Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca²âº concentration in boar spermatozoa during cryopreservation.

Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.

Progesterona plasmática e fertilidade de fêmeas caprinas submetidas à sincronização do estro com prostaglandina F2α / Plasmatic progesterone and fertility of goats submitted at estrus synchronization with Prostaglandin F2α

Avaliou-se a resposta ao protocolo de sincronização do estro com duas doses de prostaglandina F2a (22,5µg) intervaladas de 10 dias, por meio da mensuração da concentração de progesterona plasmática, bem como a taxa de concepção das cabras após a inseminação artificial, de acordo com as diferentes respostas obtidas. Utilizaram-se 23 fêmeas e dois reprodutores da raça Toggenburg. A mensuração da progesterona plasmática foi realizada no dia da primeira aplicação de PGF2a (D0), no D5, no dia da segunda aplicação de PGF2a (D10), no D15, no D20 e no D33. A resposta positiva à PGF2α foi determinada pela queda da concentração de progesterona a valores abaixo de 1,5ng/mL, mensurada nos dias cinco e 15. As fêmeas foram distribuídas em três grupos. O grupo I foi composto por fêmeas que responderam às duas aplicações; o grupo II por fêmeas que não responderam à primeira aplicação e responderam à segunda aplicação; e o grupo III por fêmeas que responderam à primeira aplicação e não responderam à segunda aplicação, foram inseminadas e não conceberam. A presença de um corpo lúteo funcional, no momento das aplicações, determinou a resposta ao protocolo. As diferentes respostas das fêmeas ao protocolo, grupo I e II, não influenciaram as taxas de concepção.

Plasmatic progesterone concentrations were evaluated during the synchronization protocol with two doses of 22.5µg of Prostaglandin F2α, 10-day interval, and the conception rate of females in accordance to the reply to the protocol. Twenty-three female goats and two sexually mature Toggenburg bucks were used. Blood was sampled on day 0 (1st PGF2α injection), and on the following days 5, 10 (2nd PGF2α injection), 15, 20, and 33. The positive reply to the PGF2α was determined when the progesterone concentrations fell to values below 1.5ng/mL at days 5 and 15. The females were divided in three groups: Group I: females that responded to the two PGF2α applications; Group II: females that responded only to the second application; Group III: females that responded the first application, did not respond to the second application, were inseminated but did not conceive. The presence of a functional corpus luteum at the moment of the applications determined the reply to the protocol. There was no difference in the conception rates between females that responded to the two PGF2α applications or responded only to the second application.

Efeito da porção do ejaculado e do método de resfriamento sobre as características físicas do sêmen suíno / Effect of ejaculate portion and conservation method on the physical characteristics of boar semen

Duas porções do ejaculado suíno - primeiros 15mL da fração espermática rica (P1) e o restante do ejaculado (P2) - foram coletadas semanalmente de cinco varrões e submetidas a dois protocolos de resfriamento, diluição no diluidor MR-A® e conservação a 17°C (T1) ou no diluidor glicina-gema de ovo e conservação a 5°C (T2). As doses foram avaliadas no que se refere à motilidade, ao vigor e à morfologia espermáticas no sêmen a fresco e em diferentes tempos de estocagem. Todos os tratamentos mantiveram uma motilidade aceitável, superior a 50 por cento, nas primeiras 24 horas de armazenamento. O grupo P2T2 manteve uma motilidade similar (P>0,05) ao longo de todo o período de resfriamento (72 horas), sendo inclusive superior aos demais neste período, enquanto os outros tratamentos apresentaram uma redução da motilidade no decorrer do tempo de armazenamento. Com relação às características morfológicas do sêmen, não se observaram diferenças (P>0,05) quanto às porcentagens de espermatozoides normais entre as duas frações do ejaculado fresco. Ainda, todos os tratamentos mantiveram-se dentro dos limites aceitáveis, independentemente do tempo de armazenamento. A P1 parece ser mais adequada à produção de doses para o transporte em virtude de seu pequeno volume e alta concentração, enquanto o restante do ejaculado (P2) pode ser utilizado com eficiência dentro da própria granja.

Two portions of boar ejaculate - first 15mL of the sperm rich fraction (P1) and the rest of the ejaculate (P2) - were collected weekly from 5 mature boars and submitted to two cooling methods, extended in MR-A® and cooled at 17°C (T1) or extended in glycine-egg yolk and cooled at 5°C (T2). Spermatozoa motility, vigor, and morphological characteristics were evaluated immediately after collection and in different storage times. All treatments kept an acceptable motility, higher than 50 percent, in the first 24h of storage. The P2T2 maintained a similar motility (P>0.05) throughout the cooling storage (72h) and was superior in that period, while the other treatments presented a decrease in motility related to time. There was no difference between the two portions regarding the total number of normal spermatozoa in the fresh semen (P>0.05). All treatments showed morphological abnormalities within the acceptable thresholds, irrespectively of the storage time. Thus, due to low volume and high concentration, P1 seems to be more adequate for sperm dose transportation. Furthermore, this methodology will allow the development of new proposals concerning the transportation of swine semen, while the rest of the ejaculate could be used in farm routines to produce conventional liquid semen doses.

Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate.

Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca(2+)]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P<0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca(2+)]i between P1 and SRF-P1 in fresh as well as in frozen-thawed semen. A higher (P<0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.

Quality of boar spermatozoa from the sperm-peak portion of the ejaculate after simplified freezing in MiniFlatpacks compared to the remaining spermatozoa of the sperm-rich fraction.

Boar sperm viability post-thaw differs depending on the ejaculate fraction used, with spermatozoa present in the first 10 mL of the sperm-rich fraction (SRF) (portion 1, P1, sperm-peak portion) displaying the best cryosurvival in vitro compared with that of spermatozoa from the rest of the ejaculate (portion 2 of the SRF plus the post-spermatic fraction), even when using simplified freezing routines. This viability apparently relates to the specific profile of seminal plasma in P1 (i.e., glycoprotein and bicarbonate concentrations, and pH). However, spermatozoa from P1 have not been compared with spermatozoa from the rest of the SRF (SRF-P1, usually 30-40 mL of the SRF), which is routinely used for freezing. We compared P1 with SRF-P1 in terms of sperm kinematics (using the QualiSperm™ system), while membrane integrity (SYBR-14/PI), acrosome integrity (FITC PNA/PI), and sperm membrane stability (Annexin-V) were explored using flow cytometry. As well, total protein concentration and the proteomics of the seminal plasma (SP) of both portions of the SRF were studied using two-dimensional electrophoresis (2DE), mass fingerprinting (MALDI-TOF), and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) on selected peptides. The SRF portions were collected weekly from four mature boars (4-5 replicates per boar, sperm concentration: P1, 1.86 ± 0.20; SRF-P1, 1.25 ± 0.14 × 10(9) spz/mL) and processed using a quick freezing method in MiniFlatPacks. Post-thaw sperm motility reached 50%, without differences between SRF portions, but with clear inter-boar variation. Neither plasma membrane nor acrosome integrity differed (ns) between fractions. These results indicate that there are no differences in cryosurvival after quick freezing of boar spermatozoa derived from either of the two SRF portions. While P1 and SRF-P1 clearly differed in relative total protein contents, as expected, they displayed very similar protein profiles as assessed using 2DE and mass spectrometry (tryptic peptide mass fingerprint analysis and CID-MS/MS), indicating a similar emission of epididymal protein content.

Parâmetros reprodutivos de cabras Toggenburg inseminadas com sêmen resfriado, após diluição em meio à base de gema de ovo / Reproductive parameters of Toggenburg goats inseminated with cooled semen diluted in egg yolk extender

Avaliaram-se a taxa de concepção, a resposta à prostaglandina, a duração do estro, a categoria reprodutiva e o tipo de muco de cabras inseminadas com sêmen diluído em meio à base de gema de ovo e resfriado a 5ºC, por 12 ou 24 horas. Foram utilizados dois reprodutores e 62 fêmeas da raça Toggenburg, que receberam duas doses de 22,5µg de PGF2α, em intervalos de 10 dias, para a sincronização do estro. A partir da primeira aplicação de PGF2α, o estro foi monitorado três vezes ao dia. Realizou-se uma única inseminação, 12 horas após o início do estro. As porcentagens de fêmeas em estro, após a primeira e segunda aplicações de PGF2α, foram de 85,5 por cento e 88,7 por cento, respectivamente. O intervalo de aplicação da primeira e segunda doses de PGF2α ao início do estro foi de 41,04±20,32 e 45,67±9,28 horas, e a duração do estro de 40,02±15,96 e 32,24±12,09 horas, respectivamente. A taxa de concepção total foi de 49,1 por cento. O período de armazenamento do sêmen e a categoria reprodutiva não influenciaram (P>0,05) a taxa de concepção. O tipo de muco observado no momento da inseminação influenciou (P<0,05) a fertilidade das fêmeas, sendo o de aspecto estriado associado aos maiores índices de concepção.

The conception rate, the prostaglandin response, the estrus duration, the reproductive class, and the mucous of goats inseminated with semen diluted in egg yolk extender and cooled at 5ºC, for 12 or 24 hours were evaluated. Sixty-two female goats and two sexually mature Toggenburg bucks were used. The females received two doses of 22.5µg of prostaglandine F2α, at 10-day intervals. After the first injection, the estrus was monitored three times a day (6:00, 12:00, and 18:00h), with a buck teaser. Only one insemination was used. The percentages of animals that showed estrus after the first and the second injection of PGF2α were 85.5 percent and 88.7 percent, respectively. The average intervals from first and second PGF2α injection to estrus were 41.04±20.32 and 45.67±9.28h, and the estrus durations for both injections were 40.02±15.96 and 32.24±12.09h, in this order. The interval from the PGF2α injection to the beginning of the estrus was longer (P<0.05), whereas the estrus duration was shorter (P<0.05) in the estrus induced after the second PGF2α injection. The total conception rate was 49.1 percent. Neither the semen storage length (12 or 24 hours) nor the reproductive class did not affect (P>0.05) the conception rate. The mucous observed at the insemination time influenced (P<0.05) the fertility of inseminated goats, with the striated aspect associated to higher fertility.

Taxa de concepção de cabras inseminadas com sêmen caprino resfriado a 5ºC, por 12 ou 24 horas, em meio diluidor à base de gema de ovo / Conception rate of goats inseminated with semen cooled in egg yolk diluent at 5ºC, for 12 or 24 hours

Avaliou-se a capacidade fecundante do sêmen caprino resfriado a 5ºC, por 12 (TI) ou 24 horas (TII), em container especial. Para tanto, utilizaram-se 62 fêmeas e dois reprodutores (B1 e B2) da raça Toggenburg, distribuídos em um esquema fatorial 2x2 (dois reprodutores e dois períodos de estocagem do sêmen). Após a coleta, o sêmen foi diluído em Tris-frutose-gema de ovo a 2,5 por cento, envasado em palhetas de 0,25mL, com 150x10(6) espermatozoides móveis e resfriado a 5ºC. As fêmeas receberam duas doses de 22,5µg de PGF2α, em intervalos de 10 dias para a sincronização do estro. A partir da primeira aplicação de PGF2α, as fêmeas foram monitoradas para ocorrência de estro, três vezes ao dia. Realizou-se uma única inseminação, pela técnica de fixação da cérvice, 12 horas após o início do estro. A motilidade e o vigor, após 12 ou 24 horas de resfriamento, foram de 66,14±0,11 por cento e 62,50±0,05 por cento, e 3,46±0,61 e 3,27±0,50, respectivamente. Não houve influência (P>0,05) do reprodutor, nem do período de armazenamento do sêmen sobre a taxa de concepção das cabras, que foi de 49,1 por cento.

The fertilizing capacity of goat semen cooled in egg yolk diluent at 5ºC, for 12 or 24 hours was evaluated. Sixty-two Toggenburg does and two sexually mature Toggenburg bucks were used in a fatorial treatment combination (two bucks and two storage periods). The semen was diluted in 2.5 percent Tris-frutose-egg yolk; envased in 0.25mL plastic straws, with 150x10(6) mobile spermatozoa; and cooled at 5ºC for 12 or 24 hours. The females received two doses of 22.5µg of prostaglandine F2α, at each 10-day intervals in order to synchronize the estrous. From the first PGF2α injection, estrous occurrence was monitored three times per day. Only one insemination was used, using the cervix fixation method, 12 hours after the estrous onset. The means of motility and strength, 12 (TI) and 24 hours (TII) after semen cooling at 5ºC, were 66.14±0.11 percent and 62.50±0.05 percent, and 3.46±0.61 and 3.27±0.50, respectively. Neither the sire nor the period of semen influenced (P>0.05) the conception rate of the does, which was 49.1 percent.

Avaliaçäo da qualidade microbiológica do leite UAT integral comercializado em Belo Horizonte / Microbiological aspects of UHT whole milk commercialized in Belo Horizonte, Brazil

Foram analisadas 80 amostras de leite UAT integral de oito marcas comercializadas em Belo Horizonte, entre janeiro e abril de 1999. Foram realizadas contagens de bactérias mesófilas aeróbicas em meio APC e ágar BHI enriquecido com vitamina B12, sendo os resultados comparados aos padröes estabelecidos pelo Regulamento Técnico de Identidade e Qualidade (RTIQ) do Serviço Federal de Inspeçäo do Ministério da Agricultura e Abastecimento do Brasil. Das 80 amostras analisadas, 33 (41,2(porcento)) apresentaram contagem de bactérias mesófilas aeróbicas entre 1,3x10 elevado a quarta potência e 1,4x10 elevado a quinta potência UFC/ml (4,1 e 5,1 log UFC/ml). De 174 amostras de microrganismos isoladas, 162 (93,1(porcento)) säo do gênero Bacillus, 3 cocos (1,7(porcento)) pertencentes ao gênero Micrococcus, 9 (5,2(porcento)) bastonetes Gram positivos pleomórficos, sugestivos de pertencerem ao gênero Corynebacterium. Cinco marcas de leite UAT foram consideradas como fora dos padröes microbiológicos estabelecidos pelo RTIQ. Bactérias do gênero Bacillus foram as mais isoladas