Influence of Lycium barbarum Extract Intake on Oxidative Stress in Wistar Rats

Abstract: This study aimed to analyze the effects of Goji Berry extract (GB, Lycium barbarum) gavage administration on liver tissue oxidative stress in Wistar rats as well as to identify and quantify the content of the major bioactive compounds of the fruit. Four diets were applied: SW - standard diet + water; SG - standard diet + Goji Berry extract (125 mg/kg of animal); PW - palatable diet + water; PG - palatable diet + Goji Berry extract (125 mg/kg of animal). Results showed a significant increase in catalase enzyme activity in the liver of rats treated with GB and also in those intaking the palatable diet without GB when compared to the SW group. An increased mRNA expression of this enzyme in the same tissue and groups was also verified. Regarding lipid peroxidation, the GB extract produced a significant decrease in the oxidation state in the SG and PG groups. The results also showed a significant amount of bioactive compounds in GB extract.

Anti-inflammatory Effect of a Goji Berry Extract (Lycium barbarum) in Rats Subjected to Inflammation by Lipopolysaccharides (LPS)

Abstract The present study aimed to evaluate the anti-inflammatory potential of a Lycium barbarum (L. barbarum) fruit extract in Wistar rats submitted to a palatable diet presenting systemic inflammation induced by lipopolysaccharides (LPS). Forty-two Wistar female rats (Rattus Novergicus) were used with 60 days old. The animals were feed for 60 days and divided in six groups (n=7): standard diet+water; standard diet+L. barbarum; palatable diet+water; palatable diet+L. barbarum; standard diet+water+LPS; standard diet+L. barbarum+LPS. A significant difference was shown between the analyzed groups concerning C-reactive protein, with the standard diet+water+LPS group presenting the highest inflammatory response in comparison to the other groups. Decreased inflammatory response was observed in the group administered a palatable diet along with the fruit extract when compared to the group that received only a palatable diet. Significant decrease in glutamic-oxaloacetic transaminase activity was observed in the standard diet+L. barbarum+LPS group compared to the standard diet+water group, as well as in the palatable diet+L. barbarum group compared to the palatable diet+water group. A significant increase in creatinine in the standard diet+water+LPS group was observed in according to the L. barbarum administration groups. The gene expression of the inflammatory markers genes in the liver showed a significant increase in TNF-α and IL-6 genes in the group treated with standard diet+L. barbarum+LPS when compared to the standard diet+LPS group. Thus, the administered L. barbarum extract displays the potential to reduce inflammatory responses induced by LPS and a palatable diet.

Clinical, hematological and histopathological aspects of experimental induction of laminitis in cattle through lipopolysaccharide infusion / Aspectos clínicos, hematológicos e histopatológicos da indução experimental por lipopolissacarídeos de laminite em bovinos

ABSTRACT: The purpose of this research was to confirm the changes occurring in the foot system of the heifers challenged with lipopolysaccharides (LPS), at the clinical, serum and histological levels. We studied 16 clinically healthy heifers, 14 months of age, placed in a confinement system. All the animals were provided with an accelerometer collar to establish their activity. They were categorized into two groups: the LPS group (n=8), or those which were administered two intravenous applications of 2 mL containing 0.5 μg/kg of body weight of LPS, with a 24-hour interval and the Control group (n=8) which were given two infusions of 2 mL of saline solution in the same time interval. General clinical examination and blood collection were done at 0, 4 and 8 hours post the LPS challenges and analyses of the hemograms and paroxonese-1 were performed. The animals were then slaughtered on day 4 and the laminar tissue was collected for histological analysis. The LPS group revealed a lower total leukocyte count with heart rate and greater activity. None of the animals revealed any abnormal signs symptomatic of foot pathology after histological analysis. Hence, the challenge with LPS failed to induce any clinical and histological changes in the foot tissue compatible with laminitis.

RESUMO: O objetivo deste trabalho foi verificar alterações do sistema podal á nível clínico, hematológico e histológico de animais desafiados com lipopolissacarídeo (LPS). Foram utilizadas 16 novilhas de corte com 14 meses de idade, clinicamente saudáveis, em sistema de confinamento. Todos os animais continham uma coleira com acelerômetro para verificar atividade, sendo divididos em 2 grupos: LPS (n= 8), que recebeu duas aplicações de 2 mL contendo 0,5 μg/kg de peso corporal de LPS via intravenosa, com intervalo de 24 horas e Controle (n= 8) que recebeu duas aplicações de 2 mL de solução salina com o mesmo intervalo de tempo. Foram realizados exame clinico geral e coletas de sangue às 0, 4 e 8 horas após os desafios com LPS para análise de hemogramas e paroxonase-1. Os animais foram abatidos aos 5 dias sendo executada a coleta de tecido laminar para análise histológica. A análise estatística foi executada com a utilização do programa NCSS(2004) através de ANOVA-medidas repetidas, considerando o efeito do tratamento do período e sua interação (tratamento*horas), sendo considerados significativos valores de P<0,05. O grupo LPS apresentou superior contagem total de leucócitos com maior frequência cardíaca e atividade. Nenhum animal apresentou sinal de anormalidade compatível com laminite através da análise histológica. Entretanto, o desafio com LPS foi capaz de gerar alterações a nível clínico, constatado pelas alterações nos parâmetros de frequência cardíaca, respiratória e temperatura corporal, além de mudanças no leucograma.

Effect of butafosfan supplementation during oocyte maturation on bovine embryo development.

Around 60-80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus-oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

Insulin treatment does not affect follicular development but alters granulosa cell gene expression in dairy cows.

The use of strategies to stimulate follicular growth are important, especially for use in timed artificial insemination (TAI) protocols, aiming to increase dairy cow's fertility. The aim of this study was to investigate the effect of insulin on follicular growth, steroid production and expression of genes related to follicular development. For this, cows were submitted to a progesterone (P4) and estradiol (E2) based synchronization protocol. In study 1, eleven primiparous lactating Holstein cows, received a single s.c. application of 0.25 IU/kg human insulin or no treatment (control) on D8 of the protocol. Blood samples were collected, and the dominant follicle diameter was assessed daily via transrectal ultrasonography, from D8 to D12. In study 2, eight multiparous non-pregnant and non-lactating Jersey cows, received a single s.c. application of 0.25 IU/kg human insulin, whereas cows from the control group received a single s.c. injection (1 mL) of saline solution (NaCl 0.9%). Blood samples were collected, and the dominant follicle diameter was assessed daily via transrectal ultrasonography from D6 to D9 of the protocol. Sixteen hours after insulin injection, follicular aspiration was performed. In study 1, insulin treatment decreased systemic glucose levels, but did not affect follicular growth. In study 2, the glucose decrease induced by insulin treatment was accompanied by a tendency of decreased progesterone levels in follicular fluid, along with a decrease in steroidogenic acute regulatory protein (STAR) and insulin like growth factor binding protein 2 (IGFBP2) mRNA abundance in granulosa cells. In conclusion, insulin treatment does not increase follicle growth and estradiol secretion in dairy cows, but decreases IGFBP2 and tends to increase pappalysin (PAPPA) mRNA abundance in granulosa cells, suggesting a positive effect on follicle development.

Intrafollicular paraoxonase 1 activity and the steroidogenic potential of the first post-partum dominant follicle in dairy cows.

Cows experiencing high levels of inflammation and specific metabolic conditions tend to have slower follicular growth and lower serum and follicular concentrations of oestradiol (E2). Paraoxonase1 (PON1) activity decreases during inflammatory processes. Therefore, the aim of this study was to evaluate the association between serum and intrafollicular (FF) PON1 activity and the serum and intrafollicular levels of E2 and progesterone (P4), as well as the mRNA expression of genes related to steroidogenesis, metabolism and inflammation in the first post-partum dominant follicle of Holstein cows. No correlation was found between PON1 activity, the expression of the analysed genes and levels of follicular E2 and P4, except for a negative correlation between serum E2 and follicular PO1 activity. Also, no correlation was found between serum and follicular PON1 during the first post-partum follicular wave.

Effect of recombinant bovine somatotropin (rbST) treatment on follicular population and development in non-lactating dairy cows.

The aim of this study was to evaluate the long-term effects of recombinant bovine somatotropin (rbST) on follicle population and ovulatory follicle development in non-lactating dairy cows. Twenty-one Jersey cows were allocated in rbST (n=11) or control (n=10) groups. On day -60, cows in rbST group received 500 mg of somatotropin (s.c. Lactotropin®, Elanco). On day 0, control and rbST cows received an intravaginal progesterone-releasing device (1.9 g, CIDR®, Zoetis) and GnRH (100 mg, IM, Factrel®, Zoetis). On day 8, cows received PGF2α (25 mg, IM, Lutalyse®, Zoetis) and the CIDR® was removed. Twelve hours after device removal (D8), serum, follicular fluid and granulosa cells samples were collected. Serum and follicular concentration of estradiol (E2) and progesterone (P4) were analyzed. Total RNA was extracted from granulosa cells to measure gene expression of LHCGR, STAR, HSD-3B1, CYP11A1, CYP19A1, CYP17A1, IGFR and PAPPA by real-time PCR. Ultrasonography was performed on days -60, -53, -46, -14, -7, 0 and 8 for antral follicle count. Results were analyzed by repeated measures ANOVA and t-test. There was no effect of rbST treatment on the number of follicles during the 60 days period, as well as no effect on serum and follicular fluid E2 and follicular fluid P4 at the moment of follicle aspiration. There was a reduction in PAPPA (P = 0.006), CYP11A1 (P = 0.04) and CYP19A1 (P = 0.002) mRNA levels in granulosa cells of the pre-ovulatory follicle of rbST treated cows. In conclusion, a single dose of rbST did not have long-term effects on antral follicle population, serum and follicular E2/P4 concentrations in non-lactating dairy cows. Despite that, rbST injection decreased granulosa cell expression of genes related to steroidogenesis in the pre-ovulatory follicle.

Effect of high-density lipoprotein on oocyte maturation and bovine embryo development in vitro.

High-density lipoprotein (HDL) is the main lipoprotein in the follicular fluid, and it has anti-inflammatory, antioxidant and cryoprotectant properties. The anti-inflammatory potential and antioxidant potential are derived from its lipid composition, especially the apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1). The aim of this study was to evaluate the effect of HDL during in vitro maturation (IVM) on oocyte maturation and early bovine embryo development. For this, cumulus-oocyte complexes (COCs) were obtained from bovine ovaries collected at a local slaughterhouse. COCs (n = 2,250) were allocated into three groups (n = 50 COCs/group) according to the addition of HDL protein (HDL-P) during IVM for 22 hr: 0 (control), 50 and 150 mg/dl. After IVM, COCs were inseminated (in vitro fertilization) and cultivated for 7 days. Total cholesterol concentration, total protein, triglycerides and ApoAI concentrations on IVM medium increased proportionally to HDL-P addition. However, PON1 activity was not detected in any treatment. The addition of HDL-P did not affect nuclear maturation rate, endogenous reactive oxygen species and glutathione levels in COCs (p > 0.05). The highest HDL-P concentration (150 mg/dl) decreased cleavage and blastocyst rate (p < 0.05). Moreover, the HDL-P 150 mg/dl group had lower cellular count/blastocyst than the 50 mg/dl group (p < 0.05). However, the addition of HDL-P did not affect relative gene expression of evaluated genes. In conclusion, the complex HDL/ApoAI obtained from human plasma, in the absence of PON1 activity during in vitro oocyte maturation, decreased initial embryo development.

Differential effects of a high-fat diet on serum lipid parameters and ovarian gene expression in young and aged female mice.

The aim of this study was to compare serum lipid profiles and ovarian gene expression between aged and younger female mice fed a control or a high-fat diet for 2 months. For this 16 female mice (C57BL/6) of 4 months (Young, n = 8) or 13 months (Old, n = 8) of age were used. The females were divided into four groups: (i) young females fed a normal diet; (ii) young females fed a high-fat diet; (iii) old females fed a normal diet; and (iv) old females fed a high-fat diet. Food intake was reduced (P < 0.05) in mice fed with a high-fat (2.9 ± 0.1 g) diet in comparison with control mice (3.9 ± 0.1 g). Body weight was higher for old females on the high-fat diet (35.1 ± 0.3 g) than for young females on the same diet (23.3 ± 0.4 g; P < 0.05). PON1 activity was lower in the high-fat than control diet group (114.3 ± 5.8 vs. 78.1 ± 6.0 kU/L, respectively) and was higher in older than younger females (85.9 ± 6.4 vs. 106.5 ± 5.3; P < 0.05, respectively). Females fed a high-fat diet had lower expression of Igf1 mRNA (P = 0.04). There was an interaction between age and diet for the expression of Gdf9 and Survivin, with lower expression in older females in both diets and young females that received the high-fat diet (P < 0.05). Concluding, the high-fat diet reduced the expression of ovarian Igf1 mRNA, and Gdf9 and Survivin mRNA in younger females, which can indicate lower fertility rates. High-density lipoprotein concentration and PON1 activity were higher in aged female mice.