Impregnation of thymol on solid carriers using supercritical carbon dioxide

Cilj ove doktorske disertacije bio je ispitivanje mogućnosti natkritične impregnacije čistog timola i ekstrakta timijana (Thymus vulgaris) na čvrste nosače kako bi se istražila mogućnost razvijanja novih, funkcionalizovanih materijala sa antimikrobnim dejstvom. Timol je odabran za procese natkritične impregnacije nosača sa polisaharidnom osnovom zbog njegovog jakog antimikrobnog, antifungalnog i antioksidativnog dejstva. Da bi se pristupilo istraživanju mogućnosti koje impregnacija timola natkritičnim ugljenik(IV)-oksidom pruža, prvo je ispitana rastvorljivost timola u natkritičnom ugljenik(IV)-oksidu u opsegu temperatura i pritisaka od interesa. Pored čistog timola, u procesu impregnacije čvrstih nosača primenjen je i ekstrakt timijana bogat timolom dobijen postupkom natkritične ekstrakcije. Čvrsti nosači za ove aktivne komponente su izabrani na osnovu njihovih poželjnih osobina (biodegradabilnost, stabilnost, biokompatibilnost itd.) i mogućnosti primene u farmaceutskoj, biomedicinskoj, kozmetičkoj i industriji hrane. Odabrani su sledeći nosači: a) polisaharidni ksero- i aerogelovi (hitozan, kukuruzni i tapioka skrob) za primenu u farmaceutskoj i prehrambenoj industriji, b) acetat celuloze za primenu u farmaceutskoj i prehrambenoj industriji i c) pamučna gaza u cilju dobijanja medicinskog tekstila sa antibakterijskim dejstvom za tretman rana. Definisani su optimalni parametri dobijanja poroznih ksero- i aerogelova (temperatura gelatinizacije, vrsta umreživača i metoda sušenja) koji daju najveći prinos impregnacije aktivne supstance. Definisani su i optimalni parametri integrisanog procesa natkritične ekstrakcije iz timijana i impregnacije dobijenog ekstrakta na čvrste nosače. U svrhu određivanja rastvorljivosti timola, impregnacije timola i izvođenja integrisanog procesa natkritične ekstrakcije iz timijana i njegove impregnacije primenjene su statička i dinamička metoda, kao i njihova kombinacija, na sledećoj opremi: ćelija za rad pod visokim pritiscima, laboratorijsko postrojenje za rad pod visokim pritiscima i laboratorijsko postrojenje za natkritičnu ekstrakciju i adsorpciju. Prisustvo timola na površini pamučnih vlakana detektovano je FTIR metodom. Karakterizacija čvrstih nosača izvršena je SEM i BET metodama. Hemijski sastav natkritičnog ekstrakta timijana određen je GC/FID metodom. Za proveru antimikrobnog dejstva impregniranih čvrstih nosača korišćene su standardne mikrobiološke metode na sojevima E. coli, S. aureus, B. subtilis, E. faecalis i C. albicans. Kinetika i Impregnacija timola na čvrste nosače natkritičnim ugljenik(IV)-oksidom Stoja Milovanović, dipl. inž. otpuštanja timola iz acetata celuloze praćena je in vitro, u vodi na sobnoj temperaturi, pomoću UV-VIS spektrofotometra merenjem apsorbance na 274 nm. Rezultati ove doktorske disertacije (Poglavlje 4) su prikazani kroz pet potpoglavlja. U delu 4.1. je ispitana rastvorljivost timola u natkritičnom ugljenik(IV)-oksidu na temperaturama 35 °C, 40 °C i 50 °C i pritiscima 7,8-25 MPa (za opseg gustina ugljenik(IV)-oksida 335,89-849,60 kg/m3) statičkom metodom. Dobijeni podaci o rastvorljivosti su korelisani upotrebom semi-empirijskih jednačina: Chrastil, Adachi i Lu i del Valle i Aguilera. Na osnovu dobijenih rezultata, temperatura od 35 °C i pritisak od 15 MPa su odabrani kao optimalni za procese ekstrakcije i impregnacije. U potpoglavlju 4.2. je predstavljena kinetika procesa impregnacije pamučne gaze timolom pomoću natkritičnog ugljenik(IV)-oksida. FTIR analiza je potvrdila prisustvo timola na površini pamučnih vlakana. Ostvareni prinos nakon 2 h impregnacije iznosio je 11,0% , a posle 24 h 19,6%. Impregnirana gaza je imala jako antimikrobno dejstvo na odabrane mikroorganizme. Rezultati su modelovani kinetikom pseudo-prvog i pseudo-drugog reda. U potpoglavlju 4.3. je optimizovan postupak pripreme polisaharidnih gelova (hitozana, kukuruznog i tapioka skroba) u odnosu na prinos impregnacije timolom. Utvrđen je uticaj botaničke vrste skroba, temperature pripreme hidrogela i metode sušenja na morfologiju i prinos impregnacije ksero- i aerogelova skroba. Specifična površina ksero- i aerogelova skrobova je bila u opsegu 0,02-5,52 m2/g. Gelovi kukuruznog skroba su imali veći prinos impregnacije (1,15-4,01%) nego gelovi tapioka skroba (0,58-3,63%). Prinos impregnacije timola je bio veći za kserogelove (1,75-4,01%) nego za aerogelove (0,58-3,31%). Rezultati su ukazali na veliki potencijal kukuruznog kserogela koji je dobijen od hidrogela geliranog na 100 °C uzimajući u obzir jednostavnost pripreme gela, njegovu morfologiju i kapacitet punjenja timolom (4,01%) na datim uslovima natkritične impregnacije. Ispitan je uticaj izbora umreživača (formaldehid i glutardialdehid) i metode sušenja na morfologiju i prinos impregnacije ksero- i aerogelova hitozana. Pri odabranim uslovima impregnacije (15 MPa, 35 °C, 24 h) najveći prinos impregnacije je postignut za aerogel hitozana (11,30%) koji je dobijen umrežavanjem zakišeljenog vodenog rastvora hitozana formaldehidom i koji je osušen natkritičnim ugljenik(IV)-oksidom na 15 MPa i 50 °C. U potpoglavlju 4.4. je utvrđena zavisnost prinosa impregnacije acetata celuloze od operativnih parametara. Prinos impregnacije acetata celuloze timolom je varirao od 4,51% do ii Impregnacija timola na čvrste nosače natkritičnim ugljenik(IV)-oksidom Stoja Milovanović, dipl. inž. 72,26% u zavisnosti od pritiska, temperature i impregnacionog vremena kada je timol u sistemu bio u višku. Morfologija uzoraka se značajno menjala sa povećanjem prinosa impregnacije, što upućuje na uticaj timola na acetat celuloze. Impregnirani uzorci su imali izraženo antimikrobno dejstvo na izabrane mikroorganizme sa mikrobnom redukcijom od 99,9%. U zavisnosti od prinosa impregnacije, uzorci su kontinualno otpuštali timol u periodu od nekoliko časova do preko 10 dana. U potpoglavlju 4.5. su definisani optimalni parametri integrisanog procesa natkritične ekstrakcije timijana i njegove impregnacije na pamučnu gazu. Optimizovani operativni uslovi integrisanog procesa su primenjeni na impregnaciju kserogela kukuruznog skroba, aerogela hitozana i acetata celuloze. Najveći prinos impregnacije ekstrakta timijana je dobijen za pamučnu gazu i iznosio je 7,18%. Doprinos ove teze se ogleda u sledećem: • Dobijene su informacije o vrednostima rastvorljivosti timola u natkritičnom ugljenik(IV)-oksidu na različitim uslovima pritiska i temperature • Razvoj i verifikacija nove metode dobijanja modifikovane pamučne gaze kao medicinskog tekstila sa antimikrobnim dejstvom • Razvoj i verifikacija procesa impregnacije polisaharidnih ksero- i aerogelova timolom • Razvoj i verifikacija procesa impregnacije acetata celuloze timolom. Naime, poznato je da na uslovima od interesa, acetat celuloze ne menja svojstva u prisustvu natkritičnog ugljenik(IV)-oksida. Međutim, prisustvo timola dovodi do uspostavljanja vodoničnih veza između molekula timola i acetata celuloze, menjajući pri tome fizička svojstva polimera. Poseban doprinos predstavlja određivanje količine impregniranog timola koji dovodi do bubrenja, odnosno promena fizičkih svojstava polimera usled formiranja vodoničnih veza između molekula timola i acetata celuloze • Razvoj integrisanog procesa natkritične ekstrakcije iz timijana i impregnacije dobijenog ekstrakta na čvrste nosače.The aim of this PhD thesis was to investigate the possibility of supercritical impregnation of pure thymol and thyme extract (Thymus vulgaris) into selected solid carriers in order to explore the possibility of developing new functionalized materials with antimicrobial properties. Thymol was selected for the supercritical impregnation process of the polysaccharide-based carrier because of its strong antimicrobial, antifungal and antioxidant effect. Before the examination of the thymol impregnation possibilities, the solubility of thymol in supercritical carbon dioxide in the range of temperatures and pressures of interest was examined first. In addition to pure thymol, in the process of solid carrier impregnation, thymol rich thyme extract obtained by the supercritical extraction was applied. Solid carriers for these active components were selected on the basis of desired properties (biodegradability, stability, biocompatibility etc.) and their potential applications in pharmaceutical, biomedical, cosmetic, and food industry. The selected carriers are: a) polysaccharide xero- and aerogels (chitosan, corn and tapioca starch) for use in pharmaceutical and food industries, b) cellulose acetate for application in pharmaceutical and food industries, and c) cotton gauze in order to obtain a medical textile with antibacterial effect in wound treatment. Optimum parameters for fabrication of porous xero- and aerogels (temperature of gelatinization, type of crosslinker and drying method) were defined with respect to the highest impregnation yield. Optimum process parameters of an integrated process of supercritical extraction and impregnation of thyme extract into solid carriers were defined as well. In order to determine the solubility of thymol, and to perform thymol impregnation and integrated process of supercritical extraction of thyme and its impregnation, static and dynamic methods and their combination were applied using following equipment: high pressure view cell, high pressure system for supercritical extraction, and laboratory installation for supercritical extraction and adsorption. The presence of thymol on the surface of cotton fiber was detected by the FTIR method. Characterization of the solid carriers was performed using SEM and BET methods. Chemical composition of the supercritical thyme extract was determined by GC/FID method. To test the antimicrobial effect of the impregnated solid carriers, the standard methods for the microbiological examination were used using the strains of E. coli, S. aureus, B. subtilis, E. faecalis and C. albicans. Kinetic of thymol release from cellulose v Impregnacija timola na čvrste nosače natkritičnim ugljenik(IV)-oksidom Stoja Milovanović, dipl. inž. acetate was followed in vitro, in water at room temperature, using UV-VIS spectrophotometer measuring the absorbance at 274 nm. The results of this doctoral dissertation (Chapter 4) are presented in five subsections. In the subsection 4.1, the solubility of thymol in supercritical carbon dioxide was investigated at temperatures of 35 °C, 40 °C and 50 °C and pressures from 7.8 to 25 MPa (for the carbon dioxide density range from 335.89 to 849.60 kg/m3) using the static method. The obtained solubility data were correlated using semi-empirical equations introduced by Chrastil, Adachi & Lu, and del Valle & Aguilera. Based on these results, temperature of 35 °C and pressure of 15 MPa were selected as the optimal for the extraction and impregnation processes. In the subsection 4.2, the kinetics of cotton gauze impregnation with thymol using supercritical carbon dioxide was presented. The FTIR analysis confirmed the presence of thymol on the surface of cotton fiber. The achieved impregnation yields after 2 h and 24 h were 11.0% and 19.6%, respectively. The impregnated gauze had strong antimicrobial activity against selected microorganisms. Results are modeled using pseudo-first and pseudo-second order kinetics. In the subsection 4.3, preparation of the polysaccharide gels (chitosan, corn and tapioca starch) is optimized referred to the yield of thymol impregnation. The influence of starch botanical origin, the hydrogel preparation temperature, and drying method on the morphology and impregnation yield of starch xero- and aerogels was determined. The specific surface area of the starch xero- and aerogels was in the range of 0.02-5.52 m2/g. Corn starch gels had higher impregnation yields (1.15-4.01%) than tapioca starch gels (0.58-3.63%). The thymol impregnation yield was higher for xerogels (1.75-4.01%) than for aerogels (0.58-3.31%). The results showed the great potential of corn xerogels obtained from hydrogel prepared at 100 °C, taking into account the easiness of gel making, its morphology and thymol loading capacity (4.01%) at the given conditions of supercritical impregnation. The influence of the crosslinking agents (formaldehyde and glutardialdehyde) and drying method on the morphology and impregnation yield of the chitosan xero- and aerogels was examined. For the selected impregnation process conditions (15 MPa, 35 °C, 24 h), the highest impregnation yield was obtained for the chitosan aerogel (11.30%) which was obtained by formaldehyde crosslinking of acidified aqueous solution of chitosan and which was dried with supercritical carbon dioxide at 15 MPa and 50 °C. vi Impregnacija timola na čvrste nosače natkritičnim ugljenik(IV)-oksidom Stoja Milovanović, dipl. inž. In the subsection 4.4, the dependence of the cellulose acetate impregnation yield on operational parameters was determined. The thymol impregnation yield of the cellulose acetate varied from 4.51% to 72.26%, depending on the pressure, temperature, and the impregnation period when thymol was in the system in exes. The morphology of the samples was significantly changed with an increase of the impregnation yield, which indicated the effect of thymol on cellulose acetate. The impregnated samples had strong antimicrobial activity against selected microorganisms with microbial reduction of 99.9%. Depending on the impregnation yield, the samples released thymol continuously over a period from several hours to 10 days. In the subsection 4.5, the optimal parameters of the integrated process of supercritical thyme extraction and its impregnation on cotton gauze were defined. The optimized operating conditions of the integrated process were applied to the corn starch xerogels, chitosan aerogel, and cellulose acetate impregnation. The highest thyme extract impregnation yield was obtained for the cotton gauze (7.18%). The contribution of this thesis is reflected in the following: • Information on the thymol solubility in supercritical carbon dioxide at different pressure and temperature conditions were obtained • Development and verification of the new method of obtaining modified cotton gauze as a medical textile with antimicrobial activity • Development and verification of the process of polysaccharide xero- and aerogels impregnation with thymol • Development and verification of the cellulose acetate impregnation process with thymol. Namely, it is known that at the conditions of interest, cellulose acetate does not change its properties in the presence of supercritical carbon dioxide. However, the presence of thymol leads to a formation of hydrogen bonds between the molecules of thymol and cellulose acetate, altering the physical properties of the polymer. A particular contribution is determination of the amount of impregnated thymol which leads to the swelling, to the change in the polymer physical properties due to forming of hydrogen bonds between thymol molecule and the cellulose acetate. • Development of the integrated process of thyme supercritical extraction and impregnation of obtained extract into the solid carriers

SUPERCRITICAL CO2 EXTRACTION OF OILS FROM RED GRAPE VARIETIES: YIELDS AND EXTRACTION PARAMETERS

In this work, the extraction by supercritical carbon (IV) oxide of grape seed oil from five red grape varieties were investigated. Apart from indigenous variety Prokupac, as a domestic, and Merlot and Cabernet Sauvignon, as  international grape varieties the most represented in Republic of Serbia, Pinot Noir and Gamay were studied as well. Extraction conditions were: temperature of 50 °C, 250 bar pressure and 0.3kg/h flow rate of CO2. It was shown that the extraction kinetics of international grape varieties, similar mutually differ significantly from domestic one. The obtained oil yields were in the range 8,3%w/w (Gamay) to 10,4%w/w (Pinot Noir) for international varieties and 4,96% w/w for domestic variety. Mathematical model “Sovova” was applied in order to define transport parameters regulating the oil mass transfer inside the seed particles and determine  agreement between experimental and model curves. The interpretation of results was done having in mind the differences of grade seed morphologies (outer surfaces and inner layers) which was investigated by SEM analysis

Impregnation of cellulose acetate with natural bioactive compounds using supercritical carbon dioxide

Porous acetate cellulose (CA) beads were tested as carriers for thymol, eugenol and carvacrol, natural compounds well known for their biological activity. For this purpose, selected compounds were impregnated into CA using the environmentally friendly medium, supercritical carbon dioxide (scCO2). Firstly, impregnation of CA with thymol was tested in the pressure range of 10, 15, and 20 MPa for 2 and 24 h at a temperature of 50 °C. The pressure increase resulted in only slight increase in thymol loading. On the other hand, an increase in operating time at 10 MPa resulted in increase in thymol loading from 4.6 to 66.2%. Following the obtained results, subsequent experiments of CA beads impregnation with eugenol and carvacrol were performed at 10 MPa during 2 h, achieving amounts of loaded eugenol and carvacrol of 1.33 and 4.6%, respectively. The presence of thymol, eugenol and carvacrol on/in CA beads as well as its interaction with polymer was confirmed by the FTIR analysis. SEM images revealed that the porous structure of CA beads remained unchanged after its exposure to neat scCO2. However, loading of CA with thymol, eugenol and carvacrol at selected conditions led to the slight change in material texture and morphology. This observation was further examined using mercury intrusion porosimetry. While neat CA had an average pore diameter of 286 nm and porosity of 58%, loaded CA beds expressed an overall reduction in these values. The results were in line with the amounts of loaded active compounds. Namely, for the higher amount of loaded active substance, the higher decrease of specific surface area was observed. These results gave the guidelines for an effective functionalization of neat CA with compounds that have pronounced antimicrobial and antioxidant activity.7th International Congress of the European Polysaccharide Network of Excellence, Nantes, 11-15.10.202

The integrated process of supercritical CO2 extraction from Helichrysum italicum and supercritical impregnation of biocompatible polymers with the obtained extract

The coupling of supercritical fluid extraction and supercritical solvent impregnation processes has been proved to be convenient in cases where the active substance, to be incorporated in the solid carrier, is supercritical extract. The integrated process enables minimizing the loss of the extract by directly using the supercritical CO2-extract solution leaving the extractor vessel for the impregnation and avoidance of the intermediate decompression step in the separate processes (decompression after extraction), and consequently leads to energy and time savings. The objective of this work was to examine the application of the integrated process for impregnation of chitosan/alginate and corn starch xerogels and aerogels with Helichrysum italicum supercritical CO2 extract. For that purpose, first chitosan/alginate and starch hydrogels were prepared by sol-gel method. Thereafter hydrogels were transformed into alcogels by water exchange with ethanol or acetone. Xerogels and aerogels were produced by air and supercritical CO2 drying of the alcogels or acetogels, respectively. Finally, impregnation of the xerogels and aerogels was performed in the laboratory unit for the integrated processes. Both chitosan/alginate and starch xerogels and aerogels showed significant affinity for the incorporation of active principles from H. italicum which was confirmed by FTIR method. SEM method indicated significant differences in materials’ porosity. One of the reason for this is pore collapse phenomenon associated the formation of the xerogels. Obtained results confirmed that it is possible to impregnate chitosan/alginate and starch xerogels and aerogels with H. italicum extract by the integrated process of supercritical CO2 extraction and impregnation

Effect of supercritical CO2 drying process conditions on starch aerogel properties

Starch is an abundant and low-cost polysaccharide obtained from renewable resources. Biocompatibility, biodegradability, and versatility of starch enable its application in several industries including food and pharmaceutical. In this work, supercritical CO2 drying process was tested for preparation of stable and porous starch-based material. First, a hydrogel was formed from an aqueous solution (cornstarch to water mass ratio 1:10) at 95 °C. Subsequently, water was replaced with acetone during several days to form an acetogel. Finally, acetogels were subjected to supercritical CO2 drying at pressures of 8, 10, 20 MPa and temperatures of 35 and 45 °C up to 3 h to form aerogels. Obtained aerogels were compared with starch xerogels prepared by air drying of acetogels. The SEM, FTIR, and porosity analysis confirmed significant effect of supercritical CO2 drying process conditions on starch aerogels properties and their suitability to form nanostructured matrices. SEM analysis showed that aerogels were filamentous with filament diameter less than 100 nm. Due to their low density (0.273-0.773 g/cm3) and high porosity (48-82%) obtained starch aerogels present promising candidates as matrices and carriers for bioactive substances

Impact of the geographic factors on the transport of lead and cadmium in the air

The aim of this study is to describe the effect of geographical varieties on the transport of lead and cadmium in the air. During period between 1997- 2006, the concentrations of lead in suspended in the air at 5 measuring sites in Serbia were measured. The minimal measured daily concentrations of lead ranged from 0,42 μg/m3 (2002.) in Kraljevo. The maximal measured daily concentrations of lead to 251,8 μg/m3 (2004.) in Belgrade. During the period between 1997-2006., the concentrations of cadmium in suspended in the air at 5 measuring sites in Serbia were measured. The minimal measured daily concentrations of cadmium ranged from 0,1 μg/m3 (1999) in Kraljevo. The maximal measured daily concentrations of lead to 42,0 μg/m3 (2000) in Belgrade. Results from our experiments demonstrated that when rapid infiltration conditions or a rainstorm exist, particle-facilitated transport of contaminants is likely to the dominant metal transport pathway influenced by acid rain.Physical chemistry 2008 : 9th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 24-28 September 200

Supercritical CO2 utilization in preparation of poorly soluble drugs solid dispersions

Formulation of solid dispersions is one of the most feasible strategies for overcoming the poor drugs’ solubility – one of the major issues affecting drug bioavailability and therapeutic outcomes. Since traditional methods used for preparation of solid dispersions often require usage of organic solvents, it is of great importance to seek for more environment-friendly methods. It has been demonstrated that supercritical (sc) CO2 may be effectively utilized for dispersion of drugs into the suitable carrier (polymer), thus obtaining solid dispersions with the improved drug dissolution rate. The aim of the presented study was to investigate the potential of scCO2 for preparation of poorly soluble antihypertensive drugs (carvedilol and valsartan) solid dispersions in the conventional pharmaceutical polymers (polyvinilypyrolidone-PVP and hypromellose-HPMC). Prepared solid dispersions were characterized by scanning electron microscopy, differential scanning calorimetry and Fourier-transform infrared spectroscopy; their porosity and density were determined and drug dissolution rate was assessed and compared to the results obtained from solid dispersions prepared by the traditional solvent casting method. Selected samples were tested for their tableting properties as well. It has been demonstrated that scCO2 may be successfully applied for preparation of carvedilol or valsartan solid dispersions with the improved drug dissolution rate. Further characterization revealed the nature of interactions between the drugs and selected polymers. Due to the low density (and high porosity), some of the prepared solid dispersions may be used for further development of the floating dosage forms. It has also been demonstrated that some of the prepared dispersions have excellent compressibility and compactibility, which is of great importance for further development of solid dosage forms. The obtained results provide framework for further development of environment-friendly methods in pharmaceutical development and production

Impregnation of cellulose acetate films with carvacrol using supercritical carbon ioxide

Cellulose acetate films were impregnated with carvacrol using supercritical carbon dioxide. The supercritical impregnation process, conducted in a static regime at pressure of 21 MPa and temperature of 50°C, was optimized by variation in the processing time (30 and 120 min) and decompression rate (from 0.3 MPa/min to 36 MPa/min). Characterization of the obtained cellulose acetate films was performed by Atomic Force Microscopy and Differential Scanning Calorimetry. Effects of glycerol and carvacrol on the properties of the films were discussed. Release kinetics from the cellulose acetate film with 31.4% of carvacrol was investigated in a physiological saline solution. In addition, the Higuchi and Korsmeyer-Peppas release models fitted the carvacrol release curve well. Obtained cellulose acetate films impregnated with carvacrol can be of interest for the application in medicine as wound dressings considering their biocompatibility and biodegradability as well as their potential antimicrobial activity or in the food industry as an active food packaging

Floating photocatalyst based on polycaprolactone foam and titanium dioxide nanoparticles

Registarski broj: 58867Broj i datum rešenja o priznanju prava: 2019/11779, 12.07.2019.Broj prijave: P2018/0703Datum objavljivanja prijave: 29.03.201

Effect of proteine content in boar seminal plasma on the sperm motility in diluted semen stored for 3 days

Recently, it was frequently demonstrated that fertility of sows after artificially inseminated is lower than after mating. This is associated with a reduced fertilization capacity of overdiluted insemination doses. The aim of this study was to investigate the sperm motility in the semen samples, forming from the ejaculates with high or low protein content, stored in vitro on 17 degrees C for 3 days. Progressive motility was significantly higher (p lt 0.01) in the ejaculates with high, compared to the ejaculates with low protein content (82% vs. 76%). After 3 days of storage, in the1:4 dilution proportion, the average progressive motility was significantly (p lt 0.01) decreased in relation to this value in native semen from the boars with high (82% to 64%), as well from the boars with low protein content in seminal plasma (76% to 48%). However, the average diluted semen progressive motility was significantly greater (p lt 0.01) in the boars with high (64%), compared to the boars with low protein content in seminal plasma (48%). The number of good diluted semen samples (>= 65% progressive motility), was also significantly (p lt 0.01) greater in the boars with high (41%), compared to the boars with low protein content in seminal plasma (12%). These results show that seminal plasma proteins play an important role in maintaining the sperm progressive motility of diluted semen in vitro stored for 3 days