Complemental tests in semen and evaluation of the anionic proteins and peptides differentially synthesized in seminal plasma of Nelore bulls classified as sound and unsound for breeding / Testes complementares em sêmen e avaliação da síntese diferencial de proteínas e peptídeos aniônicos em plasma seminal de touros da raça Nelore classificados em aptos e inaptos à reprodução

Leonardo Franco Martins

The main analyses performed routinely to assess the sperm quality are the spermatic motility and morphology for the measurement bulls fertility. Several complementary techniques are used to improve bulls fertility evaluation. Each technique is related to different aspects of sperm quality and their results are not yet fully understood. Four experiments were done. Experiment 1 was to study the relationship between the physical and morphological semen aspects with the hiposmotic test in fresh semen of young and adult bulls. Six hundred and twenty-six Nelore bulls were used, 420 young and 206 adults. Young bulls were 18 to 22 months and adult bulls were 3 to 10 years, they were subject to andrological examination. After vesicular glands palpation and testicular measurements semen was collected by eletro ejaculation method. After physical and morphological semen examination hiposmotic test was done. After the breeding soundness examination, 83% of young bulls and 94% of adults bulls were classified as sound for breeding. There was no difference between the average scrotal circumference of bulls classified as sound and unsound for breeding (P>0.05). There was difference between all semen physical and morphological aspects of bulls classified as sound and unsound for breeding (P>0.05), but there was no difference in hiposmotic test results (P>0.05). According to these results, the hiposmotic test can not be used alone to predict the reproductive potential of young and adult Nelore bulls, because there was no difference between the hiposmotic test results (P>0.05) and no relationship with the main quality semen aspects. The end point of experiment 3 was to identify proteins differentially expressed in seminal plasma of adult bulls and young Nelore bulls classified as sound and unsound for breeding. After the breeding soundness examination, the young bulls were classified as sound (117) and unsound for breeding (49) for breeding, and adults bulls (56) as sound for breeding. Samples of seminal plasma, conserved in straws under -196C, were thawed at ambient temperature to be done proteomic analysis. MDLC was performed offline, the first dimension was cation exchange chromatography and the second was reverse phase. Six fractions eluted in equilibrium conditions were collected in (pH 7.0) in cation exchange chromatography, which represent the fractions of peptides and proteins with anionic and neutral character. The fractions corresponding to peaks in the eluates reverse-phase chromatography were analyzed in a mass spectrometre for determination of protein molecular weight. Were selected and analyzed 96 eluates collected in reversephase chromatography for mass spectrometry with protein synthesis difference between the treatments. Eighteen molecular weights were identified by mass spectrometry (4233-13560 Da). The 12910 Da protein, with molecular mass and similar characteristics of aSFP (acidic seminal fluid protein), was obtained in higher concentration in adult bulls, followed by the young bulls classified as sound for breeding and unsound consecutively. The chromatographic protein profiles were different among groups, suggesting that anionic proteins can be studied in order to indentify protein markers of reproductive potential of Nelore bulls, by the seminal plasma proteomic analysis. In experiments 3 and 4 were studied the relationship between the hiposmotic test with the physical and morphological semen aspects and complemental tests in fresh and frozen/thawed semen of adult Nelore bulls. In fresh semen were done physical and morphological evaluation, supravital staining and hiposmotic test. In frozen/thawed semen were done hiposmotic test, supravital staining, slow termo resistance test and fluorescent stain. In experiment 3 were used 30 ejaculates from 6 adult Nelore bulls. It were used solutions with osmolalities of 60, 100, 150 mOsm/kg and distilled water (19 mOsm/kg) with 15, 30 and 60 minutes of incubation time at 37 C, in hiposmotic test. Only distilled water was different in relation to average values obtained in the hiposmotic test realized on fresh semen (P <0.05). There was no difference between the mean values of reactive spermatozoa incubated with different incubation time in both fresh and frozen/thawed semen (P>0.05). In Experiment 4 were used 15 ejaculates from 3 adult Nelore bulls. It was used 10, 20, 50, 100μL of semen in 1 ml of hiposmotic solution. There was no difference between the mean values of reactive spermatozoa in different concentrations of semen in hiposmotic test (P>0.05). None of spermatozoa plasma membrane integrity test was able to classified adult Nelore bulls according to their semen freezability in experiments 3 and 4. The hiposmotic test correlated with the main characteristics of semen quality and the complemental tests semen results (P <0.05). In Experiment 3 we concluded that the hiposmotic test can be accomplished with 15 minutes of incubation time and a hiposmotic solution between 60 and 150 mOsm/kg in both fresh and frozen/thawed semen. According to the results of experiment 4, the hiposmotic test can be accomplished with 20 to 100 μL in fresh semen, and 10 to 100 μL of frozen/thawed semen in 1 ml of hiposmotic solution, without interfering with their results, but it should be choose 100 μL for both fresh and frozen/thawed semen, because it improves the slides reading. According to the results of this work the hiposmotic test can not be used to predict the reproductive potential of Nelore bulls. But, because it evaluate another aspect of the spermatozoa plasma membrane and its relationships with main aspects of semen quality and others complemental tests, hiposmotic test should be studied to better understand its result. Proteomic analysis in association with breeding soundness evaluation may be a promising tool to predict bull reprodutive potential, by identification of protein molecules in seminal plasma.

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