Seasonal variation in equine follicular fluid proteome.

BACKGROUND: Proteomic studies of follicular fluid (FF) exist for several species, including the horse; however, the seasonal influence on FF proteome has not been explored in livestock. The application of high-throughput proteomics of FF in horse has the potential to identify seasonal variations of proteins involved in follicle and oocyte growth. METHODS: This study (i) profiles the proteomes of equine FF collected from dominant growing follicles during the spring anovulatory season (SAN), and spring (SOV), summer (SUM), and fall (FOV) ovulatory seasons; and (ii) identifies season-dependent regulatory networks and associated key proteins. RESULTS: Regardless of season, a total of 90 proteins were identified in FF, corresponding to 63, 72, 69, and 78 proteins detected in the SAN, SOV, SUM, and FOV seasons, respectively. Fifty-two proteins were common to all seasons, a total of 13 were unique to either season, and 25 were shared between two seasons or more. Protein-to-protein interaction (PPI) analysis indicated the likely critical roles of plasminogen in the SAN season, the prothrombin/plasminogen combination in SUM, and plasminogen/complement C3 in both SOV and FOV seasons. The apolipoprotein A1 appeared crucial in all seasons. The present findings show that FF proteome of SUM differs from other seasons, with FF having high fluidity (low viscosity). CONCLUSIONS: The balance between the FF contents in prothrombin, plasminogen, and coagulation factor XII proteins favoring FF fluidity may be crucial at the peak of the ovulatory season (SUM) and may explain the reported lower incidence of hemorrhagic anovulatory follicles during the SUM season.

Diversity and abundance of the abnormal chromosome 10 meiotic drive complex in Zea mays.

Maize Abnormal chromosome 10 (Ab10) contains a classic meiotic drive system that exploits the asymmetry of meiosis to preferentially transmit itself and other chromosomes containing specialized heterochromatic regions called knobs. The structure and diversity of the Ab10 meiotic drive haplotype is poorly understood. We developed a bacterial artificial chromosome (BAC) library from an Ab10 line and used the data to develop sequence-based markers, focusing on the proximal portion of the haplotype that shows partial homology to normal chromosome 10. These molecular and additional cytological data demonstrate that two previously identified Ab10 variants (Ab10-I and Ab10-II) share a common origin. Dominant PCR markers were used with fluorescence in situ hybridization to assay 160 diverse teosinte and maize landrace populations from across the Americas, resulting in the identification of a previously unknown but prevalent form of Ab10 (Ab10-III). We find that Ab10 occurs in at least 75% of teosinte populations at a mean frequency of 15%. Ab10 was also found in 13% of the maize landraces, but does not appear to be fixed in any wild or cultivated population. Quantitative analyses suggest that the abundance and distribution of Ab10 is governed by a complex combination of intrinsic fitness effects as well as extrinsic environmental variability.

Conjugated linoleic acids alter body composition differently according to physiological age in Moulard ducks.

Conjugated linoleic acids (CLA) have been shown to have remarkable yet inconsistent metabolic effects in mice, rats, hamsters, chickens, cattle, and humans. In particular, effects on lipogenesis vary with tissue, physiological state, and species. In this study we tested the hypothesis that CLA would differentially affect ducks of the same genetic background but of differing age. Growing (7 wk) and maintenance (11 wk) Moulard ducks were grouped by age and fed a standard diet supplemented with 5% soybean oil (control) or 5% CLA isomer mixture. Birds were slaughtered after 3 or 6 wk for assessment of body composition including adipose, liver, viscera, and empty carcass weight. Serum nonesterified fatty acid (NEFA) and glucose concentrations were evaluated, and gene targets were cloned from the duck to use in quantifying mRNA abundance for genes involved in lipogenesis (fatty acid synthase, FAS; acetyl-CoA carboxylase, ACC) and lipid oxidation (carnitine palmitoyl transferase-1, CPT-1) in liver tissue from maintenance birds. After 3 wk, the growing CLA group exhibited a 24% decrease in dissectible adipose tissue (P < 0.05), whereas maintenance birds showed no significant diet effect. After 6 wk, the growing CLA group exhibited a 20% increase in liver mass compared with the control (P < 0.05), but no diet effect on adipose tissue. Maintenance birds receiving dietary CLA had a 42% decrease in adipose tissue mass after 6 wk; increased serum NEFA, ACC, and CPT-1 mRNA after 3 and 6 wk (P < 0.05); and increased FAS mRNA after 3 wk of treatment (P < 0.05). These data indicate that CLA have potent effects on lipid metabolism in ducks, but these effects differ depending on physiological age.

Tolerance and efficacy of tribasic manganese chloride in growing broiler chickens.

These studies were designed to determine the relative bioavailability and tolerance of tribasic Mn chloride (TBMC) for growing broiler chickens. In experiment 1, birds were fed a basal diet (starter, 102 ppm; grower, 209 ppm) or the basal diet supplemented with 3,600, 4,500, or 5,400 ppm Mn from either TBMC or manganese sulfate (MnSO(4)), and BW, feed intake, and plasma Mn were measured. In experiments 2 and 3, diets included the basal diet (45 and 43 ppm Mn, respectively) and the basal diet supplemented with graded levels of either TBMC or MnSO(4) ranging from 30 to 240 ppm Mn. Body weight and feed intake were measured and tibia, bile, and liver were collected for mineral analysis; heart samples were taken for manganese superoxide dismutase activity, protein, and relative mRNA abundance. In experiment 1, BW differed among treatments, with higher Mn leading to lower BW (P < 0.05). Birds from all treatments showed higher plasma Mn than birds fed the basal diet. Birds supplemented with the highest level of MnSO(4) had the highest level of plasma Mn (P < 0.05). In experiment 2, tibia and liver Mn increased with higher dietary Mn regardless of source (P < 0.05). Liver Mn increased up to the 60 ppm diets whereas Mn in the tibia was highest with the 130 ppm diets. Bile Mn increased with increasing dietary Mn, but these differences were not significant. In experiment 3, manganese superoxide dismutase activity, protein, and relative mRNA abundance were not affected by diet. The calculated bioavailabilities of TBMC and MnSO(4) did not differ significantly (P > 0.20). Together, these results indicate that TBMC is as effective as and better tolerated than MnSO(4) and that supplementing Mn at the lowest level used in this study may be sufficient for normal development of broiler chickens.

Lutein exposure, in ovo or in the diet, reduces parameters of inflammation in the liver and spleen laying-type chicks (Gallus gallus domesticus).

These trials examined whether the demonstrated effects of embryonic and dietary carotenoid exposure on the inflammatory immune response in fast growing chickens also occur in slow growing chickens. The systemic and local inflammatory responses of chicks were examined in two experiments with two in ovo lutein levels (C+, carotenoid replete; or C-, carotenoid-deplete), two dietary lutein levels (0 or 40 mg lutein/kg diet), and two inflammatory challenges [no exposure or lipopolysaccharide (LPS)-vaccinated]. At 24 h after LPS vaccination, spleen weight was not affected by diet or in ovo lutein, but liver weight increased from C+ eggs (p < 0.01), and in LPS-vaccinated chicks fed 0 mg lutein (p < 0.05), but not in chicks fed 40 mg lutein. Plasma carotenoids and liver carotenoids were reduced post-LPS (p < 0.05). Splenic IL-6 mRNA abundance was the greatest post-LPS in C- chicks fed 40 mg lutein vs. C+ chicks fed 40 mg lutein (p < 0.05). Hepatic IL-6, iNOS and TGFß and splenic iNOS and TGFß were not affected by in ovo or dietary lutein. The systemic and local inflammatory results are similar to those observed in fast growing chickens, and support that lutein-depleted birds have greater inflammatory responses.

Localization of single- and low-copy sequences on tomato synaptonemal complex spreads using fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) is a powerful means by which single- and low-copy DNA sequences can be localized on chromosomes. Compared to the mitotic metaphase chromosomes that are normally used in FISH, synaptonemal complex (SC) spreads (hypotonically spread pachytene chromosomes) have several advantages. SC spreads (1) are comparatively free of debris that can interfere with probe penetration, (2) have relatively decondensed chromatin that is highly accessible to probes, and (3) are about ten times longer than their metaphase counterparts, which permits FISH mapping at higher resolution. To investigate the use of plant SC spreads as substrates for single-copy FISH, we probed spreads of tomato SCs with two single-copy sequences and one low-copy sequence (ca. 14 kb each) that are associated with restriction fragment length polymorphism (RFLP) markers on SC 11. Individual SCs were identified on the basis of relative length, arm ratio, and differential staining patterns after combined propidium iodide (PI) and 4', 6-diamidino-2-phenylindole (DAPI) staining. In this first report of single-copy FISH to SC spreads, the probe sequences were unambiguously mapped on the long arm of tomato SC 11. Coupled with data from earlier studies, we determined the distance in micrometers, the number of base pairs, and the rates of crossing over between these three FISH markers. We also observed that the order of two of the FISH markers is reversed in relation to their order on the molecular linkage map. SC-FISH mapping permits superimposition of markers from molecular linkage maps directly on pachytene chromosomes and thereby contributes to our understanding of the relationship between chromosome structure, gene activity, and recombination.

Effect of extruded full-fat soybeans on conjugated linoleic acid content of intramuscular, intermuscular, and subcutaneous fat in beef steers.

Crossbred Angus steers (n = 30) were used to determine whether the conjugated linoleic acid (CLA) content of beef fat could be increased by feeding varying levels of extruded full-fat soybeans as a source of polyunsaturated fatty acids for rumen biohydrogenation. Diets were as follows: 1) control, 2) 12.7% extruded full-fat soybeans (LESB), and 3) 25.6% extruded full-fat soybeans (HE SB). Steers were individually housed and fed the diets for 111 d during the finishing period. Over the experimental period, treatment groups were similar in ADG (1.7 +/- 0.1 kg/d) and had a similar slaughter weight (603 +/- 11.6 kg). Dressing percentage averaged 61.6% and carcass composition averaged 14.3% protein, 30.9% lipid, and 54.8% water. At slaughter, the intramuscular, intermuscular, and subcutaneous fat depots were sampled from the rib longissimus, eye of round, and chuck tender muscles. Across all fat depots, the CLA content differed (P < 0.05), averaging 6.6, 6.7, and 7.7 mg/g of fatty acids for the control, LESB, and HESB diets, respectively. There were significant differences in CLA content between fat depots within a cut, but differences were relatively small and the hierarchy in fat depots was not consistent among cuts. The cis-9, trans-11 isomer was the predominant CLA isomer and its content in fat was related to trans-11 C18:1 content (r = 0.53; P < 0.001). There was substantial individual variation in CLA content and this varied from 2.6 to 17.0 mg/g fatty acids across all treatments and fat depots. Overall, results demonstrated that including extruded full-fat soybeans in the diet of finishing steers increased the CLA content of beef fat. Differences were relatively small and the relationship of this to rumen fermentation and endogenous synthesis of CLA is considered.

A proteomic analysis of the effect of growth hormone on mammary alveolar cell-T (MAC-T) cells in the presence of lactogenic hormones.

The bovine mammary alveolar cell-T (MAC-T) cell line is able to uniformly differentiate and secrete casein proteins in response to dexamethasone, insulin, and prolactin and is extensively used to study bovine mammary epithelial cell (MEC) function. Somatotropin, or growth hormone (GH), has been shown to increase milk protein synthesis both in vivo and in mammary cell models and to induce cytoskeletal rearrangement in a 3T3 fibroblast cell line and a Chinese hamster ovary cell line. To identify the nature of the effects of GH in MECs cultured with lactogenic hormones, changes in global protein expression were assessed in the MAC-T cell line with the use of two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry. Forty proteins were differentially expressed in response to GH (P < 0.05) and were related to metabolism, the cytoskeleton, protein folding, RNA and DNA processing, and oxidant stress. These widespread changes in protein expression are indicative of a global role of GH in overall cellular differentiation that may underlie the direct modulation of milk component synthesis in MEC models that have been described to date.

Influence of thermal processing conditions on flavor stability in fluid milk: benzaldehyde.

Flavor loss in dairy products has been associated with enzymatic degradation by xanthine oxidase. This study was conducted to investigate the influence of milk thermal processing conditions (or xanthine oxidase inactivation) on benzaldehyde stability. Benzaldehyde was added to whole milk which had been thermally processed at 4 levels: (1) none or raw, (2) high temperature, short time (HTST) pasteurization, (3) HTST pasteurization, additionally heated to 100 degrees C (PAH), and (4) UHT sterilized. Additionally, PAH and UHT milk samples containing benzaldehyde (with and without ferrous sulfate) were spiked with xanthine oxidase. Azide was added as an antimicrobial agent (one additional pasteurized sample without) and the microbial load (total plate count) was determined on d 0, 2, and 6. The concentration of benzaldehyde and benzoic acid in all milk samples were determined at d 0, 1, 2, 4, and 6 (stored at 5 degrees C) by gas chromatography/mass spectrometry in selective ion monitory mode. Over the 6-d storage period, more than 80% of the benzaldehyde content was converted (oxidized) to benzoic acid in raw and pasteurized milk, whereas no change in the benzaldehyde concentration was found in PAH or UHT milk samples. Furthermore, the addition of xanthine oxidase or xanthine oxidase plus ferrous sulfate to PAH or UHT milk samples did not result in benzaldehyde degradation over the storage period.

Interferometry and laser control with solid Fabry-Perot etalons.

The use and analysis of solid Fabry-Perot etalons for interferometry and laser control are discussed and supported with experimental data. Low angle scattering is found to be an important factor influencing finesse and peak transmission. Thermal tuning sensitivity and wedge-angle control with thermal gradients are analyzed and illustrated. Control of laser oscillations using a solid-state etalon as a laser cavity end mirror is discussed. The use of the solid etalon as an optical cavity coupler is applied to the problem. of sideband energy removal from an internally modulated laser.

Analysis of variation in cis-9, trans-11 conjugated linoleic acid (CLA) in milk fat of dairy cows.

Conjugated linoleic acid (CLA) is a fatty acid with numerous putative health benefits and is a natural component of ruminant-derived food products. An intermediate in rumen biohydrogenation is cis-9, trans-11 CLA, the major CLA isomer in milk fat. However, the major source of cis-9, trans-11 CLA in milk is endogenous synthesis by delta 9-desaturase conversion of trans-11 C18:1, another rumen biohydrogenation intermediate. The desaturase indices serve as a proxy for delta 9-desaturase activity and are calculated from the ratios of fatty acid pairs that represent product/substrate for this enzyme. This study analyzed individual animal variation in milk fat content of cis-9, trans-11 CLA and in desaturase indices in milk fat. Thirty lactating Holstein cows were allocated to one of three treatment groups: one received a standard total mixed ration, one received a diet that produced an elevated milk fat content of CLA, and a third treatment group was alternated between these diets at 3-wk intervals over the 12-wk study. There was a two- to threefold variation among individuals on the same diet for both milk fat content of CLA and desaturase indices in milk fat. This hierarchy was maintained to a large extent over the 12-wk study even in the variable treatment group that alternated between the two diets. Within the variable diet treatment, some animals consistently had a substantial response in milk fat content of CLA to dietary shifts, whereas other cows had little or no response. We conclude that while diet is a major determinant of the CLA content in milk fat, individual animal differences also have a substantial effect. The variation among individuals includes differences related to both rumen biohydrogenation and delta 9-desaturase activity in the mammary gland.

Short communication: milk fat response to low doses of tran-10, cis-12 conjugated linoleic acid (CLA).

Trans-10, cis-12 conjugated linoleic acid (CLA) is a potent inhibitor of milk fat synthesis. We examined the effect of low doses of trans-10, cis-12 CLA using Holstein cows in a 4 x 4 Latin square design. Milk yield and milk protein were unaffected, but abomasal infusion of 1.25, 2.5, and 5.0 g/d of trans-10, cis-12 CLA reduced milk fat yield by 7, 16, and 29%, respectively. When combined with previous data, the reduction in milk fat yield was curvilinear, relating to both quantity infused and milk fat content of trans-10, cis-12 CLA (R2 = 0.99 and 0.96, respectively). Further, changes in milk fatty acid composition indicated the mechanism involved inhibition of de novo fatty acid synthesis and the utilization of circulating fatty acids.

DNA content of heterochromatin and euchromatin in tomato (Lycopersicon esculentum) pachytene chromosomes.

Lycopersicon esculentum (tomato) has a small genome (2C = 1.90 pg of DNA) packaged in 2n = 2x = 24 small acrocentric to metacentric chromosomes. Like the chromosomes of other members of the family Solanaceae, tomato chromosomes have pericentromeric heterochromatin. To determine the fraction of the tomato genome found in euchromatin versus heterochromatin, we stained pachytene chromosomes from primary microsporocytes with Feulgen and analyzed them by densitometry and image analysis. In association with previously published synaptonemal complex karyotype data for tomato, our results indicate that 77% of the tomato microsporocyte genome is located in heterochromatin and 23% is found in euchromatin. If heterochromatin is assumed to contain few active genes, then the functional genes of the tomato must be concentrated in an effective genome of only 0.22 pg of DNA (1C = 0.95 pg x 0.23 = 0.22 pg). The physical segregation of euchromatin and heterochromatin in tomato chromosomes coupled with the small effective genome size suggests that tomato may be a more useful subject for chromosome walking and gene mapping studies than would be predicted based on its genome size alone. Key words : tomato, Lycopersicon esculentum, genome size, heterochromatin, euchromatin, pachytene chromosomes, synaptonemal complex.

Infection during chronic epidural catheterization: diagnosis and treatment.

A potentially serious complication of long-term epidural catheterization in cancer patients is infection. The early signs of infection were studied in 350 patients in whom long-term epidural catheters were inserted. Three areas of the catheter track were found to be involved; exit site and superficial catheter track infection, and epidural space infection. The authors identified the early signs of infection in each area and the progress of the infection from the deep track to include the epidural space in four of these patients. All 19 patients who developed deep track or epidural infections were successfully treated with antibiotics and catheter removal. None of the patients required surgery for spinal cord decompression. Catheters were replaced in 15 of the 19 treated patients who requested them after treatment with no recurrent infections. It was concluded that use of long-term epidural catheterization is associated with a definable epidural infection rate. The use of epidural opioid analgesia is an effective and safe means of obtaining pain relief for terminally ill patients when patients are monitored for possible infection and receive prompt treatment when the diagnosis is established.

The relationship between synaptonemal complex length and genome size in four vertebrate classes (Osteicthyes, Reptilia, Aves, Mammalia).

We investigated the relationship between synaptonemal complex (SC) length and genome size in 18 species of vertebrates from the classes Osteicthyes (bony fish), Reptilia (reptiles), Aves (birds), and Mammalia (mammals). When total SC length was plotted against genome size for all 18 vertebrate species, there did not appear to be a correlation between the two variables. However, when birds were excluded from the data and a linear regression analysis was performed, variation in genome size accounted for approximately 50% of the variation in total SC length (r2 = 0.47). Dividing the average total SC length for a species by its 4C DNA amount yields the species' SC/DNA ratio. SC/DNA ratios of birds were approximately twice as high as the SC/DNA ratios of reptiles and mammals. Bony fish showed intraclass divergence in SC/DNA ratios. The sunfish (Centrarchidae) had SC/DNA ratios almost as high as those of birds, while the remaining fish in the study had SC/DNA ratios similar to those of reptiles and mammals. These observations indicate that inter and intraclass divergence in the relationship between total SC length and genome size has occurred in the vertebrates. Coupled with evidence from the literature, our results also suggest that SC/DNA ratios are positively correlated with crossover frequency.

A new permanent exteriorized epidural catheter for narcotic self-administration to control cancer pain.

The lack of an available exteriorized permanent epidural catheter led to the application of the Broviac catheter technology to the development of an epidural catheter. The resulting three-piece silicone-rubber catheter system allows specific spinal-level location of the catheter tip from a lumbar entry site, and exteriorization for ease of self-administration. Fifty-five cancer patients with metastatic disease and intractable pain received 58 catheters. No catheter infections were found during 3891 catheter-days of use, and only 18 minor side effects were noted, with no respiratory depression observed. Hospitalizations for pain control was decreased by 90% after catheter placement. Therefore, the authors recommend early placement of this silicone-rubber epidural catheter to control pain unrelieved by oral analgesics.

Toward integration of comparative genetic, physical, diversity, and cytomolecular maps for grasses and grains, using the sorghum genome as a foundation.

The small genome of sorghum (Sorghum bicolor L. Moench.) provides an important template for study of closely related large-genome crops such as maize (Zea mays) and sugarcane (Saccharum spp.), and is a logical complement to distantly related rice (Oryza sativa) as a "grass genome model." Using a high-density RFLP map as a framework, a robust physical map of sorghum is being assembled by integrating hybridization and fingerprint data with comparative data from related taxa such as rice and using new methods to resolve genomic duplications into locus-specific groups. By taking advantage of allelic variation revealed by heterologous probes, the positions of corresponding loci on the wheat (Triticum aestivum), rice, maize, sugarcane, and Arabidopsis genomes are being interpolated on the sorghum physical map. Bacterial artificial chromosomes for the small genome of rice are shown to close several gaps in the sorghum contigs; the emerging rice physical map and assembled sequence will further accelerate progress. An important motivation for developing genomic tools is to relate molecular level variation to phenotypic diversity. "Diversity maps," which depict the levels and patterns of variation in different gene pools, shed light on relationships of allelic diversity with chromosome organization, and suggest possible locations of genomic regions that are under selection due to major gene effects (some of which may be revealed by quantitative trait locus mapping). Both physical maps and diversity maps suggest interesting features that may be integrally related to the chromosomal context of DNA-progress in cytology promises to provide a means to elucidate such relationships. We seek to provide a detailed picture of the structure, function, and evolution of the genome of sorghum and its relatives, together with molecular tools such as locus-specific sequence-tagged site DNA markers and bacterial artificial chromosome contigs that will have enduring value for many aspects of genome analysis.