Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.     

Donor milk intake and infant growth in a South African neonatal unit: a cohort study

Background

Implications of donor milk feedings on infant growth in resource limited settings remain uncertain. This knowledge gap includes the impact of donor milk availability on infant intake of mother’s own milk. Therefore, this investigation aimed to measure intake and growth in infants receiving donor milk when born to women from resource limited backgrounds with high rates of human immunodeficiency virus (HIV).

Methods

A retrospective cohort study enrolled eligible infants admitted to a South African combined neonatal intensive and secondary high care unit, within a one year admission period during 2015, with signed consent for donor milk feedings. A certified milk bank provided donor milk. Daily nutritional intake during the first month was recorded. Details included proportional intake of donor milk, mother’s own milk and infant formula. The primary outcome of infant growth velocity from day back to birth weight to discharge was calculated when length of stay was ≥14 days. Analyses primarily used T-tests; mixed effects models compared weekly calorie intake.

Results

One hundred five infants with donor milk consent were born at 30.9?±?3.6 weeks of gestation, weighing 1389?±?708 g. Forty percent of mothers had HIV. Infant growth velocity did not differ based on percent of feedings as donor milk (≥ 50%: 11.8?±?4.9 g/kg/d; <?50%: 13.5?±?5.3 g/kg/d; p?=?0.3). Percent of feedings from donor milk was similar based on maternal HIV status (positive: 31?±?25%; negative: 36?±?29%; p?=?0.4), as was percent of feedings as mother’s milk (positive: 53?±?35%; negative: 58?±?30%; p?=?0.4). Calorie intake increased markedly during the first two weeks and then plateaued (p?<?0.0001).

Conclusions

Donor milk feedings in higher proportions did not further impair growth of infants managed in a South African combined neonatal intensive and secondary high care unit with growth rates already below reference ranges. The provision of donor milk contributed to feedings being composed of primarily human milk during the first month. Increasing early calorie intake may improve infant growth in this center.

     

Effects of elevated CO2 concentrations on three montane grass species: III. Source leaf metabolism and whole plant carbon partitioning

Agrostis capillaris L.⁵, Festuca vivipara L. and Poaalpina L.were grown in outdoor open-top chambers at either ambient (340 3µmol mol^–1) or elevated (6804µmol mol^–1)concentrations of atmospheric carbon dioxide (CO₂) for periodsfrom 79–189 d. Photosynthetic capacity of source leaves of plants grown atboth ambient and elevated CO₂ concentrations was measured atsaturating light and 5% CO₂. Dark respiration of leaves wasmeasured using a liquid phase oxygen electrode with the buffersolution in equilibrium with air (21% O₂, 0.034% CO₂). Photo-syntheticcapacity of P. alpina was reduced by growth at 680 µmolmol^–1 CO₂ by 105 d, and that of F. vivipara was reducedat 65 d and 189 d after CO₂ enrichment began, suggesting down-regulationor acclimation. Dark respiration of successive leaf blades ofall three species was unaltered by growth at 680 relative to340 µmol mol^–1 CO₂. In F. vivipara, leaf respirationrate was markedly lower at 189 d than at either 0 d or 65 d,irrespective of growth CO₂ concentration. There was a significantlylower total non-structural carbohydrate (TNC) concentrationin the leaf blades and leaf sheaths of A. capillaris grown at680µmol mol^–1 CO₂. TNC of roots of A. capillariswas unaltered by CO₂ treatment. TNC concentration was increasedin both leaves and sheaths of P. alpina and F. vivipara after105 d and 65 d growth, respectively. A 4-fold increase in thewater-soluble fraction (fructan) in P. alpina and in all carbohydratefractions in F. vivipara accounted for the increased TNC content. In F. vivipara the relationship between leaf photosyn-theticcapacity and leaf carbohydrate concentration was such that therewas a strong positive correlation between photosynthetic capacityand total leaf N concentration (expressed on a per unit structuraldry weight basis), and total nitrogen concentration of successivemature leaves reduced with time. Multiple regression of leafphotosynthetic capacity upon leaf nitrogen and carbohydrateconcentrations further confirmed that leaf photosynthetic capacitywas mainly determined by leaf N concentration. In P. alpina,leaf photosynthetic capacity was mainly determined by leaf CHOconcentration. Thus there is evidence for down-regulation ofphotosynthetic capacity in P. alpina resulting from increasedcarbohydrate accumulation in source leaves. Leaf dark respiration and total N concentration were positivelycorrelated in P. alpina and F. vivipara. Leaf dark respirationand soluble carbohydrate concentration of source leaves werepositively correlated in A. capillaris. Changes in source leafphotosynthetic capacity and carbohydrate concentration of plantsgrown at ambient or elevated CO₂ are discussed in relation toplant growth, nutrient relations and availability of sinks forcarbon. Key words: Elevated CO₂, Climate change, grasses, carbohydrate partitioning, photosynthesis, respiration     

HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis

The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of a hlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants.     

The Toxicity of Sulphur Dioxide towards Certain Lactic Acid Bacteria from Fermented Apple Juice

This paper describes a method for testing the effect of various concentrations of SO₂ on lactic acid bacteria from ciders. The media and methods were devised to minimize loss of SO₂ due to oxidation or binding with carbonyl compounds. Exposure of laboratory or freshly isolated strains to various concentrations of free SO₂ at pH 4·0 did not readily kill them even at high concentrations of free SO₂ ( c. 150 p/m or 0·97 p/m molecular SO₂) yet they were suppressed at low concentrations ( c. 5 p/m or 0·032 p/m molecular SO₂). Reducing the pH to 3·4 reaffirmed how much more effective SO₂ is against lactic acid bacteria at lower pH levels because more is present as molecular SO₂. As a result of this the idea of quoting SO₂ values as p/m molecular SO₂ is advocated. Addition of hydrogen peroxide or acetaldehyde to a test system containing 142 p/m free SO₂ showed that they had a similar effect in nullifying its antimicrobial properties and allowing the test bacteria to grow. There was no indication that acetaldehyde bisulphite was toxic to the test bacteria.     

Stimulation of neutrophils by tumor necrosis factor

Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H2O2 production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.     

Changes in hypothalamic noradrenergic systems during the anorexia of zinc deficiency

Pregnant Wistar rats fed control and Zn-deficient diets received daily oral doses of 0, 100, and 300 mg/kg sodium valproate from d 16 to 20 of gestation. Only the highest valproate doses induced a small reduction in fetal body weight in the normally fed group. Zinc deficiency caused a drastic reduction in maternal and only a small reduction in fetal serum Zn concentrations. Valproate treatment had no effect on maternal and fetal serum Zn concentrations. Valproate reduced fetal liver Zn content only in the normally fed group. The reduction of liver Zn content resulted from the reduction of Zn-metallothionein. Valproate did not affect total Zn and Zn-metallothionein in kidneys. Three percent of the Zn-deficient fetuses developed hydronephrosis and hydrops. Valproate treatment drastically enhanced the occurrence of fetal hydronephrosis and hydrops. Valproate induced fetal liver necroses, independent of Zn nutrition.