The in ovo administration of L-trans pyrrolidine-2,4-dicarboxylic acid regulates small intestinal growth in chicks.

Glutamate, which is one of the most important contributors to oxidative metabolism in the intestinal mucosa, is mainly transported by the excitatory amino acids transporters (EAATs) that are expressed in enterocytes. The objective of this study was to evaluate the effects of in ovo administration of l-trans pyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), a potent competitive inhibitor of glutamate uptake by EAATs, on the growth of the small intestine in chicks. Two series of experiments were conducted with hatching eggs; 100 µl of various l-trans-PDC solutions (0, 0.075 or 0.225 mg/egg for the Control group, low-dose l-trans pyrrolidine 2,4-dicarboxylic acid group (L-PDC) or high-dose l-trans pyrrolidine 2,4-dicarboxylic acid group (H-PDC), respectively) was injected into the albumen sac of these hatching eggs before incubation. Hatchlings were sacrificed by cervical dislocation to determine the embryonic development in Experiment I, whereas the birds in Experiment II were raised or sampled at hatching, days 7 and 14 (D7 and D14) for further study. Gene expression in the small intestines was determined by real-time RT-PCR; and serum concentration of free amino acids was determined by an amino acid analyzer. The results showed that the hatchability was decreased by in ovo administration of l-trans-PDC. The small intestinal weights of the H-PDC group were decreased (P<0.05) at hatching and increased (P<0.05) on D7 and D14 compared with those in the Control group. In addition, the gene expression of EAAT2 in the completed or segmental small intestines was not changed (P>0.05); EAAT3 gene expression in the duodenum (P<0.05), jejunum (P=0.084) and ileum (P=0.060) on D14 was lower in the H-PDC group than in the Control group. Furthermore, the serum concentrations of free proline, threonine and phenylalanine but not glutamate or aspartate were increased (P<0.06) in H-PDC group. In conclusion, this paper is the first to report that in ovo administration of l-trans-PDC induces small intestinal growth retardation during the embryonic period and catch-up growth after hatching.

Development of bone in chick embryos from Cobb 500 breeder hens fed diets supplemented with zinc, manganese, and copper from inorganic and amino acid-complexed sources.

Sources of Zn, Mn, and Cu (IZMC) as sulfates or as amino acid complexes (OZMC) were used to supplement Cobb 500 breeder hen diets. Experimental treatments consisted of diets supplemented with 1) 100, 100, and 10 mg/kg of Zn, Mn, and Cu, respectively, from IZMC (control); 2) 60, 60, and 3 mg/kg of Zn, Mn, and Cu, respectively, from IZMC plus 40, 40, and 7 mg/kg of Zn, Mn, and Cu, respectively, from OZMC (ISO); and 3) a diet with 100, 100, and 10 mg/kg of Zn, Mn, and Cu, respectively, from IZMC as in control plus 40, 40, and 7 mg/kg of supplemental Zn, Mn, and Cu from OZMC (on top). Ten replications of 20 females and 2 males were used per treatment. Eggs from breeders at 30, 40, 50 and 60 wk of age were incubated, and 5 embryos per replicate were collected at 10 (E10), 14 (E14), and 18 (E18) d of incubation. Midshaft width and calcification were measured for left tibia and femur stained with Alcian Blue and Alizarin Red S. At hatch, the left tibia of 5 chicks per replicate was sampled for histological evaluation of the diaphysis and distal epiphysis. Feeding the ISO treatment compared with the control diet increased the Zn (P < 0.05) but not Mn and Cu content of the yolk and albumen blend. At E14, the ISO and on-top treatments had a trend to increase tibia calcification at the rates of 1.6 and 1%, respectively (P < 0.1). The E18 ISO and on-top treatments had 2% thicker tibia compared with the control, regardless of hen age (P < 0.05). Also, at E18, calcification of tibia and femur was higher from hens fed the on-top treatment (P < 0.05). The chicks from the ISO and on-top groups had increased tibia moment of inertia (P < 0.01) at day of hatch. Broiler breeder hens consuming OZMC associated with IZMC produced embryos and hatching chicks with improvements in selected bone mineralization parameters.

Smad8 is expressed in the anterior necrotic zone: evidence for a role of bone morphogenetic proteins/SMAD signaling in the activation of a molecular cascade that culminates in cell death.

Bone morphogenetic proteins (BMPs) play a crucial role in programmed cell death (PCD), a biological process required for the sculpturing of the embryonic limbs. However, it is unknown if BMP signaling directly promotes cell death, or if it induces a molecular cascade that culminates in cell death. Given that Smad8, which encodes one component of BMP signaling, is expressed during the regression of interdigital tissue and responds to BMPs, we presumed that it may be expressed in other cell death areas during chick limb development such as the anterior and posterior necrotic zones (ANZ and PNZ). The present study found that the Smad8 expression pattern in the anterior mesoderm of the hindlimb is very similar to that observed in limbs stained to detect cell death. Also, BMPs and retinoic acid, which act as apoptosis-promoting factors, induced expression of Smad8 before the onset of cell death, while sonic hedgehog protein, acting as a survival factor, inhibited Smad8 expression in the ANZ. However, although there was correlation between Smad8 expression patterns and PCD in the ANZ, phosphorylated forms of SMAD1/5/8 and TUNEL staining did not co-localize in dying cells. Interestingly, a short pulse of BMP was sufficient to trigger cell death. On the other hand, most dying cells were located in the avascular region, while many cells expressing Smad8 were located in the vascular region of the ANZ. These results suggest that BMPs mediated by SMAD signaling activate a molecular cascade that culminates in PCD.

Effects of breeder hen age and dietary L-carnitine on progeny embryogenesis.

1. Ross 308 broiler breeder hens were given diets containing 0 or 25 mg L-carnitine/kg (8 replications per treatment) from 21 weeks of age. 2. Hens were inseminated with semen from Ross broiler breeder males. In a common facility, subsequent progeny hatchability and embryonic mortality at 25, 30, 32, and 38 weeks of breeder age were evaluated. 3. Subsequent egg component weights, incubational egg water loss, progeny embryo growth, and embryo, yolk sac and liver composition through 18 d of incubation at 27, 32, and 38 weeks of breeder age were evaluated. 4. Calculated additions of L-carnitine were in agreement with analysed contents of 3.5 and 31.1 mg free L-carnitine/kg of diet, respectively, and total L-carnitine concentrations increased by 48.6, 21.7, and 10.0% in 0-d yolk, 18-d yolk sac, and 18-d liver samples, respectively, due to the addition of dietary L-carnitine. 5. Supplemental L-carnitine resulted in increased (0.6%) relative 0-d egg yolk weight across weeks 27, 32, and 38, and reduced (0.38%) 18-d yolk sac palmitoleic acid concentration at week 27 without altering embryogenesis. 6. In conclusion, dietary L-carnitine (25 mg/kg of the diet) was deposited in the yolks of broiler breeder hens and was subsequently transferred to the embryonic liver via yolk sac absorption through 18 d of incubation. Furthermore, dietary L-carnitine supplementation increased ovarian follicle yolk deposition in 27-, 32-, and 38-week-old breeder hens, and influenced yolk sac fatty acid beta-oxidation in embryos from 27-week-old breeder hens causing yolk sac palmitoleic acid concentrations to be reduced by 18 d of incubation.

Brief report: embryonic growth and hatching implications of developmental 670-nm phototherapy and dioxin co-exposure.

OBJECTIVE: We assessed the effect of 670-nm light therapy on growth and hatching kinetics in chickens (Gallus gallus) exposed to dioxin. BACKGROUND DATA: Photobiomodulation has been shown to stimulate signaling pathways resulting in improved energy metabolism, antioxidant production, and cell survival. In ovo treatment with 670-nm light-emitting diode (LED) arrays improves hatching success and increases hatchling size in control chickens. Under conditions where developmental dioxin exposure is above the lethality threshold (100 ppt), phototherapy attenuates dioxin-induced early embryonic death. We hypothesized that 670-nm LED therapy would attenuate dioxin-induced developmental anomalies and increase hatching success. METHODS: Fertile chicken eggs were injected with control oil, 2, 20, or 200 ppt dioxin, or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prior to the start of incubation. Half of the eggs in each dose group were treated once per day from embryonic days 0-20 with 670-nm LED light at a fluence of 4 J/cm2. Hatchling size, organ weights, and energy parameters were compared between dose groups and LED treatment. RESULTS: LED therapy resulted in earlier pip times (small hole created 12-24 h prior to hatch), and increased hatchling size and weight in the 200 ppt dose groups. However, there appears to be an LED-oil interaction within the oil-treated controls that results in longer hatch times and decreased liver weight within the LED control dose groups in comparison to the non-LED control dose groups. CONCLUSION: Size and hatching times suggest that the hatching success and preparedness of chicks developmentally exposed to dioxin concentrations above the lethality threshold is improved by 670-nm LED treatment administered throughout the gestation period, but the relationship may be complicated by an LED-oil interaction.

Prenatal auditory enrichment with species-specific calls and sitar music modulates expression of Bcl-2 and Bax to alter programmed cell death in developing chick auditory nuclei.

Postnatal auditory stimulation influences early perceptual learning. Previously we reported morphological effects of prenatal auditory stimulation by species-specific and sitar musical sounds on the chick brainstem auditory nuclei-nucleus magnocellularis and nucleus laminaris. At hatching, these two nuclei of auditory enriched embryos showed higher neuronal numbers, amongst other morphological changes. There were also increases in synaptophysin and syntaxin1 expressions in the sound enriched groups and modulation of the developmental expression of transcription factors c-Fos and c-Jun. We hypothesized that prenatal auditory enrichment may have reduced embryonic apoptosis in these nuclei with possible alteration of molecular mechanisms enhancing the postsynaptic neuron's ability to survive. In the present study, therefore, we examined apoptotic cell death by TUNEL technique and Bcl-2 expression using immunohistochemistry and immunoblotting. In the controls, a peak percentage in the TUNEL-positive cells was noted in the auditory nuclei at embryonic day 12, which was reduced at embryonic day 16. Bcl-2 immunoreactivity decreased from embryonic day 8 to embryonic day 12 overlapping the period of embryonic cell death in these nuclei. The stimulated groups, however, showed fewer apoptotic neurons and higher Bcl-2 level than that in the controls. On the other hand, Bax immunohistochemistry showed correlated reverse changes compared to Bcl-2 expression. Thus prenatal extra-acoustic stimulation appears to alter Bcl-2 and Bax expression to support cell survival and differentiation, thereby augmenting the development of auditory nuclei.