydrophobic interaction and affinity chromatography. Pure preparations of the inhibitor were used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. Gene expression and immunohistochemical detection of the inhibitor were analyzed in the testis, epididymis, and ductus deferens. The inhibitor with a high molecular weight from turkey seminal plasma was identified as an ovoinhibitor, which was found in avian semen for the first time. The turkey seminal plasma ovoinhibitor was a six-tandem homologous Kazal-type domain serine proteinase inhibitor that targeted multiple proteases, including subtilisin, trypsin, and elastase, but not acrosin. Our results suggested that hepatocyte growth factor activator was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. The presence of the ovoinhibitor within the turkey reproductive tract suggested that its role was to maintain a microenvironment for sperm in the epididymis and ductus deferens. The turkey seminal plasma ovoinhibitor appeared to play a significant role in an antibacterial semen defense against Bacillus subtilis and Staphylococcus aureus.

Biochemical characterization and sperm motility parameters of ostrich (Struthio camelus) semen

The aim of the study was to obtain baseline values for biochemical parameters of ostrich seminal plasma and sperm motility parameters measured by CASA. Biochemical characteristics of ostrich semen included a high protein concentration (29.3 ± 9.1g/l) and high amidase (280.6 ± 130.8 U/l) and LDH activity (1880.0 ± 983.6 U/l). On the other hand antioxidant, superoxide dismutase, anti-proteinase and acid phosphatase activity were low. Biochemical parameters of semen were variable. Motility of ostrich sperm was characterized by low linearity (23.0 ± 6.2%). The quality of undiluted semen stored at room temperature deteriorated within an hour due to agglutination and gelation. On the other hand, ostrich semen could be stored up to 4h at 5°C without loss of motility after which loss of motility occurred but could be partially mitigated using semen extenders (EK and Ovodyl).