Impact of changes in composition of storage medium on lipid content and quality of turkey spermatozoa

Turkey semen quality is damaged by long term in vitro storage. The objective of the present study was to determine whether changes in energy substrates and antioxidants of semen extender could limit loss of quality and lipid content of turkey spermatozoa during storage. Spermatozoa were incubated in extenders based on Beltsville Poultry Semen Extender (BPSE) to which different energy substrates (acetate, pyruvate and hydroxybutyric acid) or antioxidant (Vitamin E) had been added. Semen was stored at 4 degrees C for 48 h and changes in quality, phospholipid and malondialdehyde (MDA) content of semen were evaluated. Among the different substrates studied, only acetate was able to limit the loss of motility and ATP content after 48 h in vitro storage. Losses of spermatozoal phospholipids were similar when gametes were incubated in an extender without any substrate or in normal BPSE (784-675nmol/10(9) spz versus 837-703 nmol/10(9) spz). However, motility and ATP content were significantly more affected after 48 h of storage in samples incubated without substrates than in BPSE (motility, 2.2 versus 0; ATP, 10 nmol/10(9) spz versus 3 nmol/10(9) spz). The addition of Vitamin E to the extender did not modify either the MDA or phospholipid content of fresh or stored spermatozoa, but increased the motility of stored semen. In conclusion, acetate is an essential substrate for in vitro storage. Spermatozoal phospholipids decreased during storage, but this did not seem to originate from metabolism of endogenous fatty acids. The positive effects of Vitamin E on semen storage did not originate from preservation of lipid oxidation.     

Changes in adenosine 5'-triphosphate, adenylate energy charge and adenosine 3'5'-cyclic monophosphate during the freezing of buffalo semen

In freshly ejaculated buffalo semen (N = 4) there were 24.61 +/- 5.28 nmol ATP and 40.39 +/- 5.94 nmol total adenylate/10(8) spermatozoa, and 97.75 +/- 7.06 pmol cAMP/10(9) spermatozoa. The semen was frozen in 4 steps (I, dilution; II, cooling; III, glycerolization and equilibration; IV, freezing and thawing). Motility, ATP, total adenylate and cAMP were significantly lower after Step IV than after Step I. Motility and ATP concentration were significantly correlated in egg-yolk--Tris (r = 0.530, P less than 0.05), skim milk--egg yolk (r = 0.754, P less than 0.01), egg yolk--citrate--glucose (r = 0.784, P less than 0.01) and citric acid--whey (r = 0.551, P less than 0.05). Cyclic AMP and motility in egg yolk--Tris were also correlated (r = 0.714, P less than 0.01). The adenylate energy charge was stable in all 4 freezing steps.     

Assessment of sperm viability,mitochondrial activity,capacitation and acrosome intactness in extended boar semen during long-term storage

The purpose of this study was to assess sperm quality in extended boar semen during in vitro storage in order to determine which extender should be used and how long boar semen can be stored. Freshly ejaculated boar semen was diluted with equal volumes of Beltsville thaw solution (BTS), Androhep, KIEV or Zorlesco extenders and stored at 17 degrees C for up to 15 days. Sperm quality was evaluated by examining viability using SYBR-14/PI and Hoechst 33258 staining, mitochondrial activity using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining, acrosome intactness by Coomassie blue staining, and capacitation status by chlortetracycline (CTC) staining. There were over 50% viable spermatozoa in boar semen extended with Zorlesco and Androhep extenders on Day 13 of storage. The percentage of JC-1-stained spermatozoa was 53.8 +/- 2.1% for Zorlesco and 57.7 +/- 1.60% for Androhep extenders on Day 13 of storage. The percentage of acrosome-intact spermatozoa detected by Coomassie blue staining was higher than that in the SYBR-14PI-, Hoechst 33258-, and JC-1-stained samples in our study. The results from SYBR-14/PI, Hoechst 33258, JC-1, and Coomassie blue staining were highly correlated (r > or = 0.9461). There were less than 15% capacitated spermatozoa in the semen extended with BTS, Androhep and Zorlesco extenders during 9 days of storage. However, most viable boar spermatozoa became capacitated by Day 13 of storage. The rank order of four extenders for maintaining sperm viability and mitochondrial activity was as follows: Androhep, Zorlesco, BTS, KIEV.     

Platelet-activating factor content in boar spermatozoa correlates with fertility

The objective of this study was to evaluate the level of platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] content in spermatozoa between two groups of boars that differ in farrow rate percentages. The boar farrow rate was defined as High if it was > or = 70% and Low if it was < 70%. Fresh, extended semen was collected from sexually mature boars and used in the PAF extractions. Platelet-activating factor was detected in all semen samples assayed. The amount of PAF detected in spermatozoa obtained from the High group ranged from 1.90 to 11.30 pM/10(6) cells. The level of PAF in the Low group ranged from 0.92 to 4.96 pM/10(6) cells. Regression analysis revealed a positive (R2 = 0.369) and significant (P = 0.021) relationship between PAF content in boar spermatozoa and farrow rate. Spermatozoa-derived PAF levels (mean +/- SEM) were significantly higher (P = 0.015) in the High-farrow group (6.75 +/- 1.25 pM/10(6) cells) than in the Low-farrow group (2.45 +/- 0.51 pM/10(6) cells). The PAF content in spermatozoa was significantly higher (P = 0.035) in the High-average (> or = 10.5/litter) number of piglets born group (5.78 +/- 1.24 pM/10(6) cells) than in the Low-average (< 10.5/litter) number of piglets born group (3.34 +/- 1.19 pM/10(6) cells). Additionally, PAF content in spermatozoa was significantly higher (P = 0.034) in the High-average (> or = 9/litter) number of piglets born alive group (6.82 +/- 1.35 pM/10(6) cells) than the Low-average (< 9/litter) number of piglets born alive group (3.00 +/- 0.87 pM/10(6) cells). The data demonstrate that PAF is present in boar spermatozoa and that levels are significantly higher in individuals with a high-farrow rate status and high-number of piglets born and born-alive.     

Acrosin activity in turkey spermatozoa: assay by clinical method and effect of zinc and benzamidine on the activity

We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 x 10(3) spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate, Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.     

Role of seminal plasma in damage to turkey spermatozoa during in vitro storage

In vitro storage of turkey spermatozoa is performed without consideration of the potential role of seminal plasma on sperm functions. We report the effects of seminal plasma on membrane permeability, lipid metabolism, energy status, motility and fertility of turkey spermatozoa stored at 4 or 20 degrees C. Phospholipid content (1077 nmol/10(9) spz versus 1219 nmol/10(9) spz at 48 h) and membrane permeability of spermatozoa were significantly damaged by the presence of seminal plasma after 48 h of storage at 4 degrees C, whereas damage to ATP content and fertility occurred earlier damaged by this presence (fertility after 24h storage 51% with seminal plasma versus 71% without). At 20 degrees C, seminal plasma decreased the phospholipid content of spermatozoa in the first hour of storage (1326 nmol/10(9) spz versus 1636 nmol/10(9) spz). Twenty-four hours later, this effect was masked by intense lipid peroxidation. These results show that seminal plasma is deleterious to storage of turkey spermatozoa at 4 degrees C and is involved in phospholipid metabolism of spermatozoa. Lipid peroxidation could be responsible for the acceleration of the degradation of sperm phospholipids during storage at 20 degrees C. However, lipid peroxidation seems not to be active at 4 degrees C. In this case, we suggest that phospholipase activation may contribute to sperm degradation, especially in the presence of seminal plasma.     

Evaluation of boar spermatozoa penetrating capacity using pig oocytes at the germinal vesicle stage

We evaluated the ability of immature pig oocytes (at germinal vesicle stage) to detect differences in the in vitro penetration rates of boar spermatozoa. In Experiment 1, immature and ovulated oocytes (n=303) were exposed to capacitated boar spermatozoa to determine if the penetrability of immature pig oocytes was comparable to that of ovulated oocytes. The percentages of penetrated oocytes and the mean number of spermatozoa per oocyte were similar for immature (88.82 and 7.42+/-0.41) and ovulated oocytes (90.97 and 7.95+/-0.34, respectively). In Experiment 2, immature oocytes (n=1230) were inseminated with semen from 2 boars (A and B) with satisfactory semen characteristics to establish the variability of in vitro penetrating capacity between the boars. Semen was examined for motility, movement quality, acrosome integrity and plasma membrane integrity at various stages of the in vitro procedure. Although the sperm evaluation results were similar between boars, Boar A exhibited a significantly higher (P<0.001) penetration rate (91.49%) and number of spermatozoa penetrated per oocyte (5.90+/-0.25) than Boar B (52.87% and 2.03+/-0.12, respectively). Increasing the sperm concentration at insemination from 1x10(6) to 10x10(6) cells/ml resulted in an increased penetrating capacity for both boars, and the differences in the number of spermatozoa per oocyte between boars also increased. These results indicate that immature pig oocytes can be used in a homologous in vitro fertilization assay, and that despite similarities in semen characteristics a significant boar effect is evident for parameters of in vitro penetration of oocytes.     

Radiorespirometric studies of carbohydrate metabolism by washed spermatozoa of various species.

1. The patterns of 14CO2 evolution from specifically labeled glucose substrates by washed bull, ram, boar, rabbit, dog, rooster and turkey spermatozoa were similar and indicated the Embden-Meyerhof and Kreb's cycle pathways as the major route of energy metabolism. 2. Honey bee spermatozoa metabolized glucose-3,4-[14C], glucose-[U-14C] or fructose-[U-14C], but not glucose-1-[14C], glucose-2-[14C]or glucose-6-[14C], indicating the presence of the glycolytic pathway, but the absence of respiration via the Kreb's cycle. 3. The rate of glycolysis exceeded the rate of respiration in the spermatozoa of all the species studied. 4. A preferential utilization of glucose-1-[14C] over glucose-6-[14C] was evident in some sperm samples, but no consistent indication of pentose cycle metabolism was observed, due to considerable variability between samples within each group. 5. Fructose metabolism was greater than glucose metabolism in the rooster, less in the dog, boar and turkey, and similar in the spermatozoa from the other species examined. 6. Only ram and bull spermatozoa metabolized acetate-1-[14C] to any extent.     

Differences in semen freezability and intracellular ATP content between the rooster (Gallus gallus domesticus) and the Barbary partridge (Alectoris barbara)

This study aimed to compare viability, ATP content, and DNA integrity of rooster (Gallus gallus domesticus) and Barbary partridge (Alectoris barbara) fresh and frozen spermatozoa in order to identify factors possibly related to differences in semen freezability. Ejaculates were obtained from March to May by the abdominal massage method from 3 adult roosters and 12 adult Barbary partridges. Semen was frozen with different cryoprotectants using Lake's diluents as a base medium: 1) glycerol 11%; 2) glycerol 11% and trehalose 70 mmol/L; 3) dimethylacetamide (DMA) 6%; 4) DMA 6% and trehalose 70 mmol/L. Both fresh and frozen semen showed a lower viability and higher intracellular ATP concentrations in the Barbary partridge compared with the rooster (P < 0.05). In the Barbary partridge, semen viability after thawing did not differ among the 4 media used, but glycerol showed positive effects in avoiding a significant loss of ATP after thawing, compared with DMA containing media (P < 0.05). On the other hand, in the rooster a higher viability was recorded when semen was frozen in glycerol containing media compared to DMA (P < 0.0001), while ATP values significantly decreased after thawing (P < 0.05) without showing any differences among the semen frozen in the 4 different media. DNA integrity, as evaluated by the comet assay, was assessed only in frozen semen. In the Barbary partridge, mean scored parameter did not differ significantly among semen frozen in the 4 different media. In the rooster DNA fragmentation was higher in DMA ctr medium compared with the other media and with values found in Barbary partridge semen frozen in the same medium (P < 0.001). In both species, the addition of trehalose did not show any positive effects on viability, ATP levels and DNA integrity after thawing.In conclusion, species-related differences in semen features exist between the rooster and the Barbary partridge and the wide variation observed in ATP levels may account for differences in semen freezabililty between the two species.     

Glycosphingolipid composition of human semen

Glycosphingolipids were extracted from human semen and purified. Based on the fluorometric assay of sphingosine, in spermatozoa a content of 4.4 +/- 0.9 nmol/10(8) cells of gangliosides and 22.1 +/- 1.7 nmol/10(8) cells of neutral glycosphingolipids was determined. Seminal plasma contained 4.1 +/- 0.6 nmol gangliosides and 29.3 +/- 1.5 nmol neutral glycosphingolipids per milliliter. The glycosphingolipid component patterns of human spermatozoa and seminal plasma were determined by thin-layer chromatography. Four neutral glycolipids were isolated and their carbohydrate moieties were characterized. All of these glycolipid components belonged to the globo-series. Gas chromatography, combined gas chromatography/mass fragmentography, and exoglycosidase treatments revealed the following structures for the glycosphingolipids of human semen: Glc1-Cer, Gal beta 1-4Glc1-Cer, Gal alpha 1-4Gal beta 1-4Glc1-Cer, and Gal-NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc1-Cer. In addition, the occurrence of trace amounts of lactoneotetraosyl- and lactoneohexaosylceramide was detected by immunostaining after thin-layer chromatographic separation. Human spermatozoa, as well as seminal plasma, contained the gangliosides Glac1,Glac2, a sialolactoneotetraosylceramide, and a sialolactoneohexaosylceramide. The gangliosides were identified on the basis of their running characteristics by high-performance thin-layer chromatography, exoglycosidase treatment, and immunostaining after thin-layer chromatography. The ceramide composition of the glycolipids in human spermatozoa, as well as in seminal plasma, was dominated by C22:0-behenic acid and the saturated sphingoid d18:0, sphinganine.     

Determination of acrosin activity of boar spermatozoa by the clinical method: optimization of the assay and changes during short-term storage of semen

A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen.     

Futile substrate cycles in the glycolytic pathway of boar and rat spermatozoa and the effect of alpha-chlorohydrin

In boar spermatozoa incubated with 0.1 mM-glucose about 20 nmol glucose were converted to lactate and CO2 and the rate of futile substrate cycling between glucose and glucose 6-phosphate was about 6 nmol/10(8) spermatozoa/30 min. Futile cycling was increased in the presence of 0.05 or 1 mM-alpha-chlorohydrin but not to an extent sufficient to account for the rapid decline in ATP concentration observed under these conditions. These estimates include a substantial rate of fructose formation from fructose phosphates. The addition of 10 mM-L-lactate plus 1 mM-pyruvate protected the spermatozoa against the effect of alpha-chlorohydrin and glucose on the ATP concentration but increased futile substrate cycling. Substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate could not be measured in boar spermatozoa but in rat spermatozoa its rate (nmol/10(8) spermatozoa/30 min) was about 10 under control condition and about 25 in the presence of 1 mM-alpha-chlorohydrin. This increase was insufficient to account for the decline in ATP concentration. In both species futile substrate cycling consumed a significant proportion of the ATP synthesis during lactate production but only about 5% of that produced in the oxidation of glucose to acetyl carnitine and CO2.     

New aspects of boar semen freezing strategies

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2-3% in the freezing media, cooling rates of -30 to -50 degrees C/min, and thawing rates of 1200-1800 degrees C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1x10(9) spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.     

Assay of frozen boar semen with Sephadex filtration

The suitability of filtration of frozen boar semen through Sephadex G-15-120 as a viability assay was investigated. Semen thawed on a hot plate at 38 degrees C was counted with a Coulter Counter before and after filtering through Sephadex columns with 0.1 M sodium citrate as flushing medium. More spermatozoa passed through the column when the temperature of the flushing medium was elevated from room temperature to 37 degrees C and added with 5 mM caffeine (12.3 vs. 22.8% p<0.01). The use of caffeine and 37 degrees C flushings produced a filtrate containing spermatozoa with 89+/-4% motility and 97.6+/-1.6% normal acrosome ridges with the use of frozen semen from 5 different boars. The repeatability was +/-7.3%. The unfiltered samples were judged to contain 45 +/- 8% motile spermatozoa and 66.6 +/- 7.1% spermatozoa with normal acrosome ridges. Filtering of frozen boar semen through Sephadex is proposed as a rapid, objective assay combining the benefits of differential counting of normal acrosome ridges and motility determination.     

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.     

Effects of ouabain on the response to osmotic changes in dog and boar spermatozoa

Although there is much information on the response of spermatozoa from different species to osmotic changes, little has been reported about the mechanism/s by which spermatozoa react to similar changes in the osmotic pressure of the medium. In this study we examine the effect of inhibition of Na (+)K (+), ouabain-sensitive ATP-ase on the response of canine and porcine spermatozoa when they are incubated in hypoosmotic and hyperosmotic media. The presence of ouabain slightly decreased the percentages of total and progressive motility, and increased the percentages of altered acrosomes (from 13.0 +/- 0.3% to 17.2 +/- 0.4% in the presence of 10(-4) M ouabain) and, specially, swollen tails (from 0.6 +/- 0.1% to 5.9 +/- 0.2% in the presence of 10(-4) M of ouabain) in fresh dog semen, although it did not affect these parameters in boar semen samples. Moreover, ouabain increased the percentage of both altered acrosomes and swollen tails in canine spermatozoa incubated in 100 mOsm and in 900 mOsm media at concentrations higher than 10(-5) M and 10(-7) M, respectively. The percentage of viability of canine spermatozoa was not modified by ouabain after incubation in 100, 300 or 900 mOsm media. Furthermore, ouabain did not significantly affect boar spermatozoa incubated in 100, 300 or 900 mOsm media. Although ouabain induced a significant decrease in L-lactate production in canine spermatozoa in an isoosmotic medium (from 4.7 +/- 0.4 micromol mg protein x 60 min to 2.6 +/- 0.3 micromol mg protein x 60 min in the presence of 10(-4) M ouabain), there was no significant effect on L-lactate production in boar spermatozoa. These results indicate that while dog spermatozoa acted against changes in the osmotic pressure by a mechanism(s) related to Na (+)K (+), ouabain-sensitive ATP-ase, boar spermatozoa reacted to some mechanism(s) not related to ionic pumps.     

Steady-state binding of adenine nucleotides ATP, ADP and AMP to rat liver and adipose plasma membranes

Binding of native adenine nucleotides to rat liver and adipose plasma membranes was studied under steady-state conditions using EDTA/Na for inhibition of ecto-nucleotidase activity. [3H]-labelled ATP, ADP and AMP are able to interact with specific binding sites with respective Kd values of 88 +/- 9, 278 +/- 29 and 495 +/- 40 nmol/l for liver membranes; and of 64 +/- 7, 231 +/- 36 and 2050 +/- 290 nmol/l for adipose membranes. The nucleotide-binding capacity (Bmax) varied from 15 to 18 pmol/mg protein in the case of [3H]ATP and [3H]ADP-binding studies and from 22 to 26 pmol/mg protein for [3H]AMP-binding sites. Both 2-MeSATP and ADP inhibited [3H]ATP-binding to membranes with respective IC50 values of 60 +/- 7 and 285 +/- 30 nM. Other purinergic agents suramin, Reactive blue 2, alpha,beta-MeATP and beta,gamma-MeATP were less potent competitors of [3H]ATP binding, whereas AMP, adenosine, GTP, UTP, and CTP did not cause any displacement effect at concentrations of 10(-6)-10(-5) M. It is suggested that the described ATP/ADP-binding sites are linked to G protein-coupled P2Y receptors, whereas AMP-binding sites may represent a substrate-binding component of the membrane ecto-5'-nucleotidase.     

Effect of organic selenium on turkey semen quality during liquid storage

The aim of the present study was to evaluate the effect of dietary organic selenium on the turkey semen during storage. Twenty males (BUT, Big 6, 40 weeks of age) were divided into control (n = 10) and experimental group (n = 10). The turkeys in the both groups were fed with a commercial diet containing 0.1 ppm Se in the form of sodium selenite. The experimental birds were additionally supplied with 0.3 ppm organic Se in the form Sel-Plex™ (Alltech, Inc.). After 30 days of feeding, the semen samples were collected twice a week for the 3 weeks of the study and diluted 1 + 1 (v/v) with TUR-2 diluent, and stored in a water bath (+10 to 15° C) for 6 h. The percentage of motile spermatozoa, the sperm viability (live/dead spermatozoa), total lipids, phospholipids and total cholesterol were assessed in fresh and stored semen. The fertilizing ability of semen was assessed by artificial insemination of 30 hens per group with dose containing 200 × 10⁶ spermatozoa weekly. After 6 h of semen storage, the motility of spermatozoa decreased significantly in the control group (by 8.7 relative percent, P < 0.05) and only by four relative percent (P > 0.05) in experimental group reflecting a protective effect of dietary Se supplementation. The proportion of live spermatozoa was higher in fresh semen and significantly lower in stored semen. The positive effect of Se supplementation was observed on the lipid composition of stored semen: the concentration of the total lipids and phospholipids in the seminal plasma from control group significantly increased, while in the experimental group remained constant. Better semen integrity in the experimental group was associated with an improved fertilizing ability of spermatozoa: the fertility rate of stored spermatozoa in the control group was 88%, while in the experimental group was 90.5%.     

The relationship between ATP content and motility of bovine spermatozoa

The ATP content of bovine semen was measured using a bioluminescent technique. No ATP was detected in seminal plasma or diluent. The mean concentration of ATP (+/- sd) in ejaculated semen was 76.5+/-38.21 n moles ATP/10(8) spermatozoa and in diluted, frozen and thawed semen 10.00+/-4.74 n moles ATP/10(8) spermatozoa. The observed motilities of ejaculated and of diluted, frozen and thawed semen were linearly related to the ATP content r=0.652; p<0.01 and r=0.466; p<0.001 respectively). The mean ATP content and motility calculated for each bull were similarly related (r=0.857; p<0.02 and r=0.607; p<0.05). The technique shows promise in providing an objective measure of spermatozoan activity.     

Protein kinase activity of bovine retina rod outer segments

Dark-adapted pure bovine rod outer segments (ROS) (A280/A500--2.1) can be phosphorylated in the presence of [gamma-32P]ATP and [gamma-32P]GTP. The constant levels of phosphorylation, reached within 10--15 min, are 100 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP and 2--4 pmol 32P/nmol of rhodopsin for [gamma-32P]GTP. These processes are not controlled by 10(-4)--10(-8) cAMP, cGMP or Ca2+, but are inhibited at higher concentrations of these agents. In the presence of histone the constant level of phosphorylation is increased up to 200 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP, but is not changed when [gamma-32P]GTP is used. 10(-5) M cAMP is found to activate the phosphorylation in the presence of histone and [gamma-32P]ATP by 5--6 times. All this evidences that ROS contains cAMP-dependent protein kinase, which utilizes ATP, but not GTP. Moreover, ROS contains cyclic nucleotides- and Ca2+-independent protein kinase. These protein kinases are the ROS endogenous enzymes. This is shown in experiments on separation of pure ROS in a sucrose density gradient.