Mariana Córdoba

Comparative study of functional and biochemical parameters in frozen bovine sperm

Summary. In order to correlate functional and biochemical parameters, frozen bovine sperm samples presenting diverse motility were studied. Specimens collected from four bulls (A, B, C and D) were graded according to motile spermatozoa percentage and vigour, regarding the sample from bull A as highest motility, those from bulls B and C of intermediate motility and that from bull D of low motility. In order to evaluate lipid peroxidation enhanced by the addition of sodium ascorb‐ate and ferrous sulphate, thiobarbituric acid reactive substances (TEARS) were measured. Values were 0.34 ± 0.18 and 4.95 ± 0.31 nmol TEARS/108 spermatozoa for samples A and D, respectively. Superoxide dismutase (SOD) activity was 144±16.48 and 44±4.0 IU/1010 spermatozoa for A and D, respectively, while intermediate values were recorded for samples B and C. The capability to undergo acrosome reaction induced by calcium ionophore A23187 was significantly lower for sample D, but differences were negligible for the remaining three samples inter se. Motile spermatozoa percentage correlated closely both with SOD activity (r = 0.92) and with TEARS production (r=−0.80), but not with acrosome reacted percentage (r=0.65). From correlation data, it would be inferred that motility is not entirely indicative of sperm quality.

Intracellular calcium variation in heparin-capacitated bovine sperm.

This report investigates the mechanisms by which heparin induces capacitation of sperm. Capacitation was determined with chlortetracycline and intracellular calcium concentration, [Ca2+]i, with FURA 2‐AM. Atter 15 minutes incubation with heparin, [Ca2+]i was increased 60% over basal, reaching a plateau thereafter. Sixty percent of calcium entry was inhibited by methoxy‐verapamil, suggesting that activation of voltage dependent calcium channels (VDCC) may be involved in the process. A significant correlation (R2 = 0.88, p<0.006) was found between the percentage of capacitated spermatozoa and the [Ca2+]i increase. The effects of heparin on both processes were blocked by the protein kinase C (PKC) inhibitors staurosporine (100%) and GF‐109203X (90%). It is concluded that heparin may induce sperm capacitation and calcium influx mainly through VDCC (approx. 60%), and also through other membrane systems (40%). Both systems of calcium entry as well as the capacitation process appear to involve on PKC activity.

Variations in creatine kinase activity and reactive oxygen species levels are involved in capacitation of bovine spermatozoa.

The generation of reactive oxygen species (ROS) is associated with some factors such as oxidative substrate sources, mitochondrial function and NAD(P)H oxidase activity. In bovine spermatozoa, heparin capacitation produces a respiratory burst sensitive to diphenyleneiodonium (DPI). Creatine kinase (CK) is related to extramitochondrial ATP disponibility. Our purpose was to determine the variation in ROS level and its relation with NAD(P)H oxidase sensitive to DPI and CK participation, as factors involved in redox state and energy generation in capacitation. The chlortetracycline technique was used to evaluate capacitation. CK activity and ROS level were measured by spectrophotometry and spectrofluorometry respectively. The capacitation percentage was increased by heparin or quercetin treatment (P < 0.05) and no significant differences in sperm viability were observed. Samples treated with heparin or quercetin maintained the same ROS level as control (238.62 ± 23.47 arbitrary units per 108 spermatozoa) (P > 0.05). CK activity decreased by 50% with heparin or quercetin (P < 0.05). In DPI presence, capacitation was inhibited and differential CK activities and ROS level variations were observed in heparin‐ or quercetin‐treated samples (P < 0.05). In cryopreserved bovine spermatozoa, capacitation requires equilibrium between oxidative damage susceptibility and ROS levels. CK activity is associated with redox state variation and energy sources. In conclusion, capacitation induction depends on NADPH oxidase and the shuttle creatine–creatine phosphate, both sensitive to DPI.

Capacitation and Acrosome Reaction Induction on Thawed Dama dama Deer Spermatozoa: Glycine Effect as Cryopreservation Diluent Supplement

The aim of this research was to evaluate two different diluents for sperm cryopreservation and to study functional parameters in relation to the response to heparin, lysophosphatidylcholine and progesterone, in frozen-thawed semen of fallow deer (Dama dama) during the reproductive season (brama). In this way, fallow deer can be used as a biological model of endangered cervids. Semen was obtained by electroejaculation. Heparin, progesterone and lysophosphatidylcholine were used as capacitation and acrosome reaction inducers, respectively. Capacitation and acrosome reaction were evaluated by chlorotetracycline epifluorescence technique (CTC), membrane integrity by Hypo-osmotic swelling test (HOS) and viability and acrosome integrity by trypan blue stain/DIC. Data was analyzed by ANOVA and Tukey Test (P < 0.05). Semen was cryopreserved in different diluents and Fructose-Tris-Glycine extender was selected. Capacitation with heparin at different incubation times determined that the highest capacitation percentage was obtained at 45 minutes incubation. Progesterone (1 ′M) and lysophosphatidylcholine in heparin capacitated sperm induced acrosome reaction (P < 0.05). This study contributes to improve cryopreservation methods and to increase the knowledge about capacitation and acrosome reaction in vitro in deer spermatozoa, allowing an advance in the development of reproductive biotechnologies.

Progesterone causes metabolic changes involving aminotransferases and creatine kinase in cryopreserved bovine spermatozoa

Progesterone (P4) is capable of inducing acrosome reaction in many species. The objective of this study was to determine the activity of enzymes involved in metabolism that contribute to the redox state and supply energy for acrosome reaction in cryopreserved bull spermatozoa. To accomplish this aim, acrosome reaction was induced by P4 in capacitated and non-capacitated samples. Alanine and aspartate aminotransferases (ALT, AST) and creatine kinase (CK) activities were measured spectrophotometrically at 340 nm after acrosome reaction with P4. Oxygen consumption was measured polarographically. ALT and AST activities increased by the addition of P4 capacitated and non-capacitated samples. P4 addition provoked an increase in CK activity in non-capacitated spermatozoa compared to heparin capacitated spermatozoa with or without P4 addition. P4 increased oxygen consumption, the percentage of acrosome reacted spermatozoa as well as the absence of acrosome integrity in both capacitated and non-capacitated bovine spermatozoa, but oxygen consumption in P4 samples was significantly lower than in heparin capacitated spermatozoa (P<0.05). Acrosome reaction induction by P4 required different creatine kinase activity with the same oxygen consumption and transaminases level to maintain oxidative metabolism and redox state through reducing equivalents transfer between cytosolic and mitochondrial compartment. In conclusion, P4 induces a lower oxidative metabolism during acrosome reaction in bovine cryopreserved spermatozoa, compared to heparin induced capacitation process.