EFFECT OF INITIAL SEMINAL PLASMA FRUCTOSE CONCENTRATION ON GOAT SEMEN STORAGE AT 5

extender presented inferior results. The effect of the group was observed only in the ICW-Y. CY and TY worked similarly in the two groups. No significant difference in the activity of PLA 2 was found between the groups. However, ICW-Y should not be used for the storage of goat semen.


INTRODUCTION
Many studies have been carried out in an attempt to improve the fertilizing capacity of cooled or frozen goat semen that still displays unsatisfactory results when used in vivo (Leboeuf et al., 2000).Therefore, several options have been proposed for the formulation of extenders to conserve the semen at 5 °C.One of the natural extenders used is whole cow milk (Evans and Maxwell, 1990).However, the dilution of goat semen with these extenders may be harmful to the spermatozoa, due to the PLA 2 enzyme present in the SP.PLA 2 are deleterious to sperm (Nunes et al., 1982).The use of coconut water has simplified the technology of storing liquid goat semen at 5 °C, thus avoiding the washing of SP and consequently the PLA 2 enzyme, because this extender is poor in phospholipids (Nunes, 1998).Another important aspect is that fructose concentrations ranging from 249.11 (Roca et al., 1993) to 1,420 mg/dL (Barakat et al., 1972), and supplies energy to the sperm.The objective of this study was to verify the quality of diluted goat semen in three different extenders, when the initial fructose concentration and PLA 2 activity in SP are known.

MATERIAL AND METHODS
Six crossbreed male goats two-years old were used.Extenders used were: Citrate -Egg yolk (CY): 2.37 g sodium citrate, 0.80 g glucose 2.5 % egg yolk and 100 mL distilled water (s.q.t.); TRIS-Egg yolk (TY): 3.63 g TRIS, 0.50 g fructose, 1.99 g citric acid, 2.5% egg yolk and 100 mL distilled water (s.q.t.) and industrialized coconut water1 (bottled under the UHT system) -egg yolk (ICW-Y): 50 mL coconut water, 50 mL 2.5 % sodium citrate solution, 2.5 % egg yolk and 100 mL distilled water (s.q.t.).Twenty-four samples goat semen were collected by using an artificial (four samples/per animal).After collection, the volume of each ejaculate was measured and the sperm concentration was determined by spectrophotometry.Each ejaculate collected were divided into four aliquots, three aliquots were diluted with the three extenders (CY, TY and, ICW-Y), in the concentration of 200 x 10 6 sptz/mL.The fourth aliquot was centrifuged twice at 3.450 g/ +4 °C/20 min and the supernatant was frozen at -18 °C until the analyses for fructose concentration (colorimetric method) and PLA 2 activity (Haas et al., 1968).
After dilution, a sample of 300 μL from semen (fresh) in each extender was incubated at 38 °C in a water bath and assessed by the heat resistance test as regards the vigor, sperm motility and total morphological alterations (TMA) using optical microscopy.The remainder of the diluted semen was cooled at 5 ºC and, samples of each extender were used for evaluation of sperm criteria at 2, 24 and, 48 h after storage.Semen smears were made and stained with bromophenol blue and morphological alterations, were evaluated.TMA resulted from the sum of all alterations found.After the evaluation of initial fructose concentration, the animals were divided into two groups with three goats each: group I a high initial fructose concentration on SP (>710 mg/dL) and group II a low initial fructose concentration (< 480 mg/dL).Data were evaluated using the SAS v.8 (2000), according a randomized design.Sperm motility and TMA variables expressed as percentages were submitted to angulartransformed before the statistical analysis.The data was submitted for the analysis of variance (ANOVA) to evaluate the extender type effects and the concentration of seminal fructose on sperm parameters.Differences between mean values were analysed by Tukey test (p<0.05).

RESULTS AND DISCUSSION
The initial fructose concentration did not affect in vigor, motility and TMA sperm when CY and TY extenders were used, but when the concentration of this glicide in SP were more than 710 mg/dL there was an increase in the lifetime of sperm extended in ICW-Y.This result may be probably due the presented inappropriate or unavailable energy levels in this extender to the sperm in group II, because there is high level of carbohydrates in its composition (2.5 %), than other extenders used in this study.Furthermore, our research group has shown good results in cooling with natural coconut water (Campos et al., 2004).Therefore, it is believed that the original features of natural coconut water had changed, including carbohydrate concentrations.Storage for 48 hours did not adversely affect the vigor, except in the ICW-Y from Group II that decreased within 24 h of storage (table I).In extender CY, decreased motility was observed within 24 h in Group II (table I).On the TMA such effect was observed in the CY of group I within 48 and 24 h in Group II (table I).The ability of spermatozoa to also use glucose, fructose or mannose is due to the fact that these sugars sources enter the glycolysis cycle through the hexokinase reaction with ATP, followed by formation of lactic acid from monofosfatohexose (King et al., 2006).Thus, it is believed that the depletion of sugar in animals with low fructose level is faster, and our present findings suggest that the ICW-Y extender did not sufficiently supply the energy to maintain sperm metabolic activity.In the two groups, the best motility and vigor were found in the CY and TY extenders, confirming their quality as liquid goat semen preservatives by adequately supply the energy needs of sperm (Shamsuddin et al., 1999).Significant difference between the extenders was found in Group II for vigor and motility and it was more evident for vigor within 24 h and motility within 48 h (table I), and the best results were observed in the CY and TY.The efficiency these extenders found in the present experiment for goat semen storage can be attributed to buffer capacity and appropriate sugar concentration, combined protective effect of egg yolk against cold shock (Cabrera et al., 2005).The effect of extender on the TMA was observed only in fresh semen, with the largest index in the CY in both groups (table I).The TMA averages were above 25 %, so that diluted goat semen in the CY extender presented abnormal parameters for use in artificial insemination.Blackshaw and Salisbury (1957) related that egg yolk stimulates the glycolytic activity of fresh semen.So, we believed that in fresh semen the addition of the egg yolk should be avoided, because it decreased seminal quality.PLA 2 activity was determined in both groups I (10.02 ± 2.43 U/mL) and II (12.09 ± 3.55 U/mL), and no significant differences were observed among the intensity of activity between the experimental groups (p>0.05).We found that PLA 2 activity was not related to the low quality of goat semen in Group II in this experiment.

CONCLUSION
The study concluded that when extenders are used with appropriate concentrations of carbohydrates, as the citrate and the TRIS, sperm quality remained viable regardless of the initial concentration of fructose in the goat SP.However, industrialized coconut water should not be used for storage of goat semen.