RESUMO: Os picos de LH abaixo do ideal podem bloquear ou retardar a ovulação durante programas de manejo reprodutivo em bovinos. Várias vias de sinalização podem aumentar o sinal de LH, incluindo em especial fatores de crescimento. No presente estudo, determinamos se o fator de crescimento de fibroblastos 18 (FGF18), que estimula algumas ações do pico de LH nas células da granulosa, pode potencializar a ação de uma dose sub fisiológica de LH em um sistema de cultivo de células da granulosa bovina (GC). A adição de FGF18 recombinante aumentou a concentração de progesterona (P4) no meio de cultura. A combinação de LH e FGF18 no meio de cultura aumentou a abundância de mRNA para EREG, EGR1 e EGR3, proteínas que são moduladoras precoces da cascata pré-ovulatória induzida por LH. Este estudo demonstrou que o FGF18 pode ser um cofator na regulação da cascata pré-ovulatória em células da granulosa bovina. RESUMO bovinos 1 FGF18, FGF , (FGF18) GC. GC . (GC) FGF1 P4 P (P4 EREG EGR EGR3 préovulatória pré ovulatória (FGF18 (GC (P (FGF1 (FGF

ABSTRACT: Suboptimal LH surges can lead to delayed or absent ovulation during reproductive management programs in cattle. Several signaling pathways may enhance the LH signal, potentially including growth factors. In the present study, we determined whether Fibroblast growth factor 18 (FGF18), which mimics some actions of the LH surge on granulosa cells, may potentiate the action of a suboptimal dose of LH using an established bovine granulosa cell (GC) culture system. Addition of recombinant FGF18 increased the concentration of progesterone (P4) in the culture medium. A combination of LH and FGF18 in the culture medium increased the abundance of mRNA encoding EREG, EGR1 and EGR3, proteins that are early modulators of the LH-induced pre-ovulatory cascade. This study demonstrated that FGF18 may be a co-factor in the regulation of the pre-ovulatory cascade in bovine ovarian granulosa cells. ABSTRACT cattle signal factors 1 FGF18, FGF , (FGF18) cells GC (GC system FGF1 P4 P (P4 EREG EGR EGR3 LHinduced induced preovulatory pre ovulatory cofactor co (FGF18 (P (FGF1 (FGF

Abstract We hypothesized that the hCG modulates the expression of IFNT-pathway and ISGs in bovine endometrium during early pregnancy. The aim of the current study is to evaluate the effect of hCG on IFNT-pathway signals and ISGs expression in endometrial cells. For this, 29 non-lactating cross-bread cows were used in the study and submitted to a 9-day fixed-time artificial insemination (FTAI) protocol. The day of the AI was considered Day 0 (D0), and five days (D5) after the FTAI, the cows were allocated into two groups: Control and hCG group, when a hCG group received a single dose of 2.500UI of hCG. On day 18 after FTAI (D18) cows were slaughtered and endometrial tissue samples were collected. There was no difference between the embryo recovery rate of the cows in C compared to the hCG. The hCG group increased the accessory corpus luteum formation rate. The hCG resulted in greater serum progesterone concentration in the hCG group compared to the C on Day 14. Only the expression of IFNAR2 and STAT1 were upregulated on pregnant cows of the hCG group compared to the C group. The pathway genes (JAK1, STAT2, and IRF9) were not regulated. The mRNA abundance of ISG15, MX1, MX2, and OAS1 was upregulated in pregnant cows for hCG group, compared to C group. The results show that the administration of hCG, 5 days after AI, in addition to increasing the serum progesterone, modulates the expression of IFNT-pathway and ISGs on bovine endometrium on Day 18 of pregnancy. IFNTpathway IFNT pregnancy cells this 2 nonlactating non lactating crossbread cross bread 9day 9 fixedtime fixed time (FTAI protocol D0, D0 D , (D0) D5 (D5 groups 2500UI UI 500UI 1 D18 (D18 collected 14 IFNAR STAT JAK1, JAK1 JAK (JAK1 STAT2 IRF9 IRF regulated ISG15 ISG MX1 MX MX2 OAS (D0 (D D1 (D1 (JAK ISG1

RESUMO: O objetivo deste estudo foi avaliar a relação entre balanço energético e concentração sanguínea de alguns metabólitos relevantes durante o início da lactação em vacas leiteiras primíparas com diferentes desempenhos reprodutivos. Vinte vacas leiteiras holandesas primíparas foram agrupadas de acordo com o intervalo parto-concepção em precoce (<100 dias), intermediário (100 - 150 dias) e tardio (> 150 dias); e foram avaliados no dia -7 pré-parto, e nos dias 28, 56 e 90 dias pós-parto. Todos os grupos apresentaram balanço energético positivo, concentrações séricas de HDL, N-uréia e progesterona sem diferença nos dias 28 e 56 de lactação. Aos 90 dias de lactação todas vacas apresentaram o balanço energético negativo, porém o grupo de vacas precoce foi menos intenso (P < 0.05) (-1.09 versus -2.15 e -1.86 Mcal/dia), menor concentração sérica de N-uréia (3.28 versus 4.22 e 4.88 mg/dL) e colesterol-HDL (0.84 versus 1.59 e 1.46 mmol/L) e maior concentração sérica de progesterona (8.13 versus 4.54 e 1.83 ng/mL), que vacas dos grupos intermediário e tardio, respectivamente. Em conclusão, diferente do observado usualmente em vacas multíparas, o balanço energético durante as primeiras semanas de lactação não foi um fator determinante do intervalo parto concepção das vacas primíparas do presente estudo. De fato, a condição nutricional e as concentrações séricas de N-ureia e progesterona após 56 dias de lactação sugere ter impacto no atraso da concepção destas vacas. RESUMO reprodutivos partoconcepção <100 100 (<10 dias, , (10 15 > ( 7 préparto, préparto pré parto, pré-parto 5 9 pósparto. pósparto pós parto. pós-parto positivo HDL Nuréia N uréia 2 negativo P 0.05 005 0 05 1.09 109 1 09 (-1.0 2.15 215 -2.1 1.86 186 86 -1.8 Mcal/dia, Mcaldia Mcal/dia Mcal Mcal/dia) 3.28 328 3 (3.2 422 4 22 4.2 488 88 4.8 mg/dL mgdL mg dL colesterolHDL colesterol 0.84 084 84 (0.8 159 59 1.5 146 46 1.4 mmol/L mmolL mmol L 8.13 813 8 13 (8.1 454 54 4.5 183 83 1.8 ng/mL, ngmL ng/mL ng mL ng/mL) respectivamente conclusão multíparas fato Nureia ureia <10 10 (<1 (1 0.0 00 1.0 (-1. 2.1 21 -2. 18 -1. 3.2 32 (3. 42 4. 48 0.8 08 (0. 1. 14 8.1 81 (8. 45 <1 (< 0. (-1 2. -2 -1 3. (3 (0 8. (8 (-

ABSTRACT: This study identified the relationship between energy balance and some blood metabolites in primiparous dairy cows with different calving conception interval. Twenty primiparous Holstein-Friesian dairy cows were grouped according to the interval (days) from calving to conception into early-conception (early-c; <100 days), mid-conception group (mid-c; 100 - 150 days) or late-conception (late-c; >150 days) group; and evaluated on day -7 prior to parturition, and days 28, 56 and 90 postpartum. All groups presented positive energy balance, serum concentrations of HDL, urea-N and progesterone with no difference on days 28 and 56 of lactation. On day 90 of lactation all the cows presented negative energy balance; however, the early-c group presented a less intense (P < 0.05) net energy balance (-1.09 versus -2.15 and -1.86 Mcal/day), lower concentrations of serum urea-N (3.28 versus 4.22 and 4.88 mg/dL) and HDL cholesterol (0.84 versus 1.59 and 1.46 mmol/L) and greater serum concentration of progesterone (8.13 versus 4.54 and 1.83 ng/mL) than cows of mid-c and late-c groups, respectively. In conclusion, different from those usually observed for multiparous dairy cows, the energy balance during the first weeks of lactation was not a determining factor affecting the calving conception interval of primiparous dairy cows in the present study. Instead, the nutritional status and the serum concentration of urea-N and progesterone at a later stage, after 56 days of lactation, suggest having a role in delaying these cows’ pregnant status. ABSTRACT HolsteinFriesian Holstein Friesian (days earlyconception early earlyc c (early-c <10 days, , midconception mid midc (mid-c 10 15 lateconception late latec (late-c >15 7 parturition 5 9 postpartum ureaN urea N 2 however P 0.05 005 0 05 1.09 109 1 09 (-1.0 2.15 215 -2.1 1.86 186 86 -1.8 Mcal/day, Mcalday Mcal/day Mcal Mcal/day) 3.28 328 3 (3.2 422 4 22 4.2 488 88 4.8 mg/dL mgdL mg dL 0.84 084 84 (0.8 159 59 1.5 146 46 1.4 mmol/L mmolL mmol L 8.13 813 8 13 (8.1 454 54 4.5 183 83 1.8 ng/mL ngmL ng mL respectively conclusion Instead stage <1 >1 0.0 00 1.0 (-1. 2.1 21 -2. 18 -1. 3.2 32 (3. 42 4. 48 0.8 08 (0. 1. 14 8.1 81 (8. 45 > 0. (-1 2. -2 -1 3. (3 (0 8. (8 (- (

Abstract The interaction between early embryo and maternal immune system for the establishment of pregnancy is the focus of several studies; however, it remains unclear. The maternal immune response needs to keep a balance between avoiding any damage to the conceptus and maintaining its function in combating microbes as well. When conceptus-maternal crosstalk cannot achieve this balance, pregnancy losses might occur. Intercommunication between mother and conceptus is fundamental during early pregnancy to dictate the outcome of pregnancy. In ruminants, the embryo reacts with the maternal system mainly via interferon tau (IFNT) release. IFNT can act locally on the embryo and endometrial cells and systemically in several tissues and cells to regulate their response via the expression of interferon-stimulated genes (ISGs). Also, IFNT can induce the expression of inflammatory-related genes in immune cells. Day 7 embryo induces a shift in the maternal immune response towards anti-inflammatory (Th2) immune responses. During maternal recognition of pregnancy, peripheral mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) express markers that configure an anti-inflammatory response. However, PMNs response is more sensitive to the effects of IFNT. PMNs are more likely to express interferon-stimulated genes (ISGs), transforming growth factor-beta (TGFB), interleukin 10 (IL10), and arginase-1 (ARG1), configuring one of the most rapid immune responses to early pregnancy. This review focus on the local and peripheral immune responses during early pregnancy in ruminants, mainly the PMNs function in the immune system.

Abstract The transforming growth factors beta (TGFβ) are local factors produced by ovarian cells which, after binding to their receptors, regulate follicular deviation and ovulation. However, their regulation and function during corpus luteum (CL) regression has been poorly investigated. The present study evaluated the mRNA regulation of some TGFβ family ligands and their receptors in the bovine CL during induced luteolysis in vivo. On day 10 of the estrous cycle, cows received an injection of prostaglandin F2α (PGF) and luteal samples were obtained from separate groups of cows (n= 4-5 cows per time-point) at 0, 2, 12, 24 or 48 h after treatment. Since TGF beta family comprises more than 30 ligands, we focused in some candidates genes such as activin receptors (ACVR-1A, -1B, -2A, -2B) AMH, AMHR2, BMPs (BMP-1, -2, -3, -4, -6 and -7), BMP receptors (BMPR-1A, -1B and -2), inhibin subunits (INH-A, -BA, -BB) and betaglycan (TGFBR3). The mRNA levels of BMP4, BMP6 and INHBA were higher at 2 h after PGF administration (P<0.05) in comparison to 0 h. The relative mRNA abundance of BMP1, BMP2, BMP3, BMP4, BMP6, ACVR1B, INHBA and INHBB was upregulated up to 12 h post PGF (P<0.05). On the other hand, TGFBR3 mRNA that codes for a reservoir of ligands that bind to TGF-beta receptors, was lower at 48 h. In conclusion, findings from this study demonstrated that genes encoding several TGFβ family members are expressed in a time-specific manner after PGF administration.

RESUMO: Na cartilagem saudável, os condrócitos mantêm a expressão de colágenos e proteoglicanos, sendo sensíveis a fatores de crescimento e citocinas que aumentam ou reduzem a síntese de colágeno tipo II. Na osteoartrite, citocinas pró-inflamatórias, como a IL-6, estimulam a expressão de metaloproteinases (MMP) e reduzem a síntese de agrecano. O uso de condroprotetores, como o Plasma Rico em Plaquetas (PRP) e triancinolona (TA) é uma alternativa para se reduzir a progressão do dano articular. Neste estudo foram usados condrócitos extraídos das articulações metacarpofalangeanas de equinos saudáveis. As células foram tratadas in vitro com TA ou PRP. Não foram observadas diferenças entre os tratamentos comparando-se com o grupo controle quanto à expressão genética de MMP-9, MMP-13, IL-6 e ACAN (p<0,05). Assim, pode-se sugerir que os tratamentos não foram deletérios ao cultivo de condrócitos, uma vez que não estimularam a síntese de MMP e IL-6 e nem causaram alterações no ACAN.

ABSTRACT: In healthy cartilage, chondrocytes maintain an expression of collagens and proteoglycans and are sensitive to growth factors and cytokines that either enhance or reduce type II collagen synthesis. In osteoarthritis, pro-inflammatory cytokines, such as IL-6, induce overexpression of metalloproteinases (MMP) and decreasing synthesis of aggrecan. Use of chondroprotectors agents, such as Platelet-Rich Plasma (PRP) and triamcinolone (TA) are alternatives to reduce the progression of joint damage. In this study, we used chondrocytes extracted from metacarpophalangeal joints of healthy horses as the experimental model. Cells were treated in vitro with PRP or TA. No differences were observed between these treatments in comparison to the control group when the expressions of MMP9, MMP13, IL-6 and ACAN genes were evaluated (P<0.05). With these results, we can suggest that the treatments were not deleterious to equine cultured chondrocyte, once they did not stimulate MMPs and IL-6 synthesis or caused changes in ACAN.

O objetivo deste estudo foi investigar a expressão de RNAm e a localização da proteína Grb10 em complexos cumulusoócito (CCO), oriundos de folículos bovinos em diferentes fases de desenvolvimento. Primeiramente, foi investigada a expressão de RNAm e relacionada com as taxas de maturação. CCOs oriundos de folículos com diâmetro de 1-3, 4-6, 6-8 e >8mm foram utilizados para avaliar a expressão de RNAm por PCR em tempo real e para analisar os estádios de maturação nuclear. Foi observado que os oócitos mais competentes para a progressão meiótica (oriundos de folículos com diâmetro de 6-8 e >8mm; P<0.05) tiveram menor expressão de RNAm para Grb10, quando comparados aos CCOs oriundos de folículos com 1-3 e 4-6mm (P<0.05). Posteriormente, foi realizada a técnica de imunofluorescência em CCOs oriundos de folículos de diferentes tamanhos (1-3, 4-6, 6-8 e >8mm) para investigar a localização da proteína Grb10. As amostras foram incubadas com o anticorpo primário: anti-Grb10 policlonal, produzido em coelho (1:100). O anticorpo primário foi detectado utilizando um anticorpo secundário, IgG anti-coelho, produzido em cabra, conjugado com Alexa Fluor 488 (1:500). A fluorescência foi detectada em todas as amostras analisadas, porém, menos evidente nos CCOs oriundos dos folículos maiores. Os resultados apresentados neste estudo caracterizam o gene Grb10 em CCOs de bovinos e fornecem evidências do seu envolvimento na maturação molecular do oócito.

The objective of this study was to investigate the mRNA expression and protein localization of Grb10 gene in bovine cumulus-oocyte complexes (COCs) from different follicle sizes. Firstly, it was investigated the mRNA expression to correlate with maturation rates. COCs from follicles at 1-3, 4-6, 6-8 and &gt;8mm were used to evaluate Grb10 gene expression by qRT-PCR assay and nuclear maturation rates. It was observed that more competent oocytes (from follicles at 6-8 and &gt;8mm; P&gt;0.05), had lower Grb10 mRNA expression levels when compared to the oocytes from follicles at 1-3 and 4-6mm (P&gt;0.05). After it was performed an immunofluorescence analysis in COCs from different follicle sizes (1-3, 4-6, 6-8 and &gt;8mm) to investigate Grb10 protein localization. Samples were incubated with primary antibody: Polyclonal rabbit anti-Grb10 (1:100). Primary antibody was detected using goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:500). Positive fluorescence signal was detected in all analyzed samples but less evident in COCs from largest follicles. These results characterized Grb10 gene in bovine COC and provide evidences for its involvement during oocyte molecular maturation.

O reconhecimento materno da gestação é o período em que o concepto sinaliza sua presença para a mãe. Em ruminantes, este período coincide com o alongamento do embrião e a máxima produção de interferon-tau (IFNT). O IFNT produzido pelo concepto age via parácrina no útero inibindo a expressão dos receptores de estrógenos (ESR1) e de ocitocina (OXTR) no epitélio luminal do endométrio, evitando, assim, a liberação de pulsos luteolíticos de prostaglandina F2 alfa (PGF2 ), hormonio responsável pelo início da luteólise. Além da sua ação durante o reconhecimento materno da gestação em ruminantes, o IFNT aumenta a expressão de vários genes estimulados por interferons (ISGs) no útero, no corpo lúteo (CL) e em células sanguíneas. Estudos recentes demonstraram que o IFNT possui ação endócrina no CL ovino e também estende o ciclo estral (pseudo gestação) além do dia 32 após a infusão de IFNT recombinante ovino (roIFNT) na veia uterina. A comprovação da saída de IFNT do útero pela veia uterina sugere que a ação endócrina do IFNT possa ser um mecanismo complementar ao mecanismo intrauterino de reconhecimento materno da gestação. A ação direta do IFNT em tecidos extrauterinos estimula a expressão de ISGs que, no CL, podem estar envolvidos com a resistência luteal à ação luteolítica da PGF2a.

Maternal recognition of pregnancy is the period when the conceptus signals its presence to the dam. In ruminants, it requires conceptus elongation, which coincides with maximum production of interferon-tau (IFNT). Conceptus IFNT acts in a paracrine manner silencing estrogen receptor alpha (ESR1) and oxytocin receptor (OXTR) in the luminal epithelium, thus preventing luteolytic prostaglandin F2 alpha (PGF2 ) pulses. Besides its role during maternal recognition of pregnancy, IFNT induces the expression of several interferon stimulated genes (ISGs) in the endometrium, corpus luteum (CL) and blood cells. Recently, it was suggested an endocrine role for IFNT during the period of maternal recognition of pregnancy in sheep. It was demonstrated that infusion of IFNT into the uterine vein can extend the estrous cycle beyond 32 days. This direct action of IFNT in extrauterine tissues induces ISGs expression, which might be involved in the rescue of the CL from the luteolytic effects of PGF2 pulses.

O objetivo deste estudo foi avaliar as causas de infertilidade em vacas de corte repetidoras de serviço, relacionando sinais clínicos com características laboratoriais post-mortem. A identificação dos fatores que afetam o desempenho reprodutivo pode fornecer uma fundamentação científica para auxiliar na decisão quanto ao destino de vacas repetidoras de serviço; tratamento ou descarte. Este estudo tem abordagem diferencial dos trabalhos que contemplam somente a ocorrência de alterações post-mortem do trato genital. A correlação das alterações post-mortem com o histórico clínico dos animais pode auxiliar na determinação da causa de repetição de serviço de animais sem alteração clínica. Foram utilizadas 130 vacas e novilhas cruzas que não conceberam depois de, no mínimo, três serviços. Todos os animais foram identificados e submetidos à exame clínico e coleta de sangue para cariotipagem. Foram incluídos no estudo apenas animais com histórico de falha reprodutiva. Os animais foram encaminhados para um abatedouro e foram realizados exames post-mortem como inspeção macroscópica do trato genital, cultura bacteriológica e estudo histopatológico do útero. Alterações uterinas foram predominantes seguidas de alterações de oviduto e ovário. Foi observada uma maior freqüência de alterações histopatológicas de útero que alterações clínicas. Os animais utilizados no estudo apresentaram alta freqüência de patologia uterina (95%), tanto inflamatórias (42,9%) quanto degenerativas (59,7%). Patologias diagnosticadas somente por exames post-mortem e laboratoriais como oclusão tubária (29,8%) e trissomia do par sexual também foram observadas (1/10 vacas). Portanto, este estudo evidencia a importância do exame laboratorial no diagnóstico das causas de infertilidade de vacas com histórico de repetição de serviço.

The objective of this study was to investigate the causes of the repeat breeder syndrome comparing clinical signs and postmortem findings in beef cows. The identification of factors affecting the reproductive tract can support decisions as to whether treatment of repeat breeder cows is justifiable than culling. Since all animals were submitted to clinical examination before being slaughtered, this study has a differential approach when compared with others, where genital tracts from abattoir were examined. In this study, 130 crossbred cows and heifers that have failed to conceive after three or more services were identified, submitted to a clinical examination and blood collection for karyotyping and sent to an abattoir. postmortem examinations included macroscopic evaluation of the genital tracts, bacteriology and histopathology of the uterus. Uterine alterations were predominant followed by oviduct and ovarian pathologies. Histopathological examination was more sensitive as a diagnostic tool than clinical examination. Repeat breeder cows had a predominance of uterine abnormalities (95%), such as inflammatory (42.9%) and degenerative (59.7%) conditions. Oviduct abnormalities were found in 29.8 % of animals. Furthermore, 1 out 10 karyotyped cows showed aneuploidy. Thus, this study stressed the importance of laboratory exams in the diagnosis of the causes of infertility of repeat breeder cows.