The objectives of this study were to determine fatty acid composition and acrosome status from bull sperm with different freezabilities (n = 12). We hypothesized that lipid fractions had differentiated fatty acid compositions and such differences influence sperm freezability and the sperm acrosome. Fatty acids were extracted from fresh frozen sperm and fractionated by solid-phase extraction. Thirtyour fatty acids were quantified. Saturated fatty acids were predominant, accounting for 71 to 80% of fatty acids in both fractions. Differences in composition between fractions existed (P < 0.001). Branched chain fatty acid concentrations (15 to 18 µg) were almost twice that of polyunsaturated fatty acid concentrations found in the polar fractions (8 to 9 µg; P < 0.001). Sperm with differentiated freezabilities had few differences in 22:0, 18:1 cis 9, and 14:0 13-methyl fatty acids (P ≤ 0.011). Analyses of acrosome status of sperm revealed that acrosomes were affected differently among bulls.

The objective of this study was to uncover molecular and cellular dynamics in spermatozoa from Holstein bulls with Good (11 bulls) and Poor (5 bulls) cryotolerance. Post-thaw sperm kinetics, membrane integrity, and DNA fragmentation were assessed. Data was analyzed using principal component analysis. The spermatozoa from Good bulls had a higher number of cells with intact membranes (P = 0.029), nonragmented DNA (P = 0.018), post-thaw viability (P < 0.001) in comparison to those of the Poor bulls. The Good bulls also had a faster average path velocity (VAP; P = 0.017), straight-line velocity (VSL; P = 0.036), a greater distance average path (DAP; P = 0.006) and distance straight line (DSL; P = 0.011). No differences were found in total antioxidant capacity (TAC), number of live cells or other kinetic parameters between spermatozoa from Good and Poor groups, showing that no one characteristic can be used to determine cryotolerance.

Artificial insemination (AI) using cryopreserved sperm has an important positive impact on cattle production. Fertility is the most critical trait controlling livestock production; however, molecular, cellular, and physiological determinants of bull fertility and sperm freezability are not well understood. Better understanding of molecular, cellular, and physiological underpinnings of bull fertility may increase the success rate of AI. The objective of this study was to test the hypothesis that expression dynamics of sperm nuclear proteins, post-translational modifications (PTM) of sperm Histone 4 (H4), and seminal plasma metabolome are associated with bull fertility and sperm freezability (P = 0.043). Flow cytometry experiments were conducted to quantify H4 and acetylated histone 4 (H4ac) in sperm from high and low fertility Holstein bulls. The analysis of flow cytometry experiments clarified that retained levels of H4ac in bull sperm are associated with bull fertility. In addition, gas chromatography-mass spectrometry (GC-MS) was applied to ascertain the amino acid concentration of seminal plasma from bull semen with various freezability. A total of 21 amino acids and isomers were identified, and phenylalanine was positively associated with sperm post-thaw viability (r = 0.57, P-value = 0.043). Lastly, a quantitative western blotting experiment was utilized to ascertain relative quantification of sperm nuclear proteins including protamine 1 (PRM1), protamine 2 (PRM2), Histone 3 (H3), and H4. Also, sperm functional parameters including acrosome reaction, DNA fragmentation index, PAWP expression were analyzed using flow cytometry. In addition, immunocytochemistry experiments were applied to analyze sperm chromatin decondensation ability. The analyses of western blotting experiments revealed that the relative abundance of PRM2 in poor freezability sperm (PF) was greater than those in good freezability sperm (GF) (P = 0.0259). The relative abundance of retained H3 was greater in PF bulls than in GF bulls (1.02 ± 0.005 and 0.969 ± 0.021, respectively; P = 0.0272). There was a positive correlation between the abundance of retained H4 and sperm decondensation state (r = 0.71, P = 0.05). These results are important because they can help advance fundamental andrology and the assisted reproductive technologies both for cattle and other mammals, including humans and endangered species.

Orientador: Frederico Ozanam Papa / Banca: João Carlos Pinheiro Ferreira / Banca: Alício Martins Junior / Resumo: A despeito das inúmeras variáveis que influenciam direta e indiretamente a fertilidade das fêmeas bovinas, a qualidade das amostras seminais exerce um papel importante na determinação das taxas de concepção dos programas de inseminação artificial. Os objetivos dessa pesquisa foram comparar a efetividade de dois diluidores de criopreservação de sêmen bovino no processamento de amostras seminais apresentando diferentes concentrações espermáticas em relação aos índices de congelabilidade determinados laboratorialmente (Experimento I) e as taxas de concepção proporcionadas por cada metodologia quando utilizada em programas de inseminação artificial em tempo fixo (IATF) em bovinos (Experimento II). No Experimento I foram utilizados 14 ejaculados de diferentes touros da raça Nelore. Cada ejaculado foi fracionado em oito alíquotas iguais, submetidas a criopreservação com os diluidores Tris-gema de ovo-frutose (meio TRIS) e MKA nas concentrações de 12, 25, 50 e 100 milhões de espermatozóides totais por mililitro de meio, formando oito grupos experimentais em função das variáveis diluidor e concentração. As amostras foram descongeladas a 46 ºC por 20 segundos, avaliando-se os padrões de motilidade através do método computadorizado (CASA), integridade de membrana plasmática (IMP), resistência ao teste de termorresistência rápido (TTR) e taxa de recuperação e IMP após seleção espermática pela técnica de swim-up. Para o Experimento II foram selecionados sete touros utilizados no Experimento I, obtendo-se um ejaculado de cada animal por eletroejaculação...(Resumo completo, clicar acesso eletrõnico abaixo) / Abstract: Although there are many variables which directly or indirectly influence female bovine fertility, the quality of sperm samples plays a important role in the determination of conception rates in artificial insemination programs. The aim of the present study was to compare the efficiency of two bovine semen extenders for sperm freezing with different spermatic concentrations in the freezability determined by lab tests (Experiment I), and conception rates after fixed time artificial insemination (FTAI; Experiment II). In Experiment I 14 ejaculates of different Nelore bulls were used. Each ejaculate was splitsampled in to eight equal parts and then submitted to cryopreservation with Tris-egg yolk fructose (TRIS) and MKA extenders, at concentrations of 12, 25, 50 and 100 millions spermatozoa per milliliter forming eight experimental groups. The samples were thawed at 46 ºC for 20 seconds, and the following parameters were evaluated: sperm motility and movement (by computer-assisted semen analysis - CASA), sperm membrane integrity (SMI), resistance to the fast thermoresistance test (TT), recovery rate and sperm membrane integrity after sperm selection through swim-up technique. Seven of 14 bulls used in Experiment I were selected for Experiment II, and semen was collected from each of the animals by electroejaculation. The seven ejaculates obtained were mixed (semen pool) and cryopreserved, thus forming eight experimental groups according to the freezing extenders and sperm concentrations/straws: TRIS 12, 25, 50 and 100, and MKA 12, 25, 50 and 100...(Complete abstract, click electronic address below) / Mestre

Made available in DSpace on 2014-06-11T19:29:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-26Bitstream added on 2014-06-13T19:38:31Z : No. of bitstreams: 1 crespilho_am_me_botfmvz.pdf: 507525 bytes, checksum: a1c92f545e77b50838125fbea8dfddd6 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A despeito das inúmeras variáveis que influenciam direta e indiretamente a fertilidade das fêmeas bovinas, a qualidade das amostras seminais exerce um papel importante na determinação das taxas de concepção dos programas de inseminação artificial. Os objetivos dessa pesquisa foram comparar a efetividade de dois diluidores de criopreservação de sêmen bovino no processamento de amostras seminais apresentando diferentes concentrações espermáticas em relação aos índices de congelabilidade determinados laboratorialmente (Experimento I) e as taxas de concepção proporcionadas por cada metodologia quando utilizada em programas de inseminação artificial em tempo fixo (IATF) em bovinos (Experimento II). No Experimento I foram utilizados 14 ejaculados de diferentes touros da raça Nelore. Cada ejaculado foi fracionado em oito alíquotas iguais, submetidas a criopreservação com os diluidores Tris-gema de ovo-frutose (meio TRIS) e MKA nas concentrações de 12, 25, 50 e 100 milhões de espermatozóides totais por mililitro de meio, formando oito grupos experimentais em função das variáveis diluidor e concentração. As amostras foram descongeladas a 46 ºC por 20 segundos, avaliando-se os padrões de motilidade através do método computadorizado (CASA), integridade de membrana plasmática (IMP), resistência ao teste de termorresistência rápido (TTR) e taxa de recuperação e IMP após seleção espermática pela técnica de swim-up. Para o Experimento II foram selecionados sete touros utilizados no Experimento I, obtendo-se um ejaculado de cada animal por eletroejaculação... / Although there are many variables which directly or indirectly influence female bovine fertility, the quality of sperm samples plays a important role in the determination of conception rates in artificial insemination programs. The aim of the present study was to compare the efficiency of two bovine semen extenders for sperm freezing with different spermatic concentrations in the freezability determined by lab tests (Experiment I), and conception rates after fixed time artificial insemination (FTAI; Experiment II). In Experiment I 14 ejaculates of different Nelore bulls were used. Each ejaculate was splitsampled in to eight equal parts and then submitted to cryopreservation with Tris-egg yolk fructose (TRIS) and MKA extenders, at concentrations of 12, 25, 50 and 100 millions spermatozoa per milliliter forming eight experimental groups. The samples were thawed at 46 ºC for 20 seconds, and the following parameters were evaluated: sperm motility and movement (by computer-assisted semen analysis - CASA), sperm membrane integrity (SMI), resistance to the fast thermoresistance test (TT), recovery rate and sperm membrane integrity after sperm selection through swim-up technique. Seven of 14 bulls used in Experiment I were selected for Experiment II, and semen was collected from each of the animals by electroejaculation. The seven ejaculates obtained were mixed (semen pool) and cryopreserved, thus forming eight experimental groups according to the freezing extenders and sperm concentrations/straws: TRIS 12, 25, 50 and 100, and MKA 12, 25, 50 and 100...(Complete abstract, click electronic address below)