Características seminais pós-descongelamento em garanhões da raça crioula / Post-thaw seminal characteristics of Criollo breed stallions

Este trabalho teve como objetivo avaliar as características seminais de garanhões da raça Crioula após o processo de criopreservação utilizando o BotuCrio® . Foram utilizados 24 garanhões alojados nas proximidades de Porto Alegre, RS, Brasil. As análises do sêmen foram realizadas pré e pósdescongelamento através do sistema Computer Assisted Sperm Analysis AndroVision®. Somente foram utilizadas amostras com motilidade total ≥ 60 % e vigor ≥ 3, estas foram envasadas em palhetas de 0,5 ml, dispostas horizontalmente a 5ºC durante 20 minutos. Após colocadas a 6 cm acima do nível de nitrogênio líquido durante 20 minutos. Sendo finalmente imersas em nitrogênio líquido, após descongeladas em banho-maria a 37ºC durante 30 segundos. Os valores médios e desvios padrões das variáveis pós-descongelamento foram: motilidade total (52,85 % ± 7,27); motilidade progressiva (31,15 % ± 10,21); motilidade rápida (4,78 % ± 5,37); motilidade local (22,44 % ± 8,68); velocidade curvilinear (97,67 μm/s ± 35,25); velocidade em linha reta (35,33 μm/s ± 15,29); velocidade da trajetória média (44,99 μm/s ± 17,98); linearidade (0,3 % ± 0,15); BCF (2,5 Hz ± 1,7); ALH (0,35 μm ± 0,3); integridade funcional da membrana (42,73 % ± 7,06) e integridade estrutural da membrana (48,06 % ± 8,99). Concluímos que o protocolo utilizando diluente BotuCrio® é viável para criopreservação do sêmen de cavalos da raça Crioula, pois garante motilidade acima de 30 % pós-descongelamento que é recomendado pelo Colégio Brasileiro de Reprodução Animal.(AU)

The aim of this study was to evaluate seminal characteristics of Criollo breed stallions, after cryopreservation process using the BotuCrio®. One ejaculate from twenty-four stallions located near to Porto Alegre, RS, Brazil were used. Semen analysis were performed pre and post-freezing through the Computer Assisted Sperm Analysis AndroVision® system. Only samples with total motility ≥ 60 % and vigor ≥ 3 were used. The samples were placed in 0.5 ml straws, arranged horizontally at 5ºC for 20 minutes. Then, 6 cm above the level of liquid nitrogen in vapor for 20 minutes. Finally, being immersed in liquid nitrogen, the samples thawed in a water bath at 37ºC for 30 seconds. Mean values and standard deviations of post-freeze variables were: total motility (52.85 % ± 7.27); progressive motility (31.15 % ± 10.21); rapid motility (4.78 % ± 5.37); local motility (22.44 % ± 8.68); Curvilinear Velocity (97.67 μm/s ± 35.25); Straight Line Velocity (35.33 μm/s ± 15.29); Average Path Velocity (44.99 μm/s ± 17.98); Linearity (0.3 % ± 0.15); BCF (2.5 Hz ± 1.7); ALH (0.35 μm ± 0.3) functional integrity (42.73 % ± 7.06) and physical integrity (48.06 % ± 8.99). We conclude that the protocol used for semen cryopreservation is feasible for use in the Criollo breed, because it has motility above 30 % post-freezing semen parameters, wich is the recommended by the Brazilian College of Animal Reproduction.(AU)

Características seminais pós-descongelamento em garanhões da raça crioula / Post-thaw seminal characteristics of Criollo breed stallions

Este trabalho teve como objetivo avaliar as características seminais de garanhões da raça Crioula após o processo de criopreservação utilizando o BotuCrio® . Foram utilizados 24 garanhões alojados nas proximidades de Porto Alegre, RS, Brasil. As análises do sêmen foram realizadas pré e pósdescongelamento através do sistema Computer Assisted Sperm Analysis AndroVision®. Somente foram utilizadas amostras com motilidade total ≥ 60 % e vigor ≥ 3, estas foram envasadas em palhetas de 0,5 ml, dispostas horizontalmente a 5ºC durante 20 minutos. Após colocadas a 6 cm acima do nível de nitrogênio líquido durante 20 minutos. Sendo finalmente imersas em nitrogênio líquido, após descongeladas em banho-maria a 37ºC durante 30 segundos. Os valores médios e desvios padrões das variáveis pós-descongelamento foram: motilidade total (52,85 % ± 7,27); motilidade progressiva (31,15 % ± 10,21); motilidade rápida (4,78 % ± 5,37); motilidade local (22,44 % ± 8,68); velocidade curvilinear (97,67 μm/s ± 35,25); velocidade em linha reta (35,33 μm/s ± 15,29); velocidade da trajetória média (44,99 μm/s ± 17,98); linearidade (0,3 % ± 0,15); BCF (2,5 Hz ± 1,7); ALH (0,35 μm ± 0,3); integridade funcional da membrana (42,73 % ± 7,06) e integridade estrutural da membrana (48,06 % ± 8,99). Concluímos que o protocolo utilizando diluente BotuCrio® é viável para criopreservação do sêmen de cavalos da raça Crioula, pois garante motilidade acima de 30 % pós-descongelamento que é recomendado pelo Colégio Brasileiro de Reprodução Animal.

The aim of this study was to evaluate seminal characteristics of Criollo breed stallions, after cryopreservation process using the BotuCrio®. One ejaculate from twenty-four stallions located near to Porto Alegre, RS, Brazil were used. Semen analysis were performed pre and post-freezing through the Computer Assisted Sperm Analysis AndroVision® system. Only samples with total motility ≥ 60 % and vigor ≥ 3 were used. The samples were placed in 0.5 ml straws, arranged horizontally at 5ºC for 20 minutes. Then, 6 cm above the level of liquid nitrogen in vapor for 20 minutes. Finally, being immersed in liquid nitrogen, the samples thawed in a water bath at 37ºC for 30 seconds. Mean values and standard deviations of post-freeze variables were: total motility (52.85 % ± 7.27); progressive motility (31.15 % ± 10.21); rapid motility (4.78 % ± 5.37); local motility (22.44 % ± 8.68); Curvilinear Velocity (97.67 μm/s ± 35.25); Straight Line Velocity (35.33 μm/s ± 15.29); Average Path Velocity (44.99 μm/s ± 17.98); Linearity (0.3 % ± 0.15); BCF (2.5 Hz ± 1.7); ALH (0.35 μm ± 0.3) functional integrity (42.73 % ± 7.06) and physical integrity (48.06 % ± 8.99). We conclude that the protocol used for semen cryopreservation is feasible for use in the Criollo breed, because it has motility above 30 % post-freezing semen parameters, wich is the recommended by the Brazilian College of Animal Reproduction.

Uterine nitric oxide levels and isofluopredone treatment effect in mares susceptible to persistent post-breeding endometritis

Transient endometritis is a normal consequence of breeding and results from uterine contamination with both semen and bacteria. The modulation of the inflammatory response with the use of isoflupredone has been proposed as efficient for the treatment of endometritis by increasing pregnancy rates. The aim of the current study was to determine the effects of isoflupredone on nitric oxide (NO) levels in uterine samples from mares susceptible to persistent postbreeding endometritis, presenting or not the infectious process. Seven consecutive estrous cycles were induced in 11 mares, being the first one used as control (no treatment). All mares were submitted to the following four treatments: treatment 1: control, treatment 2: glucocorticoid (GC) treatment (20 mg isoflupredone acetate) every 12 h, for three consecutive days, treatment 3: infected treatment (intrauterine infusion of 1x109 CFU/ml Streptococcus equi subsp. zooepidemicus), treatment 4: combination of GC + infected treatment (infusion of bacteria 24 h after the first GC treatment). At 12 h after the end of each treatment, uterine samples were collected by flushing and NO was determined. After nitrate reduction, total nitrite was determined by spectrophotometer. No significant differences on nitric oxide concentration were verified by analysis of variance in the different experimental groups. It is concluded that the use of isoflupredone did not alter the nitric oxide concentration in uterine flushing’s from susceptible mares 12 h after treatment.

Uterine nitric oxide levels and isofluopredone treatment effect in mares susceptible to persistent post-breeding endometritis

Transient endometritis is a normal consequence of breeding and results from uterine contamination with both semen and bacteria. The modulation of the inflammatory response with the use of isoflupredone has been proposed as efficient for the treatment of endometritis by increasing pregnancy rates. The aim of the current study was to determine the effects of isoflupredone on nitric oxide (NO) levels in uterine samples from mares susceptible to persistent postbreeding endometritis, presenting or not the infectious process. Seven consecutive estrous cycles were induced in 11 mares, being the first one used as control (no treatment). All mares were submitted to the following four treatments: treatment 1: control, treatment 2: glucocorticoid (GC) treatment (20 mg isoflupredone acetate) every 12 h, for three consecutive days, treatment 3: infected treatment (intrauterine infusion of 1x109 CFU/ml Streptococcus equi subsp. zooepidemicus), treatment 4: combination of GC + infected treatment (infusion of bacteria 24 h after the first GC treatment). At 12 h after the end of each treatment, uterine samples were collected by flushing and NO was determined. After nitrate reduction, total nitrite was determined by spectrophotometer. No significant differences on nitric oxide concentration were verified by analysis of variance in the different experimental groups. It is concluded that the use of isoflupredone did not alter the nitric oxide concentration in uterine flushings from susceptible mares 12 h after treatment.(AU)

Resposta reprodutiva de vacas de corte associada a marcadores moleculares relacionados à fertilidade / Reproductive performance of beef cattle cows associated with molecular markers related to the fertility

O objetivo deste estudo foi buscar associação entre a taxa de prenhez após inseminação e natalidade com marcadores moleculares ligados aos genes do receptor para IGF-1, LHβ, Leptina e receptores do FSH e LH. Utilizaram-se 249 vacas adultas Aberdeen Angus, das quais 199 foram submetidas a protocolos distintos para a IATF, seguida pelo repasse com touros, e 50 vacas formaram o grupo controle representado pelo acasalamento com touros. Foram avaliados o escore de condição corporal (ECC) e o escore de condição ovariana (ECO) ao início da estação reprodutiva. O ECC influenciou a taxa de natalidade, respectivamente de 55,6%, 75,8% e 82,4% (P<0,05) para os animais com ECC menor que 2,5, entre 2,5 a 2,9, e maior ou igual a 3,0, por ocasião da estação reprodutiva. Os marcadores relacionados ao gene do receptor para o IGF-1 (AFZ-1 e HEL5) mostraram associação com a taxa de natalidade. Vacas homozigóticas para o marcador AFZ-1 apresentaram 84,4% de natalidade em comparação às heterozigóticas, com 71,5% (P<0,05). A presença do alelo*161 para o marcador HEL5 foi negativa sobre a natalidade, respectivamente de 33,3% e 76,5% para vacas com e sem esse alelo (P<0,05). Esses resultados demonstram uma importante associação entre os marcadores envolvidos com o receptor para o IGF-1 e desempenho reprodutivo de vacas Angus.(AU)

The association between the reproductive performance, expressed by pregnancy rate at fixed timed artificial insemination and birth rate in the subsequent season in beef cows, and molecular markers linked to genes for IGF-1 receptor, LHβ, leptin, and FSH and LH receptors were evaluated. Data from 249 Aberdeen Angus adult cows were used in this study. One hundred and ninety-nine cows were subjected to four different protocols for FTAI, followed by clean-up bulls and 50 cows formed the control group, matted only with bulls for 90 days during the mating season. Body condition score (BCS) and ovarian condition score (OCE) were evaluated at the beginning of the breeding season. The birth rate in the following year was 75.5%, with no treatments influence. The BCS has influenced the birth rate, respectively 55.6%, 75.8% and 82.4% (P<0.05) for animals with BCS less than 2.5; 2.5 to 2.9; and greater than or equal to 3.0, at the beginning of the breeding season. The markers related to IGF-1 receptor gene (AFZ-1 and HEL5) were associated with the birth rate in beef cows. Cows homozygous for AFZ-1 marker showed 84.4% of birth rate, while heterozygous cows showed 71.5% (P <0.05). The presence of allele *161 to the HEL5 marker was negative on birth rate. Cows with this allele had only 33.3% of birth rate, while cows without this allele had 76.5% of birth rate (P <0.05). These results demonstrate a significant association between the markers involved with the IGF-1 receptor and reproductive performance of Aberdeen Angus beef cows.(AU)

Retardo da realização da IATF sobre o desempenho reprodutivo na estação de acasalamento de vacas de corte lactantes / Delay of beginning to FTAI on reproductive performance during to mating season of beef lactating cows

Compararam-se efeitos de diferentes protocolos para a IATF com o acasalamento natural (Controle) sobre o desempenho reprodutivo de 249 vacas Aberdeen Angus, distribuídas em cinco grupos: Controle (n=50); Crestar 2º uso (n=64); OvSynch (n=65); Primer 1ºuso (n=35) e Primer 2º uso (n=35). A IATF dos animais dos grupos Crestar 2º uso, OvSynch, e Primer 1º uso foi realizada 27 dias após o início da estação do grupo controle e a IATF do grupo Primer 2º uso ocorreu 38 dias após o início da estação do grupo-controle. A partir de sete dias após a IATF, os animais foram submetidos ao repasse por touros até o término da estação de acasalamento, que foi de 91 dias para o grupo-controle, 64 dias para os grupos Crestar 2º uso, OvSynch e Primer 1º uso e de 53 dias para o grupo Primer 2º uso. A taxa de gestação ao final da estação de acasalamento não se diferenciou entre os grupos (P>0,05), sendo de 85,9%; 83,1%; 82,9%; 88,6% e 80,0%, respectivamente, para Crestar 2º uso; OvSynch; Primer 1º uso; Primer 2º uso e Controle. A taxa de parição resultante da IATF foi de 23,4%; 29,2%; 48,6% e 62,9% para os grupos Crestar 2º uso, OvSynch, Primer 1º uso, Primer 2º uso, respectivamente, com diferença significativa (P<0,05) entre Crestar e Primer 1º e 2º uso. OvSynch não se diferenciou de Crestar e Primer 1º uso. Primer 1º uso não se diferenciou de Primer 2º uso. A perda gestacional, do diagnóstico de gestação ao nascimento, foi de 10,5%. O intervalo de partos estimado (IEP) não apresentou diferenças, com média de 478 dias. O escore de condição corporal (ECC) de fêmeas gestantes ao final da estação reprodutiva foi diferente do de fêmeas não gestantes (controle), mas não dos demais grupos, possivelmente pela influência do tratamento em induzir a ciclicidade dos animais com ECC inferior. O atraso da realização da IATF após 27 ou 38 dias do início da estação de acasalamento não afetou a taxa de gestação final e o IEP dos animais, quando comparado ao acasalamento por touros.(AU)

The effects of different FTAI protocols were compared to the natural mating of bulls on the reproductive performance of 249 Aberdeen Angus cows. Five groups were formed: Control (n=50); Crestar 2nd use (n=64); OvSynch (n=65); Primer 1st use (n=35) and Primer 2nd use (n=35). The FTAI of the animals in the Crestar 2nd use, OvSynch and Primer 1st use groups was accomplished 27 days after the beginning of the mating season for the control group and the FTAI in the Primer 2nd use group happened 38 days after the beginning of the mating season of the control group. From seven days after the FTAI cows were exposed to bulls until the end of the mating season. The mating season was of 91 days for the control group, 64 days for the Crestar 2nd use, OvSynch and Primer 1st use groups and 53 days for the Primer 2nd use group. The pregnancy rate at the end of the mating season didn't differ among the groups (P>0.05), being 85.9; 83.1; 82.9; 88.6 and 80.0% respectively, for Crestar 2nd use, OvSynch, Primer 1st use, Primer 2nd use and Control. The birth rate resulting from FTAI was 23.4; 29.2; 48.6 and 62.9% for the Crestar 2nd use, OvSynch, Primer 1st use, Primer 2nd use groups, with significant difference (P<0.05) among Crestar and Primer 1st and 2nd use. OvSynch didn't differ in Crestar and Primer 1st use. Primer 1st use didn't differ from Primer 2nd use. The average reproductive losses between the gestation diagnosis and the birth were10.5%. The estimated calving interval (CI) didn't present differences among the animal groups, with an average of 478 days. The body condition score (BCS) of pregnant cows at the end of the reproductive station differed from BCS of empty cows in the control group, but it didn't differ in the other groups, possibly due to the hormonal treatment influence in inducing the oestrus and ovulation in animals with lower BCS. The delay of the accomplishment of FTAI after 27 or 38 days of the beginning of the mating season didn't affect the final pregnancy rate and CI of the cows, when compared to natural mating.(AU)

Transporte espermático na égua / Sperm transport in the mare

O objetivo deste trabalho foi realizar uma revisão bibliográfica sobre o transporte espermático na égua. Um rápido transporte espermático ocorre logo após a inseminação artificial (IA), sendo que a presença de espermatozoides na ponta dos cornos uterinos é observada oito minutos após esta. Espermatozoides podem ser observados 30 minutos após a IA e permanecer nas tubas uterinas por pelo menos 24 horas. Verificou-se que os espermatozoides podem ser observados em 62,6% das éguas quando se usa microscopia de luz, tanto nas glândulas uterinas como no epitélio do útero. Glândulas uterinas podem atuar como um reservatório de espermatozoides. O número de éguas com espermatozoides no epitélio uterino, nas glândulas e na junção útero-tubárica diminui em relação ao tempo após a IA.(AU)

The objective of this study was to review the literature on sperm transport in the mare. A rapid sperm transport occurs soon after artificial insemination (AI), and the presence of sperm on the tip of the uterine horns is observed after eight minutes. Sperm can be observed 30 minutes after AI and remain in the uterine tubes for at least 24 hours. It was found that the sperm can be observed in 62.6% of the mares when using light microscopy, both in the uterine glands and the epithelium of the uterus. Uterine glands may act as a reservoir for sperm. The number of mares with spermatozoa in the uterine epithelium, glands, and utero-tubal junction decreases over time after AI.(AU)

Transporte espermático na égua / Sperm transport in the mare

O objetivo deste trabalho foi realizar uma revisão bibliográfica sobre o transporte espermático na égua. Um rápido transporte espermático ocorre logo após a inseminação artificial (IA), sendo que a presença de espermatozoides na ponta dos cornos uterinos é observada oito minutos após esta. Espermatozoides podem ser observados 30 minutos após a IA e permanecer nas tubas uterinas por pelo menos 24 horas. Verificou-se que os espermatozoides podem ser observados em 62,6% das éguas quando se usa microscopia de luz, tanto nas glândulas uterinas como no epitélio do útero. Glândulas uterinas podem atuar como um reservatório de espermatozoides. O número de éguas com espermatozoides no epitélio uterino, nas glândulas e na junção útero-tubárica diminui em relação ao tempo após a IA.

The objective of this study was to review the literature on sperm transport in the mare. A rapid sperm transport occurs soon after artificial insemination (AI), and the presence of sperm on the tip of the uterine horns is observed after eight minutes. Sperm can be observed 30 minutes after AI and remain in the uterine tubes for at least 24 hours. It was found that the sperm can be observed in 62.6% of the mares when using light microscopy, both in the uterine glands and the epithelium of the uterus. Uterine glands may act as a reservoir for sperm. The number of mares with spermatozoa in the uterine epithelium, glands, and utero-tubal junction decreases over time after AI.

Sexual and social behaviors of pony stallions and mares

Aspects related to social organization and its effects on the reproductive behavior of domestic stallions breeding under free range management systems have not been studied. This study was carried out withthe following objectives: to identify the social units established between Brazilian Pony Breed stallions and mares; to verify the effect of the stallion in harem composition and mating activity; and to verify nocturnal sexual activity. This study was carried out during two breeding seasons. In the first year animals were observed for 12 days (8.1 h/day). In the second year, another group was observed in three periods with an avarage daily duration of 9.6 hours. Stallions ranging in age from 3 to more than 12 years and a group of mares rainging in age from 3 to 20 years were used. Two social units were identified harem and bachelor group. Three-year-old stallions did not form a harem. Hierarchic relationships among stallions and inside harems were established. Dominance, fight ability, aggressive behavior and age of stallions seem to influence size and harems maintenance. The total observed matings in Years I and II was 28 and 134, respectively. Mares were mated on average 1.9 times and the average number ofdaily mating was 1.2. The average number of receptive mares and matings per day was 8.5 and 1.1, respectively. The average copulation time was 38.4 and 37 sec in Years I and II, and the average daily interval between matings was 79.8 and 104.1 min, respectively. Considering the three observation periods in Year II, the mares were mated on average 2.2 times. The average number of daily matings was 2.1. The average number of receptive and mated mares per day was 20.7 and 1.8, respectively. In the two years nocturnal sexual activity was verified. The general pregnancy rate was 79.4% in the first and 72.2% in the second year.(AU)

Sexual and social behaviors of pony stallions and mares

Aspects related to social organization and its effects on the reproductive behavior of domestic stallions breeding under free range management systems have not been studied. This study was carried out withthe following objectives: to identify the social units established between Brazilian Pony Breed stallions and mares; to verify the effect of the stallion in harem composition and mating activity; and to verify nocturnal sexual activity. This study was carried out during two breeding seasons. In the first year animals were observed for 12 days (8.1 h/day). In the second year, another group was observed in three periods with an avarage daily duration of 9.6 hours. Stallions ranging in age from 3 to more than 12 years and a group of mares rainging in age from 3 to 20 years were used. Two social units were identified harem and bachelor group. Three-year-old stallions did not form a harem. Hierarchic relationships among stallions and inside harems were established. Dominance, fight ability, aggressive behavior and age of stallions seem to influence size and harems maintenance. The total observed matings in Years I and II was 28 and 134, respectively. Mares were mated on average 1.9 times and the average number ofdaily mating was 1.2. The average number of receptive mares and matings per day was 8.5 and 1.1, respectively. The average copulation time was 38.4 and 37 sec in Years I and II, and the average daily interval between matings was 79.8 and 104.1 min, respectively. Considering the three observation periods in Year II, the mares were mated on average 2.2 times. The average number of daily matings was 2.1. The average number of receptive and mated mares per day was 20.7 and 1.8, respectively. In the two years nocturnal sexual activity was verified. The general pregnancy rate was 79.4% in the first and 72.2% in the second year.

Skim milk-egg yolk based semen extender compensates for non-enzymatic antioxidant activity loss during equine semen cryopreservation EN

Cryopreservation exposes spermatozoa to stressful conditions, leading to reduced cell viability. Several studies propose that overproduction of reactive oxygen species and decreased antioxidant capacity of semen may increase the damaging effects of the technique. The objective of this work was to evaluate the influence of a skim milk-egg yolk based semen extender on enzymatic and non-enzymatic antioxidant activity in equine semen cryopreservation. Fifteen ejaculates from six fertile Criollo stallions were cryopreserved using a commercial citrate-Hepes, egg yolk, skim milk and glycerol extender. Activities of catalase, glutathione peroxidase and superoxide dismutase and total radical-trapping antioxidant potential were assessed in raw semen, semen diluted in extender and thawed semen. All three enzymes showed higher activities in raw semen than in diluted or in thawed semen (P < 0.01), but enzyme activities did not differ significantly between diluted and thawed semen samples (P > 0.05). Non-enzymatic antioxidant defenses did not differ among any of the stages in the cryopreservation process (P > 0.05). In conclusion, the present study shows that dilution of semen with skim milk-egg yolk based extender after centrifugation compensates for the non-enzymatic antioxidant protection (but not enzymatic antioxidant defense) lost with seminal plasma removal. The absence of correlation between seminal and antioxidant parameters suggests that the compensation was enough for semen protection against oxidative stress, or antioxidant protection plays a minor role on semen from fertile stallions.(AU)

Skim milk-egg yolk based semen extender compensates for non-enzymatic antioxidant activity loss during equine semen cryopreservation

Cryopreservation exposes spermatozoa to stressful conditions, leading to reduced cell viability. Several studies propose that overproduction of reactive oxygen species and decreased antioxidant capacity of semen may increase the damaging effects of the technique. The objective of this work was to evaluate the influence of a skim milk-egg yolk based semen extender on enzymatic and non-enzymatic antioxidant activity in equine semen cryopreservation. Fifteen ejaculates from six fertile Criollo stallions were cryopreserved using a commercial citrate-Hepes, egg yolk, skim milk and glycerol extender. Activities of catalase, glutathione peroxidase and superoxide dismutase and total radical-trapping antioxidant potential were assessed in raw semen, semen diluted in extender and thawed semen. All three enzymes showed higher activities in raw semen than in diluted or in thawed semen (P 0.05). Non-enzymatic antioxidant defenses did not differ among any of the stages in the cryopreservation process (P > 0.05). In conclusion, the present study shows that dilution of semen with skim milk-egg yolk based extender after centrifugation compensates for the non-enzymatic antioxidant protection (but not enzymatic antioxidant defense) lost with seminal plasma removal. The absence of correlation between seminal and antioxidant parameters suggests that the compensation was enough for semen protection against oxidative stress, or antioxidant protection plays a minor role on semen from fertile stallions.

Recombinant expression and purification of the bovine acidic Seminal Fluid Protein

The acidic Seminal Fluid Protein (aSFP), a 12.9 kDa protein is a maker for bovine semen freezability possibly due to its antioxidant activity and effect on sperm mitochondrial function. However, its precise function on sperm preservation during freezing thaw is poorly understood. The use of recombinant DNA technology allows new approaches on the study of function and structure of proteins, and its production in procaryote systems offers several advantages. The present work describes the recombinant expression of the bovine aSFP and its binding properties. A cDNA library from the bovine seminal vesicle was used as template for amplification of the aSFP coding region. The amplicon was cloned into a pET23a (+) vector and transformed into E.coli BL21 pLysS strain. The recombinant expression was obtained in E coli. One step ion immobilized affinity chromatography was performed, resulting in high yield of purified protein. To determine the bioactivity of the r aSFP, the protein was incubated in different concentrations with 10 7 spermtozoa at 37°C for 5 h. Western blotting and fluorescence microscopy analyses showed the ability of the recombinant aSFP to attach to the spermatozoa. Based on our results, the described method can be used to obtain mg levels of recombinant aSFP.