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Purple prairie clover (PPC; Dalea purpurea Vent.) containing 84.5 g/kg dry matter (DM) of condensed tannin (CT) was ensiled without (control) or with polyethylene glycol (PEG) for 76 days, followed by 14 days of aerobic exposure. Changes in fermentation characteristics were determined, and the composition of bacterial and fungal communities were assessed using metagenomic sequencing. The addition of polyethylene glycol (PEG) that deactivated CT at ensiling increased (P < 0.05 to ∼0.001) soluble N, nonprotein N, lactic acid, total volatile fatty acids, ammonia N, deoxynivalenol (DON), and ochratoxin A (OTA) but decreased (P < 0.001) pH and water-soluble carbohydrates. The concentrations of DON and OTA increased (P < 0.001) for both silages, with the extent of increase being greater for control than for PEG-treated silage during aerobic exposure. The PEG-treated silage exhibited higher (P < 0.01 to ∼0.001) copy numbers of total bacteria, Lactobacillus, yeasts, and fungi than the control. The addition of PEG decreased (P < 0.01) bacterial diversity during both ensiling and aerobic exposure, whereas it increased (P < 0.05) fungal diversity during aerobic exposure. The addition of PEG at ensiling increased (P < 0.05) the abundances of Lactobacillus and Pediococcus species but decreased (P < 0.01) the abundances of Lactococcus and Leuconostoc species. Filamentous fungi were found in the microbiome at ensiling and after aerobic exposure, whereas Bacillus spp. were the dominate bacteria after aerobic exposure. In conclusion, CT decreased protein degradation and improved the aerobic stability of silage. These desirable outcomes likely reflect the ability of PPC CT to inhibit those microorganisms involved in lowering silage quality and in the production of mycotoxins.
IMPORTANCE The present study reports the effects of condensed tannins on the complex microbial communities involved in ensiling and aerobic exposure of purple prairie clover. This study documents the ability of condensed tannins to lower mycotoxin production and the associated microbiome. Taxonomic bacterial community profiles were dominated by Lactobacillales after fermentation, with a notable increase in Bacillus spp. as a result of aerobic exposure. It is interesting to observe that condensed tannins decreased bacterial diversity during both ensiling and aerobic exposure but increased fungal diversity during aerobic exposure only. The present study indicates that the effects of condensed tannins on microbial communities lead to reduced lactic acid and total volatile fatty acid production, proteolysis, and mycotoxin concentration in the terminal silage and improved aerobic stability. Condensed tannins could be used as an additive to control unfavorable microbial development and maybe enhanced feed safety.
Ensiling is a worldwide conservation method for green fodder because it allows high-quality forage to be conserved even under less-than-favorable weather conditions. However, conserving forage as silage has its challenges, as the quality of silage is highly dependent on the activities and types of microorganisms that contribute to the ensiling process. Therefore, understanding the microbial community involved in ensiling is central to ensuring the effective conservation of silage. Microbes involved in ensiling have been extensively studied using both culture-dependent methods and culture-independent molecular methods (1, 2). During ensiling, bacteria, such as Lactobacillus and Lactococcus strains, are known to enter into the viable-but-unculturable state (3). Hence, microbial communities being described solely on the basis of culture overlooks the contribution that these bacteria may make to the ensiling process. The recent development of high-throughput sequencing of phylogenetic gene markers for bacteria, yeasts, and fungi makes it possible to identify unculturable members of microbial communities that are associated with ensiling. Although such studies have been reported for grass and whole-crop barley silage (2, 4), they have not been undertaken for legume forages containing condensed tannins (CT). Extensive proteolysis occurs during ensiling that converts almost all plant protein to nonprotein N (NPN), thus lowering the protein nutritive value of silage (1, 5). Therefore, methods that reduce protein degradation during ensiling could improve the nutritional value of silage.
Condensed tannins are a group of naturally occurring phenolic plant secondary compounds that possess various biological activities, including protein precipitation and antimicrobial activity (1, 6). Both concentration and structure affect the biological activity of CT. It has been found that CT in temperate forage at lower than 40 g/kg dry matter (DM) generally have positive effects on ruminant performance, mainly due to the improved protein utilization via reducing extensive protein degradation in the rumen and increasing amino acid absorption in the small intestine (7–9). In contrast, CT concentrations higher than 50 g/kg DM usually depressed feed intake and reduced nutrient, especially protein, digestion (10, 11). It has also been reported that the addition of tannins decreased protein degradation during ensiling (12–14) and that ensiling CT-containing species, such as sainfoin (Onobrychis viciifolia cv. Common) and birdsfoot trefoil (Lotus corniculatus), reduce the degree of proteolysis compared to legumes that do not contain CT (1). However, the mechanisms whereby CT alter the silage microbiome to reduce proteolysis and improve silage quality during ensiling are not fully understood.
The objective of this study was to assess the effects of CT on fermentation and the bacterial and fungal microbiomes during ensiling and upon aerobic exposure.
Characteristics of terminal and aerobically exposed silage.Whole-plant purple prairie clover (PPC; Dalea purpurea Vent.) contained 84.5 g CT/kg DM at ensiling, the majority of which was extractable CT (70.2 g/kg DM; Table 1). After 76-day ensiling, extractable CT decreased to about 27.4 g/kg silage DM (Table 2). The addition of polyethylene glycol (PEG) at ensiling increased (P < 0.05 to ∼0.001) soluble N (SN), NPN, lactic acid, total volatile fatty acids (VFA), ammonia N, acetic acid, and propionic acid but decreased (P < 0.001) the contents of acid detergent-insoluble nitrogen (ADIN) and water-soluble carbohydrates (WSC). Silage ensiled with PEG also had higher (P < 0.01) concentrations of deoxynivalenol (DON) and ochratoxin A (OTA) than with the control silage.
The temperature of aerobically exposed PEG-treated silage started to increase on day 7, whereas that of the control silage did not increase until on day 8 (Fig. 1). The magnitude of the temperature increase was also greater (P < 0.01) for PEG-treated (0 to ∼11°C) than for control (0 to ∼6°C) silages. The pH after 7-day aerobic exposure was similar to day 0 silage but increased (P < 0.001) after 14 days of aerobic exposure (Table 2).
The increases in temperature and pH of silage during aerobic exposure were accompanied by a decrease in lactic acid concentration (Table 2). After 7 days of aerobic exposure, PEG-treated silage had higher (P < 0.05 to ∼0.001) concentrations of SN, NPN, ammonia N, total VFA, lactic acid, acetic acid, and propionic acid than control silage. However, this trend was observed for SN, NPN, and ammonia N after 14 days of aerobic exposure. On the contrary, the concentrations of DON and OTA increased (P < 0.001) during aerobic exposure, with concentrations of DON and OTA being greater (P < 0.01) in PEG-treated silage than in control silage.
Microbial rRNA copy numbers in terminal and aerobically exposed silage.After a 76-day ensiling, PEG-treated silage exhibited higher (P < 0.05 to ∼0.01) rRNA copy numbers of total bacteria, Lactobacillus spp., yeasts, and fungi than the control silage (Fig. 2). The rRNA copies associated with the majority of microbes increased (P < 0.001) during aerobic exposure, with the exception of copy numbers of Lactobacillus, which declined (P < 0.001) from day 7 to day 14 of aerobic exposure (Fig. 2B). The PEG-treated silage had higher (P < 0.05 to ∼0.001) copy numbers of total bacteria, yeasts, and fungi but lower (P < 0.05) copy numbers of Lactobacillus spp. than the control silage. The two silages had similar copy numbers of total bacteria, Lactobacillus spp., yeasts, and fungi after 14 days of aerobic exposure.
Bacterial and fungal microbiome in terminal and aerobically exposed silage.Across all samples, a total of 6,295,205 and 5,033,203 reads with average lengths of 450 and 380 bp were obtained for bacteria and fungi, respectively. After bioinformatics analysis, a total of 4,273,668 bacterial and 3,455,929 fungal sequences were classified. Rarefaction curves produced from bacterial and fungal sequences for the number of microbial genome operational taxonomic units (OTUs) are shown in Fig. S1A and B in the supplemental material, respectively. Rarefaction curves indicated that reasonable coverages of the bacterial and fungal communities were achieved, although the sequencing depth was insufficient to fully describe the diversity of these populations, as a plateau in the rarefaction curves was not achieved.
Bacterial microbiome.More than 96% of the bacterial amplicon sequences were assigned at the phylum level. Within the 10 different phyla identified, over 92% of the sequences were classified within the phyla Firmicutes and Proteobacteria (Fig. S2). As many as 225 different bacterial genera were further identified, representing 58.9% of total sequences, with the 15 most relatively abundant genera shown in Table 3. Overall, most of reads assigned to the Firmicutes were related to the order Lactobacillales, with Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus being the most predominant genera after 7 days of aerobic exposure. After 14 days of aerobic exposure, Bacillus, Lysinibacillus, Oceanobacillus, Paenibacillus, and Sporosarcina of the order Bacillales became more prominent than other genera.
Fungal microbiome.Similarly, more than 98% of the fungal sequences were classified into 35 fungal phyla, with Ascomycota and Basidiomycota being the most abundant, accounting for 67.6% of the total sequences (data not shown) and more than 94% of the total phyla (Fig. S3). Of the two fungal phyla, the majority of reads mapped to the Ascomycota. Fungal sequences were classified into 142 different genera, accounting for 68.7% of the total sequences, with the 15 most relatively abundant genera shown in Table 4.
Effects of CT on bacterial microbiome in terminal and aerobically exposed silage. (i) Terminal silage.Upon opening, PEG-treated silage exhibited a greater (P < 0.001) abundance of Firmicutes but a lower (P < 0.01) abundance of Proteobacteria than the control silage (Fig. S2). Moreover, PEG-treated silage had a greater (P < 0.001) abundance of Lactobacillus and Pediococcus spp., with a lower (P < 0.01) abundance of Lactococcus, Leuconostoc, Erwinia, Sphingomonas, Methylobacterium, Pseudomonas, and Agrobacterium spp. than those of control silage (Table 3). The addition of PEG at ensiling decreased (P < 0.01) the observed number of operational taxonomic units (OTUs) and the Chao 1, abundance-based coverage estimator (ACE), and Shannon bacterial diversity indices in terminal silage (Fig. 3). In addition, when the structure of bacterial communities was assessed using weighted UniFrac distance, a clear distinction between bacterial communities in the PEG-treated and control silages on day 0 was noted, as the plots cluster separately (Fig. 4A; P < 0.001; R = 0.355). Moreover, the Pearson distance analysis showed that bacterial populations in terminal silage differed (P < 0.01) between PEG-treated and control silages (Fig. S4). The higher abundance of Lactococcus spp. in control silage, along with the greater abundances of Lactobacillus and Pediococcus spp. in PEG-treated silages corresponded to the bacterial genus analysis in Table 3.
(ii) Aerobically exposed silage.The taxonomic profile of the bacterial microbiome indicated that PEG-treated silage was more abundant (P < 0.001) in Firmicutes after 7- and 14-day aerobic exposures but lower (P < 0.001) in Proteobacteria after 7 days than control silage (Fig. S2). Similar to the PEG-treated terminal silage, PEG-treated silage aerobically exposed for 7 days was less abundant (P < 0.05) in Lactococcus, Leuconostoc, Erwinia, Sphingomonas, Methylobacterium, and Pseudomonas spp. than control silage but possessed a greater abundance (P = 0.003) of Pediococcus (Table 3). Unexpectedly, the abundance of Lactobacillus declined (P = 0.007) with the addition of PEG. After 14 days of aerobic exposure, the dominant bacterial genera (i.e., Bacillus, Lysinibacillus, Paenibacillus, and Sporosarcina) did not differ (P = 0.05) between control and PEG-treated silages. In addition, the abundances of Lactococcus and Leuconostoc spp. in PEG-treated silage at day 14 remained lower (P < 0.05) than in the control silage, whereas Pediococcus, Oceanobacillus, and Sporosarcina spp. were greater (P < 0.05) in the PEG-treated silage than the control silage. Noticeably, the abundance of the majority bacteria was decreased (P < 0.001) over 14 days of aerobic exposure.
The key diversity indices of bacteria showed that Chao 1 richness was lower (P < 0.05) in PEG-treated silage than in control silage after 7 and 14 days of aerobic exposure (Fig. 3), and the same trend was also observed for number of OTUs and ACE index. The bacterial communities assessed using the weighted UniFrac distances appeared to be influenced by time (Fig. 4B) and PEG treatment (Fig. 4C) after aerobic exposure (P < 0.001). A clear separation between control and PEG treatments was observed after 7 days of aerobic exposure, although the bacterial sequences obtained after 14 days of aerobic exposure tended to cluster together (Fig. 4A and B; P > 0.05). In addition, the heatmap of bacterial taxon abundance showed that the control silage had bacterial populations that were obviously distinct from those of the PEG-treated silage either at day 7 or day 14 of aerobic exposure (Fig. S4). In addition, most of the classified bacterial species were assigned to the genus Lactobacillus on day 7 and Bacillus on day 14, which was consistent with observations in the analysis of the relative abundances of bacterial genera.
Effects of CT on fungal microbiome in terminal and aerobically exposed silage. (i) Terminal silage.The abundances of Basidiomycota (35% versus 30%) and Ascomycota (64% versus 67%) did not differ significantly (P > 0.05) between the control and PEG-treated silages (Fig. S3). The PEG-treated silage had higher (P < 0.05 to ∼0.001) abundances of Colletotrichum, Xylogone, Galactomyces, Penicillium, Fusarium, and Cryptococcus spp., with a tendency (P = 0.053 to ∼0.093) also to contain higher abundances of Aspergillus, Wickerhamomyces, Pterula, and Tremella spp. than control silage (Table 4).
The addition of PEG at ensiling did not affect the diversity of fungal communities in terminal silage (Fig. 5). In addition, Bray-Curtis distance metric assessment identified no significant (P > 0.05) distinction in fungal communities between the control and PEG-treated silages (Fig. 6A), with no distinguishing difference in the fungal compositions within silages, as indicated by the heatmap based on Pearson distance (Fig. S5).
(ii) Aerobically exposed silage.The PEG-treated silage had a lower (P < 0.001) abundance of Basidiomycota (37% versus 4%) but a higher (P < 0.001) abundance of Ascomycota (60% versus 96%) than the control silage after 7 days of aerobic exposure (Fig. S3). The abundances of Colletotrichum, Penicillium, and Fusarium spp. were higher (P < 0.05 to ∼0.001) after aerobic exposure, whereas the abundances of Candida, Wickerhamomyces, Penicillium, and Pterula spp. were lower (P < 0.05) after 14 days of aerobic exposure for PEG-treated silage than for control silage (Table 4). In contrast, PEG-treated silage contained lower (P < 0.05 to ∼0.001) abundances of Euopsis, Hannaella, Pterula, Tremella, Rhodotorula, and Cryptococcus spp. after 7 days and lower (P < 0.01 to ∼0.001) abundances of Xylogone and Tremella spp. after 14 days of aerobic exposure compared to those in the control silage.
Similar to the bacterial diversity, the total observed number of OTUs and the key diversity measures showed that Chao 1 richness, ACE, and Shannon indices for fungi were all lower (P < 0.05) in PEG-treated silage than in control silage after aerobic exposure (Fig. 5). Moreover, the addition of PEG had a greater impact on diversity than the duration of aerobic exposure, as the principal-coordinate analysis (PCoA) plots were not clustered separately (P > 0.05) between days 7 and 14 of aerobic exposure (Fig. 6B).
The decline in the abundances of majority fungal species, as shown by heatmap, also are reflected by a similar trend shown in Table 4. The composition of fungal communities in control formed a cluster either at day 7 or day 14 of aerobic exposure, while most of those PEG-treated individual samples fell into individual clades that were distinct from one another in the tree (Fig. S5).
Effect of CT on ensiling properties.It is generally recognized that legumes are difficult to ensile due to their low WSC content and high buffering capacity, which limits lactic acid production and hinders the rapid pH decline. The chemical composition and production of fermentation products (lactic acid, VFA, etc.) during ensiling in both control and PEG-treated silages were within the normal range measured in legume silages (15, 16), which indicated that PPC was ensiled well and that CT in PPC had no effect on overall silage quality. Extensive proteolysis during ensiling is recognized to reduce the nutritive value of plant protein, and it appeared that the proteolysis was decreased by the presence of CT in silage. Research has shown that other CT-containing forages, such as sainfoin and birdsfoot trefoil, undergo less proteolysis during ensiling, with transformation of their plant protein N into NPN being inhibited compared to that in forages that lack CT (17, 18). Even the direct addition of tannic acid (13) or isolated tannins (i.e., mimosa, myrabolam, quebracho, and chestnut sources) reduces proteolysis during ensiling (6, 14), likely as a result of their ability to precipitate proteins and inhibit proteolytic microorganisms (1, 19). The higher lactic acid and VFA concentrations, along with the lower WSC content and pH, in PEG-treated than in control silages indicated that CT broadly decreased the microbial activity during ensiling. In the present study, this possibility is supported by both real-time PCR and metagenomics sequencing data, which identified an increased population and abundance of lactate-producing bacteria (LAB) in PEG-treated than in control silages. Anti-LAB activity of CT during ensiling was also reported in other studies (1, 19). It was suggested that CT have ability to precipitate bacterial enzymes and bind cell membranes (20), thereby inducing changes in bacterial morphology, and to decrease the activity of bacterial enzymes (21) which led to a reduced growth of bacteria. Condensed tannins were also reported to negatively affect the growth of LAB, mainly by decreasing lactic acid production, and this effect may be due to a modification of the permeability of the bacterial cell membrane (22).
An interesting finding of this study is that control silage contained less DON and OTA than PEG-treated silage. Ochratoxin A is produced almost exclusively by Penicillium spp. (23), and DON is mainly produced by Fusarium species (24). It is generally regarded that there is little opportunity for mold/fungal growth under the proper ensiling conditions. However, there are some other commonly detected filamentous fungi during ensiling, such as Penicillium spp. (24–26) and Fusarium spp. (26–28), that are able to tolerate organic acids, carbon dioxide, and the low availability of oxygen, and their spores are always present even in healthy-looking silages (29, 30). Purple prairie clovers for both control and PEG treatment at ensiling were from the same source, and the fermentation products indicated that both silages had undergone normal ensiling fermentation. Therefore, it is difficult to explain the reason why CT reduced the production of these mycotoxins during the ensiling. Nevertheless, the lower DON and OTA concentrations were consistent with the lower copy numbers of fungi, along with the decreased abundances of Penicillium and Fusarium species in control than in PEG-treated silages in this study. This suggests that CT might have inhibited the production of these mycotoxins via inhibiting the activity of the respective mold species with an unknown mechanism during ensiling. Further research is still needed. Our study documents the ability of CT to lower mycotoxin production during ensiling. Bargiacchi et al. (E. Bargiacchi, P. Bellotti, P. Pinelli, G. Costa, S. Miele, A. Romani, P. Zambelli, and A. Scardigli A, U.S. patent application EP20150154069) also reported that chestnut tannin (a hydrolysable tannin) decreased mycotoxins in raw food materials, such as oils, oilseeds, and cereals.
Effect of CT on fermentation of silage during aerobic exposure.Once minisilos were opened, the growth of aerobic microbes resulted in increases in both temperature and pH, a response that has been well established upon aerobic exposure of silage. The delayed increase in temperature for control silage compared to that in PEG-treated silage after aerobic exposure indicates that biologically active CT improved aerobic stability. Exposure to oxygen is known to trigger the growth of undesirable aerobic microorganisms (usually yeasts and molds) and the synthesis of toxins (31, 32). Therefore, the higher temperature in aerobically exposed PEG-treated than control silages likely reflects the ability of CT to inhibit the activity of aerobic microbes. This is consistent with the real-time PCR results that showed that the majority of microorganisms were present at a higher density in aerobically exposed PEG-treated than control silages. This is also supported by the higher NPN, SN, and ammonia N in aerobically exposed PEG-treated than in control silages. It is interesting to note that the concentrations of DON and OTA did not increase over the first 7 days of aerobic exposure in PEG-treated silages and increased only slightly for control silages. This is in spite of real-time PCR results documenting a significant increase in yeast and mold populations during this time. In contrast, DON and OTA concentrations markedly increased after 14 days of aerobic exposure even though the increase in yeast and mold populations was similar to that which occurred from day 0 to day 7. This may indicate that mycotoxins in silage are mainly produced by the aerobic microbes associated with spoilage. Nevertheless, the higher DON and OTA concentrations at each sampling date (day 7 or day 14) in PEG-treated than in control silages suggests that PPC CT inhibited the activity of spoilage microbes producing these mycotoxins during aerobic exposure.
Effects of CT on bacterial microbiome in terminal and aerobically exposed silage.Although several studies have reported bacterial community changes in grass and legume silages (4, 33, 34), there is no information available about the effects of CT on the bacterial and fungal microbiome in terminal or aerobically exposed silage. In this study, higher numbers of rRNA copies associated with total bacteria, Lactobacillus, yeasts, and fungi were observed in PEG-treated than in control silages, indicating that CT in PPC likely reduce the total number of these microorganisms during ensiling. Similarly, Salawu (19) documented that CT that extracted from Calliandra calothyrsus inhibited sheep gut bacteria and rumen fungal activities using traditional culturing technology. For bacterial communities identified in terminal PPC silages, the phyla Firmicutes and Proteobacteria were the most abundant, which were also observed to be the predominant phyla in grass (4) and alfalfa (34) silages. Among the Firmicutes, the majority of sequencing reads were associated with Lactobacillus, Pediococcus, and Lactococcus spp. of homofermentative LAB. Sequences associated with Lactobacillus and Pediococcus of the homofermentative LAB have also previously been found in alfalfa silage (33). The higher population of Lactobacillus spp., as determined by real-time PCR, and higher relative abundances of Lactobacillus and Pediococcus spp. in PEG-treated than in control silages suggest that PPC CT inhibited the growth of these LAB during ensiling. This hypothesis was supported by the higher concentration of lactic acid and lower pH observed in PEG-treated than in control silages. In contrast, the lower abundance of Leuconostoc spp. in PEG-treated than in control silages suggested that CT may have promoted the growth of this genus in silage. Vivas et al. (35) also reported that oak wood tannins increased the presence of Leuconostoc strains in culture media. Unlike Lactobacillus and Pediococcus, which are homofermentative, all species within Leuconostoc are heterofermentative. It is regarded that heterofermentation is advantageous over homofermentation during ensiling in terms of improved aerobic stability of silage due to the production of acetic acid by heterofermentation. This could partially explain the enhanced aerobic stability by CT observed in this study. Condensed tannins are polyphenolics with antimicrobial activity mostly toward Gram-positive bacteria through ionophoric-like action (21, 36). In addition, previous studies have demonstrated that CT in PPC strongly inhibit Escherichia coli and E. coli O157:H7 activity (37, 38). Although all LAB are Gram-positive bacteria, the observations that LAB in different genera responded differently to CT indicate that the effects of CT on LAB might be species dependent. Tabasco et al. (39) also observed that the growth of different LAB (i.e., Lactobacillus species) responded to grape seed flavan-3-ol differently.
The greater bacterial diversity and richness indices in control silage, combined with the significant separation between control and PEG-treated silages in the microbiota structure, as demonstrated by the weighted UniFrac distances, suggest that PPC CT increased bacterial diversity in terminal silage. Zhang (40) also reported that green tea tannins increased the ruminal bacterial diversity of both sheep and cashmere goats. It has been found that bacterial population structure but not the overall levels of bacteria diversity changed significantly during ensiling (34). Therefore, it seems that the increased bacterial diversity in terminal silage by CT is likely due to the inhibitory effect of CT that inhibited some bacteria, such as LAB, resulting in niches within the silage that could be occupied by other genera, such as Erwinia, Sphingomonas, and Methylobacterium.
Upon aerobic exposure, the structure of bacterial community in silages changed substantially over time. A significant increase in Firmicutes, with a corresponding decline in the abundance of Proteobacteria, was observed after 7 and 14 days of aerobic exposure in both silages. McGarvey et al. (34) also reported a similar shift in phyla in aerobically exposed alfalfa silage. Although the populations of most microorganisms in both silages increased upon aerobic exposure, as reflected by the real-time PCR results, the extent of the increase was greater for PEG-treated than for control silages at day 7 of the aerobic exposure, indicating that the slower growth of bacteria in control than in PEG-treated silages after aerobic exposure is either from the carryover inhibitory effect of CT on bacterial growth during ensiling or PPC CT in exposed silage still exhibit antibacterial effect or both. The greater extent of the increase of Lactobacillus in control than in PEG-treated silages at the first 7-day aerobic exposure suggested that species in this genus likely proliferated faster than those in PEG-treated silage at the initial aerobic exposure, which may indicate that the action of PPC CT at the concentration used in this study on these species during ensiling was bacteriostatic, which might have been reversed under aerobic conditions. Further research is needed to confirm that Lactobacillus spp. can convert lactic acid to acetic acid under the aerobic conditions (41), and this partially explained the significant increase but significant decrease in acetic acid concentration in 7-day and 14-day aerobically exposed silage compared to terminal silage in this study.
Bacillus spp. are considered undesirable in silage, as they can produce butyric acid, which can promote the growth of less acid-tolerant spoilage microorganisms and result in reduced silage intake (2, 42). After 14 days of aerobic exposure, Bacillus was observed to be the dominant genus in both silages, followed by Paenibacillus, Lysinibacillus, and Oceanobacillus, which are belong to the same order, Bacillales. The significant increase in abundance of Bacillales from day 7 to day 14 of aerobic exposure was concomitant with the deterioration of silage after 7 days of aerobic exposure. Others have also found that Bacillales contribute to the deterioration of corn (42, 43) and small-grain silages (2) during aerobic exposure. The notable growth of Bacillus as a result of aerobic exposure is probably owed to the presence of oxygen, because these strains are aerobic bacteria. In addition, the similar extent of the increase in genus Bacillus abundance between the two silages during days 7 to 14 of aerobic exposure is likely due to the low extractable CT concentration in the exposed silage, because only 4 g was extractable of CT/kg DM at day 7, and none was detected at day 14 of the control samples.
Another undesirable group of bacteria, Clostridia, which tend to thrive in wet silage, can result in excessive protein degradation, DM losses, and the production of toxins. Kung (44) reported that wilting forage with above 30 to ∼35% DM can reduce the incidence of Clostridia. The relative abundance of genus Clostridium across the whole period of aerobic exposure was less than 0.01% (data not shown) in this study, indicating that the addition of water by the treatment did not promote the overgrowth of this organism.
Effects of CT on fungal microbiome in terminal and aerobically exposed silage.Little information is available with regard to the nature of fungal communities in legume silage, although the presence and growth of those mycotoxigenic fungi have been studied in grass and cereal silages (2, 45). This study showed that the copy numbers associated with the total fungal populations were higher in PEG-treated than in control silages in both terminal and 7-day aerobically exposed silages. Another study using culture-dependent methods showed that CT can inhibit the growth and enzyme activity of fungi (46). Fungal growth during ensiling and aerobic exposure leads to a loss of nutrients and reductions in the palatability and feed value of silage (47). Therefore, the decreased fungal populations by CT during ensiling and aerobic exposure would improve the feeding value of silage.
Candida, Tremellales, Cladosporium, Cryptococcus, Colletotrichum, and Rhodotorula spp. were the predominant fungi within the microbiome of terminal PPC silages. Similar fungal communities have been reported in alfalfa (16), barley (48), corn (49), and small-grain silages (2). The sequencing results also showed that the abundances of mycotoxigenic fungi, such as those from the genera Penicillium and Fusarium, were significantly lower in control than terminal and aerobically exposed PEG-treated silages. In addition, lower concentrations of DON and OTA in control than those in PEG-treated silages were observed in this study. These phenotypic and molecular results suggest that PPC CT potentially inhibit the growth of mycotoxigenic fungi and prevent the development of mycotoxin during ensiling and aerobic exposure. As mycotoxin in feed is currently a big problem worldwide, PPC CT could be used as a natural additive for the prevention of mycotoxin in silage production.
Yeasts, such as Candida spp., found in this study belong to the order Saccharomycetales, which have been shown to be associated with whole-crop barley (48) and corn silages (50). Besides Candida, the present study also identified some other uncultured yeast species, including those of Galactomyces and Wickerhamomyces, members of the Saccharomycetales, which dominated the fungal microbiome of silage after aerobic exposure. The higher population of yeast in terminal and 7-day aerobically exposed PEG-treated than in control silages suggests that the CT inhibited the growth of some yeasts. However, similar to those of bacteria and other fungi, the differential abundances of yeast species within the microbiome of the two silages suggested that effects of CT on yeast were also species dependent.
In contrast to these measures for the bacterial community, the diversity and richness of the fungal community were similar between the two terminal silages. This suggested that PPC CT decreased the fungal population without affecting diversity. Winder et al. (51) also reported that high levels of CT (up to 110 g/kg DM) in poplar leaves had no impact on the composition of soil fungal communities. On the contrary, it was observed that fungal communities in 7- and 14-day aerobically exposed silage have greater diversity in control than in PEG-treated silages. Furthermore, the PCoA plots between treatments were separated, suggesting that PPC CT affected silage microbiota structure during aerobic exposure. The mechanism by which CT increases fungal diversity during aerobic exposure of silage needs further study.
Conclusion.Condensed tannins inhibited the growth of total bacteria, LAB, yeast, and fungi during ensiling and aerobic exposure of silage, with the effect appearing to be species dependent, as demonstrated by the differences in their relative abundances within the microbiome. Condensed tannins increased bacterial diversity during ensiling and aerobic exposure, whereas they increased fungal diversity during aerobic exposure. The effects of CT on the microbial communities reduced lactic acid and VFA production, proteolysis, and mycotoxin concentration in terminal silage and improved aerobic stability.
Forage and ensiling.Whole-plant PPC was harvested in July 2015 at full flower from 3 irrigated Swinton silt loam soil plots at the Lethbridge Research and Development Centre, Alberta, Canada. Purple prairie clover is a native legume in the prairie region of North America and contains high levels of CT (up to 94 g/kg DM) that exhibit significant antimicrobial activity (37, 38). The plants were manually cut 5.0 cm above the ground using a pair of scissors, wilted in the field to approximately 30% DM, and chopped to a theoretical length of 1.0 cm using a paper cutter (X-Acto 26612; Westerville, OH, USA). Subsamples of harvested fresh forage from each plot were immediately stored at −40°C for chemical analysis. Equal portions of chopped PPC from of the three plots were then combined into a single lot for ensiling.
Chopped PPC was divided into 2 equal lots, with each being evenly spread on two separate plastic sheets. A chopped sample was immediately analyzed for CT, as described below. To inactivate the biological activity of CT, one lot was sprayed with a solution of 333 g/liter PEG (Sigma, molecular weight [MW], 6,000; PPC-p) at the rate of 420 ml/kg DM of PPC to achieve a CT-to-PEG ratio of 1:2 (52). Polyethylene glycol specifically binds with CT with H-bonds to form PEG-CT complexes without affecting other compounds in the plant and therefore specifically inactivates CT activity (53). The other lot was sprayed with an equivalent amount (280 ml/kg DM) of deionized water (control). Comparisons between PEG-treated (PPC-p) and control PPC were assumed to reflect the effects of CT. Once sprayed, PPC forage in each plastic sheet was manually tumbled for approximately 3 min to ensure uniform mixing. Forage in each sheet was then packed (≈2.5 kg) into polyvinyl chloride (PVC) laboratory minisilos (diameter, 10 cm; height, 35.5 cm) and compacted with a hydraulic press to achieve a packing density of approximately 890 kg (fresh weight)/m3.
Silos were sealed at both ends with rubber lids and metal bands, with one lid fitted with 7.0-cm-long rubber tubing as a vent. Five silos were prepared for each treatment and stored indoors at 22°C. Silos were opened after 76 days for an evaluation of silage quality and evaluation of aerobic stability. Upon opening, silage within 5.0 cm at both ends was discarded, and the remaining material was thoroughly mixed by hand. Subsamples of silage were either stored at −40°C for chemical analysis or at −80°C for microbial characterization. The remaining silage was used to assess aerobic stability, as outlined below.
Determination of aerobic stability.Silage (400 g) from each of 4 randomly selected minisilos of each treatment was separately transferred into four 4-liter insulated containers (diameter, 13.5 cm; height, 31.0 cm). The containers were covered with 4 layers of cheesecloth and stored at 20°C for 14 days. Two Dallas Thermochron iButtons (Embedded Data Systems, Lawrenceburg, KY, USA) were embedded in the lower and midlayers of the silage in each container to monitor temperature, and two additional iButtons were placed outside the containers to record ambient temperature. The temperature was recorded at 15-min intervals for 14 days. The contents within each container were thoroughly mixed by hand and subsampled after 7- and 14-day aerobic exposures for chemical analysis and microbial profiling. Aerobic stability was defined as the period of time between initial aerobic exposure, and the temperature of the aerobically exposed silage exceeded baseline ambient temperature by 2°C (54).
Chemical analysis.Silage samples were freeze-dried and ground to pass through a 1.0-mm screen using a Thomas Wiley cutting mill (Arthur H. Thomas Co., Philadelphia, PA, USA). The samples were subjected to analysis of DM and organic matter (OM) using an AOAC method (55), neutral detergent fiber (NDF), and acid detergent fiber (ADF) using an Ankom 200 system (Ankom Technology Corp., Fairport, NY, USA), with sodium sulfite and α-amylase added for NDF analysis. Nitrogen in NDF (NDIN) and ADF (ADIN) residues were determined with N measured by flash combustion, as described below. Samples were ball grounded in a planetary micromill (Retsch, Inc., Newtown, PA, USA) for measurement of total nitrogen (TN) by flash combustion analysis using an NA1500 nitrogen analyzer (Carlo Erba Instruments, Milan, Italy). Acid detergent lignin was measured using a method of the AOAC (55). Commercial enzyme-linked immunosorbent assay (ELISA) kits (Sigma-Aldrich, MO, USA) were used to determine DON (catalog no. SE120009) and OTA (catalog no. SE120014) concentrations, according to the manufacturer's instructions. The concentrations of extractable, protein-bound, and fiber-bound CT were determined using the method of Terrill et al. (56), with CT purified from whole-PPC plants as a standard.
For analysis of VFA, lactic acid, ammonia N, NPN, SN, and WSC content, 15 g of subsamples from fermented silage at each sampling day was immediately combined with 135 g of deionized H2O and blended in a homogenizer (Osterizer; Sunbeam, Fontana, CA, USA) for 30 s. The homogenate was strained through four layers of cheesecloth, and the supernatant was sampled and analyzed for WSC content, as described by Zahiroddini et al. (57), for VFA and lactic acid content using a 5890A gas liquid chromatograph (Phenomenex, Torrance, CA, USA), as described by Wang et al. (58), and for ammonia N content by the phenol-hypochlorite method (59). Both SN and NPN were analyzed by combustion analysis, as described above. The pH of the extract was measured using a portable pH meter (Orion; Thermo Electron Corp., Kent, WA, USA).
DNA extraction.Samples stored at −80°C were lyophilized, ground through a 1.0-mm screen, and further ball ground using a planetary micromill (Resch, Inc., Newtown, PA, USA), and DNA was extracted according to the protocol described by Healey et al. (60). This extraction method has been successfully used for the reliable isolation of high-molecular-weight genomic DNA from tannin-containing plant materials (61, 62). All samples were further purified through a QIAamp DNA stool minikit column (Qiagen Sciences, Germantown, MD, USA) and eluted in nuclease-free water. The DNA concentration was quantified using a NanoDrop 3300 (NanoDrop Technologies, Inc., Wilmington, DE, USA). High-molecular-weight samples of DNA (50 μl) at a minimal concentration of 20 ng/μl were used for sequencing.
Primer design and standard curve generation for real-time PCR.The primers used for real-time PCR are described in Table 5. The purified PCR products were cloned into One Shot DH5α Escherichia coli competent cells through TOPO TA vector (Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. Plasmids were extracted using the QIAprep Spin miniprep kit (Qiagen Science, Germantown, MD, USA), and DNA concentration was determined using a NanoDrop 3300. The copy numbers of rRNA genes were obtained based on equation 1 of Whelan et al. (63): (1)
Plasmid DNA was serially diluted 10-fold from 108 copies to 1 copy per μl and run in duplicate in a real-time PCR assay. Amplification efficiency (E) was estimated by using the slope of the standard curve and using equation 2 of Rasmussen (64): (2)
Microbial quantification by real-time PCR.For bacteria, amplifications were performed in a final volume of 20 μl containing 1 to 10 ng of DNA template, 0.3 μM each respective primer, and 10 μl of SsoAdvanced Universal SYBR Green supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For fungi, gene quantification (20 μl) consisted of 1 to 10 ng of DNA template, 0.3 μM each respective primer, 0.25 μl of bovine serum albumin (BSA; New England BioLabs, Inc., Ipswich, MA, USA), and 10 μl of iQ SYBR Green supermix (Bio-Rad Laboratories, Inc.). All amplifications were carried out in optical-grade 96-well plates on an Applied Biosystems 7500 Fast real-time PCR (RT-PCR) machine (Applied Biosystems, Foster City, CA, USA), as described in Table 5. Minor populations were quantified, and the low copy numbers were analyzed only when the coefficient of the standard curve regression was higher than 0.98 and the run efficiency was higher than 90%. The rRNA gene copy numbers quantified by real-time PCR were expressed as log10 copies/DM silage.
Microbiome characterization by metagenomic sequencing.The purified DNA samples were analyzed by Génome Québec (McGill University, Génome Québec Innovation Centre, Quebec, Canada) using Illumina MiSeq (San Francisco, CA, USA) sequencing technology. For bacteria, the V3-V4 region of 16S was targeted using universal primers 347F-CS1 (ACACTGACGACATGGTTCTACAGGAGGCAGCAGTRRGGAAT) and 803R-CS2 (TACGGTAGCAGAGACTTGGTCTCTACCRGGGTATCTAATCC) (2). Primers nu-SSU-0817 (TTAGCAT-GGAATAATRRAATAGGA) and nu-SS-1196 (TCTGGACCTGGTGAGTTTCC), originally designed by Borneman and Hartin (65), were used to target a 400-bp genomic region of the V4 and total V5 variable domains of the short subunit (SSU) ribosomal DNA (rDNA) gene from all four major phyla of fungi. The purified amplicons were paired-end sequenced with Illumina MiSeq (San Francisco, CA, USA) by Génome Québec, according to the manufacturer's recommendations.
The V3-V4 and V4-V5 regions of the Illumina data sets were initially demultiplexed using MiSeq Reporter version 2.0. The demultiplexed paired-end reads were submitted and processed using the fully automated open-source systems of the European Bioinformatics Institute (EBI) (https://www.ebi.ac.uk/metagenomics/pipelines/2.0). Briefly, overlapping reads were first merged into single contigs via SeqPrep, and low-quality ends and sequences were trimmed (>10% undetermined nucleotides) via Trimmomatic and filtered for length via Biopython. The processed reads were then computed for identification of rRNA-encoding sequences using rRNA Selector. For taxonomic classifications, 16S rRNA reads were annotated via QIIME version 1.9.1 using the Greengenes reference database (default closed-reference OTU-picking protocol with Greengenes 13.8 reference with reverse-strand matching enabled). The 18S rRNA operational taxonomic units (OTUs) were picked from the reads using a closed-reference OTU-picking protocol against the SILVA 119 database (Eukarya only) (66), at 99% identity, via the QIIME software package (version 1.9.1). The BIOM files (see http://biom-format.org/) containing phylogenetic classification information provided by EBI metagenomics were used to construct rarefaction curves using the QIIME software (version 1.9.1).
Calculations and statistical analysis.PPC-p data were calibrated by the amount of PEG present in this treatment. For an estimation of alpha diversity, the data set was rarefied to the same level (67), and the observed OTU richness, Chao 1 (68), and ACE (69) richness estimators, and Shannon (70) diversity indices were calculated and further plotted using the phyloseq package (71). The bacterial community structure (beta diversity) of each treatment and sampling time was evaluated using the weighted UniFrac distances (72) and visualized as principal-coordinate analysis (PCoA) plots using EMPeror (73). The fungal community structure was assessed using Bray-Curtis distances and PCoA plots. Analysis of similarity (ANOSIM) with 999 permutations was used to compare the weighted UniFrac and Bray-Curtis distances.
All data were analyzed by analysis of variance as a completely randomized design using the PROC MIXED procedure of SAS (74). For the minisilo study, treatment was considered a fixed effect, and individual silos (n = 5) were considered random factors. The repeated-measures model was used for analysis of data obtained from aerobic stability study, which included treatment, time (aerobic exposure time), and the treatment × time interaction. When time or time × treatment interaction effects were significant (i.e., P < 0.05), the means of the treatments were compared on each sampling date. Differences among treatments were determined using LSMEANS with the PDIFF function and declared significant at a P value of <0.05. A tendency of significant differences was described at P values between 0.05 and 0.1.
Accession number(s).All sequencing data generated in the study have been submitted to the EBI (accession numbers ERR1413924 to ERR1413975) under project accession number ERP015646.
This study was partially supported by the AAFC-Beef Science Cluster and the Alberta Livestock and Meat Agency. K.P. acknowledges the China Scholarship Council for awarding a scholarship to conduct this research at the Lethbridge Research and Development Centre (LRDC) of Agriculture and Agri-Food Canada.
Y.W., T.A.M., S.A., and S.W. conceived and designed the experiments. K.P., L.J., Q.H., and Z.X. performed the experiments. K.P., L.J., Y.D.N., H.D., and H.E.Y. analyzed the data. Y.W. and T.A.M. contributed reagents, materials, and analysis tools. K.P., Y.W., and T.A.M. wrote the paper.
We declare no competing conflicts of interest.
- Received 15 October 2017.
- Accepted 29 November 2017.
- Accepted manuscript posted online 15 December 2017.
This article is contribution 387-17038 from the Lethbridge Research and Development Centre.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02274-17.
- © Crown copyright 2018.
The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.

subset: commoncrawl


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The payments community was saddened by the tragic news last week that American Express President, Ed Gilligan died unexpectedly after becoming ill on a flight home. Gilligan had been with the company for decades, having started his career there as an intern 35 years ago, and rising to become Vice Chairman in 2007 and President in 2013.
In the statement announcing Gilligan's death, Amex CEO and Chairman Ken Chenault said Gilligan left "an indelible imprint on practically every area of our business, from commercial card and travel to international, consumer, small business, merchant services, network services, and, most recently, the group forging our digital partnerships and driving payment innovations."
Gilligan's sudden passing leaves the company without a clear successor to Chenault.
"Ed also loved American Express. He devoted his entire career to this company, starting as an intern 35 years ago and moving steadily up all the way to Vice Chairman in 2007 and President in 2013," Chenault added. "Ed was a living embodiment of our values, embracing both our heritage of service, trust and integrity, as well as our entrepreneurial spirit that has helped continually transform this company through the years. For the time being, I have asked Ed's direct leaders to report to me."
"The mark of a true leader is followership, and Ed won the hearts and minds of many thousands. We mourn his loss, and we will have opportunities to share our memories of him more fully in the coming days."
Some of those memories are captured here. PYMNTS reached out to a few executives across the payments ecosystem who knew him well to share their thoughts and pay tribute to him. Clearly, the payments ecosystem is richer for the contributions he made to it. Our thoughts and prayers are with his family and his colleagues at American Express.
Ajay Banga | MasterCard CEO
"We offer our most heartfelt sympathies to Ed Gilligan's family and the entire American Express organization. In his 35 year career at American Express, Ed not only played a major role in the success of that company, he also helped to transform the payments business into what it is today."
Dan Schulman | CEO PayPal
"I was fortunate to have worked with Ed during my tenure at American Express. He was a thoughtful partner and tremendously respected leader throughout the Blue Box; someone who was approachable and always generous with his time and his insights. He cared dearly about the success of others and about his friends. Ed's style, I believe, was forged in large part by his journey at Amex and his appreciation for its values, and for how challenging it can be to succeed in a constantly changing business environment. Ed helped me to be the best I could be. What I remember most about Ed, however, was his passion and his infectious enthusiasm, not simply for the company and its customers, but especially for his family and friends."
Hayden Reed | Senior Vice President and General Manager of Digital River World Payments
"Ed led advances in the payments industry that have enabled a more frictionless commerce experience for consumers and merchants alike. His leadership and contributions in the payments field will be greatly missed."
Guy Chiarello | President of First Data and Frank Bisignano| CEO of First Data
"Over his 35 years at Amex, Ed helped to influence, innovate and improve our industry from bottom to top, with a journey from intern to president that qualifies as a true American success story! We knew him as a great collaborator, a great competitor, and most importantly a great person, and his loss leaves us all greatly saddened."
Al Kelly | Visa Board Member and Former President, American Express
"I worked with Ed for over two decades and he certainly had a very long list of great accomplishments and he had tremendous impact on American Express. That said, I will remember Ed for his commitment to his family. There was no subject that brought a bigger smile to his face than his family. His love of Lisa and his four children was immeasurable. The business world has lost a terrific executive but the world has lost an even better husband and father."
Jud Linville | Chief Executive Officer, Citi Cards
"Ed cared for clients, and the relationship with those clients, like few we have ever seen. But beyond that, he was willing to challenge convention....to challenge what else might be possible. He did that for clients, and for colleagues, wherever he went."
The How We Shop Report, a PYMNTS collaboration with PayPal, aims to understand how consumers of all ages and incomes are shifting to shopping and paying online in the midst of the COVID-19 pandemic. Our research builds on a series of studies conducted since March, surveying more than 16,000 consumers on how their shopping habits and payments preferences are changing as the crisis continues. This report focuses on our latest survey of 2,163 respondents and examines how their increased appetite for online commerce and digital touchless methods, such as QR codes, contactless cards and digital wallets, is poised to shape the post-pandemic economy.


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Most people living in European cities are exposed to poor air quality – new report
According to a new European Environment Agency report, most people living in European cities are exposed to levels of air pollution that are harmful to their health.
Air quality in Europe – 2017 report presents air pollution levels collected at over 2,500 monitoring sites throughout Europe in 2015.
While the report shows a slow overall trend towards improvement, the figures highlight the need for more action – both at the policy and technological levels.
In particular, the report presents some concerning statistics about particulate matter 2.5 (PM2.5). PM2.5 is a common type of air pollutant emitted by diesel vehicles and the burning of fuels that can have a range of negative effects on health, and especially the lungs.
The report showed that 82% of people living in European cities are exposed to higher levels of PM2.5 that deemed acceptable for health by the World Health Organization. It also highlights that exposure to PM2.5 was responsible for 428,000 early preventable deaths in Europe in 2014.
Visit the Healthy Lungs for Life website to find out more about air pollution and lung health.


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We've helped countless high-risk merchants set up payment gateways with NMI. However, at Blue Payment Agency, we do more than that. We also offer various high-risk e-commerce tips on our blog. While we still are likely to recommend NMI as the payment gateway for your business, we'd like to take a moment to talk about MOTO card payments and whether they can benefit your business. MOTO stands for Mail-Order/Telephone-Order and is a common payment practice even in the internet age.
If you're unfamiliar with MOTO, don't worry. We'll cover some basics, and discuss some of the pros and cons of accepting MOTO card payments for your business.
While both online processing and MOTO are transaction types that fall under Card Not Present (CPN) transactions, they're still different in certain respects.
MOTO stands for Mail-Order/Telephone-Order. As the name suggests, it's a transaction type that takes place when you process credit card payments through the mail or over the phone. The big distinction from online credit card processing is that you, the merchant, are the one who enters the credit card details provided by the customer into your payment system.
These days, most high-risk business owners decide to go with online credit card processing that allows them to accept credit card payments on their website. After all, e-commerce is experiencing continuous year-on-year growth and has been for the last 15 to 20 years. We stand firm in our assessment that NMI is the payment gateway for you as a high-risk business. They offer a fantastic combination of flexibility and reliability. However, this emphasis on website credit card processing begs the question – are MOTO payments outdated? Or, if we rephrase it, is it valuable to you to start accepting MOTO card payments today?
This answer is, of course, twofold. After all, we're talking about two different means of processing credit cards: via the mail or over the phone.
It's safe to say that accepting payments over the phone is still very common here in the US. People are familiar with the concept and go through the process on a relatively frequent basis (take-out/delivery orders). On the other hand, mail order is starting to become less common by the year.
Having said that, offering more alternatives for accepting payments can be very useful. Not being able to accommodate a new client or customer because you can't meet their payment processing preferences will only make you lose out on more business.
Convenience is something that customers have come to expect from businesses, whether they're brick-and-mortar or online. This means that it's very easy to lose business if you're not catering to the various payment needs of your customers. The good news, however, is that with NMI you can accept online payments with ease. Additionally, you'll be able to accept payments manually via phone or mail order. In other words, with NMI you can accept MOTO and e-commerce credit card payments with confidence.
NMI provides the stability and security you want from a payment gateway, with PCI compliance, customer support, custom integration with plenty of platforms, etc. However, one of the biggest draws is that you can manage multiple merchant accounts through one terminal. Another great aspect is the wealth of alternatives you can offer through NMI, which include:
As you can see, there is plenty of freedom of choice when it comes to NMI – including offering MOTO payment processing.
We're very proud to be high-risk specialists that are also official NMI resellers. NMI offers many flexible, useful solutions to the many high-risk e-commerce business owners we assist. If you're looking to start accepting MOTO card payments, get in touch with us for help setting up NMI today. We have the technical expertise to guide you through the setup process with ease.
You can contact us by giving us a call at 844-253-9769 or reach out here. We look forward to helping your business successfully process MOTO payments.


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Knots Landing: Back to the Cul-de-Sac is a 1997 American television miniseries. It is a continuation of the 1979–1993 prime time soap opera Knots Landing and takes place four years after the series ended. Directed by Bill Corcoran, the four-hour miniseries was originally broadcast in two parts on CBS on May 7 and May 9, 1997.
The miniseries reunites cast members William Devane, Kevin Dobson, Michele Lee, Donna Mills, Ted Shackelford, Joan Van Ark, Michelle Phillips and Stacy Galina in starring roles. It also includes cameo appearances by cast members Tonya Crowe, Brian Austin Green, Kim Lankford, Claudia Lonow, Patrick Petersen and Nicollette Sheridan.
The mini-series opens up with Paige Matheson ending her longstanding relationship with Greg Sumner, as she wants to have a baby but he doesn't. Meanwhile, recently unemployed factory workers turn to Mack MacKenzie for help in suing Sumner for wrongful termination. When Sumner bought the factory from Gary Ewing, he fired everyone regardless of the written agreement he signed promising not to close the factory. The agreement, however, is in a box somewhere in the Ewing warehouse. Back at the Seaview Circle cul-de-sac, Valene throws Mack and Karen a party for their 15th wedding anniversary. Many of the past and present residents of Knots Landing are there, including Karen's grown children Diana and Michael, as is former neighbor Ginger Ward (who left the cul-de-sac in 1983). Karen's conniving ex sister-in-law and neighbor Abby Fairgate is also present, as well as Abby's two grown children, Olivia and Brian. During the party, Abby announces to Karen and Val that she will be moving to Malibu. But the joy is short-lived when the IRS freezes all of her assets for failure to pay over $1 million in back taxes. Abby then convinces Greg Sumner to pay her $1 million to steal the agreement from Gary's company warehouse. As she is homeless, she persuades Mack and Karen to allow her to stay with them and gets a job working at Karen and Gary's construction company so that she can find the agreement Sumner needs.
Meanwhile, Greg is surprised when Anne Matheson-Sumner (one of his ex-wives and Paige's mother) returns after four years in Paris. Mack and Karen are approached by Meg, their 12-year-old adopted daughter, who asks for the truth about the identity of her biological father. Mack will never reveal the answer and chooses to lie to Meg again. Valene, who has written a best-selling book called "Hostage" about her year-long kidnapping four years earlier, has been contracted by a major film studio to adapt the book into a screenplay. The studio assign Clay McKinney, a once promising writer whose career was plagued by his alcoholism, to be her writing partner. Meanwhile, Gary gets an unexpected visit from Kate, Greg Sumner's niece with whom he had a relationship some years earlier. Kate tells Gary that she was pregnant when she left Knots Landing four years earlier and that Gary is the father of her daughter, Molly. Clay's drinking and persistent advances towards Valene begins to compromise their working relationship. After a studio party in which Clay takes credit for Valene's work, the two argue. Later at Clay's house, he makes a drunken pass at Valene but she pushes him away and he falls down. Without looking back, Valene leaves and goes home, but the following morning, Clay's body is found floating face down in his swimming pool.
Val and Gary learn that Clay McKinney has been found dead. Because Val was the last person to see Clay alive, the police question her about an earring they found near Clay's pool. Val denies that the earring is hers and refuses to tell Gary what happened that night. Concerned about her secrecy, Gary accuses Val of having an affair with Clay. Greg Sumner decides to settle the lawsuit brought against him by Mack and the unemployed workers. This now means the deal with Abby to steal the agreement is off and now she is out the $1 million. However, this news comes too late because Abby already hired her friend Robert Simons to break into the Ewing warehouse that night. When she goes to call off the deal, Abby finds the warehouse in flames after Robert accidentally killed a security guard and then set fire to the warehouse to cover his tracks. Mack is going through a mid-life crisis but refuses to open up to Karen and moves out of their house.
Meanwhile, after Meg accidentally finds out that Greg Sumner is her father, she absconds from school and goes to visit him. Greg explains why he allowed Karen and Mack to adopt her after her real mother, Laura, died when Meg was still an infant. He plays her a videotape that Laura made for her before her death. Abby, now broke and desperate for money to pay her back taxes, begins to snoop around Greg's private business affairs, determined to find something she can use for blackmail. To her surprise, Sumner puts her in charge of an operation in Thailand which will make her rich.
Valene decides to tell Gary and the police what really happened at Clay McKinney's house the night before he was found dead, and they accept her explanation. Back at the cul-de-sac, Karen and Mack have resolved their differences. Karen's son Michael and his fiance Lori visit to announce they are expecting their first child. Abby cattily sniggers that Karen is going to be a grandmother, but her glee is short-lived when Abby's son Brian arrives with his girlfriend Kate and announce they are also expecting a baby, which means Abby is to be a grandmother too. Gary and Valene can hardly control their laughter.
Following the ratings success of the Dallas reunion movie J.R. Returns in 1996, Warner Bros. Television opted to make a reunion movie for Knots Landing which had ended its 14-year run in 1993. The miniseries was filmed in early 1997, with external filming taking place at the series' signature cul-de-sac location in the Granada Hills district of Los Angeles.
Key castmember Nicollette Sheridan (Paige Matheson) initially declined to appear, but later agreed to an uncredited cameo appearance. The video tape of Laura Avery-Sumner's message to her family (recorded before her death ten years earlier) was not shown but was heard, though the audio was rerecorded with another actress's voice as Constance McCashin, who played Laura in the original series, refused to let the original clip she filmed in 1987 be used.


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Congratulations! You have finally bought the DIY garden shed kit and you are ready to prepare the ground for the new shed. The Western Red Cedar panels will last a long time and naturally resist pests. The shed will be functional, but it also can turn into a focal point of your garden, as long as you go about the installation process correctly.
You can use your garden shed to store your gardening implements, but you also can use it as a hobby room, your private escape, game room for kids, or anything else you can think of. This multi-functional space extends your living space outdoors and if you add insulation, you can use it round the year beyond being just a storage place for your gardening tools, grass/plant seed, and implements.
Prepare the Ground for the New Shed
The first thing that you need to do to prepare the ground for the new shed is to level the surface. You can do this by first putting in concrete, timber decking, or a garden slab. If you don't do this, it could result in the garden shed warping, broken windows and even damage to the wood.
Even if you use the shed base that comes with the DIY garden shed kit, the ground would still have to be levelled and the rubble removed. It is prudent to install a weed-proof membrane before placing the shed's base to prevent weeds and grass from growing and damaging the floor and walls of the shed.
Putting the Base for Your Garden Shed
There are many ways you can create the base for your garden shed. Some of them are as follows:
- Concrete Base: If you intend installing a concrete base, make sure it levelled, flat, and dry before you erect the shed. Use string and pegs to mark the area where you want the garden shed. Dig a hole, about 15 cm deep, and place a wooden framework into which you will be pouring the concrete. Place a layer of stone, about 7 cm and then pour in the cement concrete mix. Spread it out evenly, level it, and let it set.
- Garden Paving Slabs: If you don't feel like pouring concrete in your garden, go with garden paving slabs. This too requires you to remove about 7 cm of the topsoil. Then add 4 cm of cement and place the garden slabs on the wet cement. Let the cement dry, so that the slabs are firmly fixed and then erect your garden shed.
Mistakes to Avoid When Erecting Your Garden Shed
Some homeowners make mistakes when they prepare the ground for the new shed. This can result in damage to the shed and also be dangerous for you and others. Some mistakes that you should avoid making are:
- Unstable base: Refrain from laying garden slabs on loosely arranged gravel. This will not create a stable base for your garden shed and can result in the wood warping and getting damaged. Remember, you are using a DIY garden shed kit and hence, it is necessary, you make the base firm and stable for your own safety. Think of your house, would you want your house being on a weak foundation?
- Not clearing rubble: If you don't clean the rubble, garden debris, dead leaves, and garden detritus from the ground, you will not have a hard-standing base. It is important you clear the ground completely before you start laying the base for your shed.
- Not using weed-proof membrane: Timber decking base is often the preferred base, as it gives the shed a more natural appearance. However, if you intend using a timber decking as a hard-standing for your garden shed, make it a point to first clear and level the ground and then place a weed-proof membrane between the ground and the timber base.
- Using dirt, lawn or gravel for the base: Never attempt to erect your garden shed directly on bare ground, gravel, or lawn. This could damage the wood panels due to moisture from the ground and also lead to warping and subsidence.
DIY garden shed kits come with pre-cut Western Red Cedar wood panels. This means you will not have to spend time cutting the wood and making holes in it. The kit has illustrated instructions along with the necessary hardware. However, you will need another person to help you assemble the pieces and erect the shed. And, all garden shed kits will recommend installing the base. So make sure you educate yourself on the different types of bases that can be used with your shed and then select one that best suits your needs and budget.
Remember without ground preparation, you risk damaging your garden shed and also causing injury to yourself and others, as the shed may not be as sturdy as you think it is. So prepare your garden first before you erect your shed. Choose the location carefully and then prep the surface the right way to enjoy the benefits offered by a garden shed. Don't be afraid to ask for some help. You do not want your shed slowing falling apart in the years to come. That will just invoke one conversation after another that you do not want to have with anyone who sees it.


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Parador de Puebla de Sanabria
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- Room with living room (1)
- Twin rooms (32)
- Double rooms (8)
- Capacity (82)
- Conference room
- Bar
- Restaurant
- Telephone
- Central heating
- Swimming Pool
- Disabled Facilities
- Air conditioning
- Canal plus
- Minibar
- Lift
- Garage
- Parking
- Credit cards
- Currency exchange
- Garden
- Playground
- Airport (196km)
- Station (2km)
Parador de Puebla de Sanabria - Modern hotel in beautiful North West Spain (3*)
The Parador
The Parador de Puebla de Sanabria is the perfect place for nature lovers, only 12km away from the magnificent Lake Sanabria, the largest glacial lake in Spain measuring over 900 acres. Surrounded by stunning natural scenery, this Parador offers a relaxing and tranquil environment.
The Parador is gracefully decorated with a welcoming atmosphere. Communal areas are spacious and comfortably furnished with the lounge area centred on a cosy fireplace, ideal for relaxing after a delicious evening meal in the Parador's restaurant. The large open terrace provides an atmospheric exterior space, perfect for enjoying a drink and soaking up the natural surroundings.
Bedrooms are bright with stylish décor, creating a fresh, contemporary feel. Some guest rooms benefit from excellent views of the town's prominent historical buildings such as Puebla's commandeering medieval castle.
The Parador has a seasonal pool surrounded by greenery offering cool respite during warm summer months.
Puebla de Sanbria's Parador is a wonderful environment for a relaxing and tranquil stay amidst some of Spain's most breath-taking countryside scenery.
Local Area
Puebla de Sanabria is a small, tranquil town close to the Portuguese border; in fact it is a mere 42 km from the beautiful Portuguese town of Braganca. A medieval town, Sanabria was built high-up for defensive measures and was the site of a great many battles. The town has been declared a Historic-Artistic site by the Spanish government due to its wonderful historic buildings such as the 15th century castle which measures up to 960 metres in height. This incredible building features a large keep and is one of the best preserved castles in the province of Zamora, dominating the Tera river valley. Other noteworthy historic buildings include the medieval town hall, the fine 12 century Romanesque church, and a number of ancient ancestral homes scattered throughout the town adding to the authentic atmosphere.
The surrounding area is a beautiful nature spot with plenty of hills and lakes – making it an ideal centre for outdoor activities such as riding, hiking, and mountain biking, fishing and canoeing.
Lake Sanabria is the area's most prized treasure, it being the largest glacial lake in Spain. The lake, measuring 55 feet deep, is surrounded by beautiful valleys and rolling hillsides which make up the Lake Sanabria Nature Reserve. Home to over 1500 plant varieties and 142 species of bird, this truly is a haven for nature lovers. The most striking feature is the impressive lake which holds somewhat of a mystic quality, enhanced by the fact that it dates back thousands of years.
Sanabria has plenty to offer in terms of nature, outdoor sports, and very rich in history, with something sure to interest every kind of visitor.
Click here for Lorna Robert's expert view on this Parador as she journeys through Castellon y Leon.
Restaurant meal times & typical dishes
Breakfast is served from 8.00 to 10.30 and dinner from 21:00 to 23.30.
It may be possible to arrive up to 22.00 and still enjoy a meal.
In the restaurant, try trout freshly caught from the local lakes, or "Bacalao a la Tranca" (salt cod with paprika, vinegar and roasted peppers), or "Cañas Zamoranas" (deep-fried tubes of pastry filled with cream).
Swimming Pool
The opening dates for the outdoor swimming pools are from the 14 June until the 14 September 2019.
Please note the opening and closing dates will depend on the weather and availability of lifeguards.
How to get there
Strategically located between Madrid (Exit 79) and Vigo (Exit 85) by motorway and main road, the Parador is located in a privileged spot, just 12 km. from the Sanabria Lake, in the lower part of the town. Puebla de Sanabria, capital of the region of Sanabria, between Galicia and Castilla is located 111 km. from Zamora.
Nearby Hotels
Verin - 80km
Benavente - 84km
Zamora - 111km
Leon - 153km
Santiago Airport - 320km
Region & Cuisine
CASTILLA y LEON
In 1983 the existing regions of Castilla la Vieja (Old Castile) and León were united to form Castilla y León. Occupying one-fifth of the country's territory, Castilla y León is the largest of Spain's Autonomous Communities and comprises much of the central and northern areas of the country.
Given its size – over 94,000 square kilometres – there is naturally an enormous variety of landscape within this region with mountains of varying grandeur to the north, west and south, and woodlands and fertile river plains dominating the central area. The river Duero, Spain's largest river and the country's principal source of electrical power, more or less bisects the region from east to west before flowing into Portugal – where its name changes to the Douro – and reaching the Atlantic in the city of Porto. The climate of Castilla y León is 'continental', typically with long, hard winters contrasting with moderately warm summers.
Along with the variety of terrain, the region also maintains a tremendous variety of plant and animal life. A hardy species of oak, the holm oak which can withstand both heat and cold, is found everywhere throughout this region. Chestnuts abound in the fertile areas of the Duero and its tributaries, while forests of Scots pine predominate in the Sierra de Gredos in the south. The wildest parts of the region are home to protected endangered species like wolf and brown bear; deer and wild boar are found in the mountains in the north, and mountain goat in the Sierra de Gredos, while also to be seen in this region are the imperial eagle, the tawny vulture and the ubiquitous stork.
But if only one word could be used to describe Castilla y León it would have to be 'monumental'. The region comprises nine provinces and the nine provincial capital cities are collectively a living museum of the history, heritage and culture not only of Castilla y Leon but, to a large extent, of Spain itself.
A brief note on some of these cities:
AVILA A medieval city, encircled by its hugely impressive and wonderfully preserved walls dating from the end of the 11th century, intimately linked to Saint Teresa and with a complex of palaces, churches, convents and monasteries.
BURGOS One of the key links in the cultural chain running the length of the Way of Saint James, Burgos was the capital of Castile during the Middle Ages. Its Gothic cathedral, the third largest in Spain, is the most important of the city's many monuments.
LEON Another monumental city with two thousand years of history. The cathedral is considered one of the best examples of Spanish Gothic and the Cathedral Museum is one of the most comprehensive of its kind. The Parador here, a restored 16th century monastery, is itself one of the city's most impressive monuments.
SALAMANCA A venerable city, declared by UNESCO as part of the Heritage of Mankind in recognition of its artistic legacy. Salamanca's university, founded in 1254, is one of the oldest in the world and is no doubt responsible for Salamanca's reputation as one of Spain's liveliest cities. Life here revolves around the magnificent 'Plaza Mayor' built between 1729 and 1755 and generally recognised as the finest main square in the country.
SEGOVIA A city symbolic of the old kingdom of Castile, among Segovia's innumerable treasures is the Castle in Spain 'par excellence' – the Alcazar, a medieval fortress reconstructed in 1862 after a devastating fire. The emblematic symbol of Segovia is the fantastic 2,000-year old Roman Aqueduct: 728 metres in length with 163 arches and constructed in granite blocks cut so perfectly that no mortar was needed, this is one of the best preserved monuments of Imperial Rome.
VALLADOLID The capital city of Castilla y León, Valladolid preserves some of the finest examples of Renaissance art in this region – notably the College of Santa Cruz whose library contains around 13,000 volumes printed between the 16th and 19th centuries. A university city, Valladolid enjoys the reputation as the Spanish city where the most correct Castilian (Castellano) is spoken. And for those into castles in Spain, the surrounding countryside is full of them!
The culinary tradition of Castilla y León has something for everyone, with pride of place going to roast meats prepared in traditional wood-fired brick ovens – most famously 'cochinillo' (suckling pig) and tender lamb. Also for non-vegetarians, Avila is known for its excellent beef and veal, Burgos for its 'morcilla' (blood sausage) and much of the region for its many varieties of chorizo. There is no shortage of game throughout this region either, with some less-obvious examples being quail, partridge and pigeon which provide local speciality dishes in the provinces of Valladolid, Segovia and Zamora respectively.
Pulses also figure prominently in the region's 'country' cuisine in soups and stews, particularly chick-peas in Zamora, lentils in Leon and succulent 'El Barco' large broad beans in Avila. A wide range of artisan confectionery is very much a feature of Casytilla y León, including the 'rosquillas ciegas' of Palencia, the sugared almonds of Salamanca and the delicious 'yemas de Santa Teresa', emblematic of Avila.
To go with all this, there's no shortage of wine from this region. Rueda, Toro and El Bierzo all produce distinctive wines of high quality, but for the very best (and unfortunately the most expensive) it has the be the 'denominación de origen' of Ribera del Duero, some of whose wines are nothing short of superb.
No description of Castilla and León would be complete without a mention of two of the region's smaller towns, not least because two of Spain's finest Paradors - in our opinion – are located there. LERMA, some 20 miles south of Burgos, is a town with great artistic heritage and a noble air exemplified by the Ducal Palace, begun in 1605 and now the Parador. And in LA GRANJA DE SAN ILDEFONSO, just 7 miles outside Segovia, is the magnificent Royal Palace of La Granja, built between in 1720 and 1735 in the style of the Palace of Versailles and with delightful French-style gardens complete with statues and fountains – a very popular place to visit for the people of nearby Madrid. The impressive Parador here, opened by King Juan Carlos in June 2007, occupies the restored 18th century royal summer residence.
Please be aware of the following:
- 'Special Offers' are subject to the availability of a number of rooms per night and/or a specific meal basis.
- Age restrictions apply to the 'Golden Days' Offer (for those aged 55 and over) and the 'Young Persons' Offer (for those aged between 18 and 30). All reservations made using these tariffs are checked upon your arrival at the Parador(s) booked to ensure that at least one person in a room qualifies for the restricted tariff. In the case that you do not qualify for the restricted tariff, the Parador will apply the standard rate without exception and you will be required to pay a supplement locally. However only one person (per room) needs to qualify for either of these two reductions.


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"Game of Thrones" is your classic tale of a TV series that was highly underestimated right from the beginning, by critics and avid television viewers alike. In fact, there were some game of thrones merchandise that even waited until there were a lot of people talking about it to take time out of their days and watch it simply because they thought it was another case of "déjà vu" in the world of fantasy shows. Were the assumptions right or is the "Game of Thrones" a series on HBO that would soon set records, leaving other television shows trailing in the dust?
"Game of Thrones" is an HBO production based off of a bestselling series of fantasy book s. While the books may have enjoyed massive popularity, fantasy has not been the most successful genre when it comes to television. A long history of airing mediocre fantasy shows to downright failures has perhaps unfairly earned fantasy shows a bad reputation with most viewers.
Thankfully, the "Game of Thrones" series entered this particular genre in a way that would change how people would think of it. Although many were reluctant to give the show a chance in the beginning, it quickly became clear that this was something worth paying attention to. A true masterpiece that was truly worth every bit of time it took for something of its kind to finally become aired. For those who want a non-graphic show to watch, this isn't the one as it's a very explicit series.
The "Game of Thrones" vividly details the fight that noble families had with each other in an attempt to take control of the Iron Throne. From snow and ice that made things extremely difficult all the way to violent battles, this TV show definitely doesn't hold back in showing every single bit of detail. This all leads to helping viewers see what it's like to lack a particular power in society and each episode ensures that it's built to show this each and every time without taking a lot of thinking in order to understand it.
A dwarf, dragons, and a teen girl that was bound to marriage by rape in a world where the men have the upper hand is what helped make this series very unique and breath-taking at the same time. This is also how it became a highly addictive show to watch for many people all around the world. While some may not agree with its graphic nature, this is definitely an approach that other fantasy shows haven't taken prior to the release of the "Game of Thrones."
Not only is it packed with scenes that helped craft a masterpiece in the world of fantasy, but it has also helped the Northern Ireland economy where the show's setting is located. The Northern Ireland government actually helped make this show possible by financing filming. This led to a high amount of spending within their country and also helped develop an even higher interest among tourists. From being looked at as a failure from the beginning to outperforming "The Sopranos" which was one of the most popular HBO series to winning countless awards, this is your perfect example of why one shouldn't ever underestimate the potential of anything in this modern day and age.
Article Source: http://EzineArticles.com/7326429


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Below is the genius link to sign up your scout and family for this year's Spring Camp-out. The cost is $15 per scout and $10 per adult. Siblings are also $10.
Payment Information:You can write a check out to Pack 417 at the April Pack Meeting next week. If you would like to use money in your Scout's account to cover the fees, please check in with Nikki Sandor, as she can confirm your Scout's account balance. Your fee's are covering shelter rental, food, and the shooting sports.
Weather Information:
If the weather turns bad or too cold, there will be some Leaders/Parents/Scouts that stay at camp. Please feel free to leave if you don't like the weather and return the following morning for the day's activities.
Map & Directions:
Address – 581 Ibold Road, Loveland, Ohio 45140.
Directions – see attached
Map – On the attached map see the yellow circle where you should park, and where we are camping
Check In:
Location - KPMG Campsite (see map). Best path to get there is head to the gaga pits (past the dining hall), once at the pit you will see the tee-pee looking shelter ( image in the lower left of the map), take the trail to the right of that, our camp site is right past the shower house. Parking is circled in yellow, Campsites are circled in red.
Time - Starts at 5:00 pm
All scouts and parents attending the camp-out will need to fill out A and B Medical forms and turn them in at camp the day you arrive. I suggest saving a copy of the forms so you can use them again for any summer camp you and your Scout attend. You can download the form below.
What to Bring:
See attached checklist of what to bring, as well as what not to bring
A piece of advice, less is more, as you will see by the map, we are not parking right by the campsite, meaning you will need to carry in, or wheel in if you have a wagon/cart to the campsite all of your gear, so pack smart
Friday Night:
Please bring something to share in the cracker barrel (aka potluck) of snack, treat, fruit, etc. type food. Please know this is not a full meal, we recommend eating dinner prior to arrival
Breakfast – provided by the pack, Pancake Breakfast
Lunch – provided by the pack, Peanut Butter with Jelly or Honey or a Ham and Cheese Sandwich, apples, banana, chips, and cookies
Dinner – provided by the pack, Taco Dinner, with soft taco shells, tortilla chips, salsa, corn, and bug juice to drink (kool-aid)
Breakfast – provided by the pack, donuts on the way out.
Snacks – Please know, there are no snacks planned, there is typically left over food from each meal that will be free to pick from, however if you know you or your scout will need a snack during the day, please bring.
Summary Agenda (please know the times below are simply a rough estimate)
5:00 – 7:00 pm - Check in
7:00 pm – Cracker barrel
8:00 pm – Campfire / S'mores
10:00 pm - Lights out
6:30 am - Breakfast
7:30 am – hike to Cub World
8:00 – 10:00 am Archery
10:30 am – Open Time ( bring snack if needed )
11:30 am - Lunch
12:30 pm - Field Games or Fishing or Gaga Ball
1:30 pm - Field Games or Fishing or Gaga Ball ( Adult vs Scout Gaga Game at this time )
2:30 pm – Hike to Cub World
3:00-5:00 pm – BB Shooting
5:30 pm – Dinner
7:00 pm - Craft
Skits - by den
Cub Master Minute
10:00 pm - Lights Out
Breakfast – Grab and Go Style
7:00 am – Rise and Shine
9:00 am – Final Camp Site Cleanup
*Field Games will include: Basketball, Soccer, Frisbee, KickballWe understand that schedules are busy this time of year and if you need to leave and come back during the weekend to attend practices, games, etc. that is OK.


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The chromosomes
The chromosomes are paired, so that if for example a gene for eye colour is on one chromosome in a pair then there is also a gene for eye colour on the same location, same "locus", on the other chromosome. It is not necessary the same predisposition for a certain eye color on both chromosomes, but it might be. Genes with similar functions are called homologous.
When a cell divides itself a doubling of the number of chromosomes takes place in that each and every of the chromosomes splits itself in half along the middle into a group of two. The pair is kept together in a certain place called the centromere. Since each cell has two copies of each chromosome to start with, this means that every cell, just before division, has 4 copies of each chromosome. The two original chromosomes of the pair are similar, but the two copies of each chromosome that are held together at the centromere, are identical! After this doubling, the centromeres line up in the center of the cell, where the centromeres split: the two chromosomes of the identical pair, now finally released from each other, moves to opposite sides of the cell. Since all chromosomes do this, the chromosomes are divided into two identical groups, each group moving to opposite sides of the cell to form the nucleus of the daughter cell. The cell wall is constructed between the two daughter cells and the process can start all over again. This type of cell division is called mitosis. Thus, during the mitosis the mother cell is divides into two identical daughter cells, which both have exactly the same set of chromosome as the mother cell.
There is also another type of cell division: reduction division or meiosis. During meiosis the numbers of chromosomes are reduced so that the daughter cells only contain half as many chromosomes as the mother cell, one chromosome from each pair. These cells with a single set of chromosomes are said to be haploid (Greek: haploos=single). Cells which have sets of double chromosomes, that is all chromosomes in pairs, are called diploid (Greek: diploos=double). In higher organisms only gametes, reproductive cells like eggs and sperms, are haploid. If the gametes were not haploid the number of chromosomes would double itself in each generation.
In several organisms, including mammals, there are two of the male chromosomes that do not form a homologous pair. They are called the x- and y-chromosomes. During the meiosis, two types of sperm are always formed, one with an x-chromosome and one with a y-chromosome. The female cells contain two x-chromosomes and each egg contains one of these. When an egg and sperms melt together during conception the chromosomes are mixed together and the diploid order is restored. One chromosome of each pair of chromosomes is hence from the mother and the other one from the father. The fertilized egg starts growing by mitosis. The sex of the new organism depends on whether the egg has been fertilized by a sperm with an x-chromosome, which gives a female, or a y-chromosome, which gives a male. Among certain species, birds for example, it is the other way around in that it is the egg cell that has two different gender deciding chromosomes.


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|you need money to make money
|you have to spend money to make a profit, nothing succeeds like...
|If I had money, I would invest in real estate. It's true that you need money to make money.
|you reap whatsoever you sow
|you will see the long-term effects of your actions, chickens come home...
|Youthful passion can produce a lifetime of consequences. We reap whatsoever we sow.
|you snooze, you lose
|if you are not alert you will lose an opportunity, seize the opportunity
|Store clerks sometimes charge too much for a product. If you snooze, you lose.
|you're full of it [B]
|you are a liar, you are full of shit [B]
|If you think I'm joining that cult, you're full of it.
|you're kidding
|you are joking, you are not serious
|You're kidding. Jen wouldn't take off her clothes and climb a tree.
|you're not kidding/ you aren't kidding
|you are right, you are not joking
|"It's really cold today," Jen said. "You aren't kidding!" he replied.
|you're on
|you have a deal, you have a date
|When she suggested they go out for dinner, he said, "You're on."
|you're only as good as your last shift
|you are judged by your most recent work, don't rest on your laurels
|If you believe that you're only as good as your last shift, you should do your best every day.
|you've got to be kidding
|you must be telling me a joke, get out of town, you're kidding
|"The manager wants me to give him a massage every afternoon." "You've got to be kidding!"
|young at heart
|feeling or thinking that you are young, youthful
|Sarah is over sixty, but she's young at heart.


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Funtacee Parties provide a wide range of jumping castles for parties, birthdays and other events in the West Rand.
Quality Jumping Castles
Our experienced team at Funtacee Parties only select the best jumping castles for our range of inflatables, so when you choose to hire a jumping castle from us you know you are getting a quality product. Our entire range of jumping castles are cleaned and checked prior to delivery to ensure hours of fun for the kids.
Hiring a Jumping Castle
Below is a list of some of the jumping castles available for hire. Prices exclude delivery, set up and collection for selected suburbs in the West Rand.
If you have any questions regarding our range of jumping castles please feel free to call or email us. We provide a jumping castle service in the Johannesburg West Rand Area.