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Small ruminant breeds have different reactions to climate change, which influences reproductive performance and the application of biotechnologies. The aim of the present work was to evaluate whether the reproductive characteristics superovulation response, number of Corpus luteum (CL), embryo recovery rate, following superovulation of Santa Ines breed ewes were influenced by two-years seasons and frequency of embryo collection under subtropical condition. Ewes were housed in pens with water and salt ad libitum. Reproductive characteristics were evaluated in two- years seasons: Fall and Winter (F/W) and Spring and Summer (S/S). Decreased superovulatory response and viable embryo rates were observed in S/S (33.3 % and 10 %) compared to F/W (76.5 % and 59.6 %, P

RESUMO - Objetivou-se avaliar a teofilina como agente capacitante substituto ou associado à heparina sobre a reação acrossômica dos espermatozoides e o desenvolvimento de embriões produzidos in vitro. O experimento foi realizado com quatro touros e três tratamentos, totalizando 12 grupos experimentais. O sêmen dos touros foi avaliado nos tratamentos descritos a seguir: tratamento 1 (HEP): heparina ? 10µg/mL; tratamento 2 (TEO): teofilina ? 5mM; tratamento 3 (HEP + TEO): heparina (10µg/mL) + teofilina (5mM), por zero, seis, 12 e 18 horas, corados com trypan blue/Giemsa para avaliação da reação acrossômica. Para a produção dos embriões, os agentes capacitantes foram adicionados aos meios de fertilização. Na análise espermática, a taxa de reação acrossômica verdadeira foi maior (P0,05) entre touros na análise de reação acrossômica nem na PIVE. A utilização da teofilina foi tão eficiente quanto a da heparina na indução da reação acrossômica, no entanto resultou em menores taxas de produção embrionária. ABSTRACT - The sperm capacitating process should take special attention during in vitro embryo production (IVEP) once that affects the success of embryo production. The study aimed to evaluate theophylline as substitute capacitating agent or in combination with heparin on the sperm acrosome reaction and development of embryos produced in vitro. The experiment was carried out using 4 bulls and 3 treatments, establishing 12 experimental groups. Each bull was evaluated in the following treatments: Treatment 1 (HEP): Heparin - 10mg/mL; Treatment 2 (THEO): Theophylline - 5mM; Treatment 3 (HEP + THEO), Heparin (10mg/mL) + Theophylline (5mM). The semen of bulls was incubated in each treatment for 0, 6, 12 and 18h, stained with Trypan blue / Giemsa and analyzed by electron microscopy for assessment of acrosome reaction. Using sperm of same bulls, capacitating agents were added to the fertilization media, for IVEP. In sperm analysis, the true acrosome reaction rate was higher (P0.05) between bulls in acrosome reaction analysis neither for IVEP. The use of theophylline was as effective as heparin in the induction of the acrosome reaction, although it resulted in lower embryo production rates. Made available in DSpace on 2018-01-23T16:05:24Z (GMT). No. of bitstreams: 1 Cnpgl2017ArqBrasMVZVaragoTeofilina.pdf: 618605 bytes, checksum: bfa66dc554da02ac69db9f6d25dc0dcd (MD5) Previous issue date: 2018-01-17

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-P0.05) 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

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

Contents The aim of this work was to evaluate the efficiency of the cryoprotectants dimethylformamide and ethylene glycol for cryopreservation of ovine embryos using vitrification and conventional freezing. The recovered embryos were distributed randomly in three treatment groups: Gr. 1: conventional freezing (n = 44), Gr. 2: vitrification with ethylene glycol (n = 39) and Gr. 3: vitrification with dimethylformamide (n = 38). Quality of fresh embryos in control group as well as of frozen and vitrified embryos was examined by three methodologies: staining with propidium iodide and Hoechst 33258 and evaluation under fluorescent microscopy, evaluation of re-expansion and hatching rates after culture, and determination of apoptotic index with TUNEL technique. It was established that re-expansion rate in all treatment groups was similar. In the same time, hatching rates were higher in Gr. 1 (40.5%) and Gr. 2 (35.3%) in comparison with Gr. 3 (15.5%, p

The present work aimed to characterize the uterine involution after singleton and twin parturitions in Santa Ines ewes submitted to two nutritional diets. Sixteen pregnant pluriparous Santa Ines ewes, with age ranging from 3 to 5 years, were used. The animals were randomized into four experimental groups of four animals each. The experiment was a 2 × 2 factorial with number of lambs (singleton versus twin pregnancy) and diet (maintenance diet versus maintenance diet with 15% energy deficit). To determinate the number of fetuses, all ewes were submitted to an ultrasonographic evaluation at days 30, and checked at 45 and 60 days of pregnancy. Genital tract involution was followed by vaginal smear cytology and transrectal evaluation of the uterus by ultrasonography. Changes in cells from the vaginal wall were not useful to characterize the involution process of the genital tract in ewes, however leukocyte cells were. Ultrasonographic observations pointed to a longer period for reduction and stabilization of uterine size after twin parturition, and there was no alteration of the uterine involution process due to the level of nutritional restriction.

Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in liquid nitrogen; (T2) T1 with the addition of cholesterol before cooling (the cholesterol was incorporated to the sperm membranes with the methyl-beta-cyclodextrin-cholesterol complex); (T3) T2 with post-thaw removal of the cholesterol with 0.052 mg methyl-beta-cyclodextrin/50 x 10(6) sperm; and (T4) T3 with 0.156 mg methyl-beta-cyclodextrin/50 x 10(6) sperm. Sperm progressive motility and functional integrity of sperm plasma membranes were evaluated microscopically and by the hyposmotic swelling test, respectively. Using flow cytometry, physical integrity of sperm plasma membranes was assessed with propidium iodide, acrosomal integrity with fluoresceinated lectin peanut agglutinin, and rate of sperm acrosome reaction induced with of the calcium ionophore A23187. Cholesterol inclusion (T2) increased the proportion of frozen-thawed sperm with intact plasma membrane. Nevertheless, sperm from T2 (9.3+/-5.9%) had a lower rate of acrosome reaction after induction, compared to the control group (16.5+/-11.0%). After cholesterol removal, there was no increase in the induced acrosome reaction rate (T3: 11.3+/-7.1% and T4: 11.8+/-9.9%). Perhaps the cyclodextrin concentrations used were too low to remove sufficient cholesterol from sperm membranes to restore the ability of cryopreserved sperm to undergo an acrosome reaction. Regardless, the addition of cholesterol to improve post-thaw sperm integrity, and its subsequent removal, still has potential for cryopreservation of stallion sperm.

The study aimed at testing the effectiveness of dimethylformamide, alone or combined with glycerol, as cryoprotectant for freezing ram semen. Ejaculates from nine rams were cryopreserved in Tris-based extenders, containing 5% of glycerol, association of dimethylformamide with glycerol, in four proportions achieving 5% of cryoprotectors in the media and pure dimethylformamide (2, 3, 4 and 5%) in replacement to glycerol. The samples were diluted to 100 × 10(6) sptz/ml and stored in 0.25-ml straws in liquid nitrogen. After thawing (37 °C for 30 s), motility was preserved better by the extender containing 5% of glycerol (p < 0.05). The extenders containing pure dimethylformamide, or more than 2% in combination with glycerol, provided sperm motilities close to zero. Plasma and acrosomal membrane integrity were preserved better (p < 0.05) in the extender containing 5% glycerol. It can be concluded that dimethylformamide, alone or combined with glycerol, has no beneficial effects on ovine semen cryopreservation.

The objective was to evaluate the suitability of using natural or lyophilized low density lipoproteins (LDL), in lieu of whole egg yolk, in extenders for cryopreserving ram semen. Once extragonadal sperm reserves were depleted in 10 fertile Santa Ines cross rams, two ejaculates per ram were collected for cryopreservation. Nine extenders were used: Tris-16% egg yolk extender with 5% glycerol as a control (T1), and substitution of whole egg yolk with 8, 12, 16 or 20% natural LDL (T2-T5, respectively), or with 8, 12, 16, or 20% lyophilized LDL (T6-T9). Semen was diluted to 100 × 10(6) sperm/mL, packaged into 0.25 mL straws, cooled, held at 5 °C for 3 h, and then frozen in liquid nitrogen vapor. Immediately after thawing (37 °C for 30 s), sperm total and progressive motility, and kinetic parameters were analyzed with computer assisted semen analysis (CASA). Percentage of sperm with plasma membrane functional integrity was assessed by the hypoosmotic swelling test (HOST), sperm membrane physical integrity with propidium iodide (PI), and acrosome integrity with FITC-PSA using an epifluorescent microscope. For all sperm end points, there was no difference between the control and natural LDL treatments (P > 0.05): total motility (T1: 20.9 ± 11.9 and average of T2-T5: 25.9 ± 13.6%; mean ± SD), progressive motility (T1: 6.6 ± 4.2 and average of T2-T5: 11.7 ± 7.5%), HOST(+) (T1: 23.7 ± 6.9 and average of T2-T5: 23.2 ± 8.7 %) and PI(-)/PSA(-) (T1: 13.8 ± 7.8 and average of T2-T5: 18.1 ± 7.8%). However, lyophilization was apparently unable to preserve the protective function of LDL; every sperm end point was significantly worse than in the control and natural LDL groups. We concluded that natural LDL was appropriate for cryopreserving ram semen, as it yielded results similar to those obtained with whole egg yolk.

combination of fluorescent probes propidium iodide (PI) and SYBR-14 (LIVE/DEAD Sperm viability Kit (L-7011), Molecular Probes, Eugene, Oregon) for sperm viability. In addition, acrosome integrity was evaluated using the fluorescein labelled lectin from the peanut plant, Arachis hypogea (FITC-PNA, Sigma Chemical, St. Louis, MO) and propidium iodide (PI) probe. Results were evaluated by the Cell-Quest software (Becton Dickinson, San Jose, CA, USA) and statistical differences by a one-way ANOVA, followed by post hoc comparison tests. The results indicated significant seasonal differences in testicular size and sperm production (p≤ 0.05). Ultrasonographic testicular volume was maximal in March (234.03± 15.8 cm3), decreased to a minimum in October and June (203.5± 17.0 cm3, 168.6± 10.0 cm3, respectively) and started to increase again in January (190.8± 8.8 cm3). A significant positive correlation between testicular volume and sperm production (r= 0.66; P≤ 0.01) was demonstrated, although minimum testicular volume was reached in October while minimum sperm production was registered in January. Viability in frozen-thawed semen from ejaculates collected in autumn was 37.73± 2.20% and in summer 40.99± 4.08%; the results differ significantly from other seasons: winter 54.26± 2.10% and spring 46.71± 2.32% (p≤ 0.05). The high number of dead spermatozoa after the freezing–thawing process in autumn/winter most likely indicates that seasonally related changes in seminal plasma composition may affect semen preservation. Nevertheless, no significant differences were observed for acrosome integrity in different periods of the experiment. An inter-stallion variation was observed for testicular size and sperm production, but testicular size, besides sperm production, was positively correlated in all individual stallions. At the time of semen collection, sperm concentrations determined by photometry showed that both testis size and sperm production of housed Lusitano stallions were affected by season. Testicular size and function were maximal in the spring, but testicular volume was smallest in October, while sperm production continued to decrease till January. The results indicate that in our climatic/latitude conditions, it is preferable to freeze semen during winter/spring period.