High environmental temperatures during the hot months of the year reduce reproductive performance in cattle. Summer heat stress depression in fertility is a multifactorial problem; however, there is evidence that the bovine germinal vesicle and maturing oocyte, as well as the early embryo, are major targets of the deleterious effects of heat stress. Such adverse effects are less pronounced in heat-tolerant breeds (Bos indicus) than heat-sensitive breeds (Bos taurus). This genetic variation results from the greater thermoregulatory ability and cellular thermoresistance of heat-tolerant breeds. Heat-induced oocyte cellular damage occurs in both cytoplasmic and nuclear compartments. Heat shock has been shown to reduce oocyte nuclear maturation, induce apoptosis, compromise oocyte cytoskeleton, and impair oocyte mitochondrial function and developmental competence. However, the oocyte cytoplasm is more susceptible to heat shock than the nucleus. This effect is greater for Bos taurus than Bos indicus oocytes. The detrimental effects of heat shock are also critical during the first cleavage divisions when most of the embryonic genome is inactive; however, the bovine embryo becomes more resistant to increased temperature as it proceeds through development. Several studies demonstrated that Bos indicus embryos are more thermotolerant than Bos taurus embryos. Adaptive changes involved in acquisition of thermotolerance are likely derived from changes in gene expression and (or) activity of biochemical molecules that control cellular functions against stress. Recently, molecules such as IGF-I and caspase inhibitor z-DEVD-fmk have been shown to exert a thermoprotective role, rescuing heat-induced oocyte and embryo cellular damage and developmental competence. Therefore, cattle genotype and thermoprotective molecules can be considered as an alternative to modulate the effects of increased temperature in reproductive function.
a b s t r a c tThe role of insulin-like growth factor 1 (IGF1) on cellular function and developmental capacity of heat-shocked oocytes has not been completely understood. Therefore, the objective of this study was to determine the effect of IGF1 on apoptosis, mitochondrial activity, cytoskeletal changes, nuclear maturation, and developmental competence of bovine oocytes exposed to heat shock. Cumulus-oocyte complexes were submitted to control (38.5 C for 22 hours) and heat shock (41 C for 14 hours followed by 38.5 C for 8 hours) in the presence of 0 or 100 ng/mL IGF1 during IVM. Heat shock increased the percentage of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive oocyte and reduced oocyte mitochondrial activity. However, addition of 100 ng/mL IGF1 minimized these deleterious effects of temperature. Caspase activity was affected neither by heat shock nor IGF1. Exposure of bovine oocytes to 41 C during the first 14-hour IVM affected cortical actin localization and microtubule organization at the meiotic spindle and reduced the percentage oocytes that reached the metaphase II stage. However, in the presence of IGF1, cortical actin and percentage of metaphase II oocytes were not different between control and heat-shocked oocytes, suggesting a partial beneficial effect of IGF1. There was no effect of IGF1 on microtubule organization. Heat shock also reduced the percentage of oocytes that reached the blastocyst stage, blastocyst cell number, and increased the percentage of TUNELpositive blastomeres. However, there was no effect of 100 ng/mL IGF1 on oocyte development to the blastocyst stage and blastocyst quality. Therefore, 100 ng/mL IGF1 prevented some heat shock-induced cellular damage in bovine oocytes but had no effect on oocyte developmental competence. In contrast, a low IGF1 concentration (25 ng/mL) had a thermoprotective effect on oocyte developmental competence to the blastocyst stage. In conclusion, IGF1 prevented part of the damage induced by heat shock on oocyte function. This effect was modulated by IGF1 concentration.
The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.
Germinal vesicle (GV) oocytes are susceptible to heat stress. However, neither the cellular mechanisms triggered by elevated temperature nor the thermoprotective effects of insulin-like growth factor (IGF) on GV oocytes are completely understood. Therefore, a series of experiments was conducted to determine the direct effects of IGF1 (0, 12.5, 25, 50 and 100ng mL) on heat-treated GV oocytes. Butyrolactone-arrested GV oocytes were cultured at 38.5°C (control) or 41°C (heat shock; HS) for 14h in the presence of different concentrations of IGF1. Exposure of GV oocytes to 41°C increased (P<0.05) the number of terminal deoxyribonucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling (TUNEL)-positive oocytes. At concentrations of 12.5 and 25ng mL, IGF1 tended to minimise these negative effect of HS (P=0.07). However, neither HS nor IGF1 had any effect on caspase activity. HS also decreased (P<0.05) GV oocyte mitochondrial activity and developmental competence to the blastocyst stage. These deleterious effects of HS were alleviated (P<0.05) by 12.5ng mL IGF1. This concentration of IGF1 did not affect cleavage rate, the percentage of TUNEL-positive blastomeres and total blastocyst cell number regardless of temperature. In conclusion, exposure of GV oocytes to HS triggered the apoptotic cascade and compromised oocyte developmental competence. Physiological concentrations of IGF1 had a beneficial effect on heat-shocked GV oocytes.
The series of events associated with oocyte maturation are susceptible to disruption by elevated temperature. These events are regulated by a variety of growth factors, such as insulin-like growth factor-1 (IGF-1). Exposure of bovine oocytes to heat shock compromises oocyte competence and triggers apoptosis. It has been shown that cellular stresses often alter mitochondrial function and activate the mitochondrial apoptotic cascade. Therefore, the objective of this study was to determine the effect of heat shock on bovine oocyte mitochondrial activity and the role of IGF-1 in this context. Slaughterhouse derived cumulus–oocyte complexes (COC) were subjected to control (38.5°C for 22 h) and heat shock (41°C for 14 h, followed by 38.5°C for 8 h) treatments in the presence of 0 or 100 ng mL–1 of IGF-1 during in vitro maturation (IVM). After 22 h, IVM COC were mechanically denuded and subjected to MitoTracker Red CMX-Ros assay (Invitrogen M-7512) to localize and quantify active mitochondria. Denuded oocytes were incubated in TCM-199-HEPES containing 10 μg mL–1 of polyvinyl alcohol and 50 nM MitoTracker at 37°C for 15 min. Oocytes were evaluated under fluorescence microscope and digital images were obtained and stored as TIFF files. Mitochondrial activity from each oocyte was quantified using the software Image J 1.43. This experiment was replicated 6 times using 97 to 204 COC/treatment. Data were analyzed by least-squares analysis of variance using the general linear model procedure of SAS. In the absence of IGF-1, heat shock reduced (P < 0.001) mitochondrial activity from 64.31 ± 1.91 to 56.74 ± 1.26 arbitrary units for control and heat shock groups, respectively. Addition of IGF-1 to maturation medium did not affect mitochondrial activity in the control group (66.25 ± 1.56). However, IGF-1 improved (temperature × IGF-1; P < 0.001) mitochondrial activity of bovine oocytes subjected to heat shock (70.32 ± 1.32). In conclusion, heat shock reduced bovine oocyte mitochondrial activity, suggesting activation of mitochondrial apoptotic cascade. Moreover, IGF-1 exerted a thermoprotective role, reducing the mitochondrial damage caused by elevated temperature.
Detecção imunoistoquímica de receptores de estrógeno e progesterona no endométrio de vacas Nelore (Bos taurus indicus) durante o anestro pós-parto RESUMOForam utilizadas 24 vacas Nelore P.O., em anestro pós-parto, diagnosticado pelo histórico reprodutivo, por avaliações ultrassonográficas transretais e por dosagem de progesterona plasmática, que foram submetidas à colheita de fragmento uterino via transcervical. Os animais foram divididos em dois grupos conforme o máximo diâmetro folicular: grupo 1: folículos <6mm (n=12); grupo 2: folículos ≥6mm (n=12). Para avaliar receptor de estrógeno e receptor de progesterona no epitélio glandular e no estroma, foi utilizada a técnica de imunoistoquímica. Altas contagens relativas e alta intensidade de marcação para receptor de estrógeno e progesterona no epitélio glandular e no estroma foram observadas nos dois grupos. No entanto, a intensidade de marcação para o receptor de progesterona no epitélio glandular foi mais alta no grupo 2 comparado ao grupo 1. Quando o epitélio glandular e o estroma foram comparados, o número relativo de receptor de estrógeno no grupo 1 foi mais alto no epitélio glandular comparado ao estroma, e a intensidade de marcação para o receptor de progesterona no grupo 2 foi mais alta no epitélio glandular comparado ao estroma. Os resultados sugerem que os mecanismos que controlam a expressão de receptores no anestro são semelhantes aos observados durante o ciclo estral.
ResumoCondições ambientais adversas, tais como altas temperaturas e umidade relativa, causam aumento da temperatura corporal interna (hipertermia) de vacas lactantes, que resultam em estresse térmico e diminuição dos índices de gestação. A susceptibilidade embrionária à temperatura elevada já foi bem caracterizada tanto em experimentos in vivo quanto in vitro. A exposição de embriões bovinos em estágios de zigoto e duas células à temperatura elevada diminui o desenvolvimento embrionário até o estágio de blastocisto. No entanto, o embrião torna-se mais resistente aos efeitos deletérios da temperatura elevada à medida que progride no desenvolvimento. A redução na competência de desenvolvimento embrionária causada pelo estresse térmico deve-se, em parte, às inúmeras alterações citoplasmáticas e nucleares induzidas pela temperatura elevada. No citoplasma embrionário, o choque térmico aumenta o número de mitocôndrias edemaciadas, desorganiza os microtúbulos e os filamentos de actina. No compartimento nuclear, a temperatura elevada induz a fragmentação de DNA característica de apoptose. Essa forma de morte celular é um fenômeno regulado ao longo do desenvolvimento embrionário pré-implantacional, visto que altas temperaturas não ativam a cascata de apoptose em embriões de duas ou quatro células. A apoptose embrionária induzida pelo choque térmico em embriões > 16 célu-las pode ser considerada um mecanismo de controle de qualidade para remoção dos blastômeros danificados, já que o bloqueio da apoptose nestes embriões aumenta ainda mais a susceptibilidade ao choque térmico. Além disso, o estresse térmico também pode afetar o estado redox do embrião, levando a um consequente estresse oxidativo. Palavras-chave:Estresse térmico. Embrião. Bovino. Alterações celulares. AbstractAdverse environmental conditions such as high temperature and humidity increase internal body temperature (hyperthermia) of lactating dairy cows resulting in heat stress and decreased pregnancy rates. Embryonic susceptibility to elevated temperature has been well characterized both in vivo and in vitro. Exposure of zygote and two cells stage bovine embryos to elevated temperature decreases embryonic development to the blastocyst stage. However, the bovine embryo becomes more resistant to the deleterious effects of heat stress as it proceeds in its development. The heat-induced reduction in embryonic developmental competence is due, at least in part, to the numerous cytoplasmic and nuclear changes induced by high temperature. In the embryo cytoplasm heat shock increases the number of swollen mitochondria, disrupts microtubules and microfilaments. In the nuclear compartment, elevate temperature induces DNA fragmentation characteristic of apoptosis. This form of cell death is a phenomenon regulated throughout the preimplantation embryonic development, since high temperatures do not trigger apoptosis in embryos of two or four cells. Heat-induced apoptosis in embryos > 16 cells can be seen as a quality control mechanism for removing damaged blastomeres, sinc...
O objetivo desse estudo foi avaliar a eficiência do protocolo de pré-sincronização da ovulação com progesterona (P4) injetável de longa ação em novilhas e a influência de seus efeitos sobre a taxa de prenhez do protocolo de inseminação artificial em tempo fixo (IATF) J-Synch adaptado. Novilhas Nelore (n=638; 21,5±3,1 meses e 295±23,3 kg) foram classificadas por ultrassonografia nos D-22 e D-12 em púberes (presença de corpo lúteo (CL) no D-22 e/ou D-12) ou pré-púberes (ausência de CL em ambas avaliações) e receberam 150 mg de P4 de longa ação I.M. no D-22 (tratadas) ou não (controle), perfazendo arranjo fatorial 2X2. No D-12, apenas as novilhas tratadas, receberam 150 µg de D-cloprostenol (PGF, I.M.) e 1 mg de benzoato de estradiol (BE, I.M.). No D0, avaliou-se a ciclicidade das novilhas e todas foram sincronizadas de acordo com protocolo J-Synch adaptado: D0: dispositivo intravaginal de P4 (1 g) + 2 mg BE e 75 µg PGF; D6: remoção do dispositivo + 150 µg PGF. Uma detecção de estro foi realizada 48 h após a retirada dos dispositivos e as detectadas em estro foram inseminadas 12 h depois, ou em tempo fixo no D9, com aplicação I.M. de 10,5 µg acetato de buserelina. Exames ultrassonográficos foram realizados para mensurar o diâmetro do maior folículo e foi determinado um escore uterino (EU=1-6). O diagnóstico de gestação foi realizado no D39. Após 12 dias do tratamento (D0), menor proporção de novilhas, apresentando CL, foi verificada no grupo controle, quando comparada à de novilhas tratadas. Esta diferença foi mais acentuada nas pré-púberes (controle: 11,4% (23/199) vs. tratamento: 63,7% (136/215); P<0,0001), quando comparada às púberes (controle: 79,5% (92/113) vs. tratamento: 91,2% (102/111); P=0,002). A taxa de prenhez geral foi de 42,2% e tendeu a ser influenciada por tratamento (P=0,07). Novilhas do grupo controle apresentaram menor taxa de prenhez, de manifestação de estro 48 h após a retirada do dispositivo (P=0,02) e de ovulação antecipada (P=0,007), quando comparadas às novilhas tratadas (37,3% (119/312) vs. 44,7% (150/326); 15,9% (48/312) vs. 23,3% (52/224); 7,5% (23/258) vs. 15,4% (42/244), respectivamente). Para o diâmetro do folículo dominante, no momento da IATF (D9), houve interação entre status puberal e tratamento (P<0,05), sendo que as novilhas tratadas não diferiram entre si (pré-púberes: 11,1±0,13 mm vs. púberes: 11,1±0,17 mm; P=0,99). Para as novilhas do controle, as pré-púberes apresentaram menores diâmetros foliculares (10,9±0,13 mm), quando comparados aos das púberes (11,5±0,17 mm, P=0,03). A probabilidade de ciclicidade foi influenciada pelo diâmetro do folículo no D-22 e no D-12, para as novilhas prépúberes tratadas (P<0,05). O uso da P4 aumentou o diâmetro folicular e o EU, no D-12, para as novilhas pré-púberes tratadas, quando comparadas às controles (P<0,05). A taxa de prenhez foi maior nas novilhas que manifestaram estro 48 h após a retirada do dispositivo e foram inseminadas com 60h (60h: 66,1% (85/124); IATF: 34,8% (185/515)), e nas novilhas que apresentaram CL no D0 (com CL: 51,5% (1...
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