Manipulated Mouse Embryos as Bioassay System for Water Quality Control

Mouse pronuclear stage embryos with intact slit zona pellucida (manipulated) were cultured in vitro until the hatched blastocyst stage in simplex optimized medium with higher K⁺ concentration (KSOM) prepared with three different water types: tap, deionized reverse osmosis (D‐O) water and Milli‐Q system (M‐Q) water. The culture media were supplemented with or without protein and ethylenediaminetetraacetic acid (EDTA, disodium salt). The rates of hatched blastocysts were significantly affected (p < 0.01) by micromanipulation, protein supplement and water source. The water source has no influence (p > 0.05) on development in EDTA‐supplemented protein‐free culture media, whereas in EDTA‐ and protein‐free culture media, the water quality significantly (p < 0.001) affected the rates of development, with higher rates in media prepared with M‐Q water. The micromanipulated embryos showed higher sensitivity to the water quality (p < 0.01). It worth mentioning that the rates of hatched blastocysts in protein‐free culture media were very low (0–7.5%). Furthermore, the three different water types were analysed by measuring the electrical conductivity, inorganic ions, total organic carbon and endotoxins to evaluate the purity. M‐Q water showed the lowest levels of inorganic ion, total organic carbon and endotoxin concentrations. We concluded that manipulated mouse embryos are good system to evaluate the quality of water used in biological system.     

Impact of bovine somatotropin administration beginning at day 70 of lactation on serum metabolites, milk constituents, and production in cows previously exposed to exogenous somatotropin.

Metabolic and production responses are reported for 72 cows treated with bovine somatotropin (BST) for 30 days starting at day 70 of lactation. Of these 72 cows, 48 had been exposed in the preceding lactation to long-term treatment with BST at 3 dosages and 24 (controls) had not been given BST. Approximately half of the cows in each group were parity-2 cows, the rest were older. Comparisons between groups were made separately for parity-2, and older cows. Analyses, using pretreatment values of each variable as a covariate, indicated that older cows, but not parity-2 cows, significantly (P less than 0.05) increased milk production during treatment. Parity-2 cows, however, had a significantly higher milk fat percentage than controls following treatment. Cows treated with 51.6 or 86 mg BST/d in both parity groups had significantly higher serum-free fatty acids than controls. Estimated net energy balances were significantly lower for older treated cows, but did not significantly differ from controls for parity-2 treated cows. Older cows in the 86 mg of BST/d group tended to have higher concentrations of blood glucose than did older control-group cows. Treatment with BST did not significantly increase serum ketone concentrations in any group of animals, and none of the cows developed clinical ketosis during this period. Estimated net energy balance (ENEB) during treatment was a significant (P less than 0.05) covariate for free fatty acid concentrations in older cows and for milk fat percentage in parity-2 cows. Covariate adjusted analyses, using ENEB during treatment as a covariate, indicated that lipolytic stimuli already acting may be enhanced by treatment with BST, but a negative energy balance was not a necessary precondition for free fatty acid concentrations to increase following somatotropin treatment. Similarly, milk fat percentages for parity-2 treated cows were significantly (P less than 0.05) higher during treatment than controls when ENEB during treatment was used as a covariate. Increased milk fat concentrations in parity-2 treated cows were not associated with significant increases in the ratio of C18:C4-10 milk fatty acids, indicating that increased milk fat resulted from either an increase in incorporation of C18 fatty acids into milk fat coupled with an increase in de novo mammary synthesis of C4-10 milk fatty acids or an increase in C12-16 fatty acids that may arise either from increased tissue mobilization, from diet, or from de novo mammary synthesis.     

ACE inhibition in goats under fixed‐time artificial insemination protocol increases the pregnancy rate and twin births

To assess the effect of the angiotensin‐converting enzyme (ACE) inhibition on the efficiency of the fixed‐time artificial insemination (TAI), 69 goats were divided randomly into two groups: enalapril (n = 35) and control (n = 34). In the experiment, all animals underwent the protocol of fixed‐time artificial insemination for 12 days. Enalapril group received enalapril maleate dissolved in saline (Enalapril, Lab Teuto Ltda) subcutaneously at the following doses: 0.2 mg/kg/day in D0‐D2; 0.3 mg/kg/day in D3‐D6 and 0.4 mg/kg/day in D7‐D11. The control group received the corresponding volume of 0.9% saline solution. We performed a single insemination 36 hr after sponge removal using frozen semen from two adult male goats with recognized fertility. The ultrasound pregnancy diagnosis was 30 days after the artificial insemination (AI). There was significant increase in pregnancy rates and twinning as well as a decrease in foetal loss in animals receiving enalapril (p < .01). The use of ACE inhibitors during the TAI protocol was shown to be a promising alternative to increase the efficiency of such reproductive biotechnology.     

New insights into the structural characteristics of the arabinogalactan-protein (AGP) fraction of gum arabic

The structural characteristics of the gum exudate of Acacia senegal (gum arabic) have been investigated by monitoring the composition and physicochemical properties before and after treatment with proteolytic enzyme and various alkaline systems. Molecular mass ( M w) and radius of gyration ( R g) measurements were performed using gel permeation chromatography (GPC) coupled to refractive index, UV absorbance, and multiangle light scattering detectors and indicated that the macromolecules present have a compact structure. It was found that treatment with proteolytic enzyme caused the arabinogalactan-protein component (AGP) with average molecular mass approximately 2 x 10 (6) Da to degrade, yielding material of molecular mass approximately 4 x 10 (5) Da, whereas the bulk of the material corresponding to the protein-deficient arabinogalactan component (AG) with molecular mass 4 x 10 (5) remained unaffected. Barium hydroxide was found to hydrolyze the polysaccharide component (AG) itself in addition to the proteinaceous component as demonstrated in control experiments using dextran. However, sodium borohydride/sodium hydroxide treatments were unable to hydrolyze dextran and were assumed to hydrolyze only the proteinaceous component of gum arabic. The AGP component was completely degraded, yielding material of molecular mass approximately 4.5 x 10 (4) Da. It has been concluded, therefore, that the enzyme did not fully hydrolyze all of the protein present and that the AGP component of gum arabic consists of carbohydrate blocks of approximately 4.5 x 10 (4) Da linked to a polypeptide chain consistent with the wattle blossom structure. Because the AGP was degraded to differing extents using a mild and more severe sodium borohydride/sodium hydroxide treatment, it was concluded that the polysaccharide moieties were linked through both O-serine and O-hydroxyproline residues. The gum arabic sample was deglycosylated by treatment with anhydrous hydrogen fluoride and revealed the presence of two putative core proteins of approximately 3 x 10 (4) and approximately 5 x 10 (3) Da, respectively, which correspond to proteins of approximately 250 and 45 amino acids in length. A new model for the structure of the AGP component has been proposed.     

Landscape-level naturalness of conservation easements in a mixed-use matrix

Context

With underrepresentation of habitats in publicly protected areas, attention has focused on the function of alternative land conservation mechanisms. Private conservation easements (CEs) have proliferated in the United States, yet assessing landscape-level function is confounded by varying extent, resolution, and temporal scale.

Objectives

We developed and tested an assessment tool to evaluate interacting spatial, social, and environmental attributes of easements relative to the degree of human modification (HM). We hypothesized that on both private and public conservation properties HM would be lower than on non-conserved parcels, and that for fine-scale features (most CEs), the level of HM would be driven by the variables used to create the coarser scale HM measure.

Methods

Variation in HM between private, public, and non-conserved was tested via pairwise parcel sampling. Composition was evaluated using multiple geographic bounds and edge characteristics. We assessed both environmental and social predictors using multinomial logistic regression.

Results

Privately conserved lands did not differ significantly from non-conserved lands. Publicly conserved lands had lower HM than both privately conserved and non-conserved lands. Edge contrast was similar between private and matched non-conserved patches. The level of HM was not driven by distance to roads, or by elevation in this mixed-use setting.

Conclusions

Variation in tests for differences, land characteristics, and HM variables confirmed the significantly lower HM of publicly protected lands, and opens the question as to naturalness of easements in some contexts. CEs in this location may be representative of the mixed rural-forested landscape instead of more natural land cover.

     

Experimental prevention of bitterweed (Hymenoxys odorata) poisoning of sheep

To examine the effects on bitterweed toxicity of dietary factors known to increase thiol concentrations in the body, 36 lambs were fed one of the following diets (12 lambs/diet) for a minimum of 9 days prior to bitterweed administration: diet 1, 10% crude protein; diet 2, 20% crude protein, 0.5% methionine, 0.5% sodium sulfate, and 1,102 IU of vitamin E/kg; and diet 3, diet 2 with 0.5% ethoxyquin hydrochloride added. Four lambs fed each diet were euthanatized prior to bitterweed administration (initial euthanasia group). Four lambs fed each diet were administered bitterweed (0.68% hymenoxon, air-dried basis) at a rate of 0.25% of live weight for 5 consecutive days. The remaining four lambs on each diet served as unchallenged controls. In the initial euthanasia group, diet 2 increased extracellular blood thiol concentrations (1.12 vs 0.94 mg of SH/d1, P less than 0.10), rumen fluid thiol concentrations (4.46 vs 1.88 mg of SH/d1, P less than 0.05), and liver thiol concentrations (263.6 vs 109.3 micrograms SH/g of wet wt, P less than 0.05), compared with diet 1. Ethoxyquin hydrochloride (diet 3) reduced blood thiol concentrations (0.94 vs 1.12 mg of SH/dl, P less than 0.10) and liver thiol concentrations (151.6 vs 263.6 micrograms of SH/g of wet wt, P less than 0.05), compared with diet 2. Kidney thiols were unaffected by treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ruminal and postruminal infusion of starch hydrolysate or glucose on the microbial ecology of the gastrointestinal tract in growing steers

Forty crossbred steers were used to determine the effects of carbohydrate supply site on the indigenous bacteria of the gastrointestinal tract. Steers were fitted with ruminal and abomasal infusion catheters and assigned randomly to one of eight groups in a complete randomized block design. The experimental period was 36 d. Treatments included: 1) a pelleted basal diet fed at 0.163 Mcal ME x (kg BW(0.75)) x 1 x d(-1) (LE); 2) the basal diet fed at 0.215 Mcal ME x (kg BW(0.75)) (-1) x d(-1) (HE); 3) the basal diet fed at 0.163 Mcal ME x (kg BW(0.75))(-1) x d(-1) with ruminal infusion of starch hydrolysate (SH) (RSH); 4) the basal diet fed at 0.163 Mcal ME x (kg BW(0.75))(-1) x d(-1) with abomasal infusion of SH (ASH); and 5) the basal diet fed at 0.163 Mcal ME x (kg BW(0.75))(-1) x d(-1) with abomasal infusion of glucose (AG). The total volume ofinfusate (5 kg x site(-1) x d(-1)) was equalized across treatments and infusion sites by infusion of water. Glucose and SH were infused at rates of 14.35 and 12.64 g x (kg BW(0.75)) x d(-1), respectively. Ruminal, cecal, and fecal samples were obtained on d 36. Ruminal pH was low (5.79) in LE steers and unaffected (P > 0.10) by increased energy intake or carbohydrate infusion. Cecal and fecal pH were 6.93 and 7.00, respectively, for LE steers. Increasing energy intake (P < 0.10) and the rate of carbohydrate infusion (P < 0.01) significantly decreased cecal and fecal pH compared with LE. Ruminal counts of anaerobic bacteria in LE steers were 8.99 log10 cells/g and abomasal carbohydrate infusion had no affect (P > 0.10) on these numbers. However, ASH and AG steers had approximately 1.5 log10 cells/g more (P < 0.01) cecal and fecal anaerobic populations. Ruminal, cecal, and fecal aerobic bacterial counts were 40, 22, and 23%, respectively, lower than anaerobic counts. Generally, aerobic counts responded similarly to the anaerobic counts. Less than 1% of the anaerobic bacteria enumerated in the rumen, cecum, and feces were coliforms, and 97% of the coliforms were Escherichia coli. Carbohydrate infusions resulted in only numerical increases in fecal coliform and E. coli concentrations (P > 0.10). Fecal E. coli were highly acid sensitive in all steers, with less than 1% surviving a 1-h exposure to low pH (2.0). This suggests that cecal or fecal pH is not a good indicator of acid resistance, and it supports the concept that there are other factors that may induce acid resistance.     

Activation of bovine peripheral blood gammadelta T cells for cell division and IFN-gamma production

Bovine peripheral blood gammadelta T cells have been evaluated for effector function (IFN-gamma production) and clonal expansion in a variety of systems including following activation by mitogens, IL-12, and stimulation, through the T cell receptor (TCR) with anti-CD3 monoclonal antibody (mAb), a cell-bound molecule and a soluble antigenic extract. To evaluate cell division, carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis were used, while IFN-gamma production was evaluated by intracytoplasmic staining. It was found that bovine gammadelta T cells produced IFN-gamma and clonally expanded when stimulated through the TCR/CD3 complex by a cell-associated autologous molecule on monocyte, by bacterial components following in vivo sensitization of gammadelta T cells with a leptospira vaccine or by anti-CD3 mAb. In addition, gammadelta T cells were activated efficiently for effector function but not clonal expansion by culturing with IL-12. In contrast, stimulation by Con A or PMA/ionomycin induced efficient replication but only low level IFN-gamma production which was not enhanced by the presence of IL-12. In several systems the amount of IFN-gamma produced per cell by gammadelta T cells was less than that produced by CD4 T cells in the same cultures.