Changes in sperm parameters of sex-reversed female rainbow trout during spawning season in relation to sperm parameters of normal males

The production of all-female populations has important economic benefits in commercial rainbow trout aquaculture. The procedure commonly implemented to produce all-female stocks centers on the sex reversal of rainbow trout females via the administration of androgens in the early developmental stages, followed by the egg fertilization of normal females with semen from sex-reversed females (srf). However, there is no information regarding the quality of semen from srf rainbow trout throughout the spawning season. This information is critical because the quality of srf semen is highly variable. The aim of the study was to determine the changes in the semen parameters of srf rainbow trout throughout the duration of the spawning season. Sperm concentration, sperm motility parameters, and the biochemical parameters of seminal plasma (protein concentration, antitrypsin activity, osmolality, and lactate dehydrogenase activity) from srf were monitored during the spawning season and compared with normal male rainbow trout. The observed values of sperm, protein concentration, antitrypsin activity, osmolality, and lactate dehydrogenase activity of seminal plasma were all higher in comparison with normal males. Semen from srf was therefore characterized by a lower sperm motility during each period of the spawning season, in comparison with normal males, approximately 1.8, 1.5, and 1.7 times, respectively for the beginning, middle, and end of the spawning season. The percentage of sperm motility from srf and normal males were affected by the spawning season in the same way, as the highest values in the middle of the spawning season demonstrate (60% and 91% for srf and normal males, respectively). Spermatozoa of srf are characterized by a lower speed and a more curvilinear trajectory of movement as compared with that of normal males. The patterns of changes during the spawning season in sperm concentration, sperm motility parameters, as well as osmolality, and lactate dehydrogenase activity of the seminal plasma of srf were different in comparison with normal males. Our results could be important for fish breeders in regard to the spawning control of srf rainbow trout, as well as for the development of short- and long-term sperm storage procedures.     

Improvement of Sperm Motility of Sex-Reversed Male Rainbow Trout, Oncorhynchus mykiss, by Incubation in High-pH Artificial Seminal Plasma

Changes in the motility time of spermatozoa collected from the testes and the sperm duct of normal and sex-reversed male (XX) rainbow trout in physiological balanced salt solution were examined after incubation in artificial seminal plasmas of various pHs. Although untreated spermatozoa from the sperm duct retained motility for 60–90 s in the balanced salt solution, the spermatozoa collected from the testes were immotile. During the incubation in artificial seminal plasma of pH 7.0, the spermatozoa from the sperm duct hardly moved, similar to the testicular spermatozoa in the balanced salt solution. By suspending and incubating the testicular spermatozoa in artificial seminal plasma of pH 9.9 for 2 h at 4°C, the percentage of motile spermatozoa increased from 0–5% to 80%. The spermatozoa remained motile for at least 2 min after long-term incubation (12 h). When the full-sib eggs were inseminated with untreated testicular spermatozoa or testicular sperm treated for 2 h at high pH, the percentage survival increased from 5.5% to 53.8% at the eyed stage due to the high-pH treatment. The incubation of the spermatozoa in high-pH artificial seminal plasma improved the motility of the spermatozoa from the testes of the sex-reversed male that had lost its sperm duct. By this treatment, it is possible to markedly increase the mass production efficiency of all-female or all-female triploid sterile progenies.     

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.     

The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation

The influence of permeating and of non-permeating cryoprotectants on motility, eosin permeability (sperm cells viability test) and leaking lactate dehydrogenase activity of spermatozoa of the rainbow trout ( Oncorhynchus mykiss ) was investigated. Correlations between sperm motility rate, percentage of eosinimpermeable (viable) cells and LDH activities established the three parameters as indicators for damages to rainbow trout spermatozoa. High sperm motility rates, velocities, and numbers of linear swimming sperm cells, high numbers of eosinimpermeable spermatozoa and a low extracellular LDH activity were evaluated as positive quality characteristics of semen. With a buffered physiological saline solution as basic extender we found a mixture of 0.67 mol L (5%) dimethylsulfoxide (DMSO) and 0.13 mol/L (1%) glycerol to be the most effective cryoprotectant, followed by DMSO (0.67 mol/L [5%]-1.85 mol/L [15%]) and glycerol (0.65 mol/L [5%]-1.78 mol/L [15%]) and propanediol (1.24mol L [10%]-1.78 mol/L [15%]). Addition of hen egg yolk (7%, 20%) and of 15 mmol/L (0.5%) sucrose significantly increased the semen quality in comparison to the same extender without these additives. Bovine serum albumin (1.5%, 3%) had no influence on the investigated semen parameters.     

In vitro methods of assessing the viability of rainbow trout spermatozoa

The accuracy of three in vitro methods for estimating the proportion of dead rainbow trout (Onchorhynchus mykiss ) spermatozoa was investigated. Motility rating, fluorometry using ethidium bromide, and lactate dehydrogenase (LDH) activity in seminal plasma were compared. Semen samples were prepared to contain 0, 25, 50, 75 and 100% killed spermatozoa. All three methods demonstrated highly significant relationships (P<0.001) with the percentage of killed spermatozoa. Motility rating was found to be quick and accurate but required experienced workers and the results thus could vary between evaluators. Fluorometry was rapid and relatively simple to perform and required only a small amount of semen. Measurement of LDH activity in seminal plasma was accurate but time-consuming and required large amounts of semen.     

The effect of seminal plasma on alpaca sperm function

In order to advance the development of assisted reproductive technologies in alpacas and other Camelids, the objective of this study was to explore the role of seminal plasma concentration on motility and functional integrity of alpaca sperm. Sixteen male alpacas > 3 y of age were used. In Experiment 1, epididymal sperm were incubated for 0 to 6 h in 0, 10, 25, 50, or 100% seminal plasma and motility was assessed. In Experiment 2, epididymal sperm were incubated in 0, 10, or 100% seminal plasma for 3 h and motility, acrosome integrity and DNA integrity were assessed. In Experiment 3, ejaculated sperm were incubated in 10, 25, 50, or 100% seminal plasma for 0 to 6 h and motility assessed. In Experiment 4, ejaculated sperm were incubated in 10 or 100% seminal plasma for 3 h and motility, acrosome integrity, DNA integrity, and viability were assessed. Epididymal and ejaculated sperm maintained motility longer when incubated in the presence of 10% seminal plasma compared to 0, 25, 50, or 100% seminal plasma (P < 0.001). The mean ± SEM percentage of epididymal sperm with intact acrosomes was less (P < 0.001) in samples incubated in 0% seminal plasma (39.4 ± 3.73) compared to 10% (75.3 ± 1.20) or 100% (77.4 ± 0.90) within 1 h after incubation. However, DNA integrity of ejaculated and epididymal sperm was not significantly affected by seminal plasma concentration. The mean viability of ejaculated sperm was reduced in the presence of 100 (12.7 ± 2.33) compared to 10% (36.2 ± 4.68) seminal plasma (P < 0.001) within 1 h of incubation. We concluded that alpaca semen should be diluted to a final concentration of 10% seminal plasma to prolong motility, preserve acrosome integrity, and maintain viability of sperm.     

Assessment of ability of endangered Caspian brown trout, <Emphasis Type="Italic">Salmo trutta caspius</Emphasis> (Salmonidae) in production of semen with desirable quality

To assay the effects of stripping frequency on semen characteristics of Caspian brown trout, Salmo trutta caspius, the chemical composition of seminal fluid, sperm production (semen volume, sperm density, spermatocrit,) and sperm motility characteristics (percentage and duration of motility) were investigated over four times stripping during the spawning season. According to data, semen volume, sperm density, osmolality and the concentrations of Na⁺, Cl⁻, K⁺, Ca²⁺, Mg²⁺ and total protein gradually decreased by increasing of stripping frequency. The values of glucose and triglyceride had no significant changes over four times stripping. Also, the values of semen pH, the percentage (5 s after activation) and duration of motility were statistically stable until third stripping but a decrease was recorded for these parameters in the fourth stripping. As well as, significant positive correlations were found for sperm density vs. K⁺, Cl⁻, Na⁺, Ca²⁺, Mg²⁺, total protein, spermatocrit; the percentage of motile spermatozoa vs. Ca²⁺, Mg²⁺, K⁺, Cl⁻, Na⁺, total protein, and also the duration of motility vs. K⁺, Cl⁻, total protein and pH. In text, specific objectives of this study have been expressed.     

Tissue kallikrein activity in seminal plasma of bovine ejaculates with different quality

Tissue kallikrein activity was monitored in seminal plasma from 3 groups of bovine ejaculates: those with normal total sperm motility (78.43%), with reduced sperm motility (49.29%), and with reduced sperm count (0.68 x 10(9) cells/ml). The tissue kallikrein activity was measured spectrophotometrically by using the specific chromogenic substrate S-2266. It was found that the semen samples with normal sperm motility manifested 1.083 microkat/L, on an average, or 29% higher than the activity recorded in ejaculates with reduced sperm motility (P < 0.05). After storage of a group of ejaculates of normal quality for 5 h at room temperature, sperm motility dropped by approximately 80%, expressed as a percentage of the initial motility, while the tissue kallikrein activity in the respective seminal samples decreased by 23%. No significant differences were found in kallikrein activity between ejaculates with normal and reduced sperm counts. It is concluded that a relationship exists between the level of tissue kallikrein activity in the seminal plasma of bovine ejaculates and sperm motility.     

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.     

Correlation of lactate dehydrogenase isoenzyme C4 activity with the count and motility of human spermatozoa.

The activity of lactate dehydrogenase isoenzyme C4 was determined on 90 human semen samples. The correlation between the isoenzyme activity and sperm count and motility was good (r = 0.74 for values of U/ml semen against sperm count).     

Lipid profiles of sperm and seminal plasma from boars having normal or low sperm motility

Sperm plasma membrane lipids have an important role to play in determining membrane fluidity and sperm motility. The objective of the present study was to determine whether there are differences in the lipid and fatty acid (FA) composition of boar sperm and seminal plasma in the ejaculates of boars having different sperm motilities. Semen was collected from two groups of boars having normal (> 60%; n = 53) or low (< 60%; n = 53) motility sperm and the semen was evaluated for motility, morphology and vitality. The semen was then centrifuged to separate the sperm from the seminal plasma and both were kept at −20 °C until analyzed for lipid content and FA profile by gas chromatography. Total antioxidant status (TAS) of seminal plasma was determined using a commercial kit. There were differences (P ≤ 0.05) in sperm total lipids, cholesterol, saturated fatty acids (SFA), phospholipids, n-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and the ratio of n-6:n-3 PUFA between boars with normal and low motility sperm. Total lipids, cholesterol, phospholipids, PUFA, DHA and n-3 PUFA were positively correlated with sperm motility, viability, normal morphology and normal plasma membrane. In contrast, SFA and the ratio of n-6: n-3 PUFA were negatively correlated (P ≤ 0.05) with sperm motility, viability, normal morphology and normal plasma membranes. The TAS of seminal plasma from boars having normal motility sperm was higher (P ≤ 0.05) than that of boars having low motility sperm and TAS was positively correlated (P = 0.0001) with sperm motility, viability, normal morphology and normal plasma membranes. In summary, differences in sperm motility were related to n-3 PUFA content in the sperm plasma membrane and extracellular antioxidants in seminal plasma which protect sperm plasma membranes from lipid peroxidation during periods of oxidative stress.     

Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.     

Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.)

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 μm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 μm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg⁻¹ (average: 283.88 ± 33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P < 0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg⁻¹. Osmolality above 375 mOsmol kg⁻¹ inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.     

Effects of ovarian fluid on motility characteristics of rainbow trout (Oncorhynchus mykiss Walbaum) spermatozoa

The objective of this work was to determine sperm motility characteristics in rainbow trout ( Oncorhynchus mykiss Walbaum) (nine males) and assess the motility enhancement potential of ovarian fluid of nine females in order to test gender effects on sperm motion. Spermatozoa motility characteristics were obtained using computer-assisted sperm analysis (CASA). Ovarian fluid enhanced sperm motility and this was not attributed to the percentage of motile sperm in the samples but rather to the speed and trajectory of movement. For example straight-line velocity (VSL) increased from 32.3 to 85.8 μm s⁻¹ and amplitude of lateral head displacement (ALH) decreased from 20.11 to 10.25 μm. Some females produced ovarian fluid with clearly different sperm motility-enhancement capacity. Furthermore, spermatozoa of some males performed differently regardless of the origin of the ovarian fluid. These data indirectly suggest the existence of cryptic female choice in rainbow trout.     

Quality and fertility of cooled-shipped stallion semen at the time of insemination

Stallion semen processing is far from standardized and differs substantially between AI centers. Suboptimal pregnancy rates in equine AI may primarily result from breeding with low quality semen not adequately processed for shipment. It was the aim of the study to evaluate quality and fertility of cooled-shipped equine semen provided for breeding of client mares by commercial semen collection centers in Europe. Cooled shipped semen (n = 201 doses) from 67 stallions and 36 different EU-approved semen collection centers was evaluated. At arrival, semen temperature was 9.8 ± 0.2 °C, mean sperm concentration of AI doses was 68 ± 3 x 10⁶/ml), mean total sperm count was 1.0 ± 0.1 x 10⁹, total motility averaged 83 ± 1% and morphological defects 45 ± 2%. A total of 86 mares were inseminated, overall per season-pregnancy rate in these mares was 67%. Sperm concentration significantly influenced semen motility and morphology at arrival of the shipped semen. Significant effects of month of the year on volume, sperm concentration and total sperm count of the insemination dose were found. The collection center significantly influenced all semen parameters evaluated. Semen doses used to inseminate mares that became pregnant had significantly higher total and progressive motility of spermatozoa and a significantly lower percentage of morphological semen defects than insemination doses used for mares failing to get pregnant. Results demonstrate that insemination with semen of better quality provides a higher chance to achieve pregnancy. Besides the use of stallions with good semen quality, appropriate semen processing is an important factor for satisfying results in artificial insemination with cooled-shipped horse semen.     

Association of classical semen parameters, sperm DNA fragmentation index, lipid peroxidation and antioxidant enzymatic activity of semen in ram-lambs

The objective was to determine relationships among classical semen characteristics, sperm chromatin structure assay (SCSA), lipid peroxidation and antioxidant enzymatic activity in ram-lamb semen. Fifty-seven ram-lambs were electroejaculated, and routine semen evaluation was conducted (as part of a breeding soundness evaluation). The percentage of sperm DNA fragmentation index (%DFI) and the percentage of sperm with abnormally high DNA stainability (HDS; immature spermatozoa) were determined by SCSA using the metachromatic properties of acridine orange. Semen was centrifuged at 800 x g for 15 min to separate spermatozoa and seminal plasma and the aliquots were stored at -70 degrees C until analyzed. Lipid peroxidation, superoxide dismutase (SOD), and glutathione peroxidase (GPx) levels in seminal plasma and spermatozoa were measured by spectrophotometric assays. The classical semen parameters were negatively related to lipid peroxidation and GPx activity in spermatozoa; motility and morphology were negatively related to %DFI (P < 0.05). Based on Kruskal-Wallis pair-wise comparison of median values among breeding soundness outcome groups, %DFI was lower in the satisfactory group compared to other groups (P < 0.05) and the lipid peroxidation and GPx activity in seminal plasma and spermatozoa were lower in satisfactory and questionable groups (P < 0.05). However, the SOD was lower in the unsatisfactory group (P < 0.05). In summary, classical semen parameters were negatively related to % DFI, lipid peroxidation and GPx activity in ram-lamb spermatozoa and seminal plasma. There were indications that SOD and GPx have crucial protective roles against the toxic effect of reactive oxygen species (ROS) in ram-lamb semen.     

Semen from rainbow trout produced using cryopreserved spermatozoa is more suitable for cryopreservation

Oocytes from three female rainbow trout Oncorhynchus mykiss were inseminated separately with untreated or cryopreserved semen, which had been produced using either untreated (three males) or cryopreserved (three males) spermatozoa. In half of variants, the cryopreservation did not significantly affect fertilization efficiency. Regardless of whether the sperm donors were produced from cryopreserved or intact semen, insemination of oocytes with their intact sperm resulted in the same percentage of eyed embryos (94.4 and 94.3%, respectively). When eggs were inseminated with cryopreserved semen, the use of sperm from males produced with cryopreserved spermatozoa resulted in a significantly higher percentage of eyed eggs than in case of donors produced with intact sperm (89.6 and 81.7%, respectively). The production of rainbow trout using cryopreserved sperm does not appear to negatively affect reproductive abilities of male progeny and semen from donors, which were produced using cryopreserved sperm, is more suitable for cryopreservation than the semen from donors produced with intactspermatozoa.     

Sperm quality analysis in XX, XY and YY males of the Nile tilapia (Oreochromis niloticus)

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offspring), but they also constitute major tools to study sex determinism mechanisms. In other species, sexual genotype and sex reversal procedures affect different aspects of biology, such as growth, behavior and reproductive success. The aim of this study was to assess the influence of sexual genotype on sperm quality in Nile tilapia. Milt characteristics were compared in XX (sex-reversed), XY and YY males in terms of gonadosomatic index, sperm count, sperm motility and duration of sperm motility. Sperm motility was measured by computer-assisted sperm analysis (CASA) quantifying several parameters: total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity and linearity. None of the sperm traits measured significantly differed between the three genotypes. Mean values of gonadosomatic index, sperm concentration and sperm motility duration of XX, XY and YY males, respectively ranged from 0.92 to 1.33%, from 1.69 to 2.22 ×10⁹ cells mL⁻¹ and from 18′04″ to 27′32″. Mean values of total motility and curvilinear velocity 1 min after sperm activation, respectively ranged from 53 to 58% and from 71 to 76 μm s⁻¹ for the three genotypes. After 3 min of activity, all the sperm motility and velocity parameters dropped by half and continued to slowly decrease thereafter. Seven min after activation, only 9 to 13% of spermatozoa were still progressive. Our results prove that neither sexual genotype nor hormonal sex reversal treatments affect sperm quality in male Nile tilapias with atypical sexual genotype.     

Dynamics of exogenous kallikrein-stimulated bovine sperm motility in the presence of seminal plasma kallikrein

Sperm motility is known to be activated and maintained by kallikrein contained within the seminal plasma. We studied the relationship between the levels of seminal plasma kallikrein activity and in vitro exogenous kallikrein-induced sperm motility enhancement. Semen samples were collected from Holstein-Friesian bulls and grouped on the basis of the initial total sperm motility into Group I with > 60 % (mean 75.3 +/- 1.8 %, n = 25), and Group II with < 60 % (mean 51.2 +/- 1.7%, n = 25). Seminal plasma kallikrein activity was measured with the aid of the specific chromogenic substrate S-2266. In Group I the mean activity was 0.983 +/- 0.042 microkat/L, and in Group II it was 0.805 +/- 0.063 microkat/L (P < 0.05). Then each semen sample was divided into a control and an experimental subgroup treated with 16.7 microkat/L of hog pancreatic kallikrein. Total sperm motility was monitored at 1-h intervals. It was found that the addition of exogenous kallikrein stimulated the sperm motility in both groups but in the 4th h after treatment the difference in sperm motility between the experimental and control subgroups of Group I was 12.4 % whereas in Group II it was 21.7 %. We concluded that adding exogenous kallikrein in vitro to semen samples with lower kallikrein activity in the seminal plasma enhanced total sperm motility more than adding it to ejaculates with higher levels of endogenous kallikrein activity.     

Effect of different calcium concentration on sperm motility and fertilisation capacity of rainbow trout (Oncorhynchus mykiss)

Calcium is a prerequisite factor for triggering the sperm activation in salmonids. The aim of this study was to test different concentrations of Ca²⁺ on sperm motility activation and fertility of rainbow trout (Oncorhynchus mykiss). Semen samples activated with calcium free activator (0.0 mM) and increasing concentrations of CaCl₂ (6.3, 7.9, 9.5 and 11 Mm) were evaluated by computer-assisted sperm analysis (CASA). To assess fertility, eggs from three females were fertilized with semen and activated with each of the five solutions and incubated until four-cell stage when the rate fertility and the symmetry of the first blastomeres were evaluated. The results show that eliminating calcium from the activator solution significantly reduced the motility rate and fertility in comparison with the other treatments, while increasing the CaCl₂ from 6.3 to 11 mM generated high sperm motility (78.5 ± 5.8–95.4 ± 4.0%) and fertility (85.0 ± 9.3–96.0 ± 6.51%) rates, and low symmetry rates in the first blastomeres (<20%). No significant differences were found between solutions for the latter two variables, suggesting that the calcium concentrations were in balance with the other components of the medium and within the requirements for the fertilization of rainbow trout.