Lipopolysaccharide effects on activation and apoptosis of primordial ovarian follicles in heifers / Efeitos de lipopolissacarídeos na ativação e apoptose de folículos ovarianos primordiais em novilhas

The objective of the present study was to evaluate the effect of lipopolysaccharide (LPS) administration on activation and apoptosis of primordial follicles. There was no difference in the total number of follicles as well as in the different types of follicles. Furthermore, the LPS challenge didn't modulate the expression of genes related with ovarian reserve (HAM), oocyte survival (Survivin), activation rate (Pten, KIT, KITL1, KITL2, AKT1, SIRT1), and follicular abnormalities. Therefore, the LPS exposure with 24h interval had no effect on activation rate and primordial follicles abnormalities, and also had no effect on expression of anti-apoptotic genes and genes related with ovarian reserve, oocyte survival, activation rate, and primordial follicles abnormalities.

O objetivo do presente estudo foi avaliar o efeito da administração de lipopolissacarídeo (LPS) na ativação e a apoptose de folículos primordiais. Dez novilhas saudáveis (Bos taurus taurus), com idade média de 14 meses, alojadas em sistema de confinamento e alimentadas com TMR, foram utilizadas neste experimento. Os animais foram distribuídos aleatoriamente em dois grupos: grupo LPS (LPS; n = 5), que recebeu duas injeções intravenosas de 0,5µg/kg de peso corporal de lipopolissacarídeo (Sigma Aldrich®) diluído em 2mL de solução salina (0,9% de NaCl), com intervalo de 24h; e grupo controle (CTR; n = 5), que recebeu duas injeções intravenosas de 2mL de solução salina (0,9% de NaCl), com intervalo de 24h. A primeira injeção de LPS foi realizada no d 1, e no d 5 os animais foram abatidos, os ovários foram pesados e as amostras dos ovários foram coletadas para avaliação histológica e molecular. Não houve diferença no número total de folículos, bem como nos diferentes tipos de folículos. Além disso, o desafio com LPS não modulou a expressão de genes relacionados à reserva ovariana (HAM), à sobrevivência oocitária (Survivin), à taxa de ativação (Pten, KIT, KITL1, KITL2, AKT1, SIRT1) e às anormalidades foliculares. Portanto, a exposição ao LPS com intervalo de 24h não teve efeito sobre a taxa de ativação e as anormalidades dos folículos primordiais, bem como não teve efeito sobre a expressão de genes antiapoptóticos e de genes relacionados com a reserva ovariana, a sobrevivência oocitária, a taxa de ativação e as anormalidades dos folículos primordiais.

Induction of lactation in dairy heifers: milk production, inflammatory and metabolic aspects / Indução de lactação em novilhas leiteiras: produção de leite, aspectos inflamatórios e metabólicos

The aim of this study was to evaluate the metabolic, inflammatory, and hepatic aspects, as well as the milk yield in heifers submitted to protocol for induction of lactation compared to primiparous cows. Sixty Holstein heifers were selected and enrolled into two groups: Control (n= 30), pregnant heifers and Induction heifers (n= 30), non-pregnant femeales, submitted to a lactation induction protocol. Blood samples were collected at: pre-lactation period (weeks -3, -2 and -1) and post-lactation period (weeks 1, 2 and 3), aiming to evaluate glucose, non-esterified fatty acids, paraoxonase-1, albumin, ALT, GGT and cortisol. The protocol efficiently induced lactation in all the heifers, which produced 74.54% of the total production of milk from primiparous cows. In the pre-lactation period, induced animals presented higher concentrations of non-esterified fatty acids than the Control heifers, and the opposite was observed in the post lactation period. In both moments albumin and ALT were lower in the Induction group, and paraoxonase-1 activity and GGT concentrations were higher, compared to the Control. Thus, lactation induction protocol is efficient to initiate milk production in dairy heifers with no considerable changes in energetic, metabolic and hepatic profile when compared to heifers in physiological lactation.(AU)

O objetivo deste estudo foi avaliar os perfis metabólico, inflamatório, hepático e a produção de leite de novilhas induzidas à lactação comparadas a primíparas. Sessenta novilhas da raça Holandês foram selecionadas e alocadas em grupos: controle (n=30), novilhas prenhas, e indução (n=30), novilhas vazias submetidas a um protocolo de indução de lactação. As amostras de sangue foram coletadas nas semanas -3, -2 e -1 (pré-lactação) e nas semanas 1, 2 e 3 (pós-início de lactação) para avaliação de glicose, ácidos graxos não esterificados, paraoxonase-1, albumina, ALT, GGT e cortisol. O protocolo induziu eficientemente a lactação em todas as novilhas, que produziram 74,54% da produção total de leite do controle. No período pré-lactação, o grupo indução apresentou maiores concentrações de ácidos graxos não esterificados que o controle, e o oposto foi observado pós-lactação. Em ambos os momentos, albumina e ALT foram menores no grupo indução, e a atividade da paraoxonase-1 e as concentrações de GGT foram maiores, em comparação ao controle. Assim, o protocolo de indução de lactação foi eficiente para iniciar a produção de leite em novilhas induzidas, além de terem sido observadas alterações nos perfis energético, metabólico e hepático em comparação a novilhas em lactação fisiológica.(AU)

Induction of lactation in dairy heifers: milk production, inflammatory and metabolic aspects / Indução de lactação em novilhas leiteiras: produção de leite, aspectos inflamatórios e metabólicos

The aim of this study was to evaluate the metabolic, inflammatory, and hepatic aspects, as well as the milk yield in heifers submitted to protocol for induction of lactation compared to primiparous cows. Sixty Holstein heifers were selected and enrolled into two groups: Control (n= 30), pregnant heifers and Induction heifers (n= 30), non-pregnant femeales, submitted to a lactation induction protocol. Blood samples were collected at: pre-lactation period (weeks -3, -2 and -1) and post-lactation period (weeks 1, 2 and 3), aiming to evaluate glucose, non-esterified fatty acids, paraoxonase-1, albumin, ALT, GGT and cortisol. The protocol efficiently induced lactation in all the heifers, which produced 74.54% of the total production of milk from primiparous cows. In the pre-lactation period, induced animals presented higher concentrations of non-esterified fatty acids than the Control heifers, and the opposite was observed in the post lactation period. In both moments albumin and ALT were lower in the Induction group, and paraoxonase-1 activity and GGT concentrations were higher, compared to the Control. Thus, lactation induction protocol is efficient to initiate milk production in dairy heifers with no considerable changes in energetic, metabolic and hepatic profile when compared to heifers in physiological lactation.(AU)

O objetivo deste estudo foi avaliar os perfis metabólico, inflamatório, hepático e a produção de leite de novilhas induzidas à lactação comparadas a primíparas. Sessenta novilhas da raça Holandês foram selecionadas e alocadas em grupos: controle (n=30), novilhas prenhas, e indução (n=30), novilhas vazias submetidas a um protocolo de indução de lactação. As amostras de sangue foram coletadas nas semanas -3, -2 e -1 (pré-lactação) e nas semanas 1, 2 e 3 (pós-início de lactação) para avaliação de glicose, ácidos graxos não esterificados, paraoxonase-1, albumina, ALT, GGT e cortisol. O protocolo induziu eficientemente a lactação em todas as novilhas, que produziram 74,54% da produção total de leite do controle. No período pré-lactação, o grupo indução apresentou maiores concentrações de ácidos graxos não esterificados que o controle, e o oposto foi observado pós-lactação. Em ambos os momentos, albumina e ALT foram menores no grupo indução, e a atividade da paraoxonase-1 e as concentrações de GGT foram maiores, em comparação ao controle. Assim, o protocolo de indução de lactação foi eficiente para iniciar a produção de leite em novilhas induzidas, além de terem sido observadas alterações nos perfis energético, metabólico e hepático em comparação a novilhas em lactação fisiológica.(AU)

Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

Mutations in growth and differentiation factor9 (GDF9) gene are associated to sterility or,paradoxically, increased ovulation rate in ewes. Despiteits importance, the exact function of GDF9 in ovarianphysiology is still poorly understood. This study aimedto investigate GDF9 function during dominant folliclegrowth and its regulation in follicular fluid. Theregulation of GDF9 receptors in GnRH/LH-stimulatedgranulosa cells was also investigated. In a firstexperiment, a new follicular wave was induced and theintrafollicular GDF9 treatment into the largest growingfollicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml(n = 4) had no effect on follicular growth, estrusmanifestation and ovulation compared to control (PBSinjected)follicles (n = 3). In a second experiment,follicles were obtained just after follicular deviation(day 4 after follicular emergence) and the abundance ofGDF9 in follicular fluid did not differ between healthydominant (n = 4) and atretic subordinate follicles (n =4), as assessed by western blot analysis. Finally, mRNAexpression of BMPR2 and TGFBR1 receptors wasevaluated in granulosa cells obtained from preovulatoryfollicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 hafter i.m. GnRH administration (n = 4-5follicles/moment). Both receptors were significantly upregulated 12 h after GnRH treatment. Present results donot confirm the hypothesis that GDF9 inhibits dominantfollicle growth and suggests a minor role in determiningfollicle fate. In the other hand, GDF9 receptorsregulation in GnRH/LH-stimulated granulosa cellsprovides the first in vivo evidence of its involvement inthe complex cascade of events that culminates inovulation and luteinization in cattle.(AU)

Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

Mutations in growth and differentiation factor9 (GDF9) gene are associated to sterility or,paradoxically, increased ovulation rate in ewes. Despiteits importance, the exact function of GDF9 in ovarianphysiology is still poorly understood. This study aimedto investigate GDF9 function during dominant folliclegrowth and its regulation in follicular fluid. Theregulation of GDF9 receptors in GnRH/LH-stimulatedgranulosa cells was also investigated. In a firstexperiment, a new follicular wave was induced and theintrafollicular GDF9 treatment into the largest growingfollicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml(n = 4) had no effect on follicular growth, estrusmanifestation and ovulation compared to control (PBSinjected)follicles (n = 3). In a second experiment,follicles were obtained just after follicular deviation(day 4 after follicular emergence) and the abundance ofGDF9 in follicular fluid did not differ between healthydominant (n = 4) and atretic subordinate follicles (n =4), as assessed by western blot analysis. Finally, mRNAexpression of BMPR2 and TGFBR1 receptors wasevaluated in granulosa cells obtained from preovulatoryfollicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 hafter i.m. GnRH administration (n = 4-5follicles/moment). Both receptors were significantly upregulated 12 h after GnRH treatment. Present results donot confirm the hypothesis that GDF9 inhibits dominantfollicle growth and suggests a minor role in determiningfollicle fate. In the other hand, GDF9 receptorsregulation in GnRH/LH-stimulated granulosa cellsprovides the first in vivo evidence of its involvement inthe complex cascade of events that culminates inovulation and luteinization in cattle.

Indução artificial de lactação em bovinos: histórico e evolução / Artificial induction of lactation in cattle: history and evolution

O descarte precoce de vacas leiteiras por problemas reprodutivos, por falhas no manejo e por outrosfatores que levem as vacas a encerrarem uma lactação sem estarem prenhes aumenta os custos e diminui arentabilidade da produção de leite. Como alternativa, pode-se utilizar a indução artificial de lactação, prática demanejo que mimetiza o perfil endócrino da vaca no periparto com o objetivo de induzir a síntese láctea pelaglândula mamária. Os protocolos de indução em bovinos são estudados desde a década de 40, e os primeirosestudos envolviam aplicações hormonais por até nove meses. Nos anos 70, houve uma evolução significativa,quando foi estabelecido que sete dias de aplicação de hormônios esteroides eram suficientes para induzir asvacas a lactarem. Protocolos utilizando progesterona e estradiol como base foram testados ao longo dos anos e,atualmente, os protocolos comerciais têm a duração de aproximadamente 21 dias e utilizam aplicações degrandes volumes de hormônios em manejos diários. Embora os protocolos possibilitem boa taxa de resposta e deprodução de leite, até o presente momento não foram avaliados os impactos nos animais. O objetivo destarevisão é demonstrar a evolução dos protocolos, suas aplicações, desvantagens, possíveis soluções eperspectivas.(AU)

The early culling of dairy cows due to reproductive failure, management problems and other factorsthat lead the dairy cows to be dried-off without being pregnant, increase costs and decrease profitability of milkproduction. A possible alternative is the use of artificial lactation induction, a tool that mimics peripartumendocrine profiles with the aim of inducing milk synthesis from the mammary gland. Protocols of induction oflactation in cattle have been studied since the 1940s, and the first studies consisted of hormonal applications fornine months. In the 1970s, there was a significant evolution, when it was established that seven days of steroidhormones application were sufficient to induce lactation. Since then, several protocols using progesterone andestradiol have been tested. Currently, commercial protocols lasts for 21 days and use large volumes ofhormones, requiring daily handling of animals. Although current protocols are efficient, enabling good responserate and milk production, so far the degree of discomfort to which animals are exposed is unknown. The purposeof this review is to provide background information on the evolution of protocols, their applications, drawbacks,possible solutions and prospects.(AU)

Indução artificial de lactação em bovinos: histórico e evolução / Artificial induction of lactation in cattle: history and evolution

O descarte precoce de vacas leiteiras por problemas reprodutivos, por falhas no manejo e por outrosfatores que levem as vacas a encerrarem uma lactação sem estarem prenhes aumenta os custos e diminui arentabilidade da produção de leite. Como alternativa, pode-se utilizar a indução artificial de lactação, prática demanejo que mimetiza o perfil endócrino da vaca no periparto com o objetivo de induzir a síntese láctea pelaglândula mamária. Os protocolos de indução em bovinos são estudados desde a década de 40, e os primeirosestudos envolviam aplicações hormonais por até nove meses. Nos anos 70, houve uma evolução significativa,quando foi estabelecido que sete dias de aplicação de hormônios esteroides eram suficientes para induzir asvacas a lactarem. Protocolos utilizando progesterona e estradiol como base foram testados ao longo dos anos e,atualmente, os protocolos comerciais têm a duração de aproximadamente 21 dias e utilizam aplicações degrandes volumes de hormônios em manejos diários. Embora os protocolos possibilitem boa taxa de resposta e deprodução de leite, até o presente momento não foram avaliados os impactos nos animais. O objetivo destarevisão é demonstrar a evolução dos protocolos, suas aplicações, desvantagens, possíveis soluções eperspectivas.

The early culling of dairy cows due to reproductive failure, management problems and other factorsthat lead the dairy cows to be dried-off without being pregnant, increase costs and decrease profitability of milkproduction. A possible alternative is the use of artificial lactation induction, a tool that mimics peripartumendocrine profiles with the aim of inducing milk synthesis from the mammary gland. Protocols of induction oflactation in cattle have been studied since the 1940s, and the first studies consisted of hormonal applications fornine months. In the 1970s, there was a significant evolution, when it was established that seven days of steroidhormones application were sufficient to induce lactation. Since then, several protocols using progesterone andestradiol have been tested. Currently, commercial protocols lasts for 21 days and use large volumes ofhormones, requiring daily handling of animals. Although current protocols are efficient, enabling good responserate and milk production, so far the degree of discomfort to which animals are exposed is unknown. The purposeof this review is to provide background information on the evolution of protocols, their applications, drawbacks,possible solutions and prospects.

Technique to simultaneously evaluate ram sperm morphology, acrosome and membrane integrity

Several laboratory methods to evaluate ram sperm quality have been developed. The combination of different fluorescent probes is suitable to simultaneously evaluate different sperm characteristics but the need fora fluorescent microscope restricts its use. The Aim of the present study was to evaluate the efficacy of Hypoosmotic Trypan Blue Giemsa (HTBG) staining to simultaneously detect sperm morphological abnormalities, plasma and acrosomal membrane integrity using phase contrast and fluorescence microscopy as the gold standards. Samples from twelve fresh ejaculates from three rams (4 ejaculates/ram) were used in the study. Sperm cells were evaluated using HTBG, phase contrast (PC) and fluorescent (FLUO) techniques. HTBG was more effective in detecting sperm defects when compared to PC (P < 0.05). No significant differences (P > 0.05) were observed between HTBG and FLUO in the assessment of plasmatic membrane and acrosome integrity. High correlation between HTBG and FLUO techniques was observed when assessing plasma membrane and acrosome (R = 0.97 and 0.96, respectively). In conclusion, the HTBG staining is suitable to assess ram sperm morphology, plasma and acrossomal membrane integrity simultaneously.(AU)

Technique to simultaneously evaluate ram sperm morphology, acrosome and membrane integrity

Several laboratory methods to evaluate ram sperm quality have been developed. The combination of different fluorescent probes is suitable to simultaneously evaluate different sperm characteristics but the need fora fluorescent microscope restricts its use. The Aim of the present study was to evaluate the efficacy of Hypoosmotic Trypan Blue Giemsa (HTBG) staining to simultaneously detect sperm morphological abnormalities, plasma and acrosomal membrane integrity using phase contrast and fluorescence microscopy as the gold standards. Samples from twelve fresh ejaculates from three rams (4 ejaculates/ram) were used in the study. Sperm cells were evaluated using HTBG, phase contrast (PC) and fluorescent (FLUO) techniques. HTBG was more effective in detecting sperm defects when compared to PC (P 0.05) were observed between HTBG and FLUO in the assessment of plasmatic membrane and acrosome integrity. High correlation between HTBG and FLUO techniques was observed when assessing plasma membrane and acrosome (R = 0.97 and 0.96, respectively). In conclusion, the HTBG staining is suitable to assess ram sperm morphology, plasma and acrossomal membrane integrity simultaneously.

Control of ovulation in mammals

The ovulatory L H surge induces dramatic molecular and structural changes in follicular environment, culminating with follicle rupture and release of a mature oocyte. This review addresses the pre - LH surge events associated with the ovulatory process, such as dominant fol licle differentiation and acquisition of ovulatory capacity, as well as autocrine and paracrine factors induced after the LH surge. Over the last few years, our research group has focused on studying the contribution of renin - angiotensin system in ovulatio n and the participation of Ang II and Ang - (1 - 7) and their interactions with factors essential for ovulation, which are also discussed.

Control of ovulation in mammals

The ovulatory L H surge induces dramatic molecular and structural changes in follicular environment, culminating with follicle rupture and release of a mature oocyte. This review addresses the pre - LH surge events associated with the ovulatory process, such as dominant fol licle differentiation and acquisition of ovulatory capacity, as well as autocrine and paracrine factors induced after the LH surge. Over the last few years, our research group has focused on studying the contribution of renin - angiotensin system in ovulatio n and the participation of Ang II and Ang - (1 - 7) and their interactions with factors essential for ovulation, which are also discussed.(AU)

Role of angiotensin II on follicle development and ovulation

We will address, in this review, the role of angiotensin II (AngII) on follicular development and ovulation. Over the last few years, our research group has focused on studying the contribution of renin-angiotensin system in antral follicle development and ovulation and a new concept of local regulation has been established using cattle as a model. We previously demonstrated that AT1 and AT2 receptors are expressed in both granulosa and theca cells. The abundance of AT2 mRNA in granulosa cells was higher in healthy compared with atretic follicles, whereas both receptors in theca cells and AT1 in granulosa cells did not change. Granulosa cells cultured with hormones that stimulate estradiol secretion increased AT2 mRNA and protein levels, whereas fibroblast growth factors (FGF-7 and 10) inhibited estradiol secretion and AT2 protein levels. We also found that the concentration of AngII increases in dominant follicle at expected time for follicular deviation. Transvaginal ultrasound has been used for intrafollicular injection to understand the regulation of follicular wave and ovulation. With this in vivo model, we have demonstrated that AngII-receptor blocker inhibits follicular growth and decreases estradiol concentration in follicular fluid and downregulates mRNA expression of genes involved in follicular development. Moreover, intrafollicular injection of AngII or AT2-specific agonist prevented the expected atresia of the second largest follicle, which continued to grow at a rate similar to the dominant follicle for 24 h. These findings have provided evidence that AngII plays an important role in follicle development. In regarding to ovulation, we demonstrated that AngII antagonists block ovulation in cattle when intrafollicularly injected at 0 and 6 h after applying GnRH agonist. Ovulation was also inhibited by AT2- but not by AT1-AngII receptor antagonist. Furthermore, AngII stimulates an enhancement in mRNA abundance of genes involved in ovulation. In addition, AngII stimulates genes involved in extracellular remodeling and follicular wall rupture. In conclusion, our data from in vitro and in vivo studies have demonstrated that AngII plays a pivotal role in the antral follicle development and early mechanism of ovulation via the AT2 receptor subtype in cattle.

Role of angiotensin II on follicle development and ovulation

We will address, in this review, the role of angiotensin II (AngII) on follicular development and ovulation. Over the last few years, our research group has focused on studying the contribution of renin-angiotensin system in antral follicle development and ovulation and a new concept of local regulation has been established using cattle as a model. We previously demonstrated that AT1 and AT2 receptors are expressed in both granulosa and theca cells. The abundance of AT2 mRNA in granulosa cells was higher in healthy compared with atretic follicles, whereas both receptors in theca cells and AT1 in granulosa cells did not change. Granulosa cells cultured with hormones that stimulate estradiol secretion increased AT2 mRNA and protein levels, whereas fibroblast growth factors (FGF-7 and 10) inhibited estradiol secretion and AT2 protein levels. We also found that the concentration of AngII increases in dominant follicle at expected time for follicular deviation. Transvaginal ultrasound has been used for intrafollicular injection to understand the regulation of follicular wave and ovulation. With this in vivo model, we have demonstrated that AngII-receptor blocker inhibits follicular growth and decreases estradiol concentration in follicular fluid and downregulates mRNA expression of genes involved in follicular development. Moreover, intrafollicular injection of AngII or AT2-specific agonist prevented the expected atresia of the second largest follicle, which continued to grow at a rate similar to the dominant follicle for 24 h. These findings have provided evidence that AngII plays an important role in follicle development. In regarding to ovulation, we demonstrated that AngII antagonists block ovulation in cattle when intrafollicularly injected at 0 and 6 h after applying GnRH agonist. Ovulation was also inhibited by AT2- but not by AT1-AngII receptor antagonist. Furthermore, AngII stimulates an enhancement in mRNA abundance of genes involved in ovulation. In addition, AngII stimulates genes involved in extracellular remodeling and follicular wall rupture. In conclusion, our data from in vitro and in vivo studies have demonstrated that AngII plays a pivotal role in the antral follicle development and early mechanism of ovulation via the AT2 receptor subtype in cattle.(AU)