Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue.

This study evaluated the effects of different concentrations (10, 20, or 40 µM) of eugenol (EUG 10, EUG 20, or EUG 40), ascorbic acid (50 µg/mL; AA) or anethole (300 µg/mL; ANE 300) on the in-vitro survival and development of goat preantral follicles and oxidative stress in the cultured ovarian tissue. Ovarian fragments from five goats were cultured for 1 or 7 days in Alpha Minimum Essential Medium (α-MEM+) supplemented or not with AA, ANE 300, EUG 10, EUG 20 or EUG 40. On day 7 of culture, when compared to MEM, the addition of EUG 40 had increased the rate of follicular development, as observed by a decrease in the proportion of primordial follicles alongside with an increase in the rate of normally developing follicles. Furthermore, EUG 40 significantly increased both follicular and oocyte diameters. Subsequently, ovarian fragments from three goats were cultured for 1 or 7 days in α-MEM+ supplemented or not with AA, ANE 300 or EUG 40. All tested antioxidants, except ANE 300, were able to significantly decrease the levels of reactive oxygen species in the ovarian tissue, but EUG 40 could most efficiently neutralize free radicals. All ovarian tissues cultured in the presence of antioxidants, especially EUG 40, presented a significant decrease in H3K4me3 labeling, indicating a silencing of genes that play a role in the inhibition of follicular activation and apoptosis induction. When compared to cultured control tissues, both EUG 40 and ANE 300 significantly increased the intensity of calreticulin labeling in growing follicles. The mRNA relative expression of ERP29 and KDM3A was significantly increased when the culture medium was supplemented with EUG 40, indicating a response to ER stress experienced during culture. In conclusion, EUG 40 improved in-vitro follicle survival, activation and development and decreased ROS production, ER stress and histone lysine methylation in goat ovarian tissue.

Impaired Performance of Broiler Chickens Fed Diets Naturally Contaminated with Moderate Levels of Deoxynivalenol.

Mycotoxin exposure is common in the poultry industry. Deoxynivalenol (DON) is usually detected at levels below the maximum threshold (5000 ppb), but depending on diet and age, broiler performance can be affected. We evaluated the effects of 900 ppb and 2300 ppb DON on the performance, intestinal morphometry, and lesion scores of broiler chickens. One-day-old male Ross broilers (n = 736) were divided into 4 treatments with 8 replicates each, and a pen containing 23 birds was the experimental unit. The animals were fed diets naturally contaminated with two levels of DON: 900 (Low DON-LD) or 2300 (Moderate DON-MD) ppb, with or without activated charcoal, over 28 days. After this, all birds were fed a marginally DON-contaminated diet without charcoal. During the first 28 days, body weight gain (BWG) and feed conversion ratio (FCR) were significantly impaired when broilers were fed a MD diet without activated charcoal. Even after feeding a marginally contaminated diet from D28-35, birds previously fed the MD diet presented a significantly lower performance. The villus height:crypt depth (VH:CD) ratio was significantly higher in the ileum from 14-day-old broilers fed the MD when compared with the LD diet. At D28, the MD diet caused decreased villus height (VH) and increased crypt depth (CD), affecting VH:CD ratio in both intestinal segments, with higher levels in the jejunum from 28-day-old broilers fed a non-supplemented LD diet. Broiler production was negatively affected by DON, even at moderate levels (2300 ppb).

Effect of Resin Acid and Zinc Oxide on Immune Status of Weaned Piglets Challenged With E. coli Lipopolysaccharide.

With the ban of zinc oxide (ZnO) at high dosages in piglet diets in Europe by 2022, alternative nutritional solutions are being tested to support piglet immune defence during their weaning, the most critical and stressful moment of pig production. The present study evaluated the effect of zinc oxide (ZnO; 2,500 mg/kg diet) and resin acid concentrate (RAC; 200 mg/kg diet) on the immune defence of weaned piglets challenged with lipopolysaccharide (LPS). Piglets were challenged at days 7 and 21 post-weaning, and blood was sampled 1.5 and 3.0 h after each challenge to determine serum levels of pro- and anti-inflammatory cytokines. The levels of serum tumour necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) increased at days 7 and 21, and those of IL-6 at day 21 when challenged piglets were fed a diet supplemented with ZnO. In challenged piglets fed with RAC, the serum levels of IL-1ß, IL-6, IL-8, IL-10 and TNF-α were increased at days 7 and 21, except for that of IL-1ß, which was not affected at day 21. The increased levels of these cytokines indicate the successful immune-modulatory effect of ZnO and RAC, which appears as a candidate to replace ZnO in weaned piglets' diets.

Effects of a feed additive blend on broilers challenged with heat stress.

We evaluated a blend of medium-chain fatty acids (MCFA), organic acids, and a polyphenol antioxidant on gut integrity. Eighty Ross Broilers were exposed to 20-22°C (control - normothermic) or to 35-39.5°C (heat stress) for eight hours a day for a period of 1 or 5 days. Birds were fed a standard diet, or a diet supplemented with the test blend. Thereafter, birds were euthanized, and intestinal sections were excised for morphological, morphometric and gene expression analyses. Blood samples were collected for glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GSH-Px) activity and trolox equivalent antioxidant capacity (TEAC) determination. Heart and liver tissues were used to quantify the expression of heat shock proteins 60 and 70 (HSP60 and HSP70, respectively) and inhibitor of kappa light chain gene enhancer in B cells alpha (IKBA). The jejunum was the most sensitive intestinal section, where heat stress modulated the expression of HSP70, of the inflammatory markers IKBA, interleukin 8 (IL-8), interferon gamma (IFNγ), and toll-like receptor 4 (TLR4). Moreover, expression of tight junctions (CLDN1, ZO1 and ZO2) and nutrient transporters (PEPT1 and EAAT3) was modulated especially in the jejunum. In conclusion, the feed additive blend protected intestines during heat stress from the decrease in villus height and crypt depth, and from the increase in villus width. Especially in the jejunum, heat stress played an important role by modulating oxidative stress and inflammation, impairing gut integrity and nutrient transport, and such deleterious effects were alleviated by the feed additive blend. RESEARCH HIGHLIGHTS Jejunum is the most sensitive intestinal segment during heat stress. Heat stress affects the expression of tight junctions and nutrient transporters. Feed management helps to alleviate the disturbances caused by heat stress. A blend of MCFA, organic acids and a polyphenol protects broilers under heat stress.

Extender supplementation with catalase maintains the integrity of sperm plasma membrane after freezing-thawing of semen from capuchin monkey.

We aimed to evaluate the effect of supplementation of ACP-118® extender with the antioxidant catalase (10 and 50 µg/ml) on Sapajus apella sperm motility, vigour, and plasma membrane integrity during the processes of seminal liquefaction, cooling, and freezing. Catalase did not affect any of the evaluated parameters after semen dilution or cooling. Cryopreserved sperm in the presence of 50 µg/ml catalase presented a plasma membrane integrity similar to that fresh sperm, however.

Cinnamaldehyde, Carvacrol and Organic Acids Affect Gene Expression of Selected Oxidative Stress and Inflammation Markers in IPEC-J2 Cells Exposed to Salmonella typhimurium.

Essential oils and organic acids are used as feed additives to improve health status and reduce colonization with pathogens. Although bactericidal in vitro, concentrations achieved in the animal gut are probably not lethal to pathogens. The aim of this study was to investigate the effects of cinnamaldehyde, carvacrol and cinnamic, lactic and propionic acids on the ability of Salmonella typhimurium ATCC 14028 (ST) to invade intestinal epithelial cells (IPEC-J2) and on the expression levels of immune related genes in the cells. The minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC) were determined and influence on the invasion capacity of ST was investigated. The structure of fimbriae and flagella was analysed by electron microscopy, and expression levels of HSP70, IkBa, IL-8 and IL-10 in the IPEC-J2 cells were carried out by q-PCR. Cinnamaldehyde, carvacrol and cinnamic and propionic acids inhibited ST invasion but not cell viability, bacterial viability and motility or the development of flagella. Propionic acid and cinnamaldehyde in combination with cinnamic acid caused structural impairment of fimbriae. Cinnamaldehyde up-regulated expression of HSP70 irrespective of the presence of organic acids or ST; exposure to carvacrol induced HSP70 only in the presence of propionic acid and ST. © 2016 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd.

Naringenin (NAR) and 8-prenylnaringenin (8-PN) reduce the developmental competence of porcine oocytes in vitro.

Flavanones such as naringenin (NAR) and 8-prenylnaringenin (8-PN) are increasingly used as dietary supplements despite scientific concern regarding adverse effects on female reproduction upon excessive intake. In the present study, NAR and 8-PN (0.3-1µM) significantly affected porcine oocyte maturation in vitro by decreasing cumulus expansion. In addition, NAR and 8-PN decreased percentages of meiotic spindle formation, oocyte cleavage and blastocyst formation. The effects of NAR and 8-PN were different from estradiol (3.12µM)-induced effects. Still, the flavanone-induced effects were observed at concentrations that can be found in human plasma upon supplement intake and that resemble physiological estrogen equivalence levels in follicular fluids. Considering that abnormal oocyte maturation can cause subfertility, our study warrants that precautions are in place and excessive intake of NAR and 8-PN e.g. via dietary supplements should be avoided by women.

Catalase prevents lipid peroxidation and enhances survival of caprine preantral follicles cryopreserved in a 1,2-propanediol-freezing medium.

The objectives of this study were to determine: 1) the optimal concentration (1.0 or 1.5 M) and duration of exposure (5, 10, or 20 min) of ovarian tissue to 1,2-propanediol (PROH) on morphology and viability of caprine preantral follicles; and 2) the effect of supplementing cryopreservation medium supplementation with Trolox(®) (0.1, 0.5, or 1.0 mM) or catalase (5, 10, or 20 IU/mL) on follicular morphology, viability, and lipid peroxidation. Cryopreservation decreased (p<0.05) percentages of normal follicles relative to the control (84%). Although supplementation of the cryopreservation medium (1.0 M PROH) with catalase (10 or 20 IU/mL) or Trolox(®) (0.1 mM) resulted in follicular morphology and viability similar to that in the controls (P>0.05), lipid peroxidation was reduced only when 20 IU/mL catalase was added to the cryopreservation medium.

Influences of FSH and EGF on primordial follicles during in vitro culture of caprine ovarian cortical tissue.

Factors that control the onset of folliculogenesis are critical to female gamete production, but poorly understood. The aim of the present study was to investigate the effects of FSH and EGF on the activation and growth of goat primordial follicles in vitro. To this end, pieces of goat ovarian cortex were cultured in vitro for 1, 3 or 5 days, at 39 degrees C in an atmosphere containing 5% CO(2), in minimum essential medium supplemented with insulin, transferrin, selenium, pyruvate, glutamine, hypoxanthine, BSA, penicillin, streptomycin and fungizone and with or without FSH (100 ng/ml) and/or EGF (100 ng/ml). At the end of the culture periods, the relative proportions of primordial, intermediate, primary and secondary follicles were calculated and compared with those in non-cultured tissue. In addition, mitotic activity of granulosa cells was studied by immunohistochemistry for proliferating cell nuclear antigen (PCNA). In brief, it was found that goat primordial follicles activate spontaneously during culture in vitro and, while neither FSH nor EGF affected the proportion of primordial follicles that entered the growth phase, both stimulated an increase in oocyte and follicle diameter, especially in intermediate and primary follicles cultured for 5 days. On the other hand, there was no significant effect of culture or either growth factor on the proportion of PCNA-stained growing follicles. Contrary to expectations, neither FSH nor EGF affected follicle viability or integrity during culture, since the percentages of intact follicles did not differ between control, FSH and/or EGF containing medium. In conclusion, this study demonstrated that goat primordial follicles activate spontaneously in vitro, and that both FSH and EGF stimulate an increase in follicle size by promoting oocyte growth.