Computer-assisted motion analysis of sperm from the common carp

Computer-assisted semen analysis (CASA) technology was applied to the measurement of sperm motility parameters in the common carp Cyprinus carpio. Activated sperm were videotaped at 200 frames s⁻¹ and analysed with the CellTrak/S CASA research system. The percentage of motile cells and both sperm head curvilinear velocity and straight-line velocity were measured following exposure of carp sperm to three predilution conditions and activation in media of differing ionic strengths and osmotic pressures. The highest percentage of motile sperm was obtained following predilution of sperm in seminal plasma and activation in Na-HEPES buffer pH 8.0. This level of motility was equalled after predilution in 200 m m KCl for 2 h. Straight-line velocities and curvilinear velocities of 130 μm s⁻¹ and 210 μm s⁻¹, respectively, were observed. Duration of motility was higher under seminal plasma predilution conditions (over 50% motile sperm at 55 s post-activation). The application provides a sound basis for the assessment of Sperm Characteristics in fish.     

The properties of tilapia sperm and its cryopreservation*

The specific differences between the testis, milt and sperm of six species of tilapia including Oreochromis aureus, O. mossambicus, O. niloticus, Tilapia zillii, O. nilolicus×O. aureus hybrid and red tilapia, Oreochromis sp., were studied. The shape of testis is tubular; the gonadosomatic index varied from 0.07 to 2.71. The pH values of individual milts ranged from 6.2 to 8.2 and the osmolarity from 240 to 380 mOsmol kg^?1. The quantity of milt obtained by stripping averaged only about 0.3 ml, and only in the O. niloticus×O. aureus hybrid did it exceed 3 ml. Sperm motility graded from weak to moderate was determined for the stripped tilapia milt. Sperm concentrations ranged from 7.70 × 10⁸ sperms ml^?1 in T. zillii to 2.74 × 10¹⁰ sperms ml^?1 in O. mossambicus. Tilapia sperm was active in various salinity ranges such as 0–5‰ for O. niloticus, and 0–15‰ for O. mossambicus and T. zillii. Extender containing 15% milk and 5% methanol was used to prepare milt mixture before cooling rapidly to ?35° C and then at 5° C min^?1 to ?75° C for storage in liquid nitrogen (– 196° C). Fertility tests on frozen tilapia milt resulted in a fertilization rate of 72.7% (v. control 85.7%) for the 22-day frozen milt of the O. nilolicus×O. aureus hybrid used to fertilize the eggs of O. honorum, and 93.4% (v. control 90%) for the 304-day frozen sperm of red tilapia used to fertilize eggs of red tilapia.     

Characterization of lake minnow Eupallasella percnurus semen in relation to sperm morphology,regulation of sperm motility and interpopulation diversity

Sperm morphology and regulation of sperm motility of lake minnow Eupallasella percnurus, an endangered cyprinid, were investigated. Milt characteristics from two isolated populations of E. percnurus were compared to characterize the interpopulation diversity. Electron microscopic studies revealed that E. percnurus spermatozoa comprise simple, uniflagellate, anacrosomal aquasperm with species‐specific features as an eccentrically located implantation of nuclear fossa and eccentric insertion of flagellum. Sperm motility was significantly inhibited by relatively low ion concentrations (150, 150 and 8 mM for NaCl, KCl and CaCl₂, respectively). Sperm maintained a high motility rate over a wide pH range (5·5–10·5), which may reflect adaptation to a highly variable environment. The two E. percnurus populations were markedly different in milt volume, sperm concentration, seminal plasma pH, sperm motility and beat cross frequency, which may result from genetic differences and environmental conditions.     

The influence of the length of time after hormonal treatment with [(d‐Ala6, Pro9 NEt)‐mGnRH+metoclopramide] i.e. Ovopel on barbel Barbus barbus (L.) milt quality and quantity indicators

The study purpose was the analysis of barbel Barbus barbus (L.) milt quality and quantity with regard to the time following stimulation with [(D‐Ala⁶, Pro⁹NEt)‐mGnRH+metoclopramide] i.e. Ovopel. Selected parameters such as total volume of milt (TVM, ml), volume of milt per kg of body weight (VOM, ml kg^?1 b.w.), sperm concentration (×10⁹ ml^?1), total sperm production (TSP, ×10⁹), osmolality (mOsm kg^?1) and pH of seminal plasma were determined. Sperm motility was analyzed by the CASA system, i.e. the percentage of sperm motility (MOT, %) and their progressive motility (PRG, %), curvilinear velocity (VCL, μm s^?1) and straight line velocity (VSL, μm s^?1), amplitude of lateral head displacement (ALH, μm), and beat cross frequency (BCF, Hz). Milt was collected from 12 specimens (N = 12), with the first portion obtained 12 h following treatment with Ovopel (1 granule kg^?1 b. w.). Subsequent portions of milt were taken at 24 h intervals, i.e. after 36, 60, 84, 108, and 132 h. The control group (Control, N = 12) was injected with 0.9% NaCl at 0.5 ml kg^?1b.w. from which milt was taken 12 h post injection. The highest TVM, VOM and TSP values were recorded 12 h after Ovopel treatment (3.2 ± 0.7 ml, 36.7 ± 10.5 ml kg^?1 b.w. and 39.1 ± 9.4 × 10⁹, respectively); lowest values were recorded after 132 h (0.8 ± 0.4 ml, 11.1 ± 6.5 ml kg^?1b.w. and 13.7 ± 7.5 × 10⁹, respectively). The highest seminal plasma osmolality values (300.0 ± 42.6 mOsm kg^?1) as well as the lowest sperm concentration (12.5 ± 1.5 × 10⁹ ml^?1) were observed 12 h after Ovopel treatment. No significant differences in the percentage of sperm motility (MOT) were noted during any of the periods after hormonal stimulation, however, a change in the character of their movement (PRG) was observed. The lack of significant differences (P > 0.05) in VCL and VSL values between 12 h and 60–132 h indicates that the lengthening of time does not lead to a decrease in sperm velocity and, therefore, is not likely to have a negative impact on their quality. The highest ALH (1.9 ± 0.2 μm) and BCF (11.5 ± 1.1 Hz) values were observed when the effect of stimulation was most noticeable, i.e. 12 h after Ovopel treatment. Based on the total milt volume and sperm production, the best time for milt collection from barbel is 12 h post‐hormonal treatment; 84 h post‐hormonal treatment, TVM, VOM, TSP and some CASA parameters decreased, which suggest the same aging process in sperm.     

Sperm motility and seminal fluid composition in the burbot, Lota lota

Sperm motility and composition of the seminal fluid in Lota lota were investigated. Fives after motility initiation, 88.2 ± 12.4% of the spermatozoa were motile, their mean average path swimming velocity was 61.6 ± 16.3 μm s^?1 and their principal swimming type the linear motion (77.4 ± 20.9%). In distilled water the rate of motile spermatozoa decreased to 0% in 40s. In 25–50 mosmol kg^?1 electrolyte (NaCl) or non-electrolyte (glucose, sucrose) solutions, motility was prolonged for 10s and these solutions can therefore increase the efficiency of artificial fertilization when used for sperm motility activation. When semen was diluted in electrolyte or non-electrolyte solutions with osmolalities higher than 50 mosmol kg^?1, sperm motility rates and swimming velocities decreased, and at osmolalities of 400 mosmol kg^?1 motility was completely suppressed. In the seminal fluid with an osmolality of 290.08 ± 45.22 mosmol kg^?1, sodium levels of 139.86 ± 23.79 mmol × 1^?1, potassium levels of 11.59 ± 2.45 mmol × 1^?1 and calcium levels of 0.20 ± 0.08 mmol × 1^?1, sperm motility was inhibited. Under in vitro conditions, artificial saline solutions resembling the seminal plasma composition and 400 mosmol kg^?1 NaCl or glucose solutions were useful as motility inhibiting solutions for predilution of semen. Sperm motility was not affected by pH 7.5–9.0, but at pH 6 the motility rate and the swimming velocity were reduced; seminal fluid pH was 8.47 ± 0.02. Therefore buffering of the artificial saline solutions can provide more stabile conditions for semen during storage and activation. Temperature optimum of semen was between 2 and 5°C. At higher temperatures semen became spontaneously motile. Therefore, controlled temperature conditions are an important factor for handling of semen. The qualitative, organical composition of seminal fluid was similar as in other fresh water teleosts.     

Basic physico-chemical parameters of milt from sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss)

The aim of the study was to compare the physico‐chemical parameters of milt from sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss). Milt was collected by stripping and spermatozoa concentrations, were determined and compared with sperm motility and spermatocrit values along with seminal plasma indices (pH, osmolality, sodium, potassium, chlorine, calcium, magnesium, glucose and protein concentrations). The highest spermatozoa concentration of 22.3 ± 6.7 × 10⁹ ml^?1 was found in the sea trout milt, and was significantly different of those observed in brook trout (11.9 ± 3.3 × 10⁹ ml^?1) and rainbow trout (10.7 ± 4.4 × 10⁹ ml^?1). The values for pH and K⁺ did not differ significantly among species. The mean pH was 8.0 in the milt of each species and the K⁺ concentrations ranged from 24.8 ± 7.2 to 30.5 ± 7.6 mm L^?1. Considerable differences were determined for the Ca²⁺ ions concentrations. The highest value was found in sea trout (1.7 ± 0.3 mm L^?1), while in the rainbow trout it was 0.7 ± 0.5 and in the brook trout 0.4 ± 0.1 mm L^?1. The most pronounced differences were found in the glucose concentration cause of its unnaturally low concentration in rainbow trout of the mean value of 6.0 ± 15.2 mg L^?1. The mean value in sea trout and brook trout was 185.0 ± 172.4 and 231.2 ± 148.4 mg L^?1 respectively. For all species, protein mean values were below 1.3 g L^?1. The mean osmolality was between 230.6 ± 98.6 and 272.0 ± 26.4 mOsm kg^?1 in the species studied. No correlation was found between any components determined in milt and the spermatozoa motility (P > 0.05). The sperm concentration was positively correlated with the protein content in the milt of the three species studied, other less exhibited correlation was found.     

Comparative motility of X and Y chromosome–bearing bovine sperm separated on the basis of DNA content by flow sorting

A combination of flow cytometric sperm sorting of X and Y chromosome–bearing sperm (X and Y sperm) and computer-assisted sperm analysis (CASA) for measuring sperm motility allows assessment of motion parameters in the two populations. Bull sperm were separated into X and Y populations by flow cytometry following staining with the DNA-binding dye Hoechst 33342. The motion parameters differed depending on sperm concentration. Decreasing sperm concentration resulted in higher velocities and straighter trajectories. The concentrations of control (stained-unsorted and unstained-unsorted) and flow-sorted sperm were therefore adjusted to similar numbers (5 × 10⁶ sperm per milliliter). Samples of sorted X and Y sperm and control sperm were transferred to prewarmed slides on a heated stage (37°C) and their motion video recorded for 2 min using a magnification of ×100 and a high-resolution camera. The sperm analysis was carried out on a Hobson Sperm Tracker (HST) using HST 7 software. The following motion parameters were measured: curvilinear, straight-line, and average path velocity; mean angular displacement (MAD); beat cross-frequency; amplitude of lateral head displacement; linearity (LIN); and straightness of path (STR). Sperm movement was unaffected by staining with Hoechst 33342, excitation by ultraviolet (UV) light, or the physical process of cell sorting. Significant differences were seen between X and Y sperm for MAD, LIN, and STR. No difference was observed for the other parameters. The results indicate that in a simple salts solution, Y bull sperm do not swim faster than X sperm but may be distinguished from X sperm on the basis of LIN and STR. Mol. Reprod. Dev. 50:323–327, 1998. Published 1998 Wiley-Liss, Inc.     

Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics

Transferrin (Tf) is a major protein of carp (Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.     

Fine structure and motility of spermatozoa and composition of the seminal plasma in the perch

The fine structure and motility of spermatozoa and the composition of the seminal plasma of the perch Perca fluviatilis are investigated by electron microscopy, computer assisted cell motility analysis (CMA) and biochemical methods. The spermatozoon is asymmetrical as the flagellum inserts mediolateral on the nucleus. It lacks an acrosome, has an ovoid head and a small midpiece with one mitochondrion. Sperm motility–initiated in distilled water (10° C)–is characterized as follows: 85·0 ± 2·7% of the spermatozoa are motile, the main swimming type (10 ± 1 s after motility initiation) is the linear motion (61·4 ± 24·4%) and the average swimming velocity is 122·4 ± 21·9 μm s^–1. When motility is initiated with NaCl, glucose or sucrose solutions of 100 mosmol kg^–1 the percentage of motile spermatozoa and the swimming types are similar as in water, but the swimming velocity (174·0 ± 22·3 μm s^–1) is significantly higher. Motility is inhibited by high osmolality of the diluent: when increasing the osmolality of the saline solutions to 350 mosmol kg^–1 sperm motility is totally suppressed while potassium (10–40 mmol 1^–1) does not affect motility parameters. pH optimum for sperm motility is between pH 7·0 and 8·5. The seminal fluid contains 124·01 ± 21·68 mmol 1^–1 sodium, 10·22 ± 1·11 mmol 1^–1 potassium and 0·72 ± 0·26 mmol 1^–1 calcium. pH is 8·25 ± 0·09, and osmolality 283·90 ± 37·19 mosmol kg^–1. The following organic components were determined: monosaccharides (glucose 63 ± 19 μmol 1^–1, fructose 54 ± 28 μmol 1^–1, galactose 59 ± 25 μmol 1^–1), lipids (cholesterol 5·51 ± 6·42 μmol 1^–1, triglycerides 72 ± l00 μmol l^–1, cholesteryloleate 15–150 μmol 1^–1, phosphatidylcholine 26 · 31 μmol 1^–1, glycolipids 1–10 mg 100 m1^–1), lactate 108 ± 99 μmol 1^–1, hydroxybutyrate 102 ± 99 nmol 1^–1, choline 59 ± 159 μmol 1^–1, protein 344·75 ± 59·06 mg 100m1^–1, enzymes (β-d -glucuronidase l.4 ± 0.7 μmol h^–1 100 ml^–1, protease (caseolytic activity) 1·0 ± 0·6 μmol h^–1 100 ml^–1, alkaline phosphatase 2520·0 ± 861·0 μmol h^–1 100 ml^–1, acid phosphatase 44.0 ± 16.0 μmol h^–1 100 ml^–1, glucose-6-phosphate dehydrogenase 38·9 ± 86·9 μmol h^–1 100 ml^–1, lactate dehydrogenase 134·4 ± 69·6 μmol h^–1 100 ml^–1, butyrylcholine esterase 0·014 ± 0·010 μmol h^–1 100 ml^–1, adenosine triphosphatase 562·8 ± 665·4 μmol h ^–1 100 ml^–1).     

Prostaglandins in rainbow trout (Oncorhynchus mykiss Walbaum, 1792) sperm biology – searching for answers

The purpose of this study was to determine the concentrations of prostaglandins E₂ and F_2α (PGE₂ and PGF_2α) in the blood, testis and seminal plasma of mature male rainbow trout and in the ovarian fluid to assess the effects of these prostaglandins on sperm motility parameters when present in activation media. Also prolonged incubation with prostaglandins on sperm motility and calcium influx were studied. The profile of PGE₂ and PGF_2α differed in concentration between blood, testicular supernatant and seminal plasma. PGE₂ was predominant in the blood sample (0.29 ng ml^?1) and testicular supernatant (3.1 ng ml^?1) whereas their level in seminal plasma was lower than PGF_2α (0.23 ng ml^?1). The concentrations of PGF_2α in blood, testis and seminal plasma were 0.04, 0.99, 1.3 ng ml^?1, respectively. In the ovarian fluid the concentrations of both prostaglandins were higher than in the male reproductive tract. Adding both prostaglandins to activation buffer (at concentrations 15 and 70 ng ml^?1) had no effect on any CASA parameters. Calcium influx related to rainbow trout sperm incubations with PGE₂, and PGF_2α was not detected. After 24 h incubation of sperm in artificial seminal plasma solution without and with prostaglandins all sperm samples increased their motility potential and intracellular calcium concentration. Therefore, this effect was not related to the presence of prostaglandins. In summary PGE₂, and PGF_2α were present in the rainbow trout male reproductive tract, and their profile varies from that of blood, testis and seminal plasma. The specific role of both prostaglandins in salmonid sperm biology remains unclear.     

Spermatozoa motility and variation in the seminal plasma proteome of Eurasian perch (Perca fluviatilis) during the reproductive season

This study evaluated physiological and functional sperm parameters and the seminal plasma proteome of Eurasian perch (Perca fluviatilis) over the course of their reproductive season. Spermatozoa velocity (169.56 ± 6.53 to 158.5 ± 7.4 µm sec^?1), percent motility (95.89 ± 4.28% to 89.55 ± 4.5%), and osmolality of seminal plasma (290 ± 5 to 297 ± 12 mOsmol kg^?1) remained stable throughout the reproductive season. Milt volume and protein concentration of seminal plasma gradually increased and reached the highest values late in the reproductive period. Spermatozoa concentration peaked in the mid‐reproductive season (66.90 ± 13 × 10⁹ spermatozoa ml^?1) and decreased towards the end (54 ± 10 × 10⁹ spermatozoa ml^?1). A proteomic analysis of seminal plasma using two‐dimensional polyacrylamide gel electrophoresis revealed 10 protein spots significantly altered over the course of the reproductive season. Subsequent protein characterization suggested that time in the reproductive season predominantly affected proteins involved in membrane trafficking, organization, cell motility, and oxido‐reductase activity. This study provides new data on physiological properties of sperm and protein patterns of seminal plasma over the course of the reproductive season that should be considered in the development of methods for artificial reproduction of perch. Mol. Reprod. Dev. 79: 879–887, 2012. © 2012 Wiley Periodicals, Inc.     

Optimization of handling and refrigerated storage of guppy Poecilia reticulata sperm

Sperm collection methods and the effect of osmolality, ions, sugar, temperature, pH and dilution ratio on sperm motility were investigated in guppies Poecilia reticulata. The present study revealed that the sperm was motile in a wide range of osmolalities (200–470 mOsm kg^?1) either in Hanks balanced‐salt solution (HBSS) or in non‐electrolyte solutions such as glucose or sucrose. Sperm collected from crushing testes yielded lower motility and shorter motility duration than samples collected without crushing but gentle disruption. Dilution ratios within the range of 1:50 to 1:500 of sperm to HBSS had minimal effect on sperm motility during extended refrigerated storage. Examination of storage temperature showed that refrigerated storage at 4° C was superior to room temperature (25° C). Sperm was found to tolerate a wide range of pH from 5·6 to 7·8, but motility was affected negatively by pH values >7·8.     

Different computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters

In this study, we examined different computer-assisted sperm analysis (CASA) systems (CRISMAS, Hobson Sperm Tracker, and Image J CASA) on the exact same video recordings to evaluate the differences in sperm motility parameters related to the specific CASA used. To cover a wide range of sperm motility parameters, we chose 12-second video recordings at 25 and 50 Hz frame rates after sperm motility activation using three taxonomically distinct fish species (sterlet: Acipenser ruthenus L.; common carp: Cyprinus carpio L.; and rainbow trout: Oncorhynchus mykiss Walbaum) that are characterized by essential differences in sperm behavior during motility. Systematically higher values of velocity and beat cross frequency (BCF) were observed in video recordings obtained at 50 Hz frame frequency compared with 25 Hz for all three systems. Motility parameters were affected by the CASA and species used for analyses. Image J and CRISMAS calculated higher curvilinear velocity (VCL) values for rainbow trout and common carp at 25 Hz frequency compared with the Hobson Sperm Tracker, whereas at 50 Hz, a significant difference was observed only for rainbow trout sperm recordings. No significant difference was observed between the CASA systems for sterlet sperm motility at 25 and 50 Hz. Additional analysis of 1-second segments taken at three time points (1, 6, and 12 seconds of the recording) revealed a dramatic decrease in common carp and rainbow trout sperm speed. The motility parameters of sterlet spermatozoa did not change significantly during the 12-second motility period and should be considered as a suitable model for longer motility analyses. Our results indicated that the CASA used can affect motility results even when the same motility recordings are used. These results could be critically altered by the recording quality, time of analysis, and frame rate of camera, and could result in erroneous conclusions.     

Quantification and identification of sperm subpopulations using computer-aided sperm analysis and species-specific cut-off values for swimming speed

Abstract

Motility is an essential characteristic of all flagellated spermatozoa and assessment of this parameter is one criterion for most semen or sperm evaluations. Computer-aided sperm analysis (CASA) can be used to measure sperm motility more objectively and accurately than manual methods, provided that analysis techniques are standardized. Previous studies have shown that evaluation of sperm subpopulations is more important than analyzing the total motile sperm population alone. We developed a quantitative method to determine cut-off values for swimming speed to identify three sperm subpopulations. We used the Sperm Class Analyzer^® (SCA) CASA system to assess the total percentage of motile spermatozoa in a sperm preparation as well as the percentages of rapid, medium and slow swimming spermatozoa for six mammalian species. Curvilinear velocity (VCL) cut-off values were adjusted manually for each species to include 80% rapid, 15% medium and 5% slow swimming spermatozoa. Our results indicate that the same VCL intervals cannot be used for all species to classify spermatozoa according to swimming speed. After VCL intervals were adjusted for each species, three unique sperm subpopulations could be identified. The effects of medical treatments on sperm motility become apparent in changes in the distribution of spermatozoa among the three swimming speed classes.     

Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout—Salmo trutta, perch—Perca fluviatilis, burbot—Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml⁻¹) and Ig concentrations (T-Ig: 1.11-1.61 μg ml⁻¹, IgG [measured only in brown trout]: 1.49 μg ml⁻¹) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 μg/g protein, IgG: 0.198 μg ml⁻¹) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed.     

Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 °C) on sperm quality

Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll^® centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR^®14-PI) and DNA fragmentation (TUNEL). After addition of a Tris–glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 °C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P < 0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.     

Relationships between reproductive characteristics in male Vimba vimba L. and the effects of osmolality on sperm motility

In Vimba vimba, GSI, sperm volume, and spermatozoa concentration range from 3.4-7.4 %, 0.1-1.1 ml, and 13.3-34.8 × 10⁹ spz ml⁻¹, respectively. Gonad mass (r = 0.90) and sperm volume (r = 0.35) significantly correlated with weight of males. Significant correlation was also found between gonad mass and length of males (r = 0.85). Sperm motility (r = 0.99) and velocity (r = 098) significantly decreased after activation in Tris-HCl 20 mM, pH 8.5. Osmolality of the seminal plasma was 273.2 mOsmol kg⁻¹. Sperm motility and velocity were significantly affected by the osmolality of the activation medium (P < 0.01). Hyper-osmolality compared to seminal plasma osmolality totally suppressed the sperm activation. At 15 s post-activation, the sperm motility significantly decreased at 240 mOsmol kg⁻¹ in KCl or NaCl media. The highest sperm motility and velocity (at 60 s post-activation) were observed at 200 mOsmol kg⁻¹ in NaCl, KCl, or sucrose media. In all treatments, the tip of the flagellum of spermatozoa became curled into a loop shape after activation of sperm in distilled water containing 20 mM Tris-HCl, pH 8.5 that shortened the flagellum.     

Freezing of stallion epididymal sperm

Inseminations with frozen-thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 degrees C for 24h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio, EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen-thawed epididymal sperm showing that Botu-Crio was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp+Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.     

Lifetable demography and population growth of the rotifer Brachionus angularis in Kenya: influence of temperature and food density

Lifetable demography and reproductive traits of a Kenyan strain of the rotifer Brachionus angularis were investigated using individual and small batch culture approaches. The rotifer was identified morphologically before conducting studies at 20, 25 and 30 °C, using Chlorella vulgaris at 2.5 × 10⁵ to 2.5 × 10⁷ cells ml^–1. The rotifers were highly fecund, producing 2.11 ± 0.07 offspring female^–1 day^–1 and reproductive, producing 8.43 ± 0.24 offspring female^–1 at 25 °C with 2.5 × 10⁶ algal cells ml^–1. The highest intrinsic rate of natural increase (0.74 ± 0.02 d^–1), specific population growth rate (0.49 ± 0.01), longest life expectancy at hatching (12.41 ± 0.28 d) and shortest generation time (2.87 ± 0.03 d) also occurred at 25 °C with 2.5 × 10⁶ algal cells ml^–1. The duration of hatching to first spawning was shortest (2.86 ± 0.21 h) at 30 °C with 2.5 × 10⁷ algal cells ml^–1 and longest (8.83 ± 0.39 h) at 20 °C with 2.5 × 10⁵ algal cells ml^–1. The highest population density (255.7 ± 12.6 ind. ml^–1) was realised at 25 °C with 2.5 × 10⁶ cells ml^–1 on Day 8, whereas the lowest population density (122.0 ± 3.6 ind. ml^–1) was realised at 20 °C with 2.5 × 10⁵ cells ml^–1 on Day 8. The lorica length and width of the Kenyan strain of B. angularis are 85.6 ± 3.1 µm and 75.4 ± 3.6 µm, respectively. The rotifer optimally reproduces at 25 °C when fed with 2.5 × 10⁶ algal cells ml^–1.     

Sperm motility characteristics of wild Atlantic salmon (Salmo salar L.) and sea trout (Salmo trutta m. trutta L.) as a basis for milt selection

The aim of the study was to determine the sperm motility parameters in wild Atlantic salmon and sea trout to define criteria important for selection of milt for controlled fertilisation. Parameters for these species were determined in the fish migrating into north‐western rivers of Poland at spawning time. Eight motility parameters percentage of motile sperm (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), linearity (LIN), straightness (STR), amplitude of lateral head displacement (ALH), beat cross frequency (BCF) and motility duration were subjected to computer‐assisted sperm analysis (CASA). Milt of most individuals studied representing both salmon and trout showed spermatozoa density of 12–22 × 10⁹ ml^?1 and a high percentage of motile sperm (>70%). In general, spermatozoa swim progressively with slightly curved trajectories (mean STR = 70%, LIN = 65%) and velocity VCL of 180 μm s^?1 (salmon) and 190 μm s^?1 (trout), at 10 s post‐activation. Such sperm is easily accessible in the wild populations of salmon and sea trout and is recommended for use in reproduction trials. The spermatozoa of sea trout seem to show a greater tendency to follow curvilinear trajectories than those of salmon, both in the beginning and the final phase of motion. In the first phase of motility, the values and time dependencies of the motility parameters were similar in both species. In the end phase of movement differences in LIN and BCF time dependencies were found in the samples representing the two species. In salmon the linearity and beat cross frequency remained stable in this phase, contrary to the patterns in sea trout for which LIN decreased while BCF increased in the end period of movement. Durations of movement were similar in both species (ranges of 20–40 s).