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
Ano de defesa: | 2009 |
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Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | , |
Tipo de documento: | Tese |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade Federal de Viçosa
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Programa de Pós-Graduação: |
Doutorado em Medicina Veterinária
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Departamento: |
Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de
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País: |
BR
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Palavras-chave em Português: | |
Palavras-chave em Inglês: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://locus.ufv.br/handle/123456789/1435 |
Resumo: | The main analyses performed routinely to assess the sperm quality are the spermatic motility and morphology for the measurement bull´s fertility. Several complementary techniques are used to improve bull´s 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 -196ºC, 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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Martins, Leonardo Francohttp://lattes.cnpq.br/7221736096112265Costa, Eduardo Paulino dahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787237D6Pereira, Maria Cristina Baracathttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780021E6Guimarães, José Domingoshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782270U6Fonseca, Jeferson Ferreira dahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4703690H8Torres, Ciro Alexandre Alveshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787213D42015-03-26T12:47:42Z2011-04-072015-03-26T12:47:42Z2009-10-19MARTINS, Leonardo Franco. 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. 2009. 96 f. Tese (Doutorado em Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de) - Universidade Federal de Viçosa, Viçosa, 2009.http://locus.ufv.br/handle/123456789/1435The main analyses performed routinely to assess the sperm quality are the spermatic motility and morphology for the measurement bull´s fertility. Several complementary techniques are used to improve bull´s 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 -196ºC, 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.As principais análises realizadas como rotina para avaliar a qualidade seminal são a motilidade espermática progressiva retilínea e morfologia para a mensuração da fertilidade de touros. Diversas técnicas complementares são utilizadas para melhorar avaliação da fertilidade de touros. Cada técnica está relacionada com diferentes aspectos da qualidade seminal e seus resultados ainda não são totalmente compreendidos. Foram realizados quatro experimentos. O experimento 1 teve como objetivo estudar as relações entre os aspectos físicos e morfológicos do sêmen com o teste hiposmótico em sêmen in natura de touros jovens e adultos. Foram utilizados 626 touros da raça Nelore, sendo 420 touros jovens e 206 touros adultos. Os touros jovens com idades variando de 18 a 22 meses e touros adultos com idades variando de 3 a 10 anos, foram submetidos a exame andrológico. Após a palpação das glândulas vesiculares e mensurações testiculares foi realizada a coleta de sêmen pelo método de eletroejaculação. Após a análise física e morfológica do sêmen foi realizado o teste hiposmótico. Após a classificação andrológica, 83% dos touros jovens e 94% dos touros adultos foram classificados em aptos à reprodução. Não houve diferenças entre as médias de perímetro escrotal dos touros classificados como aptos e inaptos à reprodução (P>0,05). Houve diferenças entre as médias de todos os aspectos físicos e morfológicos do ejaculado de touros inaptos e aptos à reprodução (P<0,05), mas não foram observadas no percentual de espermatozóides reativos ao teste hiposmótico (P>0,05). Com estes resultados, pode-se concluir que o teste hiposmótico não pode ser utilizado isoladamente para predizer o potencial reprodutivo de touros jovens e adultos da raça Nelore, pois não houve diferenças entre os percentuais de espermatozóides reativos do sêmen de touros aptos e inaptos à reprodução, e não houve relações com os principais aspectos de qualidade seminal. O experimento 2 teve como objetivo identificar proteínas diferencialmente sintetizadas em plasma seminal de touros jovens e adultos da raça Nelore classificados em aptos e inaptos à reprodução. Após o exame andrológico, os touros jovens foram classificados em aptos (117) e inaptos (49) à reprodução, e os touros adultos (56), em aptos à reprodução. As amostras de plasma seminal, conservadas em palhetas finas sob -196ºC, foram descongeladas à temperatura ambiente para serem realizadas as análises proteômicas. Foi realizada a MDLC offline, sendo a primeira dimensão a cromatografia de troca catiônica e a segunda a de fase reversa. Foram coletadas seis frações eluídas nas condições de equilíbrio (pH 7,0) na cromatografia de troca catiônica, que correspondem às frações de peptídeos e proteínas com caráter aniônico e neutro. As frações correspondentes aos picos eluídos obtidos na cromatografia de fase reversa foram analisadas em um espectrômetro de massa do tipo MALDI-TOF/TOF para a determinação da massa molecular das proteínas. Foram selecionados e analisados 96 eluatos coletados na cromatografia de fase reversa para a espectrometria de massa, com síntese protéica diferencial entre os tratamentos. Dezoito massas moleculares foram identificadas por espectrometria de massa, entre 4.233 e 13.560 Da. A proteína de 12.910 Da, com massa molecular e características semelhantes à aSFP (acidic seminal fluid protein), foi obtida em maior concentração nos touros aptos adultos, seguidos pelos aptos jovens e inaptos. Os perfis cromatográficos protéicos foram diferentes entre os grupos, sugerindo que a proteínas aniônicas podem ser estudadas visando identificar marcadores protéicos para a aptidão reprodutiva de touros da raça Nelore, pela análise proteômica do plasma seminal. Nos experimentos 3 e 4 foram estudadas as relações do teste hiposmótico com os aspectos físicos e morfológicos do sêmen, e testes complementares em sêmen in natura e congelado/descongelado de touros adultos da raça Nelore. No sêmen in natura foi feita a avaliação física e morfológica, a coloração supravital e o teste hiposmótico. Nas amostras congeladas/descongeladas, foram realizados os testes hiposmótico, coloração supravital, teste de termoresistência lento e a coloração fluorescente. No experimento 3 foram utilizados trinta ejaculados de seis touros adultos da raça Nelore. Foram utilizadas soluções com osmolaridades de 60, 100, 150 mOsm/kg e água destilada (19 mOsm/kg) com 15, 30 e 60 minutos de período de incubação a 37 ºC no teste hiposmótico. Apenas a água destilada apresentou diferença em relação aos valores médios obtidos no teste hiposmótico realizado no sêmen in natura (P<0,05). Não houve diferença entre os valores médios de espermatozóides reativos incubados com diferentes tempos de incubação, tanto no sêmen in natura como no congelado/descongelado (P>0,05). No experimento 4, foram utilizados 15 ejaculados de 3 touros adultos da raça Nelore. Foram utilizados 10, 20, 50 e 100μL de sêmen em 1mL de solução hiposmótica. Não houve diferença entre os valores médios de espermatozóides reativos nas diferentes concentrações de sêmen no teste hiposmótico (P>0,05). Nenhum teste de integridade de membrana plasmática dos espermatozóides foi capaz de classificar os touros quanto à sua congelabilidade do sêmen nos experimentos 3 e 4. O teste hiposmótico correlacionou-se com as principais características de qualidade seminal e com os valores obtidos nos testes complementares (P<0,05). No experimento 3, concluímos que o teste hiposmótico pode ser realizado com 15 minutos de período de incubação e uma solução hiposmótica entre 60 e 150 mOsm/kg, tanto em sêmen in natura como em congelado/descongelado. De acordo com os resultados do experimento 4, o teste hiposmótico pode ser realizado com 20 a 100μL em sêmen in natura, e 10 a 100 μL de sêmen congelado/descongelado em 1mL de solução hiposmótica, sem interferir em seus resultados, mas preferencialmente deve-se optar por 100μL, tanto para sêmen in natura como congelado/ descongelado, porque melhora a leitura das lâminas. De acordo com os resultados deste trabalho o teste hipósmótico não pode ser utilizado para predizer o potencial reprodutivo de touros da raça Nelore. Mas, o teste hiposmótico avalia outro aspecto da membrana plasmática dos espermatozóides e possue relações com os principais aspectos da qualidade seminal e outros testes complementares, por isso deve ser empregado para uma melhor compreensão dos seus resultados. A análise proteômica do plasma seminal em associação à avaliação andrológica pode ser uma ferramenta promissora para predizer a aptidão reprodutiva em touros, por meio da identificação de moléculas protéicas no plasma seminal.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de ViçosaDoutorado em Medicina VeterináriaUFVBRBiotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. deSêmenTouroProteônicaSemenBullProteomicCNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMALTestes 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 reproduçãoComplemental 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 breedinginfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf1451359https://locus.ufv.br//bitstream/123456789/1435/1/texto%20completo.pdff7f480743e2d3aa7d5917d701e99d936MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain214126https://locus.ufv.br//bitstream/123456789/1435/2/texto%20completo.pdf.txta30b7659b030f9dd61e090cd40ff5731MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3780https://locus.ufv.br//bitstream/123456789/1435/3/texto%20completo.pdf.jpg3ee723aabcc0988de98edc9fd6d9ce1eMD53123456789/14352016-04-07 23:00:36.367oai:locus.ufv.br:123456789/1435Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-08T02:00:36LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
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 |
dc.title.alternative.eng.fl_str_mv |
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 |
title |
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 |
spellingShingle |
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 Martins, Leonardo Franco Sêmen Touro Proteônica Semen Bull Proteomic CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL |
title_short |
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 |
title_full |
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 |
title_fullStr |
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 |
title_full_unstemmed |
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 |
title_sort |
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 |
author |
Martins, Leonardo Franco |
author_facet |
Martins, Leonardo Franco |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/7221736096112265 |
dc.contributor.author.fl_str_mv |
Martins, Leonardo Franco |
dc.contributor.advisor-co1.fl_str_mv |
Costa, Eduardo Paulino da |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787237D6 |
dc.contributor.advisor-co2.fl_str_mv |
Pereira, Maria Cristina Baracat |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780021E6 |
dc.contributor.advisor1.fl_str_mv |
Guimarães, José Domingos |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782270U6 |
dc.contributor.referee1.fl_str_mv |
Fonseca, Jeferson Ferreira da |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4703690H8 |
dc.contributor.referee2.fl_str_mv |
Torres, Ciro Alexandre Alves |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787213D4 |
contributor_str_mv |
Costa, Eduardo Paulino da Pereira, Maria Cristina Baracat Guimarães, José Domingos Fonseca, Jeferson Ferreira da Torres, Ciro Alexandre Alves |
dc.subject.por.fl_str_mv |
Sêmen Touro Proteônica |
topic |
Sêmen Touro Proteônica Semen Bull Proteomic CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL |
dc.subject.eng.fl_str_mv |
Semen Bull Proteomic |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL |
description |
The main analyses performed routinely to assess the sperm quality are the spermatic motility and morphology for the measurement bull´s fertility. Several complementary techniques are used to improve bull´s 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 -196ºC, 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. |
publishDate |
2009 |
dc.date.issued.fl_str_mv |
2009-10-19 |
dc.date.available.fl_str_mv |
2011-04-07 2015-03-26T12:47:42Z |
dc.date.accessioned.fl_str_mv |
2015-03-26T12:47:42Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
MARTINS, Leonardo Franco. 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. 2009. 96 f. Tese (Doutorado em Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de) - Universidade Federal de Viçosa, Viçosa, 2009. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/1435 |
identifier_str_mv |
MARTINS, Leonardo Franco. 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. 2009. 96 f. Tese (Doutorado em Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de) - Universidade Federal de Viçosa, Viçosa, 2009. |
url |
http://locus.ufv.br/handle/123456789/1435 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Viçosa |
dc.publisher.program.fl_str_mv |
Doutorado em Medicina Veterinária |
dc.publisher.initials.fl_str_mv |
UFV |
dc.publisher.country.fl_str_mv |
BR |
dc.publisher.department.fl_str_mv |
Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de |
publisher.none.fl_str_mv |
Universidade Federal de Viçosa |
dc.source.none.fl_str_mv |
reponame:LOCUS Repositório Institucional da UFV instname:Universidade Federal de Viçosa (UFV) instacron:UFV |
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LOCUS Repositório Institucional da UFV |
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LOCUS Repositório Institucional da UFV |
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