Suture Materials and Patterns

Jan M. Kümmerle , in Equine Surgery (Fourth Edition), 2012

Knots and Ligatures

Knot tying is an essential part of almost any surgical procedure. However, even a perfectly tied knot is the weakest part of a suture. 6,13 Therefore, it is of tremendous importance to perform knot tying correctly to prevent unnecessary weakening of this critical part of the suture, which could potentially leading to subsequent dehiscence.

Knot Tying Techniques

A knot is constructed by laying at least two throws on top of each other and tightening them. If the direction of the throws is reversed, a square knot results (proper); otherwise a granny knot is obtained (improper). During knot tying, opposing suture ends should be pulled perpendicular to the long axis of the incision except if sutures are placed deep in the tissues. In the latter situation, the suture ends are pulled parallel to the direction of the suture line and in doing so the tissues positioned above the knot are not pulled apart. Reversal of throw direction combined with pulling mainly on one end of the suture results in a half-hitch; if tension applied by the pulling hand is directed away from the incision by lifting this hand, a sliding half-hitch is formed (Figure 16-5 ). Granny and half-hitch knots are prone to slip. 13 However, this feature can be beneficial if the knot needs to be slid into a deep and confined space.

Generally, a superimposition of square knots is considered the most reliable knot configuration. 6 When the first throw of a square knot does not hold the wound margins in apposition, a surgeon's knot may be tied. However, the surgeon's knot should be avoided when not needed because it places more suture material into the wound and can decrease structural stiffness of a knot with some suture materials. 14 Clamping the first throw of a square knot to maintain tissue apposition after the first throw does not negatively affect mechanical properties of common multifilament suture materials; however, clamping can reduce breaking strength of monofilament sutures by 10%. 13,14 A square knot but not a surgeon's knot should be used to ligate vessels. 6 Knots can be tied using instruments or by hand. In veterinary surgery, instrument ties are more commonly used because there is less waste of suture material. If a square knot is formed at the end of a continuous suture line and a needle holder is used to tie the knot, it is important to grasp exactly at the center of the looped end to avoid asymmetric loads placed on either end of the loop. By applying tension to the suture loop with an open needle holder, the tension along the loop equalizes on its own. Hand ties are particularly useful in confined areas, when sutures have been pre-placed or to precisely adjust tension on the suture. Hand ties require that the suture ends be left longer than for an instrument tie. A one-handed or two-handed technique can be applied.

The knots of subcutaneous and intradermal suture patterns should be buried to reduce irritation caused by knots rubbing against more superficial tissue and to prevent suture extrusion.

Knot Efficiency

Loop security and knot security are ways of measuring a knot's effectiveness. 8 Loop security is the capability to maintain a tight suture loop as a knot is tied. Inadequate loop security results in loss of tissue apposition during knot tying. 15 Knot security is defined as the effectiveness of the knot at resisting slippage when load is applied. Knot security depends on the structural configuration of the knot and the type of suture material. 16 The characteristics of a suture material mainly affecting knot security are memory and coefficient of friction. Remember that body fluids come in contact with the suture material during surgery, which affects frictional behavior and thus the knot security of a suture. 17

In addition to suture material and knot configuration, the number of throws and suture end length also influence knot security. A suture end length of at least 3 mm is recommended to optimize knot integrity. 18,19 The minimal number of throws needed (including the first) for a secure square knot using No. 2-0 USP suture materials is three for polyglycolic acid, polyglactin 910, and polypropylene and four for nylon and polydioxanone. 20,21 For larger-diameter suture materials, sufficient knot security is achieved with five throws. This was demonstrated for polyglactin 910 No. 2 USP, polyglactin 910 No. 3 USP, and polydioxanone No. 2 USP. 13 Knots at the end of a continuous suture line are constructed using one looped and one free end. These knots require two or three more throws to ensure knot security than do knots constructed from two single suture strands. 22 The Aberdeen knot represents a special configuration to end a continuous suture line and is recommended in human surgery when monofilament suture material is used (the configuration of the knot can be studied in the cited publication). 23 A recent in vitro study demonstrated superior relative knot security and reduced knot volume of Aberdeen knots compared to square knots to end a continuous suture pattern of polydioxanone. 21

Another factor to consider is the wound environment. A fatty wound environment can increase the number of throws needed to achieve a secure knot. This was confirmed by the finding that fat-coated No. 2-0 USP polydioxanone requires one additional throw to form a secure square knot at the beginning of a continuous pattern compared to plasma-coated No. 2-0 USP polydioxanone. 21 Asymmetric knots like sliding half-hitch or asymmetric granny knots usually need two additional throws to achieve knot security. This was demonstrated in one study for polyglactin 910. 24 However, the superior knot security of braided lactomer (Polysorb) provided sufficient knot security even without additional throws. 24 In the clinical situation, the number of throws should be adequate to ensure knot security but not excessive to limit the amount of bulky foreign material in the tissues.

Finally, the suture diameter is also a determinant of knot security. Knot security decreases with increasing suture diameter. 13,16

Loop Sutures

As an alternative to knots, the use of a loop suture to apply a simple continuous pattern has also been described in equine surgery. 25 The use of loop sutures reduces the number of knots and the double suture strand provides a larger surface area as the suture passes through the tissue; however, they result in an increased total amount of suture material remaining in the wound and the placement of a bulky four-stranded knot at the end of the suture line. In an in vitro experiment, USP No. 2 braided lactomer loop sutures applied in a simple-continuous fashion provided sufficient security for closure of the equine linea alba based on single-cycle to failure testing, with fascial failure being the main failure mode and without occurrence of suture or knot failure. 25

Knot-Tying Techniques for Minimally Invasive Surgery

Minimally invasive surgical techniques require modifications in knot-tying techniques. In equine surgery, laparoscopy is increasing in importance, and extracorporeal knotting requires safe and efficient sliding-knot techniques. The first reliable sliding knot described for human laparoscopy was the Roeder knot. 26 Knot security was further improved by Sharp et al. 27 by developing the 4S-modified Roeder knot (Figure 16-6). In vitro studies revealed that the 4S-modified Roeder knot outperformed several other slipknot ligatures in terms of knot security. 28,29 Monofilament suture materials are suitable for laparoscopic surgery because they perform well for knot rundown, have low tissue drag, and, unlike multifilament sutures, do not loose loop characteristics when wet. With regard to suture material and size used for the 4S-modified Roeder knot, polydioxanone and polyglyconate are biomechanically superior to polyglactin 910 and polyglycolic acid and sizes USP No. 1 or 2 are superior to smaller suture sizes. 28,29

Suture Tension

Suture tension can be classified as intrinsic or extrinsic. Intrinsic tension refers to the tension on the tissue constricted within the suture loop. Excessive intrinsic tension can cause ischemic necrosis. Extrinsic tension represents the pulling tension from outside the suture loop. It depends on wound size, location, relationship to skin lines, and the amount of surrounding loose tissue. 30

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Principles of Surgery

André Desrochers , in Llama and Alpaca Care, 2014

Suture Materials: Basic Principles

The use of suture material and the performance of suturing should become second nature for every surgeon. With the recent discovery of new synthetic materials, surgeons enjoy the options of different monofilament, braided, absorbable, and nonabsorbable sutures. The optimal suture is inert, strong, and small in diameter and length; is rapidly absorbed; and is not prone to bacterial colonization. The surgeon must consider the advantages and disadvantages of each material to make a selection that is suitable to the need at hand (Table 55-1).

Despite the sophistication of today's suture materials and surgical techniques, closing a wound still involves the same basic techniques. A number of factors influence the surgeon's choice: training, professional experience, knowledge of the healing characteristics of tissues, the organ's response to suturing, knowledge of the physical and biologic characteristics of various suture materials, and patient factors (age, weight, overall health status, and the presence of infection).

Suture size denotes the diameter of the suture material. The accepted surgical practice is to use the smallest-diameter suture that will adequately hold the wounded tissue during the early phase of healing (Table 55-2). This practice minimizes trauma as the suture is passed through the tissue to achieve closure. The expectation is that the tensile strength of the suture need never exceed the tensile strength of normal tissue. However, sutures should be at least as strong as normal tissue through which they are being placed. Unfortunately, this is not always possible to achieve in large animal surgery. Wound tissue strength gradually increases during the healing process. Wound tissue strength increases to 20% of uninjured tissue by 2 to 3 weeks after injury and to 50% of uninjured tissue by 4 to 5 weeks after injury. At 3 to 6 months, wound tissue achieves its maximum strength, which is 70% to 80% that of normal tissue. Large-diameter suture materials were compared in an in vitro study using adult equine linea alba. 70 No. 2 polydioxanone (2 PD), 3 polyglactin 910 (3 PG), 6 polyglactin 910 (6 PG), and 7 polydioxanone (7 PD) linea alba constructs all failed at the knot (94 out 96 test sections). The strongest construct was with the 7 PD.

The distance between each bite and the cut edge of the incision line is important to holding power. An in vitro model of equine linea alba showed that 1.5 cm from the incision edge was optimal. 71 Braided lactomer (USP 2) was used in another in vitro study comparing distances between suture bites (1 cm versus 1.5 cm) for closure of an equine linea alba with a simple continuous loop suture. 72 Fascia failure was the main failure mode for both constructs, and no significant differences were found. In llamas, a simple continuous pattern and an inverted cruciate pattern were compared in an in vivo study of linea alba closure. 73 After days of implantation, mechanical testing on the linea alba did not show any significant differences between the two patterns, leading to the conclusion that the simple continuous pattern can be used securely while closing a normal linea alba of llamas.

Knot Security of Suture Material

Tying suture knots to obtain reliable tissue apposition is a basic but important skill in veterinary surgery. When closing a wound, the knot is usually the weakest point of the suture loop. 74 Knot failure may have disastrous consequences, and surgeons must ensure security of their knots. Knot security and strength will depend on the size and the type of suture material used, number of throws, and knot configuration. 75,76 Two in vitro studies tested the breaking strength of large USP suture materials frequently used in large animal surgery. 71,77 Trostle found that No. 5 polyester was the strongest suture tested, followed by No. 3 polyglactin 910 and No. 2 polyglycolic acid. Suture loops failed by breakage at the knot in 93% of tests. 71 Although the suture materials tested were different, another study found superior breaking strength for No. 5 polyester with an overall loop breakage rate at the knot of 93.6%. 77 Both studies used a reinforced surgeon's knot, and none of them evaluated the number of throws necessary to obtain a secure knot with these different and larger suture sizes. A secure knot is commonly defined as a knot that does not slip more than 3 mm when the loop is subjected to an increasing load. If the knot holds, eventually breakage of the suture material will occur at the knot as a result of shear stress. The symmetrical square knot is the most recommended knot. An interaction occurs between knot configuration and suture material related to knot security. 78–80 A running polypropylene suture that is initially anchored with half hitches was shown to be stronger and safer than a running suture tied down with square knots. 81

With the large-diameter suture, each surgical knot should be constituted of a minimum of five throws for an interrupted pattern (Lequient M, Desrochers A, Dubreuil P, 1996, unpublished data). If a continuous pattern is used, one additional throw at the beginning and two additional throws at the end of the suture will improve the security of the knot. Knots are difficult to tighten under excessive tension, and different knot configurations may be used to prevent this problem. Needle holders or hemostats are commonly placed on the first throw to hold it tight. Needle-holder jaws with teeth produce distinct structural changes in synthetic sutures, causing a marked reduction in the suture breaking strength. 82 The effect of the knotting method on the structural properties of large-diameter nonabsorbable monofilament sutures was studied. 83 According to the results of this study, the type of knot tied (square knot, sliding half-hitch, or surgeon's knot) did not influence the structural properties of suture materials except for the clamped square knot. Mulon evaluated, in vitro, the effect of six different knotting methods on the mechanical properties of USP 2 polydioxanone and USP 2 and USP 3 polygalactin 910. 84 The knots compared were the square knot, the surgeon knot, the clamped surgeon's knot, the sliding half hitch knot, the Delimar knot, and the self-locking knot (Figures 55-1 through 55-3). 85,86 As in the previous studies, 90% of the loops tested failed at the knot. Clamping the first throw of a surgeon knot with polydioxanone decreased the load to failure significantly compared with no clamping. The double-strand loop conferred stiffer and stronger properties compared with the self-locking knot. Different knot configurations (square, surgeon, Aberdeen, loop) with three types of 1-gauge monofilament suture (nylon, polyglyconate, and polydioxanone) were studied, and the holding capacity was greater with the loop knot, and the polyglyconate had the highest knot holding capacity. 87 The length of the suture tags may influence knot security. Suture materials with three different cut end lengths (0-mm, 3-mm, and 10-mm) were studied, and the knots with an end length of 0 mm were significantly more likely to fail by becoming untied compared with either the 3-mm or the 10-mm cut-end knots. 88

Most surgical principles can be applied across species. Differences exist with patient scrubbing techniques because of the length and density of hair, severity of body contamination with dirt and debris, and environment. Suture material properties influencing selection are similar across species, but adjustments for size, activity, and expected biomechanical forces are needed for individual patients.

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Biotechnologies for intensive production of olives in desert conditions

Zeev Wiesman , in Desert Olive Oil Cultivation, 2009

Olive knot (Pseudomonas syringae pv savastanoi)

Olive knot, a bacterial disease, requires an opening for infection. Pruning wounds, leaf scars and frost cracks are the most common wounds that become infected with olive knot. Rainfall disseminates the bacteria into these openings. Once infected, tissue grows at an uncontrolled rate, forming a gall ("knot") that girdles and kills the shoots and branches. Severe infections, usually resulting from freezing temperatures that lead to a profusion of wounds, can cause considerable fruit wood death. Growers prune olives in spring to reduce the hazard of opening pruning wounds for infection via rainfall. Pruning out of established knots is recommended in the summer to eliminate inoculum from the grove. Grove floors are managed free of weeds for optimal protection against radiation and advective freezes.

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Stem Diseases

TERRY A. TATTAR , in Diseases of Shade Trees (Revised Edition), 1989

Disease Cycle

Black knot is caused by the fungus Dibotryon morbosum (Fig. 9.10 ). The fungus overwinters in fruiting bodies on the outside of the knot or as vegetative mycelium in infected twigs and branches. During the spring, spores are forcibly ejected from fruiting bodies during wet weather and cause infections on the current year's twigs. A newly infected branch will swell slightly by fall. The fungus overwinters in the infected branch; the following spring the branch continues to swell and the bark becomes cracked and roughened. During the summer the bark splits and becomes filled with a velvety olive-green material. Summer spores are produced at this time but their role in the disease cycle is considered minor. Lack of extended wet periods during midsummer may account for the general lack of infection by summer spores. The swellings continue to enlarge, turn black, and harden into "knots" by fall. Fruiting bodies are formed on the outside of the knots during fall and winter. The disease cycle takes 2 years to complete.

Fig. 9.10. Disease cycle of black knot of plum and cherry caused by Dibotryon morbosum.

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Endoscopic Knot Tying and Suturing

Lynetta Freeman , ... David R. Stoloff , in Small Animal Endoscopy (Third Edition), 2011

Indications

Suturing and knot tying are required for closing tissues and controlling hemorrhage. Intracorporeal suturing and knot tying can be done for an intestinal resection and anastomosis during foreign body removal, but foreign body retrieval is more efficiently done as a laparoscopically assisted intestinal resection and anastomosis. Consequently, the first criterion is that suturing and knot tying be practical for the procedure. The current clinical indications are somewhat limited as veterinarians begin minimally invasive surgery using laparoscopic-assisted techniques. As veterinarians desire to perform more challenging procedures, the applications of intracorporeal knot tying and suturing will expand. Procedures using intracorporeal suturing have included laparoscopic gastropexy, thoracoscopic closure of the diaphragmatic hernias, laparoscopic closure of the urinary bladder, and laparoscopic correction of abdominal hernias including incisional hernias. Examples of procedures in which knot tying of ligatures has been necessary include vessel ligation, laparoscopic cholecystectomy, laparoscopically assisted nephrectomy, and partial amputation of a portion of lung, spleen, and liver. Although staplers, clip appliers, and energy-sealing devices can be used to appose tissue and ligate vessels, they can be expensive and difficult to maneuver in small spaces. Intracorporeal suturing and knot tying offer benefits of flexibility, decreased invasiveness as compared with laparoscopic-assisted techniques, and more secure knot holding in some procedures. Having skills and the confidence to perform laparoscopic knot tying and suturing will enable the endoscopic surgeon to attempt endoscopic correction of problems without converting to traditional open surgery.

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Presurgical Considerations

Ava M. Trent , ... Emily A. Barrell , in Farm Animal Surgery (Second Edition), 2017

Knots

Ropes are invaluable assets in animal restraint. However, a rope is only as good as the knot that is tied. There is a unique knot, with a particular advantage, for practically any situation imaginable. At the very least, every animal handler should be proficient with the three knots covered in the following discussion.

Square Knot

The square knot joins two rope ends. Joining the ends of a single rope forms a loop that allows the rope to be tied to a fixed object. Alternatively, the ends of two separate ropes can be joined to form one long rope. Once tightened, a properly tied square knot will not slip under tension. A common error is to tie a granny knot that slips when tension is applied (Figure 3-4).

Quick-Release Slip Knot

The quick-release slip knot and its modifications (see the Tail Ties section) secure a rope in a way that allows it to be easily untied at the end of a procedure or in case emergency release is required. Most commonly, the free end of a halter will be tied to a post to restrain an animal's head. When properly tied, the bow end of the knot is entirely surrounded by rope; if the bow lies against the object to which it is tied, it is not secure and will loosen as the animal struggles. Because it is a slipknot, there will always be a little play in the rope as it slips down to its anchor; tying the knot as close to the anchor as possible is important (Figure 3-5).

Bowline Knot

The bowline knot creates a permanent loop that will not tighten. It is useful to place around an animal's body, neck, or limb because it will not cinch down and compromise respiration or circulation (Figure 3-6).

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Nematodes

TERRY A. TATTAR , in Diseases of Shade Trees (Revised Edition), 1989

Treatment

Root knot is most troublesome on young trees and shrubs. Many of these plants are already infected with root knot when purchased by the homeowner, arborist, or municipal tree official. When obtaining root stock for nursery use or for outplanting, care should be taken to ensure that all plants are free of root knot and other plant parasitic nematodes. These infected trees will make poor shade trees that will probably not survive to maturity. Purchase plants only from reputable dealers who will guarantee their products. If in doubt, contact your county agent or the extension plant pathologist at your state university for advice. An added danger of purchasing nematode infected plants is the possibility of establishing these pathogens in your nursery or backyard.

High populations of root knot and other plant-parasitic nematodes already exist in many areas. Care to avoid infected plants will not be sufficient to control the problem in these areas. Many valuable shade trees and shrubs are already infected, and some type of therapy is needed to help them regain normal vigor. In these cases, efforts must be taken to decrease the population of nematodes in the soil.

Chemical control is the only practical means available to control high populations of plant-parasitic nematodes in the nursery, roadside, or backyard. Chemicals that control nematodes, called nematicides, are usually applied directly into the soil. Since the nematodes are found throughout the soil zone where most of the roots are growing (top 18 inches or 45 cm), the nematicides must be effective over a large volume of soil even around a single tree. Most nematicides are liquids that become gases (volatilize) when applied to the soil. In the gas form the nematicide can diffuse quickly throughout the soil and be quite effective in eradicating nematodes. This form of chemical control is known as soil fumigation.

Soil fumigation may be performed as a preparation for planting, such as in a nursery bed, along the roadside, or a backyard, or it may be a therapeutic treatment for an already infected tree. In planting preparation where only soil will be treated, soil fumigants, which are quite toxic to plants, are applied to sterilize the soil to remove not only nematodes but most other living pathogens as well. The soil fumigants may be applied in the soil by a variety of specific methods but most involve injection of the fumigants as liquids at least six inches (15 cm) below the soil with some type of seal placed on top of the soil (such as plastic sheets or water) to hold the fumigant in contact with the soil as long as possible. Trees and shrubs can safely be planted about 2 weeks after fumigation. Soil fumigants used for therapy, on the other hand, are not toxic to plants when used according to the instructions on the label. These fumigants can be applied as a drench over the roots within the drip line of a tree or shrub or in a granular formulation. This type of soil fumigant may also be used to protect new trees or shrubs by applications at the time of planting.

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Synaptic Plasticity Regulated by Protein–Protein Interactions and Posttranslational Modifications

Norihiko Yokoi , ... Yuko Fukata , in International Review of Cell and Molecular Biology, 2012

4.1.3.3 CKAMP44

Cysteine-knot AMPAR modulating protein (CKAMP44) was identified by immunoprecipitation of AMPARs from mouse forebrain and by subsequent quantitative mass spectrometry analysis (von Engelhardt et al., 2010). CKAMP44 is a brain-specific type I transmembrane protein that contains a cysteine-rich NTD, likely forming a cysteine knot found in ion channel toxins (Fig. 1.4; Vitt et al., 2001). The intracellular domain of CKAMP44 has a PDZ ligand (type II) at its C-terminus. Coexpression of CKAMP44 with GluA1-3 in Xenopus oocytes results in a prominent reduction in the glutamate-evoked current without any change in the amount of surface AMPARs. CKAMP44 slows deactivation less pronouncedly than TARPs but accelerates desensitization and slows recovery from desensitization of AMPARs. These activities about desensitization are in contrast to those in both TARPs and CNIHs. Dentate gyrus granule cells exhibit strong CKAMP44 expression, and CKAMP44 gene deletion increases the paired-pulse ratio of AMPA currents in perforant path–granule cell synapses, consistently with the role of CKAMP44 in slowing recovery from desensitization.

In conclusion, diverse transmembrane auxiliary proteins provide AMPARs with dynamic regulation on its trafficking and gating, which may be specific to neuron types of the different brain regions. Characterization of individual auxiliary subunits and their combination will resolve the whole picture of dynamic regulation of AMPARs. Also, based on the fact that auxiliary subunits dramatically alter the channel properties, it should be worthwhile to search for the auxiliary subunit if some channel properties in vitro (heterologous system) do not match those in vivo.

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Other food products

Kurt A. Rosentrater , A.D. Evers , in Kent's Technology of Cereals (Fifth Edition), 2018

11.2.4 Pretzels

Pretzels, crisp knot-shaped biscuits, flavoured with salt, are made from wheat flour plus shortening (1.25% on flour wt), malt (1.25%), yeasts (0.25%), ammonium bicarbonate (0.04%) and water (about 42%). Typical manufacturing stages for pretzels are shown in Fig. 11.3. A dough made from these ingredients is rolled into a rope, then twisted and allowed to relax for 10   min. The rope is passed through rollers, to set the knots, and allowed to ferment for 30  min. The starch on the surface is then gelatinized by passing the rope through a bath of caustic soda (1%) for 25   s at about 93°C. The dough pieces are then salted with 2% sodium chloride and baked in three stages: at 315°C for 10   min, then at 218°C to reduce the moisture content to 15%, and finally at 121°C for 90   min (Hoseney, 1986). Examples of traditional pretzels and novel pretzel shapes that have recently been introduced into the consumer market are shown in Fig. 11.4.

Figure 11.3. General stages in the preparation of pretzels, crackers, biscuits and cookies.

Figure 11.4. Pretzels are a popular snack food in their (a) traditional form, but nonconventional shapes are frequently developed for marketing, including (b) weave, (c) rod, and (d) crisp styles.

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The Neurobiology of Circadian Timing

Barbara Helm , ... Theunis Piersma , in Progress in Brain Research, 2012

Methods

Experiments involved eight Red Knots of the subspecies islandica (four males, three females, one sex unknown), which winters in Europe, and seven Ruffs (all males), which use the Wadden Sea for staging (Fig. 1; for details, see Buehler et al., 2009; Piersma et al., 2000). In the Dutch Wadden Sea Ruffs were caught on March 15, 1997 using a "wilsternet," while Knots were captured with mistnets near a high-tide roost between 1994 and 1997. At capture, birds were ringed, aged as older than 2 years, and transferred to outdoor aviaries (4.5   m   ×   1.5   m   ×   2.3   m high) at the NIOZ. Knots were kept in a single aviary, while Ruffs, which are more territorial, were divided over two aviaries. One-quarter of the surface contained a shallow basin with sand and running seawater (mudflat). Each aviary contained a small freshwater basin for drinking and bathing. The birds were fed ad libitum portions of protein-rich trout-food pellets and once per week were screened for physical condition, while the aviaries were disinfected and the food replenished. Birds experienced local photoperiod and temperature. Compared to wild conspecifics, Ruffs were thus exposed to identical conditions in spring and fall (Fig. 1). In summer, they would have experienced slightly longer days at more northerly latitudes and winter days would also be longer on their African winter grounds. In contrast, Knots were exposed to conditions that were identical to those experienced by wild conspecifics that stay in the Netherlands from late summer until spring. During summer, daylength was much shorter for the captive Knots than for conspecifics on their Arctic-breeding grounds. Motivation for migration should be similar in spring but differ between the species in autumn when Ruffs should be active migrants, whereas Knots would be already at their wintering grounds. Behavioral observations of Knots and other species suggest that migration may indeed be suppressed by site recognition (Ketterson and Nolan, 1988; Schwabl et al., 1991). To delineate life-cycle stages (Fig. 2), data on phenology were collected for all birds once per week. Body mass was measured on an electronic balance to the nearest gram. Moult was scored on breast plumage for body moult (0   =   no feathers growing, 1   =   feathers growing) and on the primaries for flight feather moult (scores from 1   =   feather dropped to 5   =   new feather for all 10 primaries, thus reaching values from 0 to 50; Ginn and Melville, 1983). Extent of breeding plumage was scored on a scale between 1   =   basic, indicating full gray winter plumage, and 7   =   full alternate, indicating full rusty-red breeding plumage. Experiments started for Knots on February 20 and for Ruffs on March 24, 1997 and were completed on February 11, 1998. Melatonin was sampled biweekly as explained in Fig. 3.

Fig. 2. Derivation of life-cycle stages based on phenology of (a) Knots and (b) Ruffs. Top: natural daylength, with stars marking days of blood sampling and letters indicating life-cycle stage; bottom: mean   ±   SD for body mass and scores of primary moult, plumage color, and breast moult; vertical lines indicate demarcations of life-cycle stages. Stage definitions Knot: (1) "winter": quiescent time. From end of autumn moult until start of spring preparations, that is, fattening and moult into breeding plumage; (2) "spring": fueling and prenuptial moult. From start of fattening, moult, and recoloration until reaching full breeding coloration and peak body mass; (3) "breeding state": defined by full breeding plumage. From reaching peak body mass and breeding coloration until the onset of moult; (3A) "flight phase": ca. 4   weeks of using up fuel stores, from maximum to trough of body mass. Prediction: melatonin low; (3B) "transitory phase for nonbreeders": corresponding to breeding in the wild, from end of body mass peak until start of moult. Prediction: melatonin low; (4) "moult": from start to completion of primary moult, corresponding to moult of wild conspecifics on the winter grounds slightly later in the year. Stage definitions Ruff: (1) "winter": quiescent time. From end of autumn moult until start of spring preparations, that is, moult into breeding plumage; (2) "spring": fueling and prenuptial moult. In contrast to Knots, Ruffs overlap these activities with migratory flight. From start of moult and recoloration until reaching full breeding coloration and peak body mass. Prediction: melatonin low; (3) "breeding state": defined by full breeding plumage. From peak body mass and breeding coloration until the onset of moult; (4) "moult": renewal of primaries. From start to completion of primary moult, corresponding to moult of wild birds started during migration and completed on the African winter grounds; (4A) "fuel, flight and moult phase," starting with a transitory phase in nonbreeders with slow and then progressively faster moult and fueling. Unlike in captive Knots, this corresponds to flight phases in the wild. From starting of color loss and increase of body mass until birds reach peak body mass. Prediction: melatonin low; (4B) "postmigratory completion of moult": From end of body mass peak until completion of moult, corresponding to finalizing of moult in Africa.

Fig. 3. Diel melatonin profiles across the annual cycle for Knot (left) and Ruff (right) (mean   ±   SE; [pg/ml]). Plots are arranged by corresponding time of year. Vertical curves show time of sunset and sunrise, respectively. The birds were sampled biweekly to examine diel profiles over an entire year, alternating between phases of full moon and new moon. Sampling took place over a 24-h session, starting and ending at mid-day with seven bleedings per bird at 4-h intervals (12:00, 16:00, 20:00, 00:00, 04:00, 08:00; 12:00   h; Piersma et al., 2000). On each sampling date, blood was collected for at least six birds of both species. The protocol followed local time, implying that samples were collected an hour earlier relative to the solar day during summer savings time. Blood was collected within 15   min after entering the aviary by lightly puncturing the brachial wing vein. We collected 150–250   μl of blood into heparinized capillary tubes that were stored cool. Tubes were centrifuged within 2   h of sampling at 6900   × g for 15   min, and plasma was subsequently stored at -80   °C until transfer on dry ice to the Max-Planck Institute for Ornithology in Andechs, Germany. Data collection was briefly interrupted in August 1997, and for Ruffs, two sampling periods were missing (12 November; 19 December). Individual birds were sampled up to 22 times during a total of 23 sampling periods, and overall we analyzed 1667 samples. Simultaneous monitoring of health parameters indicated no harmful effects (Piersma et al., 2000). We quantified melatonin by the established procedure at our laboratory (Silverin et al., 2009; Van't Hof and Gwinner, 1996) as described for the Knots (Buehler et al., 2009). Due to the sudden loss of Ebo Gwinner and subsequent complications, we unfortunately have only long-term information about the melatonin assay at our institute. Accordingly, mean recoveries are at 90%, and intraassay and interassay variation were 4.5% and 14%, respectively. For further details on Knots, see Buehler et al. (2009).

As a rough overview of activity patterns, in one aviary group of each species wading in the simulated mud flats was continuously recorded. In Knots, feeding activity was also surveyed by an additional sensor next to the single source of food. Ruffs, in contrast, were equipped with several feeders because of their higher aggression so that foraging was not monitored. Locomotion and feeding were surveyed by custom-made photocell systems. Beam breaks of an infrared beam were recorded in 2-min bins on a computerized data-logging system. Activity data originated from initially eight Knots (six birds from August 1997) and from initially four Ruffs (three birds from August 1997). Sensors yielded data throughout experimentation, except for brief periods of technical problems. Because of behavioral differences between the species, we interpreted their activity records from the artificial mudflats differently. Red Knots forage on mudflats, and even in our aviaries with permanent food supply elsewhere, they spent 10–20% of each day actively probing for food on the artificial mudflats. In contrast, they roost on dry land and not on mudflats. Therefore, recordings from the mudflat in Knots reflect feeding and general activity, and the additional feeding sensor narrowed down foraging motivation. Ruffs, in contrast, usually do not feed on mudflats, but they like to roost on wet ground if available. Therefore, recordings from the mudflat combine general activity and resting behavior. However, the group recordings of both species yield only tentative information.

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