The Binder of SPerm 1 (BSP1) protein is involved in the fertilization and semen cryopreservation processes and is described to be both beneficial and detrimental to sperm. Previously, the relationship of BSP1 with freezability events has not been completely understood. The objective of this work was to determine the differential abundance of the forms of the BSP1 protein in cryopreserved seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability using proteomics. A wide cohort of adult bulls with high genetic value from an artificial insemination center was used as donors of high quality, fresh semen. Nine bulls presenting different patterns of semen freezability were selected. Two-dimensional gel electrophoresis showed differential abundance in a group of seven protein spots in the frozen/thawed seminal plasma from the bulls, ranging from 15 to 17 kDa, with pI values from 4.6 to 5.8. Four of these spots were confirmed to be BSP1 using mass spectrometry, proteomics, biochemical, and computational analysis (Tukey's test at P < 0.05). The protein spot weighing 15.52 ± 0.53 kDa with a pI value of 5.78 ± 0.12 is highlighted by its high abundance in bulls with low semen freezability and its absence in bulls presenting high semen freezability. This is the first report showing that more than two forms of BSP1 are found in the seminal plasma of Nelore adult bulls and not all animals have a similar abundance of each BSP1 form. Different BSP1 forms may be involved in different events of fertilization and the cryopreservation process.

RESUMO:Extratos aquosos da planta medicinal Achillea millefolium contêm macromoléculas de interesse para desenvolver fitodefensivos para a agricultura. Duas frações de mil folhas foram obtidas por ultrafiltração, E1 (contendo moléculas maiores que 30 kDa), e E3 (peptídeos entre 1 e 10 kDa) que inibiram o crescimento das bactérias fitopatogênicas Ralstonia solanacearum, gramnegativa, e Clavibacter michiganensis subsp. michiganensis, gram-positiva, com dependência de concentração. Os valores de concentração inibitória mínima (CIM) para ambos os extratos e bactérias foram baixos, entre 20 e 80mM. A CIM relativa à proteína total evidenciou a presença de macromoléculas muito ativas em E3, embora com baixa concentração proteica. E3 se aplica à prospecção de peptídeos antimicrobianos. Estimar a CIM relativa à quantidade de amostra vegetal valorizou o potencial antimicrobiano natural de E1, que contém alta concentração proteica. E1e E3 se aplicam ao desenvolvimento de fitodefensivos para uso biotecnológico. A ultrafiltração fracionou as amostras de forma nativa, rápida, e com baixo custo; além de dessalinizar, clarificar, purificar, e concentrar E1 e E3. Esse estudo inédito sobre a separômica e a ação antimicrobiana de extratos macromoleculares aquosos de mil folhas sugere que plantas cicatrizantes podem apresentar grande potencial para desenvolver fitodefensivos agrícolas naturais não danosos, à semelhança de medicamentos fitoterápicos.Palavras-chave: Macromoléculas de defesa, peptídeos antimicrobianos, Ralstonia solanacearum, Clavibacter michiganensis subsp. michiganensis, concentração inibitória mínima relativa.ABSTRACT: Agrochemicals in medicinal plants: hydrophilic macromolecules from leaves of "mil folhas" (Achillea millefolium L.) inhibit in vitro growth of phytopathogenic bacteria. Aqueous extracts from the medicinal plant Achillea millefolium contain macromolecules of interest to develop agrochemicals for agriculture. Two fractions of "mil folhas" were obtained by ultrafiltration, E1 (containing molecules larger than 30 kDa) and E3 (peptides between 1 and 10 kDa), which inhibited the growth of phytopathogenic bacteria Ralstonia solanacearum, gramnegative, and Clavibacter michiganensis subsp. michiganensis, gram-positive, concentrationdependent. The values of minimum inhibitory concentration (MIC) for both extracts and both bacteria were low, ranging from 20 to 80mM. The MIC relative to total protein evidenced the presence of very active macromolecules in E3, although showing low protein concentration. E3 applies to the prospection of antimicrobial peptides. The estimated MIC relative to the amount of plant sample valued the natural antimicrobial potential of E1, which contains high protein concentration. E1 and E3 can be used in the development of agrochemicals for biotechnological purposes. The ultrafiltration procedure fractionated the samples in a rapid and native way and at a low cost; it also desalted, clarified, concentrated and purified both E1 and E3. This pioneering study on the separomics and on the a...