Regulation of the motility of fowl spermatozoa by calcium and cAMP

Using an objective light-scattering technique, it was confirmed that washed fowl spermatozoa become immotile as the temperature is raised from 30 degrees C to the normal body temperature of 40-41 degrees C. Motility of washed spermatozoa was restored at 40 degrees C by the addition of caffeine or calcium, both stimulating motility to a maximum in a dose-dependent manner. Neither effector stimulated the motility of spermatozoa at 30 degrees C. Caffeine, but not calcium, caused an increase in sperm cAMP levels at 40 degrees C. The concentrations of calcium and cAMP in untreated spermatozoa were not significantly different in samples incubated at 30 degrees C or 40 degrees C.     

Effects of temperature on the immobilization and the initiation of motility of spermatozoa in the male reproductive tract of the domestic fowl, Gallus domesticus

1. The motility of undiluted fowl spermatozoa taken from testis, epididymis and ductus deferens was negligible at 40 degrees C, around the normal avian body temperature. 2. The immobilization was not permanent and motility was restored by decreasing the temperature to 30 degrees C or by suspending in a NaCl/TES buffer with 2 mM Ca2+, 2 mM HCO3- or 10% seminal plasma at 40 degrees C. 3. Demembranated spermatozoa taken from testis, epididymis and ductus deferens were also immotile at 40 degrees C. However, these spermatozoa were restored the motility at 30 degrees C except testicular spermatozoa. 4. These results suggest that the capacity of movement of fowl spermatozoa can be readily obtained from testis, but that these spermatozoa are immotile due to temperature-dependent immobilization in the male reproductive tract. 5. Furthermore, it is possible that changes in environmental temperature at ejaculation are one of the important exogenous physiological factors of the initiation of fowl sperm motility.     

The mechanisms of reversible immobilization of fowl spermatozoa at body temperature

Intact fowl spermatozoa became almost immotile at 40 degrees C, but motility increased significantly at 30 degrees C. The oxygen consumption at both temperatures was 8-11 microliters O2/10(10) spermatozoa.min-1. The ATP concentration at 40 degrees C was higher than that at 30 degrees C but ADP concentration at 30 degrees C was higher than that at 40 degrees C. Consequently, the ATP/ADP ratio at 30 degrees C (1.9-2.2) increased to 3.5-3.7 at 40 degrees C. The motility of intact spermatozoa at 40 degrees C was effectively restored by 2 mM-Ca2+, 10% seminal plasma and 10% peritoneal fluid taken at the time of ovulation. In contrast, these effectors did not restore the motility of demembranated spermatozoa at 40 degrees C. Motility of demembranated spermatozoa was restored at 30 degrees C. These results suggest that the immobilization of fowl spermatozoa at 40 degrees C occurs due to a decrease in flagellar dynein ATPase activity. Furthermore, the action of effectors for motility such as Ca2+ may not be directly on the axoneme, but mediated by solubilized substances which have been removed by demembranation of the spermatozoa.     

Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa.

The principal objective of this study was to derive an improved procedure for cryopreservation of swamp buffalo (Bubalus bubalis) spermatozoa. Experiments were conducted to determine effects of cooling rate, intermediate plunge temperature and warming rate on motility and acrosome integrity of spermatozoa. Spermatozoa were obtained from three bulls (three ejaculates/bull) and were subjected to nine cooling conditions before being frozen in liquid nitrogen: cooling at 10, 20, or 30 degrees C/min each to -40, -80, or -120 degrees C before being plunged into liquid nitrogen. The spermatozoa frozen under a given condition were then thawed either at 1000 or 200 degrees C/min. Cooling rate, intermediate temperature and warming rate significantly affected survival of spermatozoa obtained from the three bulls. Cooling spermatozoa from 4 to -120 degrees C either at 20 or 30 degrees C/min yielded better progressive motility compared to other cooling conditions (50 versus 30%). Rapid warming was superior to slow warming. In an additional study, motility and fertility of spermatozoa frozen after being cooled to -120 degrees C at 20 degrees C and 30 degrees C/min and those frozen by a standard protocol used routinely for semen processing were assessed. Progressive motility of cryopreserved spermatozoa cooled at 20 degrees C and 30 degrees C/min was 40%, while that of spermatozoa cryopreserved using a standard protocol was 25%. A total of 178 buffalo cows were inseminated with cryopreserved spermatozoa obtained from one bull, and their pregnancy status was assessed 60 days later by rectal palpation. Out of the 60, 26 (43%) and 23 of 58 (40%) cows inseminated with sperm cooled at 20 and 30 degrees C/min, respectively, became pregnant, whereas 17 of 60 (28%) cows inseminated with sperm frozen by a standard protocol became pregnant. This study demonstrates that an effective cryopreservation procedure for buffalo spermatozoa can be derived by systematic examination of various cryobiological factors.     

The possible role of protein-carboxyl methylation in the regulation of flagellar movement of fowl spermatozoa

Both intact and demembranated fowl spermatozoa were incubated at 30 degrees C and 40 degrees C with adenosine, 3-deazaadenosine and homocysteine thiolactone. This combination of products is known to block intracellular protein-carboxyl methylation reaction. The motility of intact spermatozoa incubated at 30 degrees C was vigorous but decreased markedly after the addition of 100 microM adenosine+100 microM 3-deazaadenosine+100 microM homocysteine thiolactone. During this incubation period, the intracellular ATP concentrations of spermatozoa were maintained at approximately 40 nmol ATP/10(9) cells, in spite of the inhibition of motility. The motility of demembranated spermatozoa at 30 degrees C was not inhibited by the same concentrations of blocker. At 40 degrees C, the motility of intact spermatozoa without any effectors was almost negligible. The addition of blocker did not appreciably affect the motility of spermatozoa, which remained almost negligible. In contrast, motility became vigorous even at 40 degrees C when intact spermatozoa were suspended in fluid to which had been added 1 mM CaCl(2) or 100 nM calyculin A, a specific inhibitor of protein phosphatase-type 1 and -type 2. Stimulation of motility by Ca(2+) or calyculin A was inhibited by the presence of a blocker. Contrary to that of intact spermatozoa, the motility of demembranated spermatozoa stimulated by protein phosphatase inhibitor at 40 degrees C was not inhibited by the presence of a blocker. These results suggest that protein-carboxyl methylation may be involved in the regulation of fowl sperm motility. Furthermore, it appears that the methylating enzyme may be present in the cytoplasmic matrix and/or the plasma membrane but not retained in the axoneme and/or accessory cytoskeletal components.     

Regulation of flagellar motility by temperature-dependent phosphorylation of a 43 kDa axonemal protein in fowl spermatozoa.

Phosphorylation of fowl sperm proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after incubating the demembranated spermatozoa with [gamma-32P]ATP at 30 degrees C or 40 degrees C. A marked difference of phosphorylation between 30 degrees C and 40 degrees C was observed in a 43 kDa protein. This protein was slightly phosphorylated at 40 degrees C, but strongly phosphorylated at 30 degrees C in a cAMP-independent manner. The motility of demembranated spermatozoa was negligible at 40 degrees C, but vigorous movement was observed at 30 degrees C. These results showed that phosphorylation of a 43 kDa protein is likely to be a regulatory step in the maintenance of fowl sperm motility.     

Temperature-dependent inhibition of motility in spermatozoa from different avian species

This study demonstrates that the pattern of temperature-dependent inhibition of chicken sperm motility at 40 degrees C in vitro, and its release by calcium, is also found in drake spermatozoa and, partially, in turkey spermatozoa. However, no such temperature-dependent inhibition was found in spermatozoa from Japanese quail and Houbara bustard, for which physiological levels of calcium at 40 degrees C had an inhibitory and no effect on sperm motility, respectively. Thus, on the basis of this evidence on the regulation of avian sperm motility in vitro, the hypothesis that oviducal sperm storage tubules might immobilise spermatozoa by providing a calcium-free environment in vivo does not appear to be universally applicable to all species of birds.     

Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate freezers: a comparison

This study compared variation in the quality of cryopreserved boar spermatozoa and the control and accuracy of cooling rates between three semen freezers (CryoLogic Freeze Control CL3000, Planer Products Kryo Save Compact KS1.7/Kryo 10 Control module and a controlled rate 'Watson' freezing machine developed within our laboratory). Five ejaculates were collected from each of 15 boars (five boars from each of three breeds). Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% v/v glycerol) and placed into 0.5 ml straws. Three straws per treatment, from each ejaculate were cooled to -5 degrees C at 6 degrees C/min, held at -5 degrees C for 30s while ice crystal formation was induced, then further cooled from -5 to 80 degrees C at either 40 degrees C/min (Kryo Save Compact KS1.7 and Watson) or 6 degrees C/min (Freeze Control CL3000). Precise measurements of temperature fluctuations during the programmed cooling curves were made by inserting thermocouples into the semen filled straws. Semen was assessed for %motile cells, motility characteristics using computer-assisted semen analysis (CASA), plasma membrane integrity (%SYBR-14 positive stained spermatozoa) and acrosome integrity (%FITC-PNA positive stained spermatozoa). Spermatozoa cryopreserved using the Freeze Control CL3000 system (maximum rate of 6 degrees C/min) exhibited reduced post-thaw viability (14.2+/-2.8% mean plasma membrane intact spermatozoa) when compared to both the KS1.7 and Watson freezers (optimal rate of 40 degrees C/min) (18.4+/-3.2 and 25.7+/-3.7% mean plasma membrane intact spermatozoa, respectively). Differences in motility characteristics were observed between spermatozoa cryopreserved at 40 degrees C/min with the Watson apparatus preserving a larger proportion of sperm with progressive motility. Cooling curves in the CL3000 and KS1.7 were interrupted by a pronounced increase in temperature at -5 degrees C that corresponded with the latent heat of fusion released with ice crystal formation. This temperature change was significantly reduced in the cooling curves produced by the Watson freezer. These findings suggest that preserving spermatozoa using the Watson freezer improved post-thaw semen quality, with regard to sperm motility characteristics. Furthermore, that post-thaw semen viability was enhanced by minimising temperature fluctuations resulting from the release of the latent heat of fusion at ice crystal formation.     

Centrifugation and addition of glycerol at 22 degres C instead of 4 degrees C improve post-thaw motility and fertility of stallion spermatozoa

The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P<0.05). When centrifugation/glycerol-addition was performed at 22 degrees C (37 degrees C to 22 degrees C in 10 min) (10 ejaculates), post-thaw motility was lower when spermatozoa were frozen directly from 22 degrees C (23%) than when spermatozoa were cooled to 4 degrees C (22 degrees C to 4 degrees C in 1 h) before freezing (47%; P<0.0001). When centrifugation/glycerol-addition was performed at 22 degrees C (before cooling at a moderate rate), as opposed to 4 degrees C (after cooling at a moderate rate), a significant improvement of 1) recovery of spermatozoa after centrifugation (P<0,0001), 2) post-thaw motility of spermatozoa at thawing (40% vs 36% (n < or = 291 ejaculates/group), P<0.0001) and 3) per-cycle fertility (56% vs 42% (n > or = 190 cycles/group), P<0.01) was observed. In conclusion, centrifugation/glycerol-addition at 22 degrees C followed by cooling to 4 degrees C at a moderate rate results in an improvement of post-thaw motility, spermatozoa recovery rate and per cycle fertility.     

Activation of dynein adenosine triphosphatase and flagellar motility of demembranated spermatozoa by monovalent salts at 40 degrees C in the domestic fowl, Gallus domesticus

1. At 40 degrees C, around the normal avian body temperature, demembranated fowl spermatozoa with no addition of monovalent chlorides were immotile. 2. Demembranated spermatozoa become motile at 40 degrees C when 0.1-0.5 M concentrations of NH4Cl, NaCl and KCl were added to the reactivation medium, with maximum motility occurring at 0.2-0.3 M in all cases. 3. The addition of NH4Cl, NaCl and KCl also stimulated the ATPase activity of crude dynein extract. In contrast, LiCl did not appreciably affect motility and ATPase activity. 4. These results showed that the flagellar dynein ATPase activity of fowl spermatozoa could be stimulated by the addition of certain monovalent chlorides, except LiCl, and demembranated spermatozoa might be motile at 40 degrees C.     

Intracellular signal transduction pathways in the regulation of fowl sperm motility: Evidence for the involvement of phosphatidylinositol 3‐kinase (PI3‐K) cascade

The possible role of PI3‐K in the reversible temperature‐dependent immobilization of fowl sperm motility was investigated by using PI3‐K inhibitor (LY294002) and its inactive analogue (LY303511). The existence of the PI3‐K in fowl spermatozoa was also confirmed by Western blotting analysis. Fowl sperm motility in TES/NaCl buffer remained negligible at the avian body temperature of 40°C but was maintained vigorously when the temperature was decreased to 30°C. At 30°C, no stimulation or inhibition of motility was observed after the addition of 2 mM CaCl₂ and 10 µM LY294002 or LY303511: around 70–80% of spermatozoa remained motile. In contrast, at 40°C, the motility of spermatozoa was activated immediately after the addition of Ca²⁺, but the subsequent addition of LY294002 inhibited the motility again. The addition of LY303511 did not appreciably affect the Ca²⁺‐supplemented sperm motility, which was maintained for at least 15 min. The ATP concentrations of spermatozoa after the addition of LY294002 + Ca²⁺ or LY303511 + Ca²⁺ were almost the same values compared with those of Ca²⁺ alone at 40°C, suggesting that the addition of LY294002 was not simply affecting membrane damage or inhibiting energy production in the spermatozoa, but may be acting on some part of the motility‐regulating cascade. Immunoblotting of sperm extract using an antibody to PI3‐K revealed a major cross‐reacting protein of 85 kDa, which corresponds to the molecular weight of the subunit of PI3‐K. These results suggest that PI3‐K may be positively involved in the calcium‐regulated maintenance of flagellar movement of fowl spermatozoa at 40°C. Mol. Reprod. Dev. 76: 603–610, 2009. © 2008 Wiley‐Liss, Inc.     

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws

The interaction of glycerol concentrations of 0-10% and cooling rates from 1 to 1,500 degrees C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30 degrees C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0-1% glycerol after cooling at 30 degrees C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30 degrees C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1 degrees C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500 degrees C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30 degrees C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.     

Optimal physicochemical conditions for the manipulation and short-term preservation of koala (Phascolarctos cinereus) spermatozoa

Protocols for the successful manipulation and preservation of semen in a given species depend upon a fundamental knowledge of how spermatozoa respond to the physicochemical conditions of the extension media; methods developed for the preservation of eutherian spermatozoa may not necessarily be suitable for marsupial semen. The aim of this study was to investigate the effects on koala sperm motility of serial dilution, changes in temperature, diluent pH and osmolality to establish the optimal physicochemical conditions for short-term semen storage. This study showed that electroejaculated koala semen diluted 1∶1 (v/v) with PBS frequently coagulated after incubation at 35 degrees C, but that further dilution and incubation resulted in a corresponding increase in the percentage of spermatozoa swimming in a non-linear trajectory. The effect of rapid temperature change on the motility of koala spermatozoa was investigated by exposing semen, initially diluted at 35 degrees C, to temperatures of 45, 25, 15 and 5 degrees C. Although sperm motility was reduced after incubation at 45 degrees C, a rapid decrease in temperature of up to 20 degrees C did not result in a significant reduction in sperm motility. However, contrary to evidence in other marsupials, there was a small but significant decrease in sperm motility after rapid cooling of diluted semen from 35 to 5 degrees C. The effects of diluent pH and osmolality on the motility of koala spermatozoa were investigated. These experiments indicated that diluents for koala sperm manipulation should buffer in a pH range of 7-8 and have an osmolality of approximately 300 mmol kg(-1). The final experiment compared the relative effectiveness of Tris-citrate buffer (1% glucose) and PBS to maintain koala sperm motility over a range of incubation temperatures (5-35 degrees C) for up to 8 days. Reduction in sperm motility was directly related to temperature, and motility was sustained for the longest duration when stored at 5 degrees C. The Tris-citrate buffer solution was superior to PBS as a preservation diluent at all temperatures, and koala spermatozoa remained motile even after 42 days storage at 5 degrees C. Spermatozoa diluted in PBS (with Ca(2+) or Mg(2+)) and cooled to 5 degrees C showed evidence of an unusual motility pattern, similar to that of hyperactivated eutherian spermatozoa. This study showed that koala spermatozoa respond to different physicochemical conditions associated with short-term liquid storage in essentially the same way as the spermatozoa of eutherian mammals, although koala spermatozoa appear to be more tolerant of rapid temperature shock. The results of this study can be used to make informed selections with regard to appropriate diluent composition and improved short-term sperm preservation protocols and represent the first such database for any species of marsupial.     

Stimulation of sperm motility and oxygen consumption of fowl spermatozoa by a low molecular weight fraction of seminal plasma

Washed fowl spermatozoa were incubated in a phosphate buffer containing various concentrations of fowl seminal plasma at 41 degrees C, normal body temperature, and the motility and oxygen consumption of spermatozoa were determined. Immediately after the incubation, spermatozoa showed good motility in the various diluents. However, with concentrations of seminal plasma at or below 20%, spermatozoa quickly became immotile. In contrast, at concentrations higher than 40% seminal plasma, spermatozoa were motile even after 15 min. As the concentration of seminal plasma was increased, oxygen consumption of spermatozoa also increased. A filtrate of the seminal plasma, obtained by passing the fluid through an Amicon YM-2 ultra-filtration membrane (Mr less than 1000), also stimulated the motility and oxygen consumption of spermatozoa. These results suggest that some low molecular weight factor(s) in fowl seminal plasma stimulated motility and oxygen consumption of fowl spermatozoa at 41 degrees C. A physiological role of this factor(s) may be to assist passage of spermatozoa through the vagina after natural mating.     

Flagellar movement in demembranated preparations of ejaculated fowl spermatozoa

Triton X-100 at a concentration of 0.1% in the extraction medium was optimal for demembranating fowl spermatozoa. The most suitable conditions for reactivation were obtained when a medium composed of 0.5 mM-ATP, 25 mM-potassium glutamate, 10(-7) M-CaCl2, 20 mM-Tris-HCl(pH 7.9), 1 mM-MgSO4, 1 mM-dithiothreitol and 0.2 M-sucrose was used. More than 60% motile spermatozoa were obtained under these conditions. The addition of 1 or 10 microM-cAMP did not appreciably affect motility. Intact and demembranated spermatozoa were immotile at 40 degrees C, whilst at 30 degrees C motility was restored.     

Comparative semen cryopreservation in ferrets (Mustela putorius furo) and pregnancies after laparoscopic intrauterine insemination with frozen-thawed spermatozoa

A study was conducted to determine an optimum technique for semen cryopreservation and the biological competence of frozen-thawed ferret spermatozoa. Fifty-two fresh electroejaculates from 4 males were evaluated for sperm percentage motility, forward progressive motility, motility index (SMI) and acrosomal integrity. To determine the optimum temperature for maintaining sperm motility in vitro and the influence of glycerol on sperm motility, seminal aliquants were diluted in TEST diluent (containing either 0 or 4% glycerol) and maintained at 25 degrees or 37 degrees C. For cryopreservation, semen was diluted in each of 3 cryodiluents (TEST, PDV, BF5F), cooled for 30 min at 5 degrees C and pelleted on solid CO2 or frozen in 0.25 ml straws (20 degrees C/min to -100 degrees C). Following thawing, SMI and acrosomal integrity were determined. Ten females with maximum vulval swelling were given 90 i.u. human chorionic gonadotrophin and laparoscopically inseminated in utero with spermatozoa previously frozen using the optimum diluent and freeze-thaw method. The maintenance temperature of 25 degrees C was superior (P less than 0.05) to 37 degrees C for sustaining sperm motility, and glycerol did not influence (P greater than 0.05) motility for up to 11 h of culture. After thawing, motile spermatozoa were recovered in all treatment groups, but sperm motility and normal acrosomal ratings were highest using the PDV diluent, the pelleting method and thawing at 37 degrees C (P less than 0.05). Seven of the 10 ferrets (70%) inseminated with spermatozoa frozen by this approach became pregnant and produced 31 kits (mean litter size 4.4; range 1-9 kits).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of thawing method on motility, plasma membrane integrity and morphology of frozen-thawed stallion spermatozoa

Different thawing methods are used for stallion semen, however, it is unclear which method is the optimal one. To determine if the thawing temperature has an effect on semen quality, we compared 2 thawing temperatures, 75 degrees C and 37 degrees C. The following parameters were used to measure sperm quality: sperm motility, sperm viability, plasma membrane integrity and sperm morphology. Twenty-three ejaculates from 10 Dutch Warmblood stallions were thawed either at 37 degrees C for 30 sec or at 75 degrees C for 7 sec. Sperm motility was evaluated by a Hamilton Thorn Motility Analyser. Plasma membrane integrity and sperm viability were evaluated by using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-1 probe. The eosinaniline blue staining method was used to evaluate the percentage of live and dead cells, as well as sperm morphology. There was no significant difference (P = 0.84) between sperm motility after thawing at 37 degrees C and 75 degrees C. There was also no significant difference (P = 0.053) between the percentage of live spermatozoa using the calcein AM/ethidium homodimer stain after thawing at 37 degrees C and 75 degrees C. There was, however, a significant difference (P = 0.032) between the percentage of live spermatozoa using the eosin-aniline blue stain after thawing at 37 degrees C compared with that at 75 degrees C. In conclusion, our laboratory results indicated that stud farms using frozen semen should thaw the straws at 37 degrees C instead of 75 degrees C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and has to be timed very carefully to avoid damage to the spermatozoa.     

Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing.

The fertilizing capacity, motility and ultrastructure of fowl and turkey spermatozoa were examined at various stages of the freezing process. For both species, fertility and motility were depressed after equilibration with dimethyl-sulphoxide at 5 degrees C. After freezing, motility was maintained at 55% for fowl spermatozoa and 40% for turkey spermatozoa; however, fertility was 55% for the fowl and 0% for the turkey. Qualitatively, the damage to the spermatozoa of both species was nearly identical, as revealed by scanning and transmission electron microscopy. The plasmalemma was the primary site of damage. 'Bent' spermatozoa, coiled tails and swollen mitochondria were also present. Damage to the acrosome was only observed in spermatozoa which had been frozen to -180 degrees or -196 degrees C. These changes were attributed to adverse osmotic conditions. Binding of cationic ferritin to the plasmalemma of spermatozoa from both species remained unaltered.     

Resolution of the sperm motility-stimulating principle of fowl seminal plasma into Ca2+ and an unidentified low molecular weight factor

It was confirmed, using an objective assay of motility, that fowl seminal plasma restores and stimulates the motility of fowl spermatozoa at 40 degrees C in a dose-dependent manner. By separation of a 100,000 g supernatant of fowl seminal plasma with Sephadex G-15, two peaks of motility-stimulating activity were distinguished. One peak coincided with that of calcium and was absent when calcium was removed from the seminal plasma with Dowex 50. The other peak, which accounted for 44% of motility-stimulating activity, contained a low molecular weight, dialysable factor which remains to be identified.