Effects of cortisol on pregnancy rate and corpus luteum function in heifers: an in vivo study

To determine whether glucocorticoids affect the function of the bovine corpus luteum (CL) during the estrous cycle and early pregnancy, we examined the effects of exogenous cortisol or reduced endogenous cortisol on the secretion of progesterone (P4) and on pregnancy rate. In preliminary experiments, doses of cortisol and metyrapone (an inhibitor of cortisol synthesis) were established (n=33). Cortisol in effective doses of 10 mg blocked tumor necrosis factor-induced prostaglandin F(2α) secretion as measured by its metabolite (PGFM) concentrations in the blood. Metyrapone in effective doses of 500 mg increased the P4 concentration. Thus, both reagents were then intravaginally applied in the chosen doses daily from Day 15 to 18 after estrus (Day 0) in noninseminated heifers (n=18) or after artificial insemination (n=36). Pregnancy was confirmed by transrectal ultrasonography between Days 28-30 after insemination. Plasma concentrations of P4 were lower in cortisol-treated heifers than in control heifers on Days 17 and 18 of the estrous cycle (P<0.05). However, the interestrus intervals were not different between control and cortisol-treated animals (P>0.05). Moreover, metyrapone increased P4 and prolonged the CL lifespan in comparison to control animals (P<0.05). Interestingly, in inseminated heifers, cortisol increased the pregnancy rate (75%) compared with control animals (58%), whereas metyrapone reduced the pregnancy rate to 16.7% (P<0.05). The overall results suggest that cortisol, depending on the physiological status of heifers (pregnant vs. nonpregnant), modulates CL function by influencing P4 secretion. Cortisol may have a positive influence on CL function during early pregnancy, leading to support of embryo implantation and resulting in higher rates of pregnancy in heifers.     

Role of tumor necrosis factor-alpha and nitric oxide in luteolysis in cattle

Although prostaglandin (PG) F2alpha is known to be a principal luteolytic factor, its action on the bovine corpus luteum (CL) is mediated by other intra-ovarian factors. Tumor necrosis factor-alpha (TNFalpha) and its specific receptors are present in the bovine CL with the highest expressions at luteolysis. TNFalpha in combination with interferon-gamma reduced progesterone (P4) secretion, increased PGF2alpha and leukotriene C4 (LTC4) production, and induced apoptosis of the luteal cells in vitro. Low concentrations of TNFalpha caused luteolysis, which resulted in a decreased level of P4, and increased levels of PGF2alpha, LTC4 and nitrite/nitrate (stable metabolites of nitric oxide-NO) in the blood. Inhibition of local NO production counteracts spontaneous and PGF2alpha-induced luteolysis. Therefore, NO is a likely candidate for the molecule that mediates PGF2alpha and TNFalpha actions during luteolysis. Both PGF2alpha and TNFalpha increase NO concentrations in blood, and stimulate NO synthase expression on protein level in the bovine CL cells. NO stimulates PGF2alpha and LTC4 secretion, inhibits P4 production and reduces the number of viable luteal cells. TNFalpha and NO induce apoptotic death of the CL by modulating expression of bcl-2 family genes and by stimulating expression and activity of caspase-3. The above findings indicate that TNFalpha and NO play crucial roles in functional and structural luteolysis in cattle.     

Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.     

Increase in plasma total antioxidant capacity of grazing Japanese Black heifers and cows in forestland in Japan

Blood total antioxidant capacity (TAC) has become a key bio‐marker for animal health. Forest‐grazing cattle are known to forage various native plants that have high TAC. This study evaluated differences of plasma TAC between forest‐grazing (FG) and pasture‐grazing cattle (PG). Experiment 1 monitored the plasma TAC levels of 32 Japanese Black cattle. The level in PG did not change throughout the grazing period. However, that in FG, which increased from summer, was significantly higher than that in PG through fall (P < 0.05). In experiment 2, we used nine Japanese Black heifers and investigated their blood antioxidant parameters and the TAC in plants that the cattle consumed in late June and September. The plasma TAC levels in FG were significantly higher than those in PG in both periods (P < 0.05). Plasma levels of lipid peroxidation in FG tended to be lower than that in PG (P = 0.098). Furthermore, the TAC levels in various species of shrubs and trees consumed by FG were higher than those in pasture grasses. Results of this study show that plasma TAC of grazing Japanese Black cattle in forestland increase from summer through fall.     

Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.     

Effects of amino nitrogen on fermentation parameters by mixed ruminal microbes when energy or nitrogen is limited

Ruminal microbes harvested from a ruminally fistulated cow were incubated in simple batch and semicontinuous cultures with NH₃‐N or amino‐N on nitrogen‐ or energy‐excess diets in quantity (HN and LN diets, respectively, consisting of timothy hay plus soybean meal, or corn grain), based on evaluation with the National Research Council and Cornell Net Carbohydrate and Protein System models. In a batch culture experiment, supplementation with amino‐N promoted digestion and fermentation in the course of incubation (4–24 h) on both diets, but these effects mostly disappeared when the diets were sufficiently digested (at 48 h). In a semicontinuous culture experiment using Rusitec, no effect of amino‐N was exhibited after sufficient fermentation and digestion, but significant promotion of digestion was shown in the course of incubation on the HN diet, while no such effect was detected on the LN diet. The microbial yield for 24 h did not show a significant difference between the N sources of either of the two diets. These results suggest that the stimulatory effects of amino‐N are diminished when the diets are sufficiently digested after a long retention and incubation, and also that the effectiveness of amino‐N does not require a quantitatively energy‐excess status.     

Flow cytometric analysis of chicken NK activity and its use on the effect of restraint stress

Immune system is organized by the influence of both neural and endocrine systems. NK activity plays an important role in the innate immunity. In this study, we observed the effects of restraint stress on chicken peripheral blood NK activity. Viability of FITC-labeled RP9 was measured with PI after treatment with the effector cells. Chicken peripheral blood CD8alpha+ cells expressed strong cytotoxic activity, in contrast to thrombocytes, while peripheral blood CD3+ CD8alpha+ cells and CD4+ cells had little cytotoxic activity. Con A supernatant enhanced the cytotoxic activity of CD8alpha+ cells. Therefore, it is considered that these cytotoxic activities measured by flow cytometry (FCM) analysis are NK activity. When chickens were exposed to restraint stress, the levels of serum corticosterone increased transiently over a short period of time while the NK activity decreased. The decreased NK activity, however, did not recover to the intact levels for a long time, even once the serum corticosterone levels had recovered. These data indicate that chicken NK activity is able to be measured by flow cytometric analysis and that restraint stress causes severe damage to the chicken NK activity.     

Nutrient starvation affects expression of LC3 family at the feto-maternal interface during murine placentation

LC3 − the mammalian homolog of Atg8 − was found as autophagosome membrane binding protein in mammals and widely used as an autophagosomal marker. LC3A, B and C show different expression patterns in each tissue. The aim of this study was to reveal the differences of expression patterns among LC3 families in mouse placenta under normal condition and nutrient starving condition. LC3A and B were highly expressed in decidual cells. LC3A and B were increased in D14 compared with D12 and D16 in mouse placenta, while LC3C was decreased. Starvation induced increase in LC3B expression specifically. Immunohistochemistry showed different expression patterns among LC3A, B and C. LC3A expression in syncytiotrophoblast was vanished by starvation. The results of real time RT-PCR suggested differences between D12 and D16 in autophagic cascade induced by starvation. Taken together, this study suggests that autophagy could play a role in placental invasion system and that nutrient starvation affects LC3B expression.