Funding for fieldwork during which our methodologies were developed was provided by Environment Canada&#39;s Canadian Wildlife Service, Environment Canada&#39;s Strategic Technologies Application of Genomics in the Environment Research Fund, the Endangered Species Recovery Fund, the Interdepartmental Recovery Fund, the Natural Sciences and Engineering Research Council and the Ontario Ministry of Training, Colleges and Universities (scholarships to A.A.C.), Queen&#39;s University (Duncan and Urlla Carmichael Fellowship to A.A.C.) and Wildlife Preservation Canada. We wish to thank the Editor and four anonymous reviewers for their comments, which greatly improved this manuscript. Thanks to Wildlife Preservation Canada staff and the members of the North American Loggerhead Shrike Working Group for discussions that assisted in development of this methodology. We extend our thanks to all the Citizen Scientists and staff at African Lion Safari, Cambridge, Ontario, for their assistance in completing the survey. We especially thank Erin Sills, Marketing and PR Coordinator, African Lion Safari, for her assistance in producing the on-line survey and summarizing results.

The research protocol presented herein complies with African Lion Safari&#39;s Animal Care Committee guidelines.
1. Sexing Loggerhead Shrikes by Color and Patern of the Sixth Primary Wing Feather
NOTE: Shrikes can be sexed in hand based on the coloration and pattern in the sixth primary wing feather (P6). In brief, the technique requires the observer to visually extrapoloate a line along the lower edge of the primary wing coverts, and then to assess how far the brown extends in the rachis (shaft) through the white portion of the feather visible below the primary coverts.
<ol>
	<li>Hold the bird firmly in the banders grip and carefully extend one wing to allow the sixth primary wing feather to be viewed (Figure 1). Do not over-extend as this can cause harm to the bird&#39;s musculature.</li>
	<li>Locate the P6 feather: Loggerhead Shrikes have 10 primary feathers, with the last (10th) being the most distal (outermost) feather and reduced in size compartive to the other 9 primary feathers. Unlike the secondary feathers, and with the exception of the reduced 10th primary feather, all primaries have a degree of white coloration. It may be easier to count backwards from the 10th primary than to locate the first primary and count forward to locate the P6.</li>
	<li>Assess the brown in the rachis (hereafter shaft): Does it extend at least half way through the white, and touch, or nearly touch the distal end (furthest from the body and point of emergence) of the primary covert feathers as they lay naturally over the primary feathers? If &#34;yes&#34;, the bird is female (Figure 2). If &#34;no&#34;, then the bird is male (Figure 2).</li>
	<li>The pattern and extent of coloration in the primary feathers varies among individuals. If unsure of whether the brown in the shaft extends at least half way through the white patch to the distal tips of the primary coverts, or if the brown is somewhat indistinct, use a secondary technique. Specifically, assess the symmetry of the brown to white transition in the feather vane on each side of the shaft (Figure 2). If the brown at the point where the coloration changes to white in the vane meets at the same spot on either side of the shaft, the bird is male. If there the line meets asymetrically at the shaft, creating a step or notch, the pattern indicates that the bird is a female (Figure 2).</li>
	<li>If there is still uncertainty as to the individual&#39;s sex, examine the angle of the transition between the brown and the white coloration. If the brown in the feather vane has a steep angle where it meets the white in the vane at the shaft, forming an upside down &#34;V&#34;, the pattern indicates that the bird is a female (Figure 2). If the pattern is more that of an &#34;M&#34;, it indicates a male bird (Figure 2).</li>
</ol>

The authors have nothing to disclose.

Herein, we describe a simple and efficient method whereby loggerhead shrike can be sexed based only on visual cues, and provide an assessment of the method&#39;s accuracy. Our simple method is easily and quickly undertaken, with results indicating a high accuracy rate that increases with a small amount of training. Our results support those of previous work13 that indicated the method originally developed for use in northern shrike12 had utility for sexing adult loggerhead ...

The loggerhead shrike (Lanius ludovicianus) is a North American passerine with a broad geographical range encompassing most of North America and a variety of habitats that can generally be described as grassland1. It is one of only two species of shrikes (Order Passeriformes) that occur in North America. Shrikes are best known for the unique raptor-like bill, which allows them to take vertebrate prey, and their unique behavior of impaling food items on thorns or other sharp objects. The loggerhead shrike is the only species of &#39;true shrike&#39; (Family Laniidae) endemic to the continent. Shrikes breeding above 40&#176;N are generally obligate migrants1,2,3, with wintering grounds almost entirely encompassed within that of non-migratory conspecifics1,4.
North American Breeding Bird Survey data5 for loggerhead shrike indicate a significant (3.18% yr-1) range-wide population decline. The loggerhead shrike is one of Partners in Flight&#39;s 24 &#34;Common Birds in Steep Decline&#34;-i.e., those that have lost more than 50% of their populations over the past 40 years, but which lack other elevated vulnerability factors that would warrant higher &#34;Watch List&#34; status6. Habitat loss due to succession and human development likely contributed to the initial declines4,7, but continued population declines are outpacing habitat loss in the breeding season, suggesting other limiting factors, in particular in areas where the species is an obligate migrant4,8. Results of a population viability analysis conducted for the critically endangered population of loggerhead shrike in Ontario suggests that over-wintering success of birds in their first year of life is a driver of the population trends9,10. Results further indicate that the conservation breeding effort, which is augmenting the wild population, has kept the species from extirpation in this area9,10.
Understanding sex differences is an important component of both ecological and evolutionary hypotheses. The plumage of loggerhead shrikes (Laniusludovicianus) is sexually monochromatic and therefore individuals cannot reliably be sexed in the hand. However, based on a method applicable to the northern shrike (Lanius excubitor)11,12, it has been shown to be possible to sex at least some populations of adult loggerhead shrikes using the coloration pattern in the sixth primary wing feather13. We have revised this methodology13 to include consideration of a second variable, specifically the extent of pigmentation in the rachis of the sixth primary, which allows reliable identification of sex in the majority of individuals in eastern populations, and tested its application (previously only applied to adult birds) to fledged young of the year. The method requires no specialized equipment or costly lab assays, and no measurements are required that would be subject to observer bias. Based on our results, the method is easily learned and, once mastered, highly accurate. Here, we present detailed instructions on how to sex shrike in the hand using our method and discuss the wider implications of including sex assessment in future research and conservation efforts for this unique and enigmatic species of conservation concern.

visually sexing loggerhead shrike (lanius ludovicianus) using plumage coloration and pattern

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, the species has undergone a drastic decline since the mid-1960s. The cause of decline is unknown, and research is actively underway to address this knowledge gap. These efforts are hindered by an inability to sex the species in hand, which to date was only possible using molecular markers. Here, we present a protocol to sex loggerhead shrikes by visually analyzing the coloration and pattern in the sixth primary feather. The application of the method will facilitate our ability to identify threats on a finer scale than has been possible to date and to address various ecological and evolutionary hypotheses. The methodology is simple and results reliable&#8211;we encourage including this method for research of both in situ and ex situ populations.

Male and female plumage is, overall, monomorphic in the loggerhead shrike. However, it has been established that sex can be discerned based on the pattern of coloration in the 6th primary in the population that occurs on mainland California13 and northern shrike12. We tested Sustaita et al.&#39;s (2014) protocol13 to determine if it was applicable to northeastern populations of loggerhead shrike and to youn...

Watch this Scientific Journal Video about Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern at JoVE.com

Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern

We present a protocol to characterize the sex of loggerhead shrike visually based on the coloration and pattern of the sixth primary wing feather.

Visually Sexing Loggerhead Shrike (Lanius Ludovicianus) Using Plumage Coloration and Pattern

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, ...

visually-sexing-loggerhead-shrike-lanius-ludovicianus-using-plumage

Research

JoVE Journal

Biology

6.0K Views.  African Lion Safari. We present a protocol to characterize the sex of loggerhead shrike visually based on the coloration and pattern of the sixth primary wing feather.

Video: Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Understanding Cerebellar Pattern Formation

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...