Different signaling pathways in bovine sperm regulate capacitation and hyperactivation

Hyperactivated sperm motility is characterized by high-amplitude and asymmetrical flagellar beating that assists sperm in penetrating the oocyte zona pellucida. Other functional changes in sperm, such as activation of motility and capacitation, involve cross talk between the cAMP/PKA and tyrosine kinase/phosphatase signaling pathways. Our objective was to determine the role of the cAMP/protein kinase A (PKA) signaling pathway in hyperactivation. Western blot analyses of detergent extracts of whole sperm and flagella were performed using antiphosphotyrosine antibody. Bull sperm capacitated by 10 microg/ml heparin and/or 1 mM dibutyryl-cAMP plus 100 microM 3-isobutyl-1-methylxanthine exhibited increased protein tyrosine phosphorylation without becoming hyperactivated. Procaine (5 mM) or caffeine (10 mM) immediately induced hyperactivation in nearly 100% of motile sperm but did not increase protein tyrosine phosphorylation. After 4 h of incubation with caffeine, sperm expressed capacitation-associated protein tyrosine phosphorylation but hyperactivation was significantly reduced. Sperm initially hyperactivated by procaine or caffeine remained hyperactivated for at least 4 h in the presence of Rp-cAMPS (cAMP antagonist) or PKA inhibitors H-89 or H-8. Pretreatment with inhibitors also failed to block induction of hyperactivation; however, the inhibitors did block protein tyrosine phosphorylation when sperm were incubated with capacitating agents, thereby verifying inhibition of the cAMP/PKA pathway. While induction of hyperactivation did not depend on cAMP/PKA, it did require extracellular Ca(2+). These findings indicate that hyperactivation is mediated by a Ca(2+) signaling pathway that is separate or divergent from the pathway associated with acquisition of acrosomal responsiveness and does not involve protein tyrosine phosphorylation downstream of the actions of procaine or caffeine.     

In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa.

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.     

Semen coagulum liquefaction, sperm activation and cryopreservation of capuchin monkey (Cebus apella) semen in coconut water solution (CWS) and TES-TRIS

The objectives of the present study were to test the effect of coconut water solution and TES-TRIS on the seminal coagulum liquefaction, sperm activation in fresh diluted semen, and on the cryopreservation of semen from capuchin monkeys (Cebus apella). Semen was collected from six males by electro-ejaculation, diluted in TES-TRIS or coconut water solution (CWS), and incubated at 35°C until the coagulated fraction of the semen was completely liquefied. In the experiment I, after liquefaction, samples were diluted in TES-TRIS or CWS, plus 6 and 10mM/mL of caffeine. Sperm motility and vigor were evaluated during 5h. For experiment II, after liquefaction, semen samples were extended in TES-TRIS (3.5% glycerol in the final solution) or CWS (2.5% glycerol in the final solution), cryopreserved and stored in liquid nitrogen for 1 week. The seminal coagulum was liquefied in (mean±SDM) 4.5±1.7 and 2.8±1.1h in TES-TRIS and CWS, respectively. Sperm were motile in TES-TRIS and CWS for 5.0±1.4 and 1.0±0.5h, respectively. The mean motility in this period was 38±22% (TES-TRIS) and 22.0±16.0 (CWS). Motility increased after caffeine addition only in samples diluted in CWS containing 6mM (22.5±16.0) or 10mM (28.0±19.0) caffeine. Post-thaw live sperm percentage was 26.2% in TES-TRIS and 13.2% in CWS. For cryopreservation of semen from C. apella TES-TRIS (3.5% glycerol) was more appropriate than CWS (2.5% glycerol). CWS+caffeine potentially increase sperm motility and may be useful in artificial insemination of fresh diluted semen.     

Paradoxical stimulation of human sperm motility by 2-deoxyadenosine

Exposure of cryostored human spermatozoa to 2-deoxyadenosine resulted in significant increases in percentage motility, the linear velocity of progression and the frequency of sperm head rotation, which were maximal at a dose of 2.5 mM. At the same dose both adenosine and caffeine significantly increased percentage motility, although neither compound influenced the quality of sperm movement as assessed by time-exposure photomicrography. 2-Deoxyadenosine was also significantly more effective than caffeine in sustaining the motility of cryostored spermatozoa as well as in enhancing the motility of fresh and washed preparations of human spermatozoa. The ability of caffeine and 2-deoxyadenosine to influence sperm motility was counteracted by the presence of calcium in the external medium although the latter was less susceptible to such inhibition and still enhanced motility in the presence of calcium levels (1.7 mM) typical of media used for in-vitro fertilization. The mechanism of action of 2-deoxyadenosine was associated with an increase of intracellular cAMP levels, which were sustained over a time course lasting from 5 to 180 min and exhibited significant dose dependency over the range 1-10 mM. The response to 2-deoxyadenosine did not involve any changes in the steady state levels of ATP and was augmented by the presence of the phosphodiesterase inhibitors, IBMX and caffeine. We conclude that 2-deoxyadenosine is a powerful stimulator of human sperm motility and that this effect involves an increase of intracellular cAMP levels via mechanisms which do not involve the classical 'R'-site receptor mediated pathway.     

Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.     

In vivo study of cecal fermentation activity in the rabbit. Completion and validation of a new technique for cecal cannulation]

A new technique for caecal cannulation in adult rabbit was validated in order to perform in vivo studies of caecal fermentation under a physiological status. The cannulation did not modify the digestibility of the diet, and it was possible to collect digesta (10 g fresh matter) after a few minutes without stress for animals only 10 days post-surgery. Volatile fatty acid (VFA) (60 mM/l) and ammonia (11 mM/l) concentration of caecal digesta collected in vivo was not different from that obtained from slaughtered rabbits. VFA measurements were reproducible (CV = 6.8%) and were not dependent on the daily collection frequency (1-5/day). Caecal VFA level in the starved rabbit was very low (23 mM/l) while the butyrate/propionate rate was the inverse (C4/C3 = 0.6). Five h after feeding, the in vivo VFA level reached 96 mM/l and the butyrate proportion (12%) over-reached those of propionate (3.5%). The caecal fermentations of a 7-week-old rabbit differed from those of an adult by a higher VFA level and by a greater post-feeding variation in the fermentation pattern.     

Protonmotive force and motility of Bacillus subtilis.

Motility of Bacillus subtilis was inhibited within a few minutes by a combination of valinomycin and a high concentration of potassium ions in the medium at neutral pH. Motility was restored by lowering the concentration of valinomycin or potassium ions. The valinomycin concentration necessary for motility inhibition was determined at various concentrations of potassium ions and various pH's. At pH 7.5, valinomycin of any concentration did not inhibit the motility, when the potassium ion concentration was lower than 9 mM. In the presence of 230 mM potassium ion, the motility inhibition by valinomycin was not detected at pH lower than 6.1. These results are easily explained by the idea that the motility of B. subtilis is supported by the electrochemical potential difference of the proton across the membrane, or the protonmotive force. The electrochemical potential difference necessary for motility was estimated to be about -90 mV.     

Effect of antioxidants on preservation of motility,viability and acrosomal integrity of equine spermatozoa during storage at 5 degrees C

Preservation of liquid semen at 5 degrees C is an important technique in the breeding management of horses. Oxidative damage to spermatozoa during storage is a potential cause of the decline in motility and fertility during hypothermic storage of liquid semen. The objective of this study was to evaluate the use of water-soluble and lipid-soluble antioxidants to improve the maintenance of motility of equine spermatozoa at 5 degrees C during storage for 72 to 96 h. In Experiment 1, the effect of addition of catalase on the maintenance of motility, viability and acrosomal integrity was determined. Semen was collected, and these treatments were applied: catalase (0, 100 or 200 U/mL) in nonfat, dried skim milk extender (NFDSM; with or without seminal plasma) or 10% seminal plasma + NFDSM. Motility was determined by computerized semen analysis (CASA) at 0, 24, 48 and 72 h. Viability and acrosomal integrity were determined at 72 h of storage. There was no significant treatment effect on the maintenance of sperm motility during 72 h storage. In Experiment 2, the effect of adding lipid-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted to a final concentration of 25 x 10(6) sperm/mL in NFDSM containing butylated hydroxytoluene (BHT; 2.0, 1.0, or 0.5 mM), Vitamin E (4.0, 2.0, 1.0 mM), or Tempo (2.0, 1.0, or 0.5 mM). Although the addition of BHT significantly reduced (P < 0.05) progressive motility during storage compared to the control, there were no positive treatment effects of either Vitamin E or Tempo on maintenance of motility. In Experiment 3, the effect of adding water-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted in NFDSM containing these treatments: Trolox (2.0 mM), Tempo (1.0 mM), Vitamin C (0.45 mg/mL), BSA (3% w/v), combinations of these antioxidants, or control. Adding these water-soluble antioxidants did not significantly improve the maintenance of motility during cooled storage at 5 degrees C. In conclusion, adding the enzyme scavenger, catalase, or a variety of lipid- and water-soluble antioxidants did not significantly improve the maintenance of motility during liquid semen storage at 5 degrees C.     

The effects of carnitine,glycerylphosphorylcholine, caffeine,and egg yolk on the motility of rat epididymal spermatozoa

Experiments were performed to further the understanding of epididymal processes involved in the acquisition of sperm motility. Samples of luminal contents were collected by micropuncture from four regions of the rat epididymis. These samples were incubated in various diluents to observe the effects of the diluents on sperm motility. Consonant with previous reports, 40 mM glycerylphosphorylcholine (GPC) and 60 mM DL-carnitine reduced overall motility scores of cauda epididymidal spermatozoa but did not prevent normal initiation of motility. Additionally, control sperm cells and cells treated with carnitine could reinitiate full motility after becoming immotile. Spermatozoa treated with GPC could not reinitiate motility. The sperm cells in our system thus react to GPC and carnitine in fundamentally different ways, the exact nature of which remains to be determined. Spermatozoa from the distal caput epididymidis evidenced high motility scores when diluted in a 5% egg yolk + 10 mM caffeine diluent. It was demonstrated, however, that the subjective appearance of full motility in these immature cells was not supported by actual progressive motility as measured in an assay of linear distance traveled. It was concluded that neither 10 mM caffeine, 5% egg yolk, nor their combination was sufficient to induce progressive motility in immature rat spermatozoa.     

Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa

We have carried out a study on the influence of prolonged cold storage (5 degrees C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 degrees C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 degrees C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 degrees C (P<0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h.     

Characteristics of Iberian red deer (Cervus elaphus hispanicus) spermatozoa cryopreserved after storage at 5 degrees C in the epididymis for several days

The aim of this study was to assess the influence of prolonged cold storage of Iberian red deer epididymides on post-thaw sperm characteristics. Thirty-seven pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control group). The remaining epididymides were cooled to 5 degrees C and stored for 12, 24, 48, 72 and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. After thawing, sperm motility, membrane and acrosome integrities, mitochondrial function and DNA damage were evaluated. The motility of spermatozoa stored in the epididymis for up to 96 h did not decrease significantly (P>0.05) but, after cryopreservation, a decline in sperm motility was seen in spermatozoa stored for 48 h, or later. A slower decrease in sperm membrane and acrosome integrities after cryopreservation were seen as storage time progressed. Some differences were seen when different methods were used to assess the same sperm parameter although changes followed similar patterns. This was the case for acrosome integrity (phase contrast microscopy versus fluorescent lectin) or membrane integrity (hypo-osmotic swelling test or nigrosin-eosin stain versus propidium iodide). We conclude that frozen-thawed spermatozoa of Iberian red deer recovered from epididymides stored at 5 degrees C have a good sperm quality (including motility) during less than 48 h of storage for most of the sperm parameters assessed.     

The disruption of sodium balance in brook charr, Salvelinus fontinalis (Mitchill), by manganese and iron

A primary toxic action of manganese to brook charr, Sulvelinusjonfinalis, at concentrations near or above the 96 h LC₅₀ was the disruption of sodium regulation. Body and plasma sodium concentrations of brook charr declined by 52 and 40%, respectively, during exposure to 10.9 mM manganese (in 250 PM CaCI), and all fish died within 36 h. Sodium balance was less severely affected by 2.7 and 5.5 mM manganese. An increase in the external calcium concentration from 0.05 to 1.0 mM raised the LC₅₀ for manganese from 4.9 to 5.8 mM, and a further increase to 2.5 mM calcium almost doubled it to 10.2 mM. An examination of stable manganese uptake by the gills revealed that accumulation was inversely correlated with body sodium concentration (r =−0.77). Radioactive J4Mn entered the bloodstream in low levels and accumulated in the liver. Thus manganese may have systemic effects as well as those attributable to surface binding on the gill. Studies of the mechanism ofdissolved iron toxicity were less conclusive, but it did not appear to involve extensive disruption of sodium balance. There was about a 15% drop in body sodium concentration when the trout were exposed for 48 h to the 96 h LC₅₀ level of iron, but plasma sodium was unaffected. Also, an iron concentration at twice the LC₅₀ did not escalate the loss of body sodium, and increasing the water calcium concentration did not raise the LC₅₀.     

Effects of fluoride and caffeine on the metabolism and motility of ejaculated bovine spermatozoa

Treatment of washed, ejaculated bovine sperm with 30 mM sodium fluoride immobilized the cells in a characteristically rigid form. In cells metabolizing endogenous substrates, fluoride decreased respiration by about 60%, but did not inhibit the cells' ability to produce adenosine-5'-triphosphate (ATP) via oxidative phosphorylation and did not block access to endogenous substrates. Fluoride-immobilized sperm maintained maximal ATP titers for at least 60 min, but oligomycin treatment rapidly depleted ATP, indicating that ATP synthesis and metabolism was occurring in immobilized sperm. The putative phosphodiesterase inhibitor caffeine (2.5 mM) restored motility and increased respiration in fluoride-treated sperm, but 8-bromo-adenosine-3',5'-monophosphate (8-bromo-cAMP) did not, even though 8-bromo-cAMP stimulated respiration in control (untreated) sperm. Carboxyfluorescein analysis of the intracellular pH of untreated sperm indicated a normal pH of 6.3. Fluoride addition decreased the apparent intracellular pH slightly, but this effect was attributable to dilution. Caffeine did not change internal pH in untreated or fluoride-immobilized sperm. Fluoride did not appear to affect cAMP metabolism, but caffeine increased intracellular cAMP titers by about 35% in both untreated and fluoride-inhibited sperm. However, caffeine treatment did not mimic 8-bromo-cAMP, as analyzed by electrophoresis and autoradiography of sperm proteins labeled with 32P from endogenously generated [32P]ATP. Clearly, caffeine is not stimulating motility in fluoride-treated sperm by affecting the cyclic AMP system. Fluoride also inhibited motility in digitonin-permeabilized sperm by a mechanism that may have involved magnesium depletion, but caffeine had no stimulatory effect on either untreated or fluoride-immobilized, permeabilized sperm.     

Influence of caffeine on movement characteristics, fertilizing capacity and ability to penetrate cervical mucus of human spermatozoa

When added to frozen-thawed human semen, the 3 doses of caffeine tested (2, 5 and 10 mM) induced a significant increase in the percentage of motile spermatozoa but did not influence the quality of movement. Considerable variability was noted between samples in their responsiveness to caffeine which, at the 5 and 10 mM doses, was significantly correlated with the degree of motility lost during cryostorage. Caffeine treatment of frozen-thawed human spermatozoa also increased the number of spermatozoa penetrating cervical mucus in unit time, by increasing the frequency rather than the success of collisions between spermatozoa and the cervical mucus interface. When caffeine-stimulated spermatozoa were washed free of seminal plasma containing this compound they were no longer at an advantage with respect to their motility or fertilizing ability. When 2 mM-caffeine was added to washed suspensions of capacitated spermatozoa it failed to stimulate motility but did significantly enhance the fertilizing ability of the spermatozoa, indicating a possible clinical role for this compound in in-vitro fertilization therapy.     

Developmental and cytogenetic effects of caffeine on mouse blastocysts, alone or in combination with benzo(a)pyrene

Mouse blastocysts were treated with caffeine and/or benzo(a)pyrene (BP), and the effects on development and on induction of sister chromatid exchanges (SCEs) were examined. Caffeine interfered with blastocyst development in a dose-related manner. At 4 mM, the highest concentration tested, caffeine interfered with development of blastocysts to all four endpoints: hatching, trophoblast outgrowth, inner cell mass (ICM) growth, and two-layer (primary endoderm and ectoderm) differentiation of ICMs. At 2 mM, caffeine reduced the incidence of both ICM growth and differentiation but did not affect hatching or formation of trophoblast outgrowths. At 1 mM, caffeine interfered only with ICM differentiation. Cell proliferation was least sensitive to caffeine and was reduced at concentrations of greater than or equal to 2 mM. Induction of SCEs was most sensitive to caffeine exposure; an increase in SCE frequency was observed at 0.1 and 0.5 mM. When caffeine was added to cultures with BP (1 microM, a concentration that was not embryotoxic and did not induce SCEs), both embryotoxic effects and SCE frequency were increased. The enhancing effect on SCE induction was particularly marked; as little as 0.1 mM caffeine was sufficient to cause doubling of induced SCE frequencies when added to cultures with BP.     

Effect of various extenders and taurine on survival of stallion sperm cooled to 5 degrees C

Stallion semen was diluted in five different extenders (dimitro-poulus onze (Dimitro's), Kenney's modified tryode (Kenney's), modified INRA82 (INRA82), egg yolk-citrate-taurine (Citrate) and EZ-Mixin) and evaluated for motility after cooling and storage at 5 degrees C for 0, 24, 48, 72 and 96 h. EZ-Mixin extender was used as control while 70 and 100 mM of taurine were added to Dimitro's, Kenney's and INRA82 to study its effect under conditions of storage at 5 degrees C and varying processing modifications. Motility in INRA82 was 57.0, 58.4, 61.1, and 56.1% after 24, 48, 72 and 96 h, respectively and was higher (P < 0.05) than in other extenders after 48, 72, and 96 h. Motility decreased over time in Dimitro's (P < 0.05) and Kenney's (P < 0.01). Motility in INRA82 and EZ-Mixin decreased (P < 0.05) after 24 h and then remained unchanged until 96 h. In taurine (70 mM) containing extender Citrate, motility decreased throughout storage. Motility in INRA82 and Kenney's with taurine decreased (P < 0.05 and P < 0.01, respectively) after 48 h, and then remained stable until 96 h only in INRA82 with taurine. Motility in INRA82 with taurine was higher (P < 0.05) than in Citrate throughout incubation, whereas motility in INRA82 with taurine was higher than in EZ-Mixin and Kenney's with taurine after 24 and 48 h, respectively. Motility in INRA82 and Kenney's with taurine improved (P < 0.05) when osmotic pressure was increased with taurine (100 mM) but not when osmotic pressure was increased with Na (+) and K (+) salts. Motility was always higher (P < 0.01) in taurine (70 mM or 100 mM) containing extenders than in non-taurine extenders, Dimitro's, INRA82, and Kenney's, when sperm were incubated for 24 h at 5 degrees C in these extenders, then washed and incubated at 39 degrees C in Sp-TALP for 12 or 24 h. In conclusion 1) INRA82 was a better extender than the other extenders tested. 2) inclusion of taurine (100 mM) in INRA82 and Kenney's improved sperm survival until 96 and 48 h, respectively, and 3) sperm preincubation for 24 h in taurine containing extenders resulted in better sperm survival when washed and stored in Sp-TALP for further 12 or 24 h.     

Effect of caffeine on the post-thaw motility of buffalo spermatozoa

Buffalo semen was diluted (1:2) with lactose diluent containing caffeine (2, 4 and 6 mM). Diluted semen samples were frozen in a pellet form (0.15 ml), thawed 24 h after freezing in 2.9% sodium citrate for 30 sec and incubated at 37 degrees C for 3 h. Addition of caffeine to diluted buffalo semen before freezing resulted in a significant increase in the post-thaw motility of spermatozoa over the 3-h incubation period. When caffeine was added to the thawing medium, the post-thaw motility was further improved. Thus, the increase in motility due to caffiene treatment was even more pronounced than in samples treated with caffiene before freezing.     

Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 x 10(6) sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4 degrees C). Three cooling rates (-1.0, -0.3 and -0.1 degrees C/min) were also investigated at the lowest storage temperature (4 degrees C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4 degrees C than at 35 or 22 degrees C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4 degrees C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (-1.0 and -0.3 degrees C) than at the slow cooling rate (-0.1 degrees C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4 degrees C. Additional studies are needed to evaluate the fertility of semen stored in this manner.     

Further evidence for a different mode of action of caffeine and benzamide on mammalian cells

Post-treatment with 2 mM caffeine or 2 mM benzamide increased the lethality of MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) treated V79 cells; in the presence of 50 microM deoxycytidine, the caffeine effect was eliminated whereas the benzamide effect remained the same. Combined treatment with caffeine/benzamide alone produced a large amount of cell lethality which was eliminated by 50 microM deoxycytidine. Benzamide produced a strong inhibition of the poly(ADP-ribose)polymerase activity present in cell-free extracts prepared from V79 cells with greater than 90% inhibition at 2 mM concentration; caffeine on the other hand did not produce any substantial inhibition of this activity in the 2-5 mM range. These results further substantiate our earlier hypothesis that the mode of action of caffeine and benzamide on eukaryotic cells containing DNA damage are not identical [S.K. Das, C.C. Lau and A.B. Pardee (1984) Mutation Res., 131, 71-79].     

Activation of bovine epididymal sperm respiration by caffeine: Its transient nature and relationship to the utilization of acetyl carnitine

Caffeine, which stimulates the motility of freshly extruded bovine epididymal spermatozoa, caused a large but transient increase in the respiratory activity of these cells incubated in a modified Ringer buffer without exogenously added substrate. In spermatozoa that were incubated without added substrate for 2 h at 30 °C or for 15 min at 37 °C, caffeine addition failed to increase respiratory activity even transiently. However, subsequent addition of pyruvate to these aged and caffeine-treated cells resulted in a rapid increase in the respiratory rate, nearly equal to that observed after caffeine addition to fresh cells or to cells stored at 4 °C. These observations indicate that the loss in metabolic response to caffeine is a result of the active metabolism of the spermatozoa.In freshly prepared sperm that were incubated without added substrate, the acetyl carnitine content declined and the free carnitine content of the sperm increased in amounts sufficient to account for the entire respiratory increment produced by caffeine addition. Respiratory stimulation by caffeine was sustained in the presence of those exogenously added substrates that are capable of entering the acetyl carnitine pool, such as acetate, pyruvate, l(+)-lactate, glucose, fructose or β-hydroxybutyrate. Tricarboxylic acid cycle intermediates were not effective.These observations clarify the relationship between the stimulatory effects of caffeine and the metabolic state of the spermatozoan and suggest the importance of the acetyl carnitine pool to the activation of sperm motility and oxidative metabolism.