O objetivo deste estudo foi avaliar os efeitos de três diluidores diferentes na criopreservação do sêmen bovino sobre a motilidade, hiperativação espermática, reação acrossomal, capacitação e peroxidação lipídica das membranas espermáticas. Foram realizadas seis colheitas de sêmen em intervalos quinzenais de dez touros zebuínos da raça Nelore. O sêmen in natura foi avaliado, dividido em três tratamentos: uma fração foi diluída em meio a base de gema de ovo (Botu-Bov® - diluidor 1), a segunda fração diluída em meio a base de lecitina de soja (Botu-Bov® - diluidor 2) e a terceira fração em meio AndroMed® (diluidor 3) também a base de lecitina de soja. Logo após as amostras foram submetidas à congelação. As avaliações do sêmen após descongelação consistiram na análise computadorizada das características de motilidade e nas análises pela citometria de fluxo quanto à reação acrossomal (PI/FITC-PSA/H33342); peroxidação lipídica (PI/C11-BODIPY581/591/H33342) e capacitação espermática através da estabilidade da membrana plasmática (Merocianina 540/Yo-Pro1/H33342). As variáveis foram submetidas à análise de variância e ao teste LSD para comparação das médias, ao nível de 5% de significância. O diluidor a base de gema de ovo preservou melhor a motilidade espermática, a população de células com integridade de membrana plasmática e acrossomo não reagido no sêmen bovino pós-descongelação que os diluidores a base de lecitina de soja. Os espermatozóides criopreservados com o diluidor a base de gema de ovo apresentou menor sub população de células hiperativas e com membrana plasmática desestabilizada quando comparados com os diluidores a base de lecitina de soja e o diluidor a base de gema de ovo possibilitou uma diminuição da peroxidação lipídica das membranas espermáticas / The objective of this experiment was evaluate the effects of three diferent extenders in bovine semen cryopreservation about motility, sperm hiperactivation, acrosome reaction, capacitation, and sperm membrane lipid peroxidation. It was made six collection of semen samples each 15 days of 10 Nelore bulls. Semen samples in natura was verify and divided in three treatments: The first one was extended in egg yolk base (Botu-Bov® Extender 1), second one was extended in soy lecithin base (Botu-Bov® Extender 2) and the third was extended in AndroMed® soy lecithin base too. After that, semen samples were submitted to freeze process. Semen evaluations after thawing were made with computer assisted sperm analysis (CASA) and flow citometry for acrosome reaction (PI/FITC-PSA/H33342), lipid peroxidation (PI/C11-BODIPY581/591/H33342) and sperm capacitation by plasma membrane stability (Merocianina 540/Yo-Pro1/H33342). Data obtained from experimental proceeding were submitted to variance analysis and LSD test to compare means with 5% of significance. Egg yolk base extender preserved better sperm motility, cell population with plasma membrane integrity and non-reacted acrosome in bovine semen after thawing than soy lecithin extenders. Cryopreserved sperm with egg yolk base extender displayed less subpopulation of hiperactivated cells, less destabilized plasma membrane and permitted a decrease of lipid peroxidation of membranes when it was compared with soy lecithin extender

Com os processos de preservação do sêmen, os espermatozoides sofrem danos devido à indução de alterações estruturais e funcionais na membrana e, aliada a estrutura anatômica do cérvix da ovelha, repercute na redução de fertilidade após a inseminação artificial. Muitos protocolos de congelação e de refrigeração aliados a inúmeros diluentes, vêm sendo estudados, a fim de diminuir os danos causados a essas células e aumentar sua capacidade fertilizante. Para otimizar os processos de congelação e de refrigeração, os objetivos deste estudo foram determinar (1) a influência do método de congelação na motilidade, viabilidade, potencial de membrana mitocondrial e nos defeitos acrossomais do semen ovino criopreservado e (2) a influência de três diluentes comerciais na motilidade, viabilidade, potencial de membrana mitocondrial e nos defeitos acrossomais do sêmen ovino refrigerado. Doze ejaculados foram coletados, diluídos e congelados utilizando (A) uma curva manual de congelação (em vapor de nitrogênio liquido) e (B) um congelador programável (CL-8800, Cryologic Freeze Control®). Na curva A, as amostras foram refrigeradas e congeladas à 30°C/min. Na curva B, houve uma redução de temperatura de 20°C até -50°C durante 37min. Imediatamente após as curvas de criopreservação, as amostras foram submersas em nitrogênio líquido (-196ºC). Para o descongelamento, as palhetas foram submetidas à 37°C por 20seg e analisadas. Para os protocolos de refrigeração, o sêmen diluído em Botubov®, Bovimix® e TYB® foi armazenado à 5°C e à 15°C, e avaliado em 0, 24, 48 e 72h pós coleta. As análises de viabilidade e potencial de membrana mitocondrial (MMP) foram realizadas por citometria de fluxo, e a motilidade e os defeitos acrossomais foram avaliados microscopicamente. Motilidade (29%), viabilidade (18%) e MMP (26%) dos espermatozoides criopreservados com o diluente Bovimix® e a curva B foram significativamente (P<0,05) superiores do que todos os outros tratamentos. As amostras refrigeradas e diluídas com o diluente Bovimix® apresentaram resultados superiores, tanto à 5°C quanto à 15°C, para MMP (44% e 51%, respectivamente) e viabilidade (60% e 53%, respectivamente) 72h pós coleta, quando comparado com as amostras diluídas com Botubov® e TYB®. Em conclusão, os protocolos de congelação/descongelação e de refrigeração utilizados nestes experimentos, reduziram a motilidade, viabilidade, MMP e aumentaram os defeitos acrossomais quando comparados com o sêmen fresco. Os protocolos de congelação/descongelação prejudicaram mais a função espermática quando comparado com os protocolos de refrigeração. Por fim, os resultados sugerem que as amostras espermáticas diluídas com o diluente Bovimix® causou menos danos às células espermáticas do que as amostras diluídas com os diluentes Botubov® e TYB®, tanto nos protocolos de congelação, quanto nos protocolos de refrigeração. / The process of sperm preservation damages spermatozoa due to induction of structural and functional changes in the membrane, and allied to the ewe cervix anatomy, reflects the reduction in fertility after artificial insemination. Many semen preservation protocols including freezing and cooling rates coupled with different extenders have been studied in order to reduce sperm cryo-damage, increase the fertilizing capacity and increase the time of storage. To improve freezing and cooling processes, the objectives of this study were to determine (1) the influence of freezing method on sperm motility, viability, mitochondrial membrane potential and defected acrosome of cryopreserved sperm and (2) the influence of three commercial extenders on the motility, viability, mitochondrial membrane potential and defected acrosome of liquid storaged and frozen-thawed sperm. Twelve semen samples were collected from two mature rams, pooled, and diluted in Botubov®, Bovimix® and TYB®, and frozen using (A) a manual freezing method (liquid nitrogen vapor) and (B) an automated programmable freezer (CL-8800, Cryologic Freeze Control®). For curve A, semen samples were maintained at 37°C and then cooled/frozen at 30°C/min. For curve B, the temperature reduced from 20°C to -50°C during 37min. Immediately, after cryopreservation curves, samples were plunged and stored into liquid nitrogen (-196ºC). Then, the straws were thawed at 37°C for 20sec, and analyzed. For cooling studies, pooled ram semen was diluted in Botubov®, Bovimix® and TYB®, stored at 5°C and at 15°C, and evaluated at 0, 24, 48 and 72 h post collection. For viability and mitochondrial membrane potential (MMP) assessments, semen samples were stained and analyzed by flow cytometry. Motility and defected acrosome assessments were analyzed microscopically. Motility (29%), viability (18%) and MMP (26%) of spermatozoa cryopreserved in Bovimix® coupled to curve B were significantly (P<0.05) higher than all other treated groups. Semen samples diluted with Bovimix® and stored at 5°C and at 15°C yielded better MMP (44% and 51%, respectively) than the samples diluted in Botubov® and in TYB®, and also showed higher viability characteristics at 5°C and at 15°C (60% and 53%, respectively) 72h post collection. In conclusion, freezing/thawing and cooling protocols used in these experiments reduced sperm motility, viability, MMP and increased the defects of acrosomes when compared to fresh semen. Freezing/thawing protocols harmed more spermatozoa function than cooling protocols. Furthermore, our results suggest that the semen samples diluted in the extender Bovimix® caused the least cellular injury than the samples diluted in Botubov® and in TYB® on freezing and on cooling protocols.

Cryopreservation of germplasm is widely used in agriculture, biotechnology, conservation of threatened species and human reproductive medicine. There is a need however to improve the reproductive efficiency of breeding with cryopreserved semen, which may involve increasing the post-thaw quality of sperm through improvements in cryopreservation extenders. Extenders including egg yolk from chickens are successfully used worldwide for cryopreservation of bovine semen, whereas the protective agent in the egg yolk is believed to be the low-density lipoprotein (LDL) fraction. Egg yolks of different avian species vary in their cholesterol, phospholipid and polyunsaturated fatty acid content which have been shown to have important effects on sperm’s freezing capability. The purpose of this study was to determine the cryoprotective effect of clarified egg yolk and LDLs extracted from different egg yolk sources (chicken, chicken omega-3, pigeon, quail and turkey) on bovine sperm. Semen from six bulls was collected four times each by electroejaculation, split and diluted with the 10 following extenders: chicken clarified (Ccl), chicken omega-3 clarified (O3cl), pigeon clarified (Pcl), quail clarified (Qcl), turkey clarified (Tcl), chicken LDL (CLDL), chicken omega-3 LDL (O3LDL), pigeon LDL (PLDL), quail LDL (QLDL) and turkey LDL (TLDL). The extended semen was evaluated, cryopreserved and examined directly after thawing (0h) and after two hours at 37 ˚C (2h). Computer assisted sperm analysis (CASA) was used to determine total sperm motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP). Intact plasma membrane (IPM) and intact acrosomes (IA) were measured by flow cytometry. The percentage change (loss; Δ%) of each sperm characteristic was calculated and used to compare the effect of the extenders. From extending to 0h post-thaw, the pigeon LDL extender lead to greater losses in sperm total and progressive motility, as well as of intact acrosomes, than the other nine extenders tested (P < 0.05). During 0h to 2h post-thaw, the sperm in PLDL extender experienced greater losses in total and progressive motility (P < 0.0001), as well as in curvilinear velocity (P < 0.05), than in all the other nine extenders. Sperm in turkey clarified extender had a greater loss in the velocity parameters (VSL, VAP, VCL) than sperm in several of the other extenders such as O3cl, CLDL, O3LDL, QLDL and TLDL from 0h to 2h (P < 0.05). Concomitantly, sperm in the Tcl extender had a greater loss in the velocity parameters and of intact acrosomes compared to sperm in its counterpart, the turkey LDL extender, from 0h to 2h post-thaw (P < 0.05). The differences produced in post-thaw quality of cryopreserved bovine sperm in the pigeon LDL and turkey clarified extenders were attributed to methodological differences in these egg yolk preparations compared with the other eight extenders. Importantly, the results demonstrate that with most egg yolk preparations derived from a variety of species, there are equivalent cryoprotective effects afforded by the use of omega-3 chicken, pigeon, quail, or conventional chicken egg yolk in a clarified form in freezing extenders for bovine semen. We further proved that the freezing capabilities of bovine semen extenders containing the low-density lipoprotein fraction of omega-3 chicken, quail, turkey and conventional chicken egg yolk were similar.

Modular polyketide synthases (PKSs) are the largest enzymes known to man and are responsible for synthesizing some of the most important human medicines. Their ability to construct stereochemically-rich carbon chains containing diverse substituents has inspired the biosynthetic community to engineer these factories for the in vitro synthesis of a small library of polyketide compounds. New complex polyketides are discovered every year, yet the lack of compound prohibits characterization and testing of these new compounds for medicinal properties. Smaller polyketide compounds generated in vitro could be organically manipulated to generate larger, more complex polyketide natural products and natural product analogs. Chemoenzymatic approaches like this would be extremely beneficial to the scientific community; however, there are still obstacles that must be overcome before the use of PKS for the preparative synthesis of an in vitro generated polyketide library would prove fruitful: purchasing substrates such as methylmalonyl-CoA is cost-prohibitive, PKSs are often difficult to express and purify, and the products generated are typically nonchromophoric. The use of a malonyl-CoA ligase from Streptomyces coelicolor (MatB) was investigated for the enzymatic synthesis of polyketide extender units such as methylmalonyl-CoA (Chapter 2). MatB synthesized a total of 5 CoA-linked extender units in vitro: malonyl-, methylmalonyl-, ethylmalonyl-, hydroxymalonyl- and methoxymalonyl-CoA. Two ternary complex structures of MatB with bound product and leaving group were also solved to sub-2Å resolution. MatB generated extender units were employed in the module-catalyzed synthesis of a triketide pyrone. The selectivity of a PKS module to incorporate a variety of side chains into triketide pyrones was also investigated (Chapter 3). A total of 10 triketide pyrone compounds were synthesized, 5 produced via modular "stuttering" and one possessing a terminal alkyne chemical handle. Lastly, nonchromphoric polyketide products were made visible upon copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) with fluorescent sulforhodamine B azide revealing insights into in vitro reactivites of a PKS module (Chapter 4). The work described in this dissertation has helped advance the scientific community towards procuring an in vitro synthesized polyketide library for future synthetic applications. / text

Novel solutions to offset thermal degradation of polyamide-6 (PA-6) and organoclay (organically modified layered silicates) nanocomposites during melt compounding have been investigated. In this research, a novel chain extender (Joncryl ADR 3400) has been used to improve thermal stability of PA-6 and PA- 6/organoclay nanocomposites during melt compounding. The materials were compounded using a linear twin extruder and various laboratory scale mixers. The effects of organoclay and chain extender were studied using both processing methods. In order to replicate large scale production used in industry, a comprehensive plan of experimental work was carried out under different processing conditions (extrusion temperature and screw speed), organoclay and chain extender loading using a linear twin screw extruder. Rheology, mechanical and thermal properties were analysed and selected samples were also characterised by TEM and FTIR. Process induced degradation of PA-6 during the melt compounding was found to have significant influence on the rheological and mechanical properties. Rheological and mechanical characterisation clearly showed showed that incorporation of the chain extender minimised thermal degradation of PA-6 and nanocomposites during melt processing. Visual analysis of selected nanocomposites using TEM confirmed that chain extender increased the dispersion of nanoclays in the PA- 6 matrix. The crystallinity of the PA-6 was slightly affected by addition of organoclay and chain extender. The samples obtained by linear twin screw extrusion showed higher rheological properties than the samples from laboratory scale mixers suggesting better mixing and less thermal degradation during extrusion.

Com os processos de preservação do sêmen, os espermatozoides sofrem danos devido à indução de alterações estruturais e funcionais na membrana e, aliada a estrutura anatômica do cérvix da ovelha, repercute na redução de fertilidade após a inseminação artificial. Muitos protocolos de congelação e de refrigeração aliados a inúmeros diluentes, vêm sendo estudados, a fim de diminuir os danos causados a essas células e aumentar sua capacidade fertilizante. Para otimizar os processos de congelação e de refrigeração, os objetivos deste estudo foram determinar (1) a influência do método de congelação na motilidade, viabilidade, potencial de membrana mitocondrial e nos defeitos acrossomais do semen ovino criopreservado e (2) a influência de três diluentes comerciais na motilidade, viabilidade, potencial de membrana mitocondrial e nos defeitos acrossomais do sêmen ovino refrigerado. Doze ejaculados foram coletados, diluídos e congelados utilizando (A) uma curva manual de congelação (em vapor de nitrogênio liquido) e (B) um congelador programável (CL-8800, Cryologic Freeze Control®). Na curva A, as amostras foram refrigeradas e congeladas à 30°C/min. Na curva B, houve uma redução de temperatura de 20°C até -50°C durante 37min. Imediatamente após as curvas de criopreservação, as amostras foram submersas em nitrogênio líquido (-196ºC). Para o descongelamento, as palhetas foram submetidas à 37°C por 20seg e analisadas. Para os protocolos de refrigeração, o sêmen diluído em Botubov®, Bovimix® e TYB® foi armazenado à 5°C e à 15°C, e avaliado em 0, 24, 48 e 72h pós coleta. As análises de viabilidade e potencial de membrana mitocondrial (MMP) foram realizadas por citometria de fluxo, e a motilidade e os defeitos acrossomais foram avaliados microscopicamente. Motilidade (29%), viabilidade (18%) e MMP (26%) dos espermatozoides criopreservados com o diluente Bovimix® e a curva B foram significativamente (P<0,05) superiores do que todos os outros tratamentos. As amostras refrigeradas e diluídas com o diluente Bovimix® apresentaram resultados superiores, tanto à 5°C quanto à 15°C, para MMP (44% e 51%, respectivamente) e viabilidade (60% e 53%, respectivamente) 72h pós coleta, quando comparado com as amostras diluídas com Botubov® e TYB®. Em conclusão, os protocolos de congelação/descongelação e de refrigeração utilizados nestes experimentos, reduziram a motilidade, viabilidade, MMP e aumentaram os defeitos acrossomais quando comparados com o sêmen fresco. Os protocolos de congelação/descongelação prejudicaram mais a função espermática quando comparado com os protocolos de refrigeração. Por fim, os resultados sugerem que as amostras espermáticas diluídas com o diluente Bovimix® causou menos danos às células espermáticas do que as amostras diluídas com os diluentes Botubov® e TYB®, tanto nos protocolos de congelação, quanto nos protocolos de refrigeração. / The process of sperm preservation damages spermatozoa due to induction of structural and functional changes in the membrane, and allied to the ewe cervix anatomy, reflects the reduction in fertility after artificial insemination. Many semen preservation protocols including freezing and cooling rates coupled with different extenders have been studied in order to reduce sperm cryo-damage, increase the fertilizing capacity and increase the time of storage. To improve freezing and cooling processes, the objectives of this study were to determine (1) the influence of freezing method on sperm motility, viability, mitochondrial membrane potential and defected acrosome of cryopreserved sperm and (2) the influence of three commercial extenders on the motility, viability, mitochondrial membrane potential and defected acrosome of liquid storaged and frozen-thawed sperm. Twelve semen samples were collected from two mature rams, pooled, and diluted in Botubov®, Bovimix® and TYB®, and frozen using (A) a manual freezing method (liquid nitrogen vapor) and (B) an automated programmable freezer (CL-8800, Cryologic Freeze Control®). For curve A, semen samples were maintained at 37°C and then cooled/frozen at 30°C/min. For curve B, the temperature reduced from 20°C to -50°C during 37min. Immediately, after cryopreservation curves, samples were plunged and stored into liquid nitrogen (-196ºC). Then, the straws were thawed at 37°C for 20sec, and analyzed. For cooling studies, pooled ram semen was diluted in Botubov®, Bovimix® and TYB®, stored at 5°C and at 15°C, and evaluated at 0, 24, 48 and 72 h post collection. For viability and mitochondrial membrane potential (MMP) assessments, semen samples were stained and analyzed by flow cytometry. Motility and defected acrosome assessments were analyzed microscopically. Motility (29%), viability (18%) and MMP (26%) of spermatozoa cryopreserved in Bovimix® coupled to curve B were significantly (P<0.05) higher than all other treated groups. Semen samples diluted with Bovimix® and stored at 5°C and at 15°C yielded better MMP (44% and 51%, respectively) than the samples diluted in Botubov® and in TYB®, and also showed higher viability characteristics at 5°C and at 15°C (60% and 53%, respectively) 72h post collection. In conclusion, freezing/thawing and cooling protocols used in these experiments reduced sperm motility, viability, MMP and increased the defects of acrosomes when compared to fresh semen. Freezing/thawing protocols harmed more spermatozoa function than cooling protocols. Furthermore, our results suggest that the semen samples diluted in the extender Bovimix® caused the least cellular injury than the samples diluted in Botubov® and in TYB® on freezing and on cooling protocols.

Dois experimentos foram conduzidos para verificar a viabilidade do uso do leite UHT como diluente para sêmen equino refrigerado. O objetivo principal deste estudo foi avaliar o uso de leite UHT nas apresentações desnatado, semidesnatado e integral como diluente de sêmen e posteriormente foi avaliada a viabilidade espermática com o uso de leite como diluente comparado a outros dois diluentes industrializados. No experimento I foram utilizados 31 ejaculados de 3 garanhões, as amostras de cada ejaculado foram diluídas em leite UHT nas apresentações integral, semidesnatado e desnatado, numa proporção de 3:1 (3 ml de diluente e 1ml de sêmen). Foram realizadas análises de viabilidade espermática com testes de motilidade espermática, vigor espermático, integridade de membrana (CFDA/PI) e funcionalidade de membrana espermática (HOST) pós-diluição, os quais foram repetidos nas amostras refrigeradas a 4ºC após 24 e 48h. Neste experimento, não foram observadas diferenças significativas entre os 3 diluentes, quanto a motilidade espermática, vigor, CFDA/PI e HOST. No entanto, houve uma diminuição na qualidade do sêmen armazenado por 48h (P < 0,01), sendo mais acentuada entre 24 e 48h de refrigeração. No experimento II foram utilizados 30 ejaculados de 3 garanhões onde, logo após a coleta do sêmen, as amostras de cada ejaculado foram diluídas com leite UHT desnatado (LD), Botusemen® (BS) e Botusemen Special® (BSP). As amostras foram examinadas a fresco, após a diluição (0h) e em resfriamento de 24h e 48h a 4°C. Foram realizados os mesmos testes de análise espermática do primeiro experimento para avaliação comparativa dos diluentes. As amostras de sêmen refrigeradas com BSP obtiveram maior porcentagem de motilidade total (51,1%) após 24h de refrigeração quando comparado aos outros dois diluentes testados, LD (45,8%) e BS (47,3%). Com base nos resultados obtidos nos dois experimentos, o leite UHT, independente da apresentação, pode ser utilizado como diluente de sêmen para as primeiras 24h de refrigeração, mantendo a viabilidade espermática adequada, sendo também de fácil acesso e manuseio. Se for necessário a manutenção do sêmen por mais de 24h, o BSP se mostrou superior em manter a qualidade do sêmen até 48h. / Two experiments were conducted to verify the feasibility of the use of UHT milk as an extender for chilled equine semen. The main objective of this study was to evaluate the use of UHT milk in skimmed, semi-skimmed and whole milk as extender for semen and subsequently assess sperm viability using milk as a diluent compared to two other industrial extenders. In the first experiment were used 31 ejaculates from 3 stallions of known fertility, samples of each ejaculate were diluted in the whole UHT milk, semi-skimmed milk and skim milk shows a ratio of 3:1 (3ml diluent and 1ml of semen). Sperm viability analyzes were performed, including sperm motility, sperm vigor, membrane integrity (CFDA/PI) functionality and sperm membrane (HOST) post-dilution tests which were repeated in the samples cooled to 4°C after 24 and 48h. In this experiment, no significant differences in sperm motility, vigor, CFDA/IP and HOST were observed among the 3 extenders. However, there was a decrease in the quality of semen stored for 48h (P < 0.01), being more pronounced between 24 and 48h of cooling. In the second experiment, 30 ejaculates from 3 stallions were used soon after collection of semen samples, each ejaculate was diluted with UHT skim milk (LD), Botusemen® (BS) and Botusemen Special® (BSP). The samples were analyzed fresh, immediately after the dilution (0h) and after cooling for 24h and 48h at 4°C. The same tests of sperm analysis used in the first experiment for comparative evaluation of extenders were performed. Semen samples cooled with BSP obtained the highest percentage of total motility (51.1%) after 24h of cooling when compared to the two other extenders tested, LD (45.8%) and BS (47.3%). Based on the results from the two experiments, UHT milk, regardless of the presentation, can be used as a diluent of semen during the first 24h of cooling maintaining adequate sperm viability and is easy to access and handle. If maintenance of semen for more than 24h is required, the BSP was superior in maintaining the quality of semen until 48h.

Dois experimentos foram conduzidos para verificar a viabilidade do uso do leite UHT como diluente para sêmen equino refrigerado. O objetivo principal deste estudo foi avaliar o uso de leite UHT nas apresentações desnatado, semidesnatado e integral como diluente de sêmen e posteriormente foi avaliada a viabilidade espermática com o uso de leite como diluente comparado a outros dois diluentes industrializados. No experimento I foram utilizados 31 ejaculados de 3 garanhões, as amostras de cada ejaculado foram diluídas em leite UHT nas apresentações integral, semidesnatado e desnatado, numa proporção de 3:1 (3 ml de diluente e 1ml de sêmen). Foram realizadas análises de viabilidade espermática com testes de motilidade espermática, vigor espermático, integridade de membrana (CFDA/PI) e funcionalidade de membrana espermática (HOST) pós-diluição, os quais foram repetidos nas amostras refrigeradas a 4ºC após 24 e 48h. Neste experimento, não foram observadas diferenças significativas entre os 3 diluentes, quanto a motilidade espermática, vigor, CFDA/PI e HOST. No entanto, houve uma diminuição na qualidade do sêmen armazenado por 48h (P < 0,01), sendo mais acentuada entre 24 e 48h de refrigeração. No experimento II foram utilizados 30 ejaculados de 3 garanhões onde, logo após a coleta do sêmen, as amostras de cada ejaculado foram diluídas com leite UHT desnatado (LD), Botusemen® (BS) e Botusemen Special® (BSP). As amostras foram examinadas a fresco, após a diluição (0h) e em resfriamento de 24h e 48h a 4°C. Foram realizados os mesmos testes de análise espermática do primeiro experimento para avaliação comparativa dos diluentes. As amostras de sêmen refrigeradas com BSP obtiveram maior porcentagem de motilidade total (51,1%) após 24h de refrigeração quando comparado aos outros dois diluentes testados, LD (45,8%) e BS (47,3%). Com base nos resultados obtidos nos dois experimentos, o leite UHT, independente da apresentação, pode ser utilizado como diluente de sêmen para as primeiras 24h de refrigeração, mantendo a viabilidade espermática adequada, sendo também de fácil acesso e manuseio. Se for necessário a manutenção do sêmen por mais de 24h, o BSP se mostrou superior em manter a qualidade do sêmen até 48h. / Two experiments were conducted to verify the feasibility of the use of UHT milk as an extender for chilled equine semen. The main objective of this study was to evaluate the use of UHT milk in skimmed, semi-skimmed and whole milk as extender for semen and subsequently assess sperm viability using milk as a diluent compared to two other industrial extenders. In the first experiment were used 31 ejaculates from 3 stallions of known fertility, samples of each ejaculate were diluted in the whole UHT milk, semi-skimmed milk and skim milk shows a ratio of 3:1 (3ml diluent and 1ml of semen). Sperm viability analyzes were performed, including sperm motility, sperm vigor, membrane integrity (CFDA/PI) functionality and sperm membrane (HOST) post-dilution tests which were repeated in the samples cooled to 4°C after 24 and 48h. In this experiment, no significant differences in sperm motility, vigor, CFDA/IP and HOST were observed among the 3 extenders. However, there was a decrease in the quality of semen stored for 48h (P < 0.01), being more pronounced between 24 and 48h of cooling. In the second experiment, 30 ejaculates from 3 stallions were used soon after collection of semen samples, each ejaculate was diluted with UHT skim milk (LD), Botusemen® (BS) and Botusemen Special® (BSP). The samples were analyzed fresh, immediately after the dilution (0h) and after cooling for 24h and 48h at 4°C. The same tests of sperm analysis used in the first experiment for comparative evaluation of extenders were performed. Semen samples cooled with BSP obtained the highest percentage of total motility (51.1%) after 24h of cooling when compared to the two other extenders tested, LD (45.8%) and BS (47.3%). Based on the results from the two experiments, UHT milk, regardless of the presentation, can be used as a diluent of semen during the first 24h of cooling maintaining adequate sperm viability and is easy to access and handle. If maintenance of semen for more than 24h is required, the BSP was superior in maintaining the quality of semen until 48h.

Com os processos de preservação do sêmen, os espermatozoides sofrem danos devido à indução de alterações estruturais e funcionais na membrana e, aliada a estrutura anatômica do cérvix da ovelha, repercute na redução de fertilidade após a inseminação artificial. Muitos protocolos de congelação e de refrigeração aliados a inúmeros diluentes, vêm sendo estudados, a fim de diminuir os danos causados a essas células e aumentar sua capacidade fertilizante. Para otimizar os processos de congelação e de refrigeração, os objetivos deste estudo foram determinar (1) a influência do método de congelação na motilidade, viabilidade, potencial de membrana mitocondrial e nos defeitos acrossomais do semen ovino criopreservado e (2) a influência de três diluentes comerciais na motilidade, viabilidade, potencial de membrana mitocondrial e nos defeitos acrossomais do sêmen ovino refrigerado. Doze ejaculados foram coletados, diluídos e congelados utilizando (A) uma curva manual de congelação (em vapor de nitrogênio liquido) e (B) um congelador programável (CL-8800, Cryologic Freeze Control®). Na curva A, as amostras foram refrigeradas e congeladas à 30°C/min. Na curva B, houve uma redução de temperatura de 20°C até -50°C durante 37min. Imediatamente após as curvas de criopreservação, as amostras foram submersas em nitrogênio líquido (-196ºC). Para o descongelamento, as palhetas foram submetidas à 37°C por 20seg e analisadas. Para os protocolos de refrigeração, o sêmen diluído em Botubov®, Bovimix® e TYB® foi armazenado à 5°C e à 15°C, e avaliado em 0, 24, 48 e 72h pós coleta. As análises de viabilidade e potencial de membrana mitocondrial (MMP) foram realizadas por citometria de fluxo, e a motilidade e os defeitos acrossomais foram avaliados microscopicamente. Motilidade (29%), viabilidade (18%) e MMP (26%) dos espermatozoides criopreservados com o diluente Bovimix® e a curva B foram significativamente (P<0,05) superiores do que todos os outros tratamentos. As amostras refrigeradas e diluídas com o diluente Bovimix® apresentaram resultados superiores, tanto à 5°C quanto à 15°C, para MMP (44% e 51%, respectivamente) e viabilidade (60% e 53%, respectivamente) 72h pós coleta, quando comparado com as amostras diluídas com Botubov® e TYB®. Em conclusão, os protocolos de congelação/descongelação e de refrigeração utilizados nestes experimentos, reduziram a motilidade, viabilidade, MMP e aumentaram os defeitos acrossomais quando comparados com o sêmen fresco. Os protocolos de congelação/descongelação prejudicaram mais a função espermática quando comparado com os protocolos de refrigeração. Por fim, os resultados sugerem que as amostras espermáticas diluídas com o diluente Bovimix® causou menos danos às células espermáticas do que as amostras diluídas com os diluentes Botubov® e TYB®, tanto nos protocolos de congelação, quanto nos protocolos de refrigeração. / The process of sperm preservation damages spermatozoa due to induction of structural and functional changes in the membrane, and allied to the ewe cervix anatomy, reflects the reduction in fertility after artificial insemination. Many semen preservation protocols including freezing and cooling rates coupled with different extenders have been studied in order to reduce sperm cryo-damage, increase the fertilizing capacity and increase the time of storage. To improve freezing and cooling processes, the objectives of this study were to determine (1) the influence of freezing method on sperm motility, viability, mitochondrial membrane potential and defected acrosome of cryopreserved sperm and (2) the influence of three commercial extenders on the motility, viability, mitochondrial membrane potential and defected acrosome of liquid storaged and frozen-thawed sperm. Twelve semen samples were collected from two mature rams, pooled, and diluted in Botubov®, Bovimix® and TYB®, and frozen using (A) a manual freezing method (liquid nitrogen vapor) and (B) an automated programmable freezer (CL-8800, Cryologic Freeze Control®). For curve A, semen samples were maintained at 37°C and then cooled/frozen at 30°C/min. For curve B, the temperature reduced from 20°C to -50°C during 37min. Immediately, after cryopreservation curves, samples were plunged and stored into liquid nitrogen (-196ºC). Then, the straws were thawed at 37°C for 20sec, and analyzed. For cooling studies, pooled ram semen was diluted in Botubov®, Bovimix® and TYB®, stored at 5°C and at 15°C, and evaluated at 0, 24, 48 and 72 h post collection. For viability and mitochondrial membrane potential (MMP) assessments, semen samples were stained and analyzed by flow cytometry. Motility and defected acrosome assessments were analyzed microscopically. Motility (29%), viability (18%) and MMP (26%) of spermatozoa cryopreserved in Bovimix® coupled to curve B were significantly (P<0.05) higher than all other treated groups. Semen samples diluted with Bovimix® and stored at 5°C and at 15°C yielded better MMP (44% and 51%, respectively) than the samples diluted in Botubov® and in TYB®, and also showed higher viability characteristics at 5°C and at 15°C (60% and 53%, respectively) 72h post collection. In conclusion, freezing/thawing and cooling protocols used in these experiments reduced sperm motility, viability, MMP and increased the defects of acrosomes when compared to fresh semen. Freezing/thawing protocols harmed more spermatozoa function than cooling protocols. Furthermore, our results suggest that the semen samples diluted in the extender Bovimix® caused the least cellular injury than the samples diluted in Botubov® and in TYB® on freezing and on cooling protocols.

Dois experimentos foram conduzidos para verificar a viabilidade do uso do leite UHT como diluente para sêmen equino refrigerado. O objetivo principal deste estudo foi avaliar o uso de leite UHT nas apresentações desnatado, semidesnatado e integral como diluente de sêmen e posteriormente foi avaliada a viabilidade espermática com o uso de leite como diluente comparado a outros dois diluentes industrializados. No experimento I foram utilizados 31 ejaculados de 3 garanhões, as amostras de cada ejaculado foram diluídas em leite UHT nas apresentações integral, semidesnatado e desnatado, numa proporção de 3:1 (3 ml de diluente e 1ml de sêmen). Foram realizadas análises de viabilidade espermática com testes de motilidade espermática, vigor espermático, integridade de membrana (CFDA/PI) e funcionalidade de membrana espermática (HOST) pós-diluição, os quais foram repetidos nas amostras refrigeradas a 4ºC após 24 e 48h. Neste experimento, não foram observadas diferenças significativas entre os 3 diluentes, quanto a motilidade espermática, vigor, CFDA/PI e HOST. No entanto, houve uma diminuição na qualidade do sêmen armazenado por 48h (P < 0,01), sendo mais acentuada entre 24 e 48h de refrigeração. No experimento II foram utilizados 30 ejaculados de 3 garanhões onde, logo após a coleta do sêmen, as amostras de cada ejaculado foram diluídas com leite UHT desnatado (LD), Botusemen® (BS) e Botusemen Special® (BSP). As amostras foram examinadas a fresco, após a diluição (0h) e em resfriamento de 24h e 48h a 4°C. Foram realizados os mesmos testes de análise espermática do primeiro experimento para avaliação comparativa dos diluentes. As amostras de sêmen refrigeradas com BSP obtiveram maior porcentagem de motilidade total (51,1%) após 24h de refrigeração quando comparado aos outros dois diluentes testados, LD (45,8%) e BS (47,3%). Com base nos resultados obtidos nos dois experimentos, o leite UHT, independente da apresentação, pode ser utilizado como diluente de sêmen para as primeiras 24h de refrigeração, mantendo a viabilidade espermática adequada, sendo também de fácil acesso e manuseio. Se for necessário a manutenção do sêmen por mais de 24h, o BSP se mostrou superior em manter a qualidade do sêmen até 48h. / Two experiments were conducted to verify the feasibility of the use of UHT milk as an extender for chilled equine semen. The main objective of this study was to evaluate the use of UHT milk in skimmed, semi-skimmed and whole milk as extender for semen and subsequently assess sperm viability using milk as a diluent compared to two other industrial extenders. In the first experiment were used 31 ejaculates from 3 stallions of known fertility, samples of each ejaculate were diluted in the whole UHT milk, semi-skimmed milk and skim milk shows a ratio of 3:1 (3ml diluent and 1ml of semen). Sperm viability analyzes were performed, including sperm motility, sperm vigor, membrane integrity (CFDA/PI) functionality and sperm membrane (HOST) post-dilution tests which were repeated in the samples cooled to 4°C after 24 and 48h. In this experiment, no significant differences in sperm motility, vigor, CFDA/IP and HOST were observed among the 3 extenders. However, there was a decrease in the quality of semen stored for 48h (P < 0.01), being more pronounced between 24 and 48h of cooling. In the second experiment, 30 ejaculates from 3 stallions were used soon after collection of semen samples, each ejaculate was diluted with UHT skim milk (LD), Botusemen® (BS) and Botusemen Special® (BSP). The samples were analyzed fresh, immediately after the dilution (0h) and after cooling for 24h and 48h at 4°C. The same tests of sperm analysis used in the first experiment for comparative evaluation of extenders were performed. Semen samples cooled with BSP obtained the highest percentage of total motility (51.1%) after 24h of cooling when compared to the two other extenders tested, LD (45.8%) and BS (47.3%). Based on the results from the two experiments, UHT milk, regardless of the presentation, can be used as a diluent of semen during the first 24h of cooling maintaining adequate sperm viability and is easy to access and handle. If maintenance of semen for more than 24h is required, the BSP was superior in maintaining the quality of semen until 48h.