Inhibition of lactate dehydrogenase C4 (LDH-C4) blocks capacitation of mouse sperm in vitro

Lactate dehydrogenase C4 (LDH-C4) is a tissue-specific enzyme in the mammalian testis and the only lactate dehydrogenase isozyme of sperm. Inhibitors of LDH activity were used to determine whether this enzyme plays a role in sperm capacitation, the acrosome reaction and/or fertilization. Oxamate or its derivative was used to inhibit sperm LDH activity in a medium promoting capacitation. Complete inhibition of LDH activity blocked capacitation. This effect could be reversed partially by the addition of dbcAMP or pentoxifylline to the culture medium. Western blotting showed that oxamate and N-isopropyl oxamate inhibited the tyrosine phosphorylation of proteins during the sperm capacitation process. Presumably, glycolysis is the primary energy pathway for sperm metabolism. The oxidation of reduced NAD with the conversion of pyruvate to lactate by LDH provides ATP necessary for protein kinase A (PKA) activity. Our data indicate that LDH-C4 plays an important metabolic role in sperm capacitation.     

Excess exogenous pyruvate inhibits lactate dehydrogenase activity in live cells in an MCT1-dependent manner

Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-¹³C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging. However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECARs) as a measure of lactate production, and hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various heterotetrameric isoforms comprising LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates in cellulo. Of importance, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and monocarboxylate transporter-1–dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-¹³C]pyruvate magnetic resonance spectroscopic imaging and stable isotope tracer experiments.     

Lactate dehydrogenase C and energy metabolism in mouse sperm

We demonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD(+) cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC.     

Functional interrelations between isozymes of dehydrogenases in the intact and denervated rabbit muscles]

A correlation is shown to exist between malate dehydrogenase (MDH), lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (glycerol-3-PDH activity values, lactate/pyruvate and malate/oxaloacetate coefficients, MDH and LDH isozyme spectra and kinetic properties of LDH isozymes in soluble fractions of cytoplasm from intact rabbit m. soleus (red), m. gastrocnemius (mixed) and m. quadratus lumborum (white). In denervated soleus and gastrocnemius the cytoplasmic MDH/LDH, mitochondrial MDH/LDH, MDH mitochondrial/MDH cytoplasmic activity ratios, concentrations of substrates and isozyme spectra of MDH and LDH tend to equalize. The obtained results indicate the importance of isozyme composition and total activity ratios of the dehydrogenases for regulation of pyruvate and NADH metabolic pathways.     

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

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

Lactate Dehydrogenase Activity is Inhibited by Methylmalonate in vitro

Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K _i=3.02±0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.S. R. Mirandola and E. N. Maciel contributed equally to this work.     

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

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

Biochemical and physiological characteristics of semen of sex-reversed female rainbow trout (Oncorhynchus mykiss, Walbaum)

This works studies the biochemical (protein concentration, osmolality, antitrypsin activity, lactate dehydrogenase activity) and physiological characteristics (sperm motility characteristics) of semen of sex-reversed female rainbow trout (n = 42) obtained with the application of 11β-hydroksyandrostendione for sex reversal. All data were arbitrarily divided into three classes depending on the percentage of sperm motility: I XX < 25%; II XX 25-50% and III XX > 50%. The average percentage of sperm motility was 18 ± 7% n = 12 (group I XX); 42 ± 6% n = 15 (group II XX) and 65 ± 12% n = 15 for group III XX, respectively) to link the values of semen parameters to the maturation stage of semen. Semen from 12 normal males of the same age was used as a reference group. Sperm concentration as well as protein concentration, osmolality, antitrypsin activity, and lactate dehydrogenase activity in seminal plasma of sex-reversed females were higher compared with the values obtained for normal male rainbow trout. The values of these parameters declined with the increasing percentage of sperm motility toward values established for normal males. The fertilization success of semen (3 × 10⁶ spermatozoa/egg) of sex-reversed females was very high (above 90%) for both the percentage of eyed embryos and hatched larvae and was related to sperm motility classes. Correlations between the quality parameters of sex-reversed females semen corresponded to those established previously for the semen of normal male rainbow trout. Antitrypsin activity, lactate dehydrogenase, protein concentration, and osmolality were found to be characteristic of seminal plasma of sex-reversed females. The maturity of sex-reversed female spermatozoa seems to be associated with the decline in the values of those parameters toward the values characteristic for seminal plasma of normal males.     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.     

Cryogenic changes in proteases and antiprotease activities of buffalo (Bubalus bubalis) and cattle (Bos taurus) semen

The postthaw motility and fertility of buffalo and cattle semen is reduced when they are cryopreserved for artificial insemination. In the present study, an attempt was made to characterize the cryogenic changes in proteases and antiprotease activities (APA) of buffalo and cattle semen because these proteolysis regulators have been reported to be associated with sperm motility and fertility. Buffalo sperm demonstrated at least two major proteases of 45 and 42 kDa and three minor proteases of 95, 52, and 33 kDa. Similarly, cattle sperm demonstrated three major proteases of 62, 45, and 42 kDa and two minor proteases of 85 and 78 kDa. Buffalo seminal plasma demonstrated at least three major proteases of 78, 68, and 62 kDa and one minor protease of 98 kDa and cattle seminal plasma demonstrated one major protease of 68 kDa and two minor proteases of 78 and 75 kDa. Except for the 45 kDa protease, most of the previously mentioned proteases were found to be metalloproteinases. Compared with fresh sperm, cryopreserved buffalo and cattle sperm demonstrated a major protease band of 52/49 kDa and the activity of this protease reduced progressively with the duration of cryopreservation. On the contrary, compared with the fresh seminal plasma, cryopreserved buffalo and cattle semen extenders displayed the presence of a new protease band of 45 kDa and demonstrated that this protease activity was leaked from buffalo and cattle cryopreserved spermatozoa. Buffalo and cattle seminal plasmas displayed at least two major APA of 86 and 26 kDa. Compared with buffalo, cattle seminal plasma demonstrated significantly greater APA. Thus, the present study demonstrated the presence of an array of proteases and APA in buffalo and cattle semen and the activities of which changed during cryopreservation. The leakage of the specific protease activity and changes in the proteases and APA might be attributed to reduced motility and fertility of cryopreserved semen in these species.     

In vitro methods of assessing the viability of rainbow trout spermatozoa

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

Cloacal gland foam enhances motility and disaggregation of spermatozoa in Japanese quail (Coturnix japonica)

The adult male Japanese quail produces white foam from the cloacal gland, which is transferred to the female proctodeum during natural mating. The physiological role of foam on quail spermatozoa is still unclear. Therefore, attempts have been made to understand the effect of cloacal foam on motility and metabolism of quail spermatozoa. The profile of various biochemical constitutes in the foam extract was investigated. The addition of foam extract to neat semen completely disaggregated the clumps of spermatozoa leading to vigorous motility. The metabolic rate (MBRT) of the spermatozoa was significantly increased with the addition of foam extract. The foam extract was sub fractionated into seven different fractions by using the molecular cut off devices. Among all the seven sub-fractions from the foam extract, the addition of < 1 KDa sub-fraction contained lactate and has enhanced sperm motility and metabolism. Another fraction (3-10 KDa) has non-protein and non-heparin components which completely disaggregated the clumped quail spermatozoa. However, the remaining fractions did not show any effect on quail spermatozoa. It can be concluded from the present investigation that the lactate present in foam might be a fuel for sperm metabolism and motility. Furthermore, low molecular weight (3-10 KDa) components in the foam may responsible for sperm disaggregation.     

Plasma LDH and CK activities after 400 m sprinting by well-trained sprint runners

Blood lactate concentration and the activities of plasma LDH and CK were determined in 13 well-trained middle distance runners after a 400-m sprint. It was found that there is a significant relationship between mean velocity in the 400-m sprint and plasma CK activity (r = -0.56, P less than 0.05), but the mean sprint velocity did not correlate with peak blood lactate concentration (r = -0.09) or plasma LDH activity (r = -0.40). There was a significant negative correlation between mean sprint velocity and H type LDH isozyme activity (r = -0.66, P less than 0.05), and a significant positive correlation with M type LDH isozyme activity (r = 0.66, P less than 0.05). These results suggest that the magnitude of enzyme efflux from tissue into blood may be depressed by training, and that in well-trained sprinters plasma CK and LDH isozyme activities may be better indicators of physical training and/or physical performance than peak blood lactate or plasma LDH activities.     

Determination of some enzymes and macro- and microelements in stallion seminal plasma and their correlations to semen quality

Seminal plasma is very important for sperm metabolism as well as sperm function and survival and transport in the female genital tract. Analysis of enzyme activities and concentrations of elements can estimate integrity and function of sperm cell membranes. In man much data are available about biochemical analyses of seminal plasma. However, not many studies have been conducted in horses yet. We collected ejaculates from 72 stallions, measured the volume, obtained seminal plasma by centrifugation and examined spermatozoa with light microscopy for motility, concentration, for dead sperm and morphology. Of seminal plasma fluid, we measured activities of aspartate-amino-transferase (AST), gamma-glutamyl-transferase (GGT), alkaline phosphatase (AlP), acid phosphatase (AcP) and lactate-dehydrogenase (LDH) as well as concentrations of sodium (Na(+)), potassium (K(+)), total and ionised calcium (Ca(TOTAL)/Ca(2+)), magnesium (Mg(2+)), phosphate (P), chloride (Cl), copper (Cu), iron (Fe) and zinc (Zn). In addition, correlations among different parameters in light microscopy and seminal plasma were statistically examined by using the Spearman rank correlation coefficient. Median enzyme activities for AST, GGT, AlP, AcP and LDH were 80.0, 7,500, 30,200, 20.0, 81.0 IU/L, respectively. Concentrations of Na(+), K(+), Ca(TOTAL), Ca(2+), Mg(2+), P, Cl were 110.5, 22.1, 2.9, 1.7, 3.1, 1.1 and 114.5 mmol/L, and of microelements Cu, Fe and Zn were 17.8, 1.9 and 13.2 micromol/L, respectively. Furthermore, we found significant correlations between semen volume as well as sperm concentration and AST, GGT, AlP, AcP and LDH as well as Fe and Zn. This made us propose a primary testicular and epididymal origin of these parameters. Significant correlation between GGT and motility may be a sign for its function for cell protection against free radicals. LDH activity significantly correlates with motility and progressive motility, live:dead-ratio and pathomorphology. In our study, LDH seems to be the most predictive enzyme for semen quality. This is the first report about GGT, AcP and LDH activities as well as iron in equine seminal plasma.     

Influence of seminal plasma on the fecundity of chicken spermatozoa

This study was undertaken to investigate the influence of seminal plasma on the fecundity of chicken sperm. Sperm diluted with either incubated seminal plasma (5 or 37 degrees C for 24 h) or seminal plasma from incubated whole semen (5 or 37 degrees C for 24 h) had lower fertility levels and motility scores than sperm diluted in either fresh seminal plasma or a synthetic diluent. The number of sperm with damaged membranes increased with seminal plasma derived from 37 degrees C incubation. The depressive effect of incubated seminal plasma on semen fertility was eliminated by microfiltering .(0.22 mum) the seminal plasma either before or after incubation. Filtration of seminal plasma was only effective in eliminating the depressive effect on sperm motility when filtering was done after incubation. Filtration of seminal plasma reduced the percentage of damaged sperm in all treatments. It can be concluded that there are factors in seminal plasma that are deleterious to the fecundity of chicken spermatozoa and they may be derived from degenerating sperm and/or various fluids, cells and debris collected with the semen during manual semen collection.     

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

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

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

High quality sperm for nonhuman primate ART: Production and assessment

Factors that affect sperm quality can include method of semen collection, conditions for capacitation and whether or not agglutination is present. Media and procedures for sperm washing can also impair or improve sperm function in assisted reproductive technologies. For example, the removal of seminal fluid through large volume washing is required to eliminate decapacitation activity of seminal plasma. The forces involved with centrifugation and the metabolic stress of tightly pelleting sperm during washing procedures can have deleterious results. In contrast to human sperm, sperm from the most commonly used species of nonhuman primates, rhesus and cynomolgus macaques, do not spontaneously capacitate in vitro; rather, chemical activation with dibutryl cyclic AMP and caffeine is required. Recognizing motility patterns of non-activated and activated sperm can be accomplished with simple observation. After activation, sperm agglutination sometimes occurs and can interfere with sperm binding to the zona pellucida. Because nonhuman primate oocytes require a large investment to produce and currently, each animal can be hormonally stimulated a limited number of times, it is important to have means to evaluate quality prior to using sperm from a new male for in vitro fertilization. Methods for producing live, acrosome reacted sperm may also have application for ICSI. Because many genetically valuable males are now being identified, it may be necessary to individualize sperm preparation to accommodate male-to-male variation.     

ATP contents of human semen, seminal plasma and isolated sperm at time intervals after ejaculation

1. ATP was estimated in 105 samples of human semen, seminal plasma and sperm of normozoospermic and oligozoospermic origins at time intervals after ejaculation. 2. In semen with sperm counts up to 40 millions per ml and in seminal plasma, ATP levels were lower than in specimens with higher sperm counts. 3. In isolated sperm, the ATP content decreased with the increase in sperm density. 4. The decrease in ATP after 24 hr was the highest in sperm, lower in semen and the lowest in seminal plasma, being maximal in specimens with high sperm counts.     

Utilization of citrate and lactate through a lactate dehydrogenase and ATP-regulated pathway in boar spermatozoa

Incubation of boar spermatozoa in Krebs-Ringer-Henseleit medium with either 10 mM lactate or 10 mM citrate induced a fast and robust increase in the intracellular levels of ATP in both cases, which reached a peak after 30 sec of incubation. Utilization of both citrate and lactate resulted in the export of CO(2) to the extracellular medium, indicating that both substrates were metabolized through the Krebs cycle. Incubation with citrate resulted in the generation of extracellular lactate, which was inhibited in the presence of phenylacetic acid. This indicates that lactate is produced through the pyruvate carboxylase step. In addition, there was also a significant increase in tyrosine phosphorylation induced by both citrate and lactate. Boar sperm has a sperm-specific isoform of lactate dehydrogenase (LDH), mainly located in the principal piece of the tail. Kinetic studies showed that boar sperm has at least two distinct LDH activities. The major activity (with an estimated Km of 0.51 mM) was located in the supernatants of sperm extracts. The minor LDH activity (with an estimated Km of 5.9 mM) was associated with the nonsoluble fraction of sperm extracts. Our results indicate that boar sperm efficiently metabolizes citrate and lactate through a metabolic pathway regulated by LDH.