Dietary supplementation with Lactobacillus fermentum I5007 improves the anti-oxidative activity of weanling piglets challenged with diquat.

AIMS: To determine the effects of Lactobacillus fermentum I5007 on the redox state of piglets oxidatively stressed with diquat. METHODS AND RESULTS: Twenty-four, 28-day-old barrows were used in a 2 × 2 factorial design experiment with the main effects being Lact. fermentum supplementation and diquat challenge. Half of the pigs (n = 12) were orally administered with 20 ml of a solution containing 10(8 ) CFU ml(-1) of Lact. fermentum each morning of the 21-day trial, while the remainder received saline. On day 8, these two groups were further subdivided so that half of the pigs in each group (n = 6) were intraperitoneally injected with 10 mg kg(-1) BW diquat, while the remainder received saline. The diquat-injected pigs had significantly poorer performance and increased levels of plasma cortisol, adrenaline, carbonyl and malondialdehyde. Lactobacillus fermentum supplementation significantly increased superoxide dismutase and glutathione and increased the ability to inhibit superoxide anion production in liver and muscle. CONCLUSIONS: Lactobacillus fermentum improved the anti-oxidative defence system and alleviated damage caused by diquat. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus fermentum has the potential to alleviate oxidative stress and improve weaning pig performance.

Elevation in tumour necrosis factor-alpha (TNF-alpha) messenger RNA levels in the uterus of pregnant gilts after oestrogen treatment.

In pigs, induction of embryonic degeneration, by exogenous oestrogens given early in gestation, has been long recognised. However, the underlying mechanisms responsible for this degeneration remain unclear. The present study was conducted to determine whether oestrogen-induced early porcine embryonic mortality was associated with changes in the levels of tumour necrosis factor-alpha (TNF-alpha) messenger RNA in the uterine endometrium. Prepubertal gilts were induced into oestrus with PG600 and artificially inseminated at their second natural oestrus and again 24 h later. After insemination, gilts were randomly assigned to treatment and given 0.5 ml intramuscular injections of either oestradiol valerate (10 mg ml-1) or corn oil on day 9 and 10 of gestation. The gilts were slaughtered on day 12, 15 or 18 of gestation. The reproductive tract was removed from each gilt and the uterine horns were flushed to check for the presence and integrity of embryos. Samples of uterine endometrial tissues were collected, snap-frozen in liquid nitrogen and stored at -80 degrees C. Total cellular RNA was isolated from frozen tissues using a guanidine isothiocyanate-cesium chloride method. The abundance of TNF-alpha messenger RNA was determined by Northern blot hybridisation analysis. Treatment of pregnant gilts with oestrogen resulted in severe fragmentation of embryos on days 15 (2/3) and 18 (2/2), confirming the embryocidal effect of exogenous oestrogen. Uterine TNF-alpha messenger RNA level was elevated in oestrogen-treated gilts compared with controls (P < 0.05). This observation of an association between increased levels of TNF-alpha mRNA in the uterus and embryonic degeneration in oestrogen-treated gilts suggests that TNF-alpha may be involved in mediating oestrogen-induced early embryonic mortality in the pig.

Growth hormone as an amplifier of insulin-like growth factor-I action: potentiated oestradiol accumulation.

The interaction between GH and IGF-I in modulating oestradiol biosynthetic capacity was examined in cultured porcine granulosa cells. Granulosa cells (2 x 10(6) viable cells/culture) were initially cultured for 96 h (treatment period) in an androstenedione-free medium in the absence or presence of IGF-I (0 or 50 ng/ml), with or without GH at 100 ng/ml. At the conclusion of this period, culture media were discarded and the cells were washed twice with the androstenedione-free medium and reincubated for an additional 24 h (test interval) in an androstenedione (10(-7) M)-supplemented medium for oestradiol accumulation. GH alone induced no stimulation (P > 0.05) of basal oestradiol accumulation. In contrast, concurrent treatment with IGF-I produced a 4.3-fold increase (26 vs 112 ng/ 2 x 10(6) cells per 24 h, P < 0.001) in oestradiol accumulation. GH amplified IGF-I-induced oestradiol production in a dose (minimal dose requirement of 0.3 ng/ml)- and time (minimal time requirement of 24-48 h)-dependent manner. Studies on the site(s) of action indicated that GH exerts its amplifying effects on IGF-I-induced oestradiol production both proximal and distal to cAMP generation. As the specificity study and the inhibitory study indicated, GH amplification of IGF-I-induced oestradiol production is a process involving gene transcription and/or translation and the synergism is not solely specific to IGF-I as IGF-II-induced oestradiol production was also amplified in the presence of GH.

Effects of GH on IGF-II-induced progesterone accumulation by cultured porcine granulosa cells.

A total of seven experiments were conducted to investigate the potential facilitative interaction of growth hormone (GH) and insulin-like growth factor II (IGF-II) in stimulating steroidogenesis by cultured porcine granulosa cells and to examine the possible nature of this action. Porcine granulosa cells were cultured in serum-free medium in the presence or absence of GH or prolactin, with or without IGF-II or IGF-I. IGF-II by itself dose (with peak progesterone production of 498 ng/mg DNA/24 h being observed at 100 ng of IGF-II/mL) and time- (with minimum time requirement of 24-48 h) dependently increased progesterone accumulation (P < 0.01). Neither GH (dose range 0, 5, 10, 50, 100, and 150 ng/mL) nor prolactin (dose range 0, 0.01, 0.1, 1, 5, 10 micrograms/mL) alone stimulated progesterone accumulation when compared with the control (P > 0.05). However, in the presence of IGF-II, GH proved to be a potent amplifier of IGF-II in progesterone production (P < 0.01) with a minimum GH time requirement of 24-48 h. In contrast, prolactin did not influence IGF-II-induced progesterone accumulation (P > 0.05). An inhibitory study showed that the presence of cycloheximide (3 micrograms/mL) or actinomycin D (1 microgram/mL) blocked both the stimulatory effect of IGF-II on progesterone accumulation and the amplification of GH on IGF-II induced production (P > 0.01), suggesting GH amplification of IGF-II-induced progesterone accumulation is a process involving gene transcription and translation. Northern blot analysis further demonstrated that GH amplification of IGF-II-induced steroidogenesis can be attributed, at least partially, to enhanced IGF-II-induced cytochrome P450 cholesterol side-chain cleavage mRNA by GH.

Growth hormone amplifies insulin-like growth factor I induced progesterone accumulation and P450scc mRNA expression.

The interaction of growth hormone (GH) and insulin-like growth factor I (IGF-I) in the acquisition of progesterone biosynthetic capacity were examined in cultured porcine granulosa cells. Basal progesterone production was not affected (P > 0.05) by GH treatment. However, concurrent treatment with GH produced a 4.1-fold increase (539 versus 2214 ng/culture) in the IGF-I-stimulated accumulation of progesterone. GH potentiated IGF-I induced progesterone production in a dose and time dependent manner, with a time requirement of 48 h or less. The amplified effect of GH was not attributable to changes in cellular protein, DNA content, cell number, plating efficiency or cell viability. Moreover, Northern blot analyses revealed that GH enhanced IGF-I induced expression of the gene encoding cytochrome P450 side chain cleavage. These observations provide further evidence to support the role of GH in the regulation of ovarian steroidogenesis.

Comparative effects of insulin and insulin-like growth factor-1 on follicle-stimulating hormone-induced responses in porcine granulosa cells.

The effect of insulin and insulin-like growth factor-1 (IGF-1) on progesterone secretion by porcine granulosa cells and their modulatory effect on follicle-stimulating hormone (FSH)-induced responses were examined. For comparative purposes, growth hormone (GH), previously shown to stimulate IGF-1 secretion, was also included. Granulosa cells from ovarian follicles (3 to 5 mm) were cultured in multiwell plates for the first 48 hours, either in the presence or absence of 1% fetal bovine serum (FBS). Following plating, all cultures were maintained in serum-free media. The addition of only insulin, but not IGF-1 or GH, enhanced progesterone secretion under both culture conditions. When low-density lipoprotein was provided as steroid substrate, a stimulatory effect of insulin on progesterone accumulation was observed with a minimum dose of 10 ng/ml. Granulosa cells cultured in serum-free media from the time of plating secreted less progesterone and were less responsive to FSH compared with cultures plated with 1% FBS. Only insulin, but not IGF-1, enhanced FSH responses to threefold in cells cultured with 1% FBS. However, when cells were cultured in serum-free media from the time of plating, both insulin and IGF-1, but not GH, potentiated the responses to FSH, but insulin was more potent than IGF-1. Insulin-like growth-factor-1 binding studies with granulosa cells indicate the presence of specific high-affinity binding sites (Kd 3.96 nM). A dose of 100 ng/ml of insulin had negligible cross-reactivity with IGF-1 receptors.

The influence of exogenous pituitary growth hormone on the ovulatory response to PMSG and hCG and the LH response to estradiol benzoate in prepubertal gilts.

Two experiments were performed to examine the influence of exogenous growth hormone on the reproductive axis in gilts. Experiment one employed 26 Yorkshire X Landrace prepubertal gilts, which were selected at 150 d and 86.5 +/- 1.5 kg bodyweight (BW) and assigned equally to two treatments. Gilts received injections of either porcine growth hormone at 90 micrograms/kg BW, or vehicle buffer, from 150 to 159 d. At 154 d gilts received 500 IU PMSG, followed 96 hr later by 250 IU hCG. Gilts were slaughtered at 163 days and their ovaries recovered to determine ovulatory status. In each treatment, 2/13 gilts failed to show any ovarian response to PMSG/hCG. All remaining control gilts ovulated and their ovaries appeared morphologically normal. In gilts receiving exogenous growth hormone, fewer ovaries (4/11, P less than .01) appeared morphologically normal. The ovaries of all other growth hormone injected gilts had very large (12-25 mm) non-luteinized follicles. In experiment two, 20 prepubertal Yorkshire X Landrace gilts were selected at 138 days and 85 kg BW. These gilts received injections of growth hormone at 90 micrograms/kg BW (n = 9) or vehicle (n = 11) from 138 to 147 days. At 143 days, all gilts were given an injection of estradiol benzoate (EB) at 15 micrograms/kg BW. Blood samples were taken at the time of EB injection, at 24 and 36 hr and then at 6 hr intervals until 78 hr. All samples were assayed for serum LH concentrations. The EB induced LH peak height was lower (P less than .04) in gilts receiving exogenous growth hormone than in controls.(ABSTRACT TRUNCATED AT 250 WORDS)