Pigs weaned from the sow at 10 days of age respond to dietary energy source of manufactured liquid diets and exogenous porcine somatotropin.

Previous research indicates that the neonatal pig does not alter feed intake in response to changes in the energy density of manufactured liquid diets. Also, the limited response of IGF-I to exogenous porcine ST (pST) previously observed in young pigs may be influenced by the source of dietary energy. Our objectives were to 1) determine the effect of a high-fat (HF; 25% fat and 4,639 kcal/kg ME; DM basis) or low-fat (LF; 2% fat and 3,481 kcal/kg ME; DM basis) manufactured liquid diet on pig performance; and 2) determine whether the limited response to exogenous pST in young pigs depends on the source of dietary energy. Two replicates of 60 pigs (n = 120; barrows and gilts distributed evenly), with an initial BW of 4,207 +/- 51 g, were weaned from the sow at 10 d of age and used in a randomized complete block design. Pigs were assigned by BW to one of six pens. Diets were formulated to provide a constant lysine:ME ratio and were fed on a pen basis for a duration of 9 d. On d 5, barrows and gilts within a pen were assigned randomly to receive either 0 or 120 microg of pST.kg BW(-1).d(-1) for 4 d. Pigs gained 336 +/- 9 g/d, which resulted in an ending BW of 7,228 +/- 120 g, regardless of dietary treatment (P > 0.15). Pigs fed the LF diet consumed 17% more DM per pen daily than pigs fed the HF diet (2,777 +/- 67 vs. 2,376 +/- 67 g/d, P < 0.01), but calculated ME intake did not differ between dietary treatments (P > 0.20). The G:F was 24% greater in HF- than in LF-fed pigs (P < 0.01). Plasma urea N concentrations were higher in the HF-fed pigs (11.0 +/- 0.6 mg/dL) than in pigs fed the LF diet (6.2 +/- 0.6 mg/dL; P < 0.05). Treatment with pST increased circulating IGF-I (P < 0.01) and decreased PUN (P < 0.01) concentration 32 and 25%, respectively, regardless of dietary treatment (P > 0.30). Circulating leptin averaged 1.8 +/- 0.1 ng/mL and was not affected by dietary treatment (P > 0.35) or pST (P > 0.40). These results suggest that the ST/IGF axis is responsive in the young pig and the increase in circulating IGF-I and growth is independent of the source of dietary energy. Also, young pigs respond to a lower energy density liquid diet with increased feed intake, without altering growth performance, apparently utilizing a mechanism other than circulating leptin.

In vitro interactions of cryopreserved stallion spermatozoa and oviduct (uterine tube) epithelial cells or their secretory products.

Formation of a spermatozoa ('sperm') reservoir in the mare is thought to occur through lectin-mediated sperm attachment to the oviductal epithelium. Once attached, prefertilization sperm survival is supported by oviductal factors. Cryopreservation of stallion sperm decreases the number of sperm attaching to oviduct epithelial cells (OEC) and the length of time these sperm survive. Quantification of in vitro interactions between sperm and OEC in a co-culture system may provide an assay for functional integrity of cryopreserved or fresh sperm samples. Additionally, superior additives for in vitro handling of stallion sperm may be isolated from OEC secretory products. Experiment 1 compared first service conception (FSC) rates resulting from the use of cryopreserved sperm of seven stallions, with sperm function in co-culture such as attachment to OEC and subsequent survival time. Stallions were grouped by cumulative FSC rates observed over three seasons as having average (44 +/- 3%) or high (65 +/- 2%) fertility over a total of 217 first services (31 +/- 9 per stallion). Samples from stallions in the high fertility group had more (P = 0.04) sperm attached to OEC and longer subsequent sperm survival in co-culture (P = 0.05) as compared with those from the average fertility group. FSC rates correlated with numbers of sperm attaching to OEC and their survival time in co-culture (r > or = 0.71). In Experiment 2, the function of cryopreserved stallion sperm was evaluated in culture with OEC secretory products from three different sources. After 5 h of culture, sperm incubated with medium conditioned by bovine OEC which had been 'bioactivated' (e.g. previously exposed to sperm in culture) were found to be more (P < or = 0.05) motile and capacitated as compared to sperm in basal TALP medium alone. Sperm in this conditioned medium also survived longer (P = 0.05; 27 +/- 5 h vs. 17 +/- 4 h) than did those in control medium.

Effects of bovine serum albumin on function of cryopreserved stallion spermatozoa during medium culture and uterine tube epithelial cell coculture.

OBJECTIVE: To compare function of cultured cryopreserved stallion spermatozoa in a modified Tyrode's medium (TM), with or without bovine serum albumin (BSA), or in uterine tube (oviduct) epithelial cell (OEC) coculture in TM, with or without BSA. SAMPLE POPULATION: Cryopreserved spermatozoa from 6 proven stallions and OEC from bovine reproductive tracts in follicular phase. PROCEDURE: Thawed spermatozoa were cultured in TM, with or without BSA, or cocultured with OEC monolayers in TM, with or without BSA. Percentages of capacitated and acrosome-reacted spermatozoa were measured at 5 hours for TM cultures. Spermatozoal survival and motility characteristics were observed over time for all culture methods. Number of spermatozoa attaching to OEC were compared for cocultures. RESULTS: Use of TM without BSA altered spermatozoal function in cell-free medium culture and OEC coculture. A higher percentage of spermatozoa were acrosome reacted in TM with BSA, although percentages of capacitated spermatozoa did not differ. Spermatozoa survived longer and maintained superior motion in TM culture without BSA and in OEC cocultures. More spermatozoa were able to attach to OEC in TM without BSA. CONCLUSIONS: Incubation of cryopreserved spermatozoa in media with BSA resulted in rapid decrease in percentage of intact, motile spermatozoa and limited their ability to interact with OEC. CLINICAL RELEVANCE: Current culture media used for assisted reproduction techniques in horses do not provide functionally capacitated spermatozoa. Removal of BSA from such media improves spermatozoal quality and survival.

Direct measurement of unfractionated heparin using a biochemical assay.

A number of investigations have noted that functional biological assays for heparin are not always reliable and may not reflect the actual biochemical level of heparin in patients receiving anticoagulant therapy. This creates the possibility that patients receiving anticoagulant treatment may have an excess or deficiency of circulating levels of heparin. To address this problem, we have developed a direct biochemical measurement of heparin. The heparin assay uses fluorophore-assisted carbohydrate electrophoresis (FACE) to directly measure the predominate disaccharide of unfractionated heparin. In this study, unfractionated heparin was measured in vitro throughout a wide range of heparin concentrations in plasma. Seven in vivo pharmacokinetic studies in five normal subjects given 3,000 USP units of unfractionated heparin intravenously showed a three-phase elimination process with higher peak plasma levels and shorter elimination times than predicted from previous studies. At these doses, heparin is largely eliminated intact through urinary excretion. Body weight has a significant effect on heparin kinetics. When we compared the direct biochemical assay with two biological clotting assays, we found the latter can overestimate biochemical heparin concentrations. The FACE assay, due to its sensitivity, is also able to measure circulating levels of endogenous heparin in plasma and urine. Direct heparin measurement using the FACE technique is practical and useful for studies of the correlation of biochemical and biological activities.

Light propagation in a planar dielectric waveguide with a gyrotropic layer.

The nature of the magneto-optic Kerr effect in a planar dielectric waveguide geometry has been investigated by calculation of the Jones matrix for a planar waveguide structure with a gyrotropic magnetic material as one wall. The intensity of the component of the field that is in the polarization state orthogonal to the input was calculated as a function of length of the gyrotropic material and input polarization state. The degree of polarization rotation depends on the relative orientation of the magnetization in the magnetic material and the direction of propagation. It is found that there exists an optimal waveguide length and input polarization at which the output signal is maximized and that a significant enhancement in polarization rotation is available with respect to free-space reflection. These results indicate that a magnetic-film-bounded planar waveguide can be used for device applications such as magnetic field sensors or magneto-optic modulators.

Salivary mesenchyme cells that induce mammary epithelial hyperplasia up-regulate EGF receptors in primary cultures of mammary epithelium within collagen gels.

Salivary mesenchyme is a potent stimulator of mammary epithelial hyperplasia and carcinogen-induced tumor formation in vivo. We have utilized a three-dimensional collagen gel culture system, which mimics the in vivo growth environment, to identify growth stimulatory molecules produced by salivary mesenchyme cells. In this report we describe the development and characteristics of salivary mesenchyme cell lines, and we present further evidence that these cells produce growth factor(s) which could account for the effect by interacting with epidermal growth factor (EGF) receptors on primary mouse mammary epithelial cells isolated from midpregnant mice. Using a receptor assay with isolated cell membranes, we characterized [125I]-EGF binding to mammary epithelial cells cultured within collagen gels. Scatchard analysis revealed one class of high affinity EGF receptors with a Kd ranging from 8.3 x 10(-11) M on day one to 5.1 x 10(-11) M on day 10 of the culture period. Addition of 10 ng/ml purified EGF to the culture medium progressively up-regulated the expression of EGF receptors during a 10-day culture period. Scatchard analysis showed that the increase in specific [125I]-EGF binding was due predominantly to an increase in EGF receptor number. We also demonstrated that conditioned medium collected from salivary mesenchyme cells competed effectively for EGF receptor sites on mammary epithelial cells, and chronic exposure to conditioned medium up-regulated EGF receptor expression. Thus, EGF-related growth factor(s) released by salivary mesenchyme cells may induce hyperplasia of adjacent mammary epithelium in vivo, both by directly activating EGF receptors, and by provoking long term up-regulation of EGF receptors.

A transforming growth factor related to epidermal growth factor is expressed by fetal mouse salivary mesenchyme cells in culture.

Fetal mouse salivary mesenchyme cells secrete a protein with an apparent MW of 15 Kd that is immunologically related to epidermal growth factor (EGF). Conditioned medium collected from these cells in culture stimulates the growth of primary mouse mammary epithelial cells cultured within collagen gels, competes for binding to EGF receptor sites on these mammary epithelial cells and stimulates the anchorage-independent growth of normal rat kidney fibroblast cells within soft agarose. Prior immunoprecipitation of salivary mesenchyme cell conditioned medium with anti-EGF antibodies effectively removes or attenuates all of these effects confirming that an EGF-like factor is involved in these responses.

Resistance of herpes simplex virus to 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine: physical mapping of drug synergism within the viral DNA polymerase locus.

A herpes simplex virus type 2 (HSV-2) mutant TS6 (strain HG52) induces a heat-labile viral DNA polymerase at the nonpermissive temperature and is markedly resistant to 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-guanine [2'-nor-2'-deoxyguanosine; 2'NDG]. This antiviral drug requires HSV thymidine kinase for phosphorylation to an active inhibitor (2'NDG-triphosphate), and thymidine kinase-deficient mutants of HSV exhibit varying degrees of resistance to 2'NDG, with the HSV type 1 (HSV-1) B2006 mutant (Kit) being markedly resistant. The ts6 mutation and the 2'ndgR-1 mutation within the viral DNA polymerase locus have been physically mapped by marker rescue and generation of HSV-1/HSV-2 intertypic recombinants. The physical map limits for the ts6 mutation and 2'ndgR-1 mutation are closely linked within a 2.2-kilobase-pair region of DNA sequences and are physically separate from the paaR-1 and acvR-1 mutations. Resistance to 2'NDG by HSV-2 ts6 can be overcome in the presence of combinations of 2'NDG and phosphonoacetic acid, indicating drug synergism within the viral DNA polymerase locus. These physical mapping studies expand the limits of DNA sequences defining an active center in the viral polymerase to 3.5 kilobase pairs, indicating that regions spanning the entire polymerase polypeptide may contribute to a specialized surface able to interact with nucleotides of different structure.