Efficacy and limitations of different approaches to anticipate the diagnosis of pregnancy in cattle / Eficácia e limitações de diferentes abordagens para antecipar o diagnóstico de gestação em bovinos

The study evaluated sonographic and serologic exams performed for early (20 to 30d) diagnosis of pregnancy. One hundred-twenty (n= 120) bovine recipients were synchronized (estrous=D0) and timed embryo transferred (TET, D7) with fresh in vitro produced embryos. In the first trial (n= 46), diagnosis of pregnancy was performed on day 20 (D20) by detecting CL blood flow (BF) and by Pregnancy-Associated Glycoproteins (PAGs) serology. In the second trial (n= 30), pregnancy diagnosis was performed on D25 by ultrasound visualization of uterine contents and by PAGs serology. In the last trial, PAG's serology was performed on D30. Ultrasonographic detection of the uterine contents and embryo viability performed on D30 (DG30) was considered the gold standard. The PROC FREQ procedure was used to test the agreement between diagnostic methods. On D20, the Doppler ultrasonography of the CL had showed high sensitivity (100%), but only moderate specificity (53.3%). On the same day, serologic diagnostic had no agreement (k= -0.08, P< 0.46) with the gold standard, with very low sensitivity (6.3%). However, the sensitivity of the serologic exam increased dramatically (from 6.3 to 100%) from D20 to D25, and it contributed to detect false negatives from the ultrasound diagnosis, improving the overall accuracy from 90% to 96.7%.(AU)

O estudo foi planejado para correlacionar exames ultrassonográficos e sorológicos realizados para o diagnóstico precoce (20 a 30d) de gestação. Cento e vinte (n= 120) receptoras bovinas foram sincronizadas (estro=D0), e embriões frescos produzidos in vitro foram transferidos em tempo fixo (TETF, D7). No experimento 1 (n= 46), o diagnóstico de gestação foi realizado no D20, pela detecção do fluxo sanguíneo do CL e pela sorologia de glicoproteínas associadas à gestação (PAGs). No experimento 2 (n= 30), a detecção da gestação foi realizada por meio da visualização do conteúdo do útero e também pela sorologia para PAGs. No experimento 3, a sorologia para PAGs foi realizada no D30. Em todos os experimentos, a visualização ultrassonográfica da vesícula e da viabilidade embrionária, realizada no D30, foi considerada padrão-ouro. O procedimento PROC FREQ testou o nível de concordância dos métodos diagnósticos. No D20, o diagnóstico baseado na vascularização do CL mostrou alta sensibilidade (100%) e apenas moderada especificidade (53,3%). Nesse mesmo dia, o diagnóstico sorológico não apresentou concordância (k=-0,08, P<0,46) com o padrão-ouro, além de baixa sensibilidade (6,3%). No entanto, a sensibilidade do exame sorológico aumentou drasticamente (6,3 para 100%) do D20 para o D25, contribuindo para detectar falsos negativos diagnosticados pela ultrassonografia, melhorando a acurácia (90 para 96,7%).(AU)

Efficacy and limitations of different approaches to anticipate the diagnosis of pregnancy in cattle / Eficácia e limitações de diferentes abordagens para antecipar o diagnóstico de gestação em bovinos

The study evaluated sonographic and serologic exams performed for early (20 to 30d) diagnosis of pregnancy. One hundred-twenty (n= 120) bovine recipients were synchronized (estrous=D0) and timed embryo transferred (TET, D7) with fresh in vitro produced embryos. In the first trial (n= 46), diagnosis of pregnancy was performed on day 20 (D20) by detecting CL blood flow (BF) and by Pregnancy-Associated Glycoproteins (PAGs) serology. In the second trial (n= 30), pregnancy diagnosis was performed on D25 by ultrasound visualization of uterine contents and by PAGs serology. In the last trial, PAG's serology was performed on D30. Ultrasonographic detection of the uterine contents and embryo viability performed on D30 (DG30) was considered the gold standard. The PROC FREQ procedure was used to test the agreement between diagnostic methods. On D20, the Doppler ultrasonography of the CL had showed high sensitivity (100%), but only moderate specificity (53.3%). On the same day, serologic diagnostic had no agreement (k= -0.08, P< 0.46) with the gold standard, with very low sensitivity (6.3%). However, the sensitivity of the serologic exam increased dramatically (from 6.3 to 100%) from D20 to D25, and it contributed to detect false negatives from the ultrasound diagnosis, improving the overall accuracy from 90% to 96.7%.(AU)

O estudo foi planejado para correlacionar exames ultrassonográficos e sorológicos realizados para o diagnóstico precoce (20 a 30d) de gestação. Cento e vinte (n= 120) receptoras bovinas foram sincronizadas (estro=D0), e embriões frescos produzidos in vitro foram transferidos em tempo fixo (TETF, D7). No experimento 1 (n= 46), o diagnóstico de gestação foi realizado no D20, pela detecção do fluxo sanguíneo do CL e pela sorologia de glicoproteínas associadas à gestação (PAGs). No experimento 2 (n= 30), a detecção da gestação foi realizada por meio da visualização do conteúdo do útero e também pela sorologia para PAGs. No experimento 3, a sorologia para PAGs foi realizada no D30. Em todos os experimentos, a visualização ultrassonográfica da vesícula e da viabilidade embrionária, realizada no D30, foi considerada padrão-ouro. O procedimento PROC FREQ testou o nível de concordância dos métodos diagnósticos. No D20, o diagnóstico baseado na vascularização do CL mostrou alta sensibilidade (100%) e apenas moderada especificidade (53,3%). Nesse mesmo dia, o diagnóstico sorológico não apresentou concordância (k=-0,08, P<0,46) com o padrão-ouro, além de baixa sensibilidade (6,3%). No entanto, a sensibilidade do exame sorológico aumentou drasticamente (6,3 para 100%) do D20 para o D25, contribuindo para detectar falsos negativos diagnosticados pela ultrassonografia, melhorando a acurácia (90 para 96,7%).(AU)

Differential expression of LHCGR and its isoforms is associated to the variability in superovulation responses of Gir cattle.

The aim of this study was to evaluate the pattern of expression of LHCGR isoforms in Gir heifers characterized as good (10.3 ±â€¯1.2 ova/embryos per flush, n = 5) or poor responders (1.1 ±â€¯0.3 ova/embryos per flush, n = 5) to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system was used to collect granulosa cells from 8 mm follicles formed either during a synchronized, non-stimulated follicular wave (no stimulation control, NS) or on the fourth day of a superovulation protocol (SOV) induced with 200 IU of pFSH. The recovered follicular fluid was centrifuged and granulosa cells were washed with NaCl 0.9% and kept in RNAlater®. RNA extraction was performed using a commercial RNeasy Micro Kit and eluted samples were quantified and reverse transcribed using the commercial Superscript III kit. cDNA samples were amplified by real-time PCR using a primer to target LH/hCG receptor gene - not selective for LHCGR isoforms (total LHCGR) - and four sets of isoforms selective primers (S1, S10, S10 + 11, and S11). Analyses were performed using the REST software and expression levels are shown as mean ± SEM. Under physiological conditions (NS), poor responders had a higher expression of total LHCGR (4.9 ± 1.7 fold-change, P < 0.01) as well as isoforms S10, S11 and S10 + 11, compared to good responders. In both phenotypes, superovulation down-regulated total LHCGR expression (-0.5 ± 0.2 and -0.9 ± 0.0 for good and poor responders, respectively; P < 0.05). However, in poor responders the exogenous FSH treatment up-regulated the S10 (2.4 ± 2.0; P < 0.05), S10 + 11 (3.8 ± 3.2; P < 0.01), and S1 isoforms (1.8 ± 1.3; P < 0.05), compared to good responders We conclude that down-regulation of total LHCGR, associated to up-regulation of their inactive isoforms, may have compromised follicle development and thus contributed to the low efficiency of superovulation in heifers with a poor responder phenotype.

Esteroides e análogos da prostaglandina F2α na prevenção de endometrites pós-parto em vacas girolandas / Steroids and prostaglandin F2α analogs in the prevention of endometritis postpartum in giroland cows

Objetivou-se avaliar a ocorrência de endometrites, a taxa de prenhez (TP) e o intervalo parto concepção (IPC) de vacas leiteiras tratadas 10 dias pós-parto com esteroides ou análogo da prostaglandina F2α. Dividiu-se 207 animais clinicamente sãos em 4 tratamentos: T1 (0,5 mg de Cipionato de Estradiol; T2 (70 mg de progesterona); T3 (0,5 mg de cloprostenol) e T4 2 mL de solução salina. 30 a 45 dias pós-parto realizou-se o exame ginecológico e nos animais saudáveis realizou-se a citologia endometrial. Por fim, estes animais foram submetidos a um protocolo de IATF. Analisou-se o IPC por ANOVA e as variáveis qualitativas pelo teste do Qui-Quadrado. O IPC não diferiu (P < 0,05) entre os tratamentos (104,7 ± 72,6; 93,5 ± 63,0; 98,2 ± 77,8; 112,8 ± 67,8, respectivamente). O Cloprostenol reduziu (P < 0,01) o número de animais não gestantes (10%, 5/50) em relação ao controle (26%, 13/50). Não houve diferença (P < 0,09) entre os tratamentos na incidência de endometrites. A TP não diferiu entre os animais diagnosticados com endometrite subclínica. Conclui-se que a utilização de esteroides ou análogos da prostaglandina F2α neste período não é eficiente na prevenção de endometrites e na melhoria da TP e IPC. O Cloprostenol foi o único a reduzir o número de animais não gestantes em comparação ao controle.

This study was designed to evaluate steroids or prostaglandin F2α analogue treatment performed 10 days after parturition postpartum over the following reproductive indexes: occurrence of endometritis, the pregnancy rate (PR) and parturition conception interval (PCI). Two-hundred seven (n = 207) cows without reproductive abnormalities were assigned in four treatments performed 10 days after parturition: T1 (0.5 mg estradiol cypionate, T2 (progesterone 70 mg), T3 (0.5 mg cloprostenol, prostaglandin F2α analogue) and T4 (2 ml saline). Thirty to fourty-five days after parturition all animals were gynecologically examined, and the endometrial cytology was performed only in cow without clinical endometritis. Finally, these animals were submitted to an IATF protocol. The PCI was analyzed by ANOVA and the qualitative variables were analyzed by the Chi-Square. The PCI did not differ (P < 0.2) between treatments (104.7 ± 72.6, 93.5 ± 63.0, 98.2 ± 77.8, 112.8 ± 67.8, respectively). Cloprostenol treatment reduced (P < 0.01) the number of non-pregnant animals (10%, 5/50) compared to the control (26%, 13/50). The steroids or Cloprostenol treatment is not effective to prevent endometritis or improve PR and PCI indexes, when injected at this period. Cloprostenol was the only one to reduce the number of non-pregnant animals in comparison to the control.

Esteroides e análogos da prostaglandina F2α na prevenção de endometrites pós-parto em vacas girolandas / Steroids and prostaglandin F2α analogs in the prevention of endometritis postpartum in giroland cows

Objetivou-se avaliar a ocorrência de endometrites, a taxa de prenhez (TP) e o intervalo parto concepção (IPC) de vacas leiteiras tratadas 10 dias pós-parto com esteroides ou análogo da prostaglandina F2α. Dividiu-se 207 animais clinicamente sãos em 4 tratamentos: T1 (0,5 mg de Cipionato de Estradiol; T2 (70 mg de progesterona); T3 (0,5 mg de cloprostenol) e T4 2 mL de solução salina. 30 a 45 dias pós-parto realizou-se o exame ginecológico e nos animais saudáveis realizou-se a citologia endometrial. Por fim, estes animais foram submetidos a um protocolo de IATF. Analisou-se o IPC por ANOVA e as variáveis qualitativas pelo teste do Qui-Quadrado. O IPC não diferiu (P < 0,05) entre os tratamentos (104,7 ± 72,6; 93,5 ± 63,0; 98,2 ± 77,8; 112,8 ± 67,8, respectivamente). O Cloprostenol reduziu (P < 0,01) o número de animais não gestantes (10%, 5/50) em relação ao controle (26%, 13/50). Não houve diferença (P < 0,09) entre os tratamentos na incidência de endometrites. A TP não diferiu entre os animais diagnosticados com endometrite subclínica. Conclui-se que a utilização de esteroides ou análogos da prostaglandina F2α neste período não é eficiente na prevenção de endometrites e na melhoria da TP e IPC. O Cloprostenol foi o único a reduzir o número de animais não gestantes em comparação ao controle.(AU)

This study was designed to evaluate steroids or prostaglandin F2α analogue treatment performed 10 days after parturition postpartum over the following reproductive indexes: occurrence of endometritis, the pregnancy rate (PR) and parturition conception interval (PCI). Two-hundred seven (n = 207) cows without reproductive abnormalities were assigned in four treatments performed 10 days after parturition: T1 (0.5 mg estradiol cypionate, T2 (progesterone 70 mg), T3 (0.5 mg cloprostenol, prostaglandin F2α analogue) and T4 (2 ml saline). Thirty to fourty-five days after parturition all animals were gynecologically examined, and the endometrial cytology was performed only in cow without clinical endometritis. Finally, these animals were submitted to an IATF protocol. The PCI was analyzed by ANOVA and the qualitative variables were analyzed by the Chi-Square. The PCI did not differ (P < 0.2) between treatments (104.7 ± 72.6, 93.5 ± 63.0, 98.2 ± 77.8, 112.8 ± 67.8, respectively). Cloprostenol treatment reduced (P < 0.01) the number of non-pregnant animals (10%, 5/50) compared to the control (26%, 13/50). The steroids or Cloprostenol treatment is not effective to prevent endometritis or improve PR and PCI indexes, when injected at this period. Cloprostenol was the only one to reduce the number of non-pregnant animals in comparison to the control.(AU)

Corpus luteum blood flow evaluation on Day 21 to improve the management of embryo recipient herds.

The aim of the present study was to use blood flow evaluation of the CL at 14 days after embryo transfer to detect nonpregnant animals and optimize the management of bovine recipients. The estrous cycle was synchronized in 165 recipients, and the day of expected ovulation was considered to be Day 0. Embryo transfer was performed 7 days later, on Day 7. On Day 21, pregnancy was diagnosed on the basis of blood flow evaluation of the CL (DG21-predictive diagnostic). To validate this methodology, visual scores for blood flow were compared to objective data extracted from CL ultrasound images recorded in the Doppler mode. The size was also evaluated using recorded images of the CL in the B mode. Blood samples were also collected for further analysis of the progesterone (P4) concentration. The diagnosis of pregnancy was confirmed at 35 days after estrus (DG35-definitive diagnostic). The DG21 showed that 55.2% (90 of 163) of the animals were presumptively pregnant, and this value was higher (P < 0.04) than that obtained at DG35 (43.6%, 71 of 163). The predictive diagnostic achieved moderate specificity (79.3%) for the detection of pregnancy, but most importantly, high sensitivity (100%) for the detection of nonpregnant recipients. The overall accuracy of the diagnosis was 88.3%. The P4 concentrations were different (P < 0.02) and correlated with each visual score assigned for the CL size. Visual scores for CL blood flow were also efficient (P < 0.0001) to distinguish animals with different levels of P4; however, P4 concentrations were higher for scores 1 and 2 (high and regular blood flow, respectively) than those for score 3 (low blood flow). This technique showed high sensitivity and facilitated the early detection of nonpregnant animals. The DG21 would allow about 79.3% of nonpregnant animals to be resynchronized 9 to 14 days earlier, when compared to conventional management based on pregnancy diagnosis at Days 30 to 35.

Perfil de progesterona em novilhas com diferente atividade luteal e tratadas com implantes vaginais / Serum progesterone in heifers with different luteal activity treated with vaginal devices

O dispositivo de liberação de progesterona (DLP) é muito importante em protocolos de Inseminação Artificial em Tempo Fixo (IATF). Representa cerca de 43% dos custos e é objeto de estudos sobre a eficiência da sua reutilização. No entanto, perfis de liberação de progesterona (P4) em animais com diferentes concentrações endógenas desse esteroide não são claramente descritos. Este estudo teve como objetivo avaliar a concentração sérica de P4 em fêmeas com diferentes situações de atividade luteal, tratadas com DLP novo (1g de P4) por 8 dias. Trinta novilhas mestiças cíclicas foram divididas em três grupos: em G1 e G2, o DLP foi inserido (D0) sete dias após a ovulação induzida. Adicionalmente, 0,15mg de D-cloprostenol foi administrado três dias depois para promover a luteólise em G2. Para G3, o corpo lúteo foi lisado antes da inserção do DLP para que a P4 exógena fosse a única fonte desse hormônio. O sangue foi coletado no D0, D3, D5 e D8, e a P4 avaliada por RIA. Médias de P4 foram comparadas entre os grupos em cada dia e dentro do grupo, entre os dias, utilizando o teste Tukey. Antes da inserção do implante (D0), os níveis de P4 foram, nos grupos, semelhantes em G1 e G2, e superiores a G3 (5,3±3,1a e 5,3±1,4a vs 0,6±0.3bng/mL, respectivamente-P<0,05). No D3, ocorreu o mesmo perfil (5,7±2,6a e 5,4±2,0a e 3,6±0.8bng/mL, respectivamente para G1 e G2 vs G3, P<0,05). Trinta e seis horas (D5) após a PGF, a P4 no G2 caiu para níveis semelhantes aos do grupo G3 e ambos diferiram (P<0,05) de G1 (3,3±1,6b vs 2,4±0,9b e 2,1±0.7bng/mL). Essa diferença se manteve (P<0,05) em D8 (3,1±1,3a, 1,8±0,8b e 1,6±0.6b ng/mL). O aumento da P4 após a inserção (D3 - D0) foi maior (P<0,05) em G3 que em G1 e G2 (2,8±0,9a vs 0,4±1,8b e 0,2±1.4bng/mL). Os animais com maior P4 endógena levam a menor liberação de P4 exógena a partir do DLP. Portanto, os níveis remanescentes de P4 no DLP após o uso dependem da concentração endógena de P4 do animal e possíveis alterações durante a permanência.(AU)

The progesterone (P4) device is a very important step in the ovulation control in Timed Artificial Insemination (TAI) protocols. It represents about 43% of the hormone costs, thus it has been the subject of several studies on efficiency of the reused device as an alternative to reduce TAI costs. However, to our knowledge, profiles for P4 release in animals with different endogenous concentrations of P4 are not clearly described. This study aimed to evaluate serum concentration of P4 in females with different ovarian conditions - related to luteal activity - and treated with a new intravaginal device (1g of P4) for 8 days. Thirty normally cyclic crossbred heifers were divided into three groups: for G1 and G2, P4 device was inserted (D0) seven days after ovulation (7 day old CL). Additional PGF (0.15 mg of D-cloprostenol) was given three days later to promote luteolysis in the G2 group. For G3, CL was killed before P4 insertion and the exogenous progesterone was the only source of this hormone. Blood samples were collected on D0, D3, D5 and D8 and P4 concentration was measured by radioimmunoassay (RIA). Means for P4 concentration were compared among groups in each day and within the group among days using the Tukey test. Before P4 device insertion (D0), P4 levels were higher (P<0.05) in G1 and G2 when compared to G3 (5.3±3.1 and 5.3±1.4 vs. 0.6±0.3ng/mL, respectively). Three days later (D3), the same pattern was observed (5.7±2.6 and 5.4±2.0 and 3.6±0.8ng/mL, respectively for G1 and G2 vs. G3, P<0.05). Thirty-six hours (D5) after PGF injection (G2), P4 in G2 dropped to levels similar to the G3 group and both differed (P<0.05) from G1 (3.3±1.6 vs. 2.4±0.9 and 2.1±0.7ng/mL, G1 vs. G2 and G3, respectively). There were no differences (P>0.05) among groups on D8 (3.1±1.3, 1.8±0.8 and 1.6±0.6ng/mL, respectively, for G1, G2 and G3). Progesterone increase after P4 insertion (D3 - D0) was higher (P<0.05) in G3 compared to G1 and G2 (2.8±0.9 vs. 0.4±1.8 and 0.2±1.4ng/mL, respectively). The interpretation was that animals with higher endogenous P4 promote less release of the exogenous P4 from the device. Therefore, the remaining P4 levels from used progesterone devices depend on the physiological condition of the animal at the time of insertion and possible changes during the treatment.(AU)

Perfil de progesterona em novilhas com diferente atividade luteal e tratadas com implantes vaginais / Serum progesterone in heifers with different luteal activity treated with vaginal devices

O dispositivo de liberação de progesterona (DLP) é muito importante em protocolos de Inseminação Artificial em Tempo Fixo (IATF). Representa cerca de 43% dos custos e é objeto de estudos sobre a eficiência da sua reutilização. No entanto, perfis de liberação de progesterona (P4) em animais com diferentes concentrações endógenas desse esteroide não são claramente descritos. Este estudo teve como objetivo avaliar a concentração sérica de P4 em fêmeas com diferentes situações de atividade luteal, tratadas com DLP novo (1g de P4) por 8 dias. Trinta novilhas mestiças cíclicas foram divididas em três grupos: em G1 e G2, o DLP foi inserido (D0) sete dias após a ovulação induzida. Adicionalmente, 0,15mg de D-cloprostenol foi administrado três dias depois para promover a luteólise em G2. Para G3, o corpo lúteo foi lisado antes da inserção do DLP para que a P4 exógena fosse a única fonte desse hormônio. O sangue foi coletado no D0, D3, D5 e D8, e a P4 avaliada por RIA. Médias de P4 foram comparadas entre os grupos em cada dia e dentro do grupo, entre os dias, utilizando o teste Tukey. Antes da inserção do implante (D0), os níveis de P4 foram, nos grupos, semelhantes em G1 e G2, e superiores a G3 (5,3±3,1a e 5,3±1,4avs 0,6±0.3bng/mL, respectivamente-P<0,05). No D3, ocorreu o mesmo perfil (5,7±2,6a e 5,4±2,0a e 3,6±0.8bng/mL, respectivamente para G1 e G2 vs G3, P<0,05). Trinta e seis horas (D5) após a PGF, a P4 no G2 caiu para níveis semelhantes aos do grupo G3 e ambos diferiram (P<0,05) de G1 (3,3±1,6b vs 2,4±0,9b e 2,1±0.7bng/mL). Essa diferença se manteve (P<0,05) em D8 (3,1±1,3a, 1,8±0,8b e 1,6±0.6b ng/mL). O aumento da P4 após a inserção (D3 - D0) foi maior (P<0,05) em G3 que em G1 e G2 (2,8±0,9a vs 0,4±1,8b e 0,2±1.4bng/mL). Os animais com maior P4 endógena levam a menor liberação de P4 exógena a partir do DLP. Portanto, os níveis remanescentes de P4 no DLP após o uso dependem da concentração endógena de P4 do animal e possíveis alterações durante a permanência.(AU)

The progesterone (P4) device is a very important step in the ovulation control in Timed Artificial Insemination (TAI) protocols. It represents about 43% of the hormone costs, thus it has been the subject of several studies on efficiency of the reused device as an alternative to reduce TAI costs. However, to our knowledge, profiles for P4 release in animals with different endogenous concentrations of P4 are not clearly described. This study aimed to evaluate serum concentration of P4 in females with different ovarian conditions - related to luteal activity - and treated with a new intravaginal device (1g of P4) for 8 days. Thirty normally cyclic crossbred heifers were divided into three groups: for G1 and G2, P4 device was inserted (D0) seven days after ovulation (7 day old CL). Additional PGF (0.15 mg of D-cloprostenol) was given three days later to promote luteolysis in the G2 group. For G3, CL was killed before P4 insertion and the exogenous progesterone was the only source of this hormone. Blood samples were collected on D0, D3, D5 and D8 and P4 concentration was measured by radioimmunoassay (RIA). Means for P4 concentration were compared among groups in each day and within the group among days using the Tukey test. Before P4 device insertion (D0), P4 levels were higher (P<0.05) in G1 and G2 when compared to G3 (5.3±3.1 and 5.3±1.4 vs. 0.6±0.3ng/mL, respectively). Three days later (D3), the same pattern was observed (5.7±2.6 and 5.4±2.0 and 3.6±0.8ng/mL, respectively for G1 and G2 vs. G3, P<0.05). Thirty-six hours (D5) after PGF injection (G2), P4 in G2 dropped to levels similar to the G3 group and both differed (P<0.05) from G1 (3.3±1.6 vs. 2.4±0.9 and 2.1±0.7ng/mL, G1 vs. G2 and G3, respectively). There were no differences (P>0.05) among groups on D8 (3.1±1.3, 1.8±0.8 and 1.6±0.6ng/mL, respectively, for G1, G2 and G3). Progesterone increase after P4 insertion (D3 - D0) was higher (P<0.05) in G3 compared to G1 and G2 (2.8±0.9 vs. 0.4±1.8 and 0.2±1.4ng/mL, respectively). The interpretation was that animals with higher endogenous P4 promote less release of the exogenous P4 from the device. Therefore, the remaining P4 levels from used progesterone devices depend on the physiological condition of the animal at the time of insertion and possible changes during the treatment.(AU)

Redução da resposta superovulatória de fêmeas bovinas superestimuladas com FSH em doses split / Reduced superovulatory response of bovine females overstimulated with FSH in split doses

Objetivou-se avaliar a superovulação (SOV) de vacas zebuínas, utilizando protocolo convencional ou protocolo com número menor de aplicações e similar dosagem (dose split). Utilizaram-se 16 fêmeas (total 32 SOV), com idade entre 17-42 meses e escore de condição corporal 2,5-4 (escala de 1-5), em delineamento tipo cross-over. No início do tratamento (D0), os animais receberam um dispositivo de progesterona e 2mg de benzoato de estradiol. As fêmeas do grupo convencional receberam 250UI de FSH/LH divididas em oito doses decrescentes administradas em intervalos de 12h (FSH/LH no D4, D5, D6 e D7 no período da manhã e tarde, nas respectivas dosagens: 50,0 UI; 37,5 UI; 25,0 UI; 12,5 UI). No D7 pela manhã, as fêmeas foram tratadas com 150μg de D+cloprostenol, e a remoção da progesterona foi realizada no D7 à tarde. As fêmeas do grupo split também receberam 250 UI de FSH/LH. No D4 de manhã, administraram-se 62,5 UI de FSH/LH via IM e 125 UI por via SC. Quarenta e oito horas após (D6) administraram-se 62,5 UI via SC e na manhã do D7 foi removida a progesterona e aplicaram-se 150μg de D+cloprostenol. As fêmeas de ambos os grupos receberam 50μg de análogo de GnRH no D8 pela manhã e foram inseminadas 12 e 24 horas após. No D15 realizou-se a colheita dos embriões em ambos os tratamentos. Avaliou-se a resposta superovulatória pela contagem do número de folículos e CLs de cada ovário, com auxílio de ultrassom. Todas as variáveis foram submetidas ao teste T de Student para amostras pareadas. Houve diferença (P<0,05) na quantidade de folículos acima de 8mm no D8 (9,06±4,54 e 5,50±4,59); número de CLs no dia da colheita (8,12± 3,26 e 4,69±3,46), número de embriões totais (6,69±3,05 e 3,37±2,50) e de embriões viáveis (5,25±2,29 e 2,37±1,78) nas vacas do grupo convencional em relação às do split, respectivamente. Conclui-se que o protocolo split tem pior resposta superovulatória e de produção in vivo de embriões, em vacas zebuínas, quando comparado ao protocolo convencional.(AU)

The aim of this study was to evaluate the superovulation (SOV) response of zebu cows, using conventional protocol or other protocol with similar dose but smaller number of applications (split dose). 16 females (32 SOV), aged 17-42 months, and body condition score of 2.5-4 (1-5 scale) were used in randomized cross-over. At the start of treatment (D0), the animals received progesterone device and 2mg of estradiol benzoate. The females from the conventional group received 250 IU of FSH/LH divided into eight decreasing doses administered at intervals of 12 hours (FSH/LH in D4, D5, D6 and D7, with their respective strengths: 50.0 IU, 37.5 IU, 25.0 IU, 12.5 IU). In D7, the females were treated with 150μg of D+cloprostenol, and the removal of progesterone device was held in the afternoon. The females from the split group also received 250 IU of FSH/LH. In the morning D4 was administered in 62.5 IU FSH/LH IM and 125 IU subcutaneously. Forty eight hours later (D6) 62.5 IU was administered subcutaneously in the morning and on D7, the progesterone device was removed and 150 μg of D+cloprostenol was applied. The females in both groups were given 50 μg of GnRH in the morning and on D8 were inseminated after 12 and 24 hours. On D15 the embryo collection was performed in both treatments. The evaluation of superovulatory response was done by counting the number of follicles and corpus luteum (CL) in each ovary, with the aid of ultrasound. The variables were assessed by Student's t test for paired samples. There were differences (P<0.05) in the number of follicles over 8mm in D8 (9.06±4.54 and 5.50±4.59); in the amount of CL at the time of collection (8.12±3.26 and 4.69±3.46), total number of embryos (6.69±3.05 and 3.37±2.50) and viable embryos (5.25±2.29 and 2.37±1.78) for cows in the conventional group compared to the split group, respectively. It is concluded that the split protocol has worse superovulatory response and in vivo production of embryos in zebu cows compared with the conventional protocol.(AU)

Redução da resposta superovulatória de fêmeas bovinas superestimuladas com FSH em doses split / Reduced superovulatory response of bovine females overstimulated with FSH in split doses

Objetivou-se avaliar a superovulação (SOV) de vacas zebuínas, utilizando protocolo convencional ou protocolo com número menor de aplicações e similar dosagem (dose split). Utilizaram-se 16 fêmeas (total 32 SOV), com idade entre 17-42 meses e escore de condição corporal 2,5-4 (escala de 1-5), em delineamento tipo cross-over. No início do tratamento (D0), os animais receberam um dispositivo de progesterona e 2mg de benzoato de estradiol. As fêmeas do grupo convencional receberam 250UI de FSH/LH divididas em oito doses decrescentes administradas em intervalos de 12h (FSH/LH no D4, D5, D6 e D7 no período da manhã e tarde, nas respectivas dosagens: 50,0 UI; 37,5 UI; 25,0 UI; 12,5 UI). No D7 pela manhã, as fêmeas foram tratadas com 150μg de D+cloprostenol, e a remoção da progesterona foi realizada no D7 à tarde. As fêmeas do grupo split também receberam 250 UI de FSH/LH. No D4 de manhã, administraram-se 62,5 UI de FSH/LH via IM e 125 UI por via SC. Quarenta e oito horas após (D6) administraram-se 62,5 UI via SC e na manhã do D7 foi removida a progesterona e aplicaram-se 150μg de D+cloprostenol. As fêmeas de ambos os grupos receberam 50μg de análogo de GnRH no D8 pela manhã e foram inseminadas 12 e 24 horas após. No D15 realizou-se a colheita dos embriões em ambos os tratamentos. Avaliou-se a resposta superovulatória pela contagem do número de folículos e CLs de cada ovário, com auxílio de ultrassom. Todas as variáveis foram submetidas ao teste T de Student para amostras pareadas. Houve diferença (P<0,05) na quantidade de folículos acima de 8mm no D8 (9,06±4,54 e 5,50±4,59); número de CLs no dia da colheita (8,12± 3,26 e 4,69±3,46), número de embriões totais (6,69±3,05 e 3,37±2,50) e de embriões viáveis (5,25±2,29 e 2,37±1,78) nas vacas do grupo convencional em relação às do split, respectivamente. Conclui-se que o protocolo split tem pior resposta superovulatória e de produção in vivo de embriões, em vacas zebuínas, quando comparado ao protocolo convencional...

The aim of this study was to evaluate the superovulation (SOV) response of zebu cows, using conventional protocol or other protocol with similar dose but smaller number of applications (split dose). 16 females (32 SOV), aged 17-42 months, and body condition score of 2.5-4 (1-5 scale) were used in randomized cross-over. At the start of treatment (D0), the animals received progesterone device and 2mg of estradiol benzoate. The females from the conventional group received 250 IU of FSH/LH divided into eight decreasing doses administered at intervals of 12 hours (FSH/LH in D4, D5, D6 and D7, with their respective strengths: 50.0 IU, 37.5 IU, 25.0 IU, 12.5 IU). In D7, the females were treated with 150μg of D+cloprostenol, and the removal of progesterone device was held in the afternoon. The females from the split group also received 250 IU of FSH/LH. In the morning D4 was administered in 62.5 IU FSH/LH IM and 125 IU subcutaneously. Forty eight hours later (D6) 62.5 IU was administered subcutaneously in the morning and on D7, the progesterone device was removed and 150 μg of D+cloprostenol was applied. The females in both groups were given 50 μg of GnRH in the morning and on D8 were inseminated after 12 and 24 hours. On D15 the embryo collection was performed in both treatments. The evaluation of superovulatory response was done by counting the number of follicles and corpus luteum (CL) in each ovary, with the aid of ultrasound. The variables were assessed by Student's t test for paired samples...

Florfenicol associado ou não ao cloprostenol no tratamento de retenção de placenta em vacas leiteiras / Florfenicol and sodium cloprostenol for the treatment of retained placenta in dairy cows

The aim of this study was to compare the reproductive efficiency in dairy cows with retained placenta (RP), treated with Florfenicol associated or not with Cloprostenol. One hundred and eight cows with RP from dairy herds in southern Minas Gerais state and northern São Paulo state, were included for this purpose. The experiment was designed in factorial 2X2 . The four treatments encompassed two levels of each factor : 1 ) Florfenicol - 4 0mg/kg ( IM) divided in two injections or 40mg / kg ( SC) in one injection after calving; 2) Cloprostenol (0.530mg) - two injections associated with florfenicol treatment or without cloprostenol. The analysis of discrete variables used the Kruskal - Wallis (fou r groups) or Wilcoxon (two groups) statistic to compare groups. The Student "T" test was used to compare means of continuous variables. The overall occurrence of RP was 20.7%. The florfenicol groups had no effects on postpartum uterine health or subsequent reproductive efficiency of the cows. The cloprostenol injections of RP hastened the uterine involution and decreased the occurrence of uterine infection, and increased the percentage of animals with corpus luteum between 25 and 40 days after parturition. The intervals from parturition to first artificial insemination (AI) and to conception, as well as the number of AI per conception were reduced in the cloprostenol treatment.(AU)

Florfenicol associado ou não ao cloprostenol no tratamento de retenção de placenta em vacas leiteiras / Florfenicol and sodium cloprostenol for the treatment of retained placenta in dairy cows

The aim of this study was to compare the reproductive efficiency in dairy cows with retained placenta (RP), treated with Florfenicol associated or not with Cloprostenol. One hundred and eight cows with RP from dairy herds in southern Minas Gerais state and northern São Paulo state, were included for this purpose. The experiment was designed in factorial 2X2 . The four treatments encompassed two levels of each factor : 1 ) Florfenicol - 4 0mg/kg ( IM) divided in two injections or 40mg / kg ( SC) in one injection after calving; 2) Cloprostenol (0.530mg) - two injections associated with florfenicol treatment or without cloprostenol. The analysis of discrete variables used the Kruskal - Wallis (fou r groups) or Wilcoxon (two groups) statistic to compare groups. The Student "T" test was used to compare means of continuous variables. The overall occurrence of RP was 20.7%. The florfenicol groups had no effects on postpartum uterine health or subsequent reproductive efficiency of the cows. The cloprostenol injections of RP hastened the uterine involution and decreased the occurrence of uterine infection, and increased the percentage of animals with corpus luteum between 25 and 40 days after parturition. The intervals from parturition to first artificial insemination (AI) and to conception, as well as the number of AI per conception were reduced in the cloprostenol treatment.

Efficiency of the spermatogenesis in zebu bulls (Bos taurus indicus).

The objective of the research was to evaluate the efficiency of the spermatogenesis through the morphology of the testicular parenchyma in bulls of different zebu breeds. We used testicular fragments from bull of the breeds Nelore (n = 10), Polled Nelore (n = 6), Gyr (n = 5), Guzerat (n = 5) and Tabapuã (n = 5). The tissue was perfused with Karnovsky solution, included in glycol methacrylate and stained with toluidine blue-sodium borate 1%. Animals of the Nelore breed presented higher population of primary spermatocyte in pre-leptotene/leptotene (38.30) and in pachytene (38.14) and round spermatids (113.30), higher yield of spermatogonia mitosis (21.2) and higher daily spermatic production per gram of testicular parenchyma (32.8 × 10(6) ) than those from breeds Gyr, Guzerat and Tabapuã and higher general yield of spermatogenesis (62.4) than breeds Gyr and Tabapuã. There was no significant difference in any of the evaluated parameters between breeds Nelore and Polled Nelore. The rate of Sertoli cells did not vary between the studied breeds. Apparently, the genetic selection applied to the breeds has been improving the yield in the spermatogenic process by decreasing cellular loss, although it did not increase the support capacity of the Sertoli cells.

Inseminação artificial em tempo fixo em primíparas Nelore lactantes acíclicas / Fixed-time artificial insemination in primiparous lactating Nelore acyclic cows

Avaliou-se a eficiência de um protocolo de inseminação artificial em tempo fixo (IATF), para melhorar o desempenho reprodutivo de vacas Nelore primíparas, lactantes e acíclicas. Utilizaram-se 350 vacas com escore de condição corporal médio de 2,65±0,01 e média de dias pós-parto de 67,35±1,0 dias. A estação de monta (EM) foi de 180 dias. Os animais foram distribuídos em: G1(n=61) e G3(n=116) - vacas com presença de folículos ovarianos ≤8mm de diâmetro e sem corpo lúteo (CL); G2(n=57) e G4(n=116) - vacas com folículos ≥8mm e sem CL. As vacas do G1 e do G2 permaneceram em monta natural (MN) a partir do dia zero da EM. G3 e G4 foram tratados (dia zero) com 2mg de benzoato de estradiol (BE), IM, e dispositivo com 1,0g de P4 por oito dias. Retirou-se a P4, administraram-se 400UI de eCG e 75µg de cloprostenol, IM, 24h após, e 1mg BE, IM. A IATF foi realizada 51-54h após retirar P4. Aos cinco dias após término da IATF, as vacas do G3 e do G4 também foram mantidas em MN. Avaliou-se, por ultrassonografia, a presença de CL e realizou-se o diagnóstico de gestação, aos 45 e 210 dias após início da EM. Aos 45 dias, mais animais do G3, 42,2%, e do G4, 51,7%, estavam gestantes (P<0,05), em relação aos do G1, 0%, e do G2, 3,5%. A porcentagem de CL foi de 6,6%; 28,1%; 50,9%; 62,9% em G1, G2, G3 e G4, respectivamente. Aos 210 dias, 22,9% do G1; 45,6% do G2; 40,5% do G3 e 65,5% do G4 estavam gestantes. Os períodos de serviço foram: em G1, 134,00±11,70 dias; em G2, 119,65±8,27 dias; em G3, 79,91±5,38 dias; e em G4, 81,40±4,59 dias. A IATF foi eficiente para melhorar o desempenho reprodutivo de vacas Nelore primíparas lactantes acíclicas em relação ao manejo apenas da MN.(AU)

The aim of this experiment was to evaluate the efficiency of a fixed-time artificial insemination protocol to improve the reproductive performance of lactating primiparous acyclic Nelore cows. A total of 350 cows with average body condition score of 2.65±0.01 and average postpartum days of 67.35±1.0 were used. The breeding season (MS) was 180 days. The animals were distributed into G1 (n = 61) and G3 (n = 116) - cows with ovarian follicles ≤8mm in diameter and without corpus luteum (CL). G2 (n = 57) and G4 (n = 116) groups - cows with follicles ≥8mm and without CL. Cows in G1 and G2 remained in natural mating (NM) from day 0 of MS. G3 and G4 were treated (Day 0) with 2mg of oestradiol benzoate (EB), IM, and 1.0g P4 device for eight days. The withdrawal of P4 was administered with 400IU of eCG and cloprostenol 75μg, IM, after 24h, and 1mg BE, IM. The artificial insemination was performed at 51-54h after removing the P4 device. Five days after the end of the artificial insemination, cows from G3 and G4 were also held in MN. Ultrasonography was performed to evaluate the presence of CL and the pregnancy diagnosis at days 45 and 210 after the onset of MS. At 45 days more animals in G3, 42.2% and G4, 51.7% were pregnant (P <0.05) than in G1, 0% and G2, 3.5%. The CL percentage was 6.6%, 28.1%, 50.9%, 62.9% in G1, G2, G3 and G4, respectively. At 210 days 22.9% in G1, 45.6% in G2, 40.5% in G3 and 65.5% in G4 were pregnant. The service periods were: G1, 134.00±11.70 days; G2, 119.65±8.27 days, in G3, 79.91±5.38 days, and G4, 81.40±4.59 days. The fixed-time artificial insemination was efficient to improve the reproductive performance of primiparous lactating Nelore acyclic cows compared to animals that remained only with natural mating.(AU)

Inseminação artificial em tempo fixo em primíparas Nelore lactantes acíclicas / Fixed-time artificial insemination in primiparous lactating Nelore acyclic cows

Avaliou-se a eficiência de um protocolo de inseminação artificial em tempo fixo (IATF), para melhorar o desempenho reprodutivo de vacas Nelore primíparas, lactantes e acíclicas. Utilizaram-se 350 vacas com escore de condição corporal médio de 2,65±0,01 e média de dias pós-parto de 67,35±1,0 dias. A estação de monta (EM) foi de 180 dias. Os animais foram distribuídos em: G1(n=61) e G3(n=116) - vacas com presença de folículos ovarianos ≤8mm de diâmetro e sem corpo lúteo (CL); G2(n=57) e G4(n=116) - vacas com folículos ≥8mm e sem CL. As vacas do G1 e do G2 permaneceram em monta natural (MN) a partir do dia zero da EM. G3 e G4 foram tratados (dia zero) com 2mg de benzoato de estradiol (BE), IM, e dispositivo com 1,0g de P4 por oito dias. Retirou-se a P4, administraram-se 400UI de eCG e 75µg de cloprostenol, IM, 24h após, e 1mg BE, IM. A IATF foi realizada 51-54h após retirar P4. Aos cinco dias após término da IATF, as vacas do G3 e do G4 também foram mantidas em MN. Avaliou-se, por ultrassonografia, a presença de CL e realizou-se o diagnóstico de gestação, aos 45 e 210 dias após início da EM. Aos 45 dias, mais animais do G3, 42,2%, e do G4, 51,7%, estavam gestantes (P<0,05), em relação aos do G1, 0%, e do G2, 3,5%. A porcentagem de CL foi de 6,6%; 28,1%; 50,9%; 62,9% em G1, G2, G3 e G4, respectivamente. Aos 210 dias, 22,9% do G1; 45,6% do G2; 40,5% do G3 e 65,5% do G4 estavam gestantes. Os períodos de serviço foram: em G1, 134,00±11,70 dias; em G2, 119,65±8,27 dias; em G3, 79,91±5,38 dias; e em G4, 81,40±4,59 dias. A IATF foi eficiente para melhorar o desempenho reprodutivo de vacas Nelore primíparas lactantes acíclicas em relação ao manejo apenas da MN.

The aim of this experiment was to evaluate the efficiency of a fixed-time artificial insemination protocol to improve the reproductive performance of lactating primiparous acyclic Nelore cows. A total of 350 cows with average body condition score of 2.65±0.01 and average postpartum days of 67.35±1.0 were used. The breeding season (MS) was 180 days. The animals were distributed into G1 (n = 61) and G3 (n = 116) - cows with ovarian follicles ≤8mm in diameter and without corpus luteum (CL). G2 (n = 57) and G4 (n = 116) groups - cows with follicles ≥8mm and without CL. Cows in G1 and G2 remained in natural mating (NM) from day 0 of MS. G3 and G4 were treated (Day 0) with 2mg of oestradiol benzoate (EB), IM, and 1.0g P4 device for eight days. The withdrawal of P4 was administered with 400IU of eCG and cloprostenol 75μg, IM, after 24h, and 1mg BE, IM. The artificial insemination was performed at 51-54h after removing the P4 device. Five days after the end of the artificial insemination, cows from G3 and G4 were also held in MN. Ultrasonography was performed to evaluate the presence of CL and the pregnancy diagnosis at days 45 and 210 after the onset of MS. At 45 days more animals in G3, 42.2% and G4, 51.7% were pregnant (P <0.05) than in G1, 0% and G2, 3.5%. The CL percentage was 6.6%, 28.1%, 50.9%, 62.9% in G1, G2, G3 and G4, respectively. At 210 days 22.9% in G1, 45.6% in G2, 40.5% in G3 and 65.5% in G4 were pregnant. The service periods were: G1, 134.00±11.70 days; G2, 119.65±8.27 days, in G3, 79.91±5.38 days, and G4, 81.40±4.59 days. The fixed-time artificial insemination was efficient to improve the reproductive performance of primiparous lactating Nelore acyclic cows compared to animals that remained only with natural mating.

Occurrence and characteristics of residual follicles formed after transvaginal ultrasound-guided follicle aspiration in cattle.

Ultrasound-guided transvaginal follicle aspiration is used to recover cumulus-oocyte complexes (for IVF) and to synchronize follicular wave emergence (ablation of dominant follicle). Although aspirated follicles are generally supposed to undergo immediate atresia, there are indications that they may remain active. The objective was to evaluate the occurrence and characteristics of residual follicles (RF) after transvaginal follicle aspiration in cattle. Ovarian follicular wave emergence was synchronized in Holstein cows (N = 13) in the presence (groups 1 and 3) or absence (groups 2 and 4) of norgestomet implants. The largest follicle was aspirated at a diameter of 8 mm (groups 1 and 2) or 12 mm (groups 3 and 4). Ovarian follicles were visualized (transrectal ultrasonography) every 12 h after wave emergence. Follicular fluid samples were collected from the largest follicle and from the ensuing RF and concentrations of estradiol and progesterone were determined. After aspiration, 73.2% (52/71) of the follicles refilled with fluid, and a new antrum was detected 12 to 24 h later. Norgestomet did not affect (P > 0.05) RF occurrence or diameter, but in RF from group 4, concentrations of estradiol decreased (-530.7 ± 133.9 ng/mL; P < 0.01) whereas progesterone increased (+429.6 ± 171.7 ng/mL; P < 0.05) relative to preaspiration. In RF, there were three steroidogenesis patterns: (1) high estradiol concentration and high estradiol:progesterone ratio (estradiol-active RF); (2) low estradiol, but high progesterone concentrations (luteinized RF); and (3) low estradiol and low progesterone concentrations (inactive RF). Estradiol-active RF were more likely (P < 0.05) from follicles with high estradiol concentrations (regardless of diameter). In conclusion, fluid-filled structures (RF) with variable steroid production patterns are frequently formed after ultrasound-guided follicle aspiration. The occurrence and features of these RF depended on the diameter and status of these follicles before aspiration.

Desenvolvimento de embriões de camundongas após manutenção em diferentes soluções de manipulação / Development of mice embryos after maintenance in different manipulation solutions

Avaliou-se a eficácia de duas soluções de manipulação (SM) de embriões de camundongas nos estádios de blastocisto inicial (Bin), mórula compacta grau I (McI) e II (McII), distribuídos aleatoriamente em três tratamentos (T), de acordo com a solução de manutenção. No T1 usou-se PBS modificado (controle); no T2, SME e no T3, SME enriquecida. Os embriões foram mantidos durante quatro horas na solução de manutenção e posteriormente classificados quanto ao estádio de desenvolvimento e à qualidade embrionária. Logo após, foram cultivados em meio TCM 199 e classificados novamente quanto ao estádio de desenvolvimento e à qualidade embrionária. A taxa de desenvolvimento dos embriões após manutenção por quatro horas em solução de manipulação foi menor (P<0,05) nos embriões do controle, comparada à de embriões do SME e SME enriquecida, diferença esta não observada (P>0,05) após o cultivo in vitro. Os embriões McII do T3 tiveram maior desenvolvimento (P<0,05) em relação aos embriões do T1 e T2, indicando o efeito benéfico do enriquecimento da solução SME. Conclui-se que as soluções de manipulação SME e SME enriquecida influenciaram beneficamente o desenvolvimento de embriões.

The effect of embryo manipulation solution followed by in vitro culture in mice embryos was studied. The embryos at early blastocyst (Bin), and compact morula grades I (McI) and II (McII) were randomly assigned into three treatments. T1 used modified PBS (control), T2 used EMS, and T3 used EMS supplemented. In each treatment, the embryos were kept in manipulation solution for four hours. Finishing the manipulation period, the embryos were classified according the development stage and quality. Following, the embryos were cultured in TCM 199. After the culture period, the embryos were evaluated according to quality and development stage. The development rate for Bin, McI, and McII after maintenance for four hours in manipulation solution was lower for control embryo (P>0.05) as compared to EMS and EMS supplemented embryos. After in vitro culture, no differences (P>0.05) on embryo development rate among control, EMS, and EMS supplemented were observed. Moreover, McII from EMS supplemented had a higher development (P<0.05) (93 percent) as compared to control (82.5 percent) and EMS (83.9 percent), suggesting a beneficial effect of EMS supplemented. EMS and EMS supplemented embryos had a positive effect on embryo development, showing higher embryo development than those in PBS solution.

Desenvolvimento de embriões de camundongas após manutenção em diferentes soluções de manipulação / Development of mice embryos after maintenance in different manipulation solutions

Avaliou-se a eficácia de duas soluções de manipulação (SM) de embriões de camundongas nos estádios de blastocisto inicial (Bin), mórula compacta grau I (McI) e II (McII), distribuídos aleatoriamente em três tratamentos (T), de acordo com a solução de manutenção. No T1 usou-se PBS modificado (controle); no T2, SME e no T3, SME enriquecida. Os embriões foram mantidos durante quatro horas na solução de manutenção e posteriormente classificados quanto ao estádio de desenvolvimento e à qualidade embrionária. Logo após, foram cultivados em meio TCM 199 e classificados novamente quanto ao estádio de desenvolvimento e à qualidade embrionária. A taxa de desenvolvimento dos embriões após manutenção por quatro horas em solução de manipulação foi menor (P<0,05) nos embriões do controle, comparada à de embriões do SME e SME enriquecida, diferença esta não observada (P>0,05) após o cultivo in vitro. Os embriões McII do T3 tiveram maior desenvolvimento (P<0,05) em relação aos embriões do T1 e T2, indicando o efeito benéfico do enriquecimento da solução SME. Conclui-se que as soluções de manipulação SME e SME enriquecida influenciaram beneficamente o desenvolvimento de embriões.(AU)

The effect of embryo manipulation solution followed by in vitro culture in mice embryos was studied. The embryos at early blastocyst (Bin), and compact morula grades I (McI) and II (McII) were randomly assigned into three treatments. T1 used modified PBS (control), T2 used EMS, and T3 used EMS supplemented. In each treatment, the embryos were kept in manipulation solution for four hours. Finishing the manipulation period, the embryos were classified according the development stage and quality. Following, the embryos were cultured in TCM 199. After the culture period, the embryos were evaluated according to quality and development stage. The development rate for Bin, McI, and McII after maintenance for four hours in manipulation solution was lower for control embryo (P>0.05) as compared to EMS and EMS supplemented embryos. After in vitro culture, no differences (P>0.05) on embryo development rate among control, EMS, and EMS supplemented were observed. Moreover, McII from EMS supplemented had a higher development (P<0.05) (93 percent) as compared to control (82.5 percent) and EMS (83.9 percent), suggesting a beneficial effect of EMS supplemented. EMS and EMS supplemented embryos had a positive effect on embryo development, showing higher embryo development than those in PBS solution.(AU)

Pregnancy rates and corpus luteum-related factors affecting pregnancy establishment in bovine recipients synchronized for fixed-time embryo transfer.

The objective was to investigate the influence of corpora lutea physical and functional characteristics on pregnancy rates in bovine recipients synchronized for fixed-time embryo transfer (FTET). Crossbred (Bos taurus taurus x Bos taurus indicus) nonlactating cows and heifers (n=259) were treated with the following protocol: 2mg estradiol benzoate (EB) plus an intravaginal progesterone device (CIDR 1.9g progesterone; Day 0); 400 IU equine chorionic gonadotropin (eCG; Day 5); prostaglandin F(2alpha) (PGF(2alpha)) and CIDR withdrawal (Day 8); and 1mg EB (Day 9). Ovarian ultrasonography and blood sample collections were performed on Day 17. Of the 259 cattle initially treated, 197 (76.1%) were suitable recipients; they received a single, fresh, quality grade 1 or 2 in vivo-derived (n=90) or in vitro-produced (n=87) embryo on Day 17. Pregnancy rates (23 d after embryo transfer) were higher for in vivo-derived embryos than for in vitro-produced embryos (58.8% vs. 31.0%, respectively; P<0.001). Mean (+/-SD) plasma progesterone (P(4)) concentration was higher in cattle that became pregnant than that in nonpregnant cattle (5.2+/-5.0 vs. 3.8+/-2.4 ng/mL; P=0.02). Mean pixel values (71.8+/-1.3 vs. 71.2+/-1.1) and pixel heterogeneity (14.8+/-0.3 vs. 14.5+/-0.5) were similar between pregnant and nonpregnant recipients (P>0.10). No significant relationship was detected between pregnancy outcome and plasma P(4), corpus luteum area, or corpus luteum echotexture. Embryo type, however, affected the odds of pregnancy. In conclusion, corpus luteum-related traits were poor predictors of pregnancy in recipients. The type of embryo, however, was a major factor affecting pregnancy outcome.

Neisseria meningitidis PorA variable regions: rapid detection of P1.7 and P1.19 variants by PCR.

AIM: Rapid characterization of variable region (VR)1 variants of the porA gene among invasive strains is crucial for outbreak management and epidemiology studies. Recent sequence analysis studies in Brazil showed that the VR1 P1.7 and P1.19 variants are highly prevalent, accounting for 68%, of the total number of VR1 variants characterized. The aim of this work is to develop a rapid polymerase chain reaction (PCR)-based method for genosubtyping Neisseria meningitidis by detection of porA variable regions P1.7 and P1.19. METHODS AND RESULTS: PCR primers for the detection of porA VR1 P1.7 and P1.19 were designed and tested using 198 clinical N. meningitidis isolates that had been previously evaluated by porA sequencing. All 50 strains with VR1 P1.7 and all 65 strains with VR1 P1.19 were positively identified by the respective VR-specific PCR and no false-positive reactions occurred. CONCLUSIONS: VR-specific PCR amplification accurately identified VR P1.7 and P1.19 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: To overcome the disadvantages of serosubtyping and sequencing for typing the porA VR1 segment of N. meningitidis, we developed a PCR-based method to rapidly and accurately detect VR1 P1.7 and P1.19 variants. This approach is highly specific and sensitive; moreover it may allow for genotype determination of culture-negative samples.