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      Efecto de la adición de cafeína y lactato sobre la motilidad del semen equino diluido en leche descremada-glucosa Translated title: Effect of caffeine and lactate addition on the motility of equine semen diluted in skim-glucose milk

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          Abstract

          En equinos la inseminación artificial se practica mayormente con semen refrigerado por las dificultades que plantea la criopreservación. Para mejorar las condiciones de conservación a 5ºC se debe considerar el deterioro espermático post-recolección, puesto que componentes del plasma seminal complican la supervivencia de los espermatozoides con procesos oxidativos. Algunos compuestos tienen propiedades antioxidantes y mejoran notablemente la motilidad y la supervivencia espermática. En esta experiencia se utilizó lactato de sodio (2mM) y cafeína (10 mM) incorporados al momento de la dilución del semen y a las 48 h de almacenaje a 5ºC, en un extender de base leche descremada-glucosa, con el propósito de estudiar los efectos de estos compuestos sobre los espermatozoides. Incorporados al momento de la dilución, el lactato y la cafeína indujeron movimientos más vigorosos que las muestras sin aditivos desde el inicio. Cuando se agregaron a las 48 h de almacenaje a 5ºC, ambos aditivos produjeron una notable recuperación en la motilidad (49% vs. 31%). Cuando estas mismas muestras fueron cultivadas a 37ºC, a los 30 minutos de incubación aquellas sin aditivos tuvieron escasas formas móviles (5%), frente a las adicionadas con lactato (29%) y cafeína (40%). A los 60 minutos las muestras sin aditivos casi no registraron movimiento, en tanto que las restantes mantuvieron porcentajes elevados. En los tres casos se encontraron diferencias estadísticas (P<0,01) entre los tratamientos con aditivos y el testigo. La incorporación de aditivos al inicio o a las 48 h no presentó, en general, diferencias significativas. Se concluye que tanto el lactato como la cafeína pueden ser utilizados en diluyentes de semen equino base leche descremada-glucosa, resultando interesante la adición tanto a la dilución como a las 48 h de conservación.

          Translated abstract

          In equines, artificial insemination is practiced mostly with the use of refrigerated semen due to the difficulties that comes with the preservation of frozen semen. To improve the conservation conditions at 5ºC (refrigerated semen) it is necessary to consider the spermatic deterioration after the gathering, because components of the seminal plasma complicate the survival of the sperm with oxidative processes. Some components have antioxidant properties and improve notably the spermatic motility and survival. In this experience sodium lactate (2 mM) and caffeine (10 mM) were incorporated at the moment of the dilution of the semen, and at 48 h of conservation at 5ºC in a skim milk - glucose bases extender, with the purpose of studying their effects on the sperm. Incorporated at the moment of the dilution, the lactate and the caffeine induced more vigorous movements than the samples without additives. When they were added at 48 h of preservation at 5ºC, both additives produced a remarkable recovery in the motility (49% vs. 31%). When these same samples were cultivated at 37ºC, at 30 minutes of incubation those without additives had scarce mobile forms (5%), and different from those added with lactate (29%) and caffeine (40%). At 60 minutes, the samples without additives hardly registered movement while the rest maintained the former percentages. In the three cases, there were found statistical differences (P<0.01) among treatments with additive and the control. The additive added at beginning or at 48 h did not show significant differences. It is concluded that the lactate and the caffeine can be used as diluters of equine semen skim milk-glucose bases.

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          Most cited references18

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          Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing.

          Growing evidence suggests that the generation of reactive oxygen species (ROS) and their detoxification by antioxidants plays a very important role in fertility. However, the relationship between the level of antioxidants in spermatozoa and the decreased fecundity following a freeze/thaw cycle remains poorly understood. We assessed the activities of antioxidant enzymes such as catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD), and levels of reduced/oxidized glutathione (GSH/GSSG) in bovine semen. Sperm cells were isolated using a Percoll gradient to avoid contamination from seminal plasma, cellular debris, and other cell types. We found that bovine spermatozoa are poorly adapted to metabolize the toxic hydrogen peroxide (H(2)O(2)). Indeed, very low levels of GPx and an absence of catalase were observed. We also studied the effect of freezing and thawing bovine spermatozoa in a egg yolk-Tris-glycerol extender (EYTG). Cryopreservation significantly reduced sperm GSH levels by 78% and SOD activity by 50%. We also investigated whether the decrease in GSH level could be linked to oxidative metabolism and found that a greater reduction in intracellular GSH level occurred when fresh sperm cells were incubated in EYTG for 6 hr at 38.5 degrees C under aerobic conditions than when incubated under restricted oxygen availability. Our results strongly suggest the involvement of an oxidative stress during a freeze/thaw cycle and are consistent with the hypothesis that ROS generated during such a cycle are detrimental to sperm function. Copyright 2000 Wiley-Liss, Inc.
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            Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility.

            Under moderate conditions, reactive oxygen species (ROS) have been shown to inhibit sperm motility after several hours of incubation. The rapid decrease in flagellar beat frequency observed within the first hour of contact between ROS and spermatozoa was associated with a rapid loss of intracellular adenosine triphosphate (ATP). Motility of intact spermatozoa ceased when their ATP concentration was reduced by 85 +/- 5%. Axonemal damage was confirmed when ROS-treated spermatozoa could not reactivate motility after demembranation in a medium containing magnesium adenosine triphosphate (Mg.ATP). However, in conditions allowing rephosphorylation of the axonemes (addition of cyclic adenosine monophosphate, or cAMP, and protein kinase or sperm extracts to the demembranation medium), the motility could reactivate. Three lines of evidence suggested that ATP depletion induced by ROS treatment was responsible for the effects observed in spermatozoa. First, the rapid decrease in intracellular ATP observed after ROS treatment was closely followed by a decrease in beat frequency, loss of intact sperm motility, and axonemal damage due to insufficient phosphorylation. Second, incubation of spermatozoa with the combination pyruvate-lactate allowed maintenance of sperm ATP at a normal level and prevented the effects of ROS; furthermore, spermatozoa immobilized after ROS treatment, then supplemented with pyruvate-lactate, were able to reinitiate motility in parallel with an increase in their ATP level. Third, treatment of spermatozoa with rotenone, an ATP depleting agent, produced effects similar to ROS treatment and could also be reversed by the addition of pyruvate-lactate. These data are consistent with the conclusion that ROS treatment produced axonemal damage mostly as a result of ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
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              Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage

              The objective of this study was to determine if centrifugation and partial removal of seminal plasma would improve spermatozoal motility in semen from stallions whose whole ejaculates have poor tolerance to cooling and storage. Stallions were divided into two groups (n = 5/group) based on the ability of their extended semen to maintain spermatozoal motility after cooling and storage. Group 1 stallions ("good coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by or = 40% of progressive motility prior to storage. The sperm-rich portion of each ejaculate was divided into 4 aliquots. Two aliquots underwent standard processing for cooled transported semen and were examined after 24 and 48 h of cooling and storage in an Equitainer. The remaining two aliquots were diluted 1:1 with semen extender, then centrifuged at 400 x g for 12 min at room temperature. After centrifugation, approximately 90% of the seminal plasma was removed, and the sperm pellet was resuspended in extender to a final concentration of 25 to 50 x 10(6) sperm/mL. These aliquots were then packaged as for the non-centrifuged aliquots and examined after 24 and 48 h of storage. The spermatozoal motion characteristics in fresh semen and after 24 and 48 h of cooling and storage was determined via computer-assisted semen analysis. Centrifugation and partial removal of seminal plasma increased the percentage of progressively motile spermatozoa and limited the reduction in progressive spermatozoal motility of "poor cooling" stallions after 48 h of cooling and storage. Results of this study indicate that centrifugation and partial removal of seminal plasma is beneficial for stallions whose ejaculates have poor tolerance to cooling and storage with routine semen dilution and packaging techniques, especially if the semen is stored for > 24 h.
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                Author and article information

                Journal
                zt
                Zootecnia Tropical
                Zootecnia Trop.
                Instituto Nacional de Investigaciones Agricolas INIA, Maracay, Venezuela. (Maracay, Aragua, Venezuela )
                0798-7269
                January 2004
                : 22
                : 1
                : 101-112
                Affiliations
                [01] Tucumán orgnameUniversidad Nacional de Tucumán orgdiv1Facultad de Agronomía y Zootecnia orgdiv2Cátedra de Zootecnia General I Argentina owilde@ 123456manant.unt.edu.ar
                Article
                S0798-72692004000100007 S0798-7269(04)02200107
                d851c21f-4c17-4cb6-9485-a866c667fdf9

                http://creativecommons.org/licenses/by/4.0/

                History
                : 26 June 2003
                : 21 April 2004
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 15, Pages: 12
                Product

                SciELO Venezuela

                Categories
                Artículos Científicos

                lactato,cafeína,motilidad,equino,semen,lactate,caffeine,equine,motility
                lactato, cafeína, motilidad, equino, semen, lactate, caffeine, equine, motility

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