אני רוצה להודות מייק דיואי ממרכז במלאי Peromyscus הגנטי של תובנה חשובה שלו, סבלנות וידע על רבייה מכרסמים.

הזרעה מלאכותית הזרעה מלאכותית משמש במקום הזדווגויות טבעי כדי להגדיל את הסיכוי שכל הנקבות יש רצון להיכנס להריון. 2 זכר שימש כל 1 הנקבות superovulated או נקבה ידוע להיות הייחום. Epididymides caudae מעכברים זכרים בוגרים הוצבו השעיה חלב 9% ופרוסים לפתוח עם 18 מחטים מד. הזרע היה מותר לשחות בחופשיות במשך כמה דקות ואז epididymides נלקחו מתוך התערובת. המתלים הוערך באמצעות מיקרוסקופ עבור מפגש והזרע ניעתי. מד בוטה 22 ½ 1 אינטש מחט ארוכה שימש לנהל את הזרע. לכופף 120 ° הוצב על ה 3 / 4 לאורך המחט. המחט המוחדרת לנרתיק עד לעיקול ו .025 - 0.05mls מוזרק. אם עוזר משמש להחזיק את הנקבה בעמדה המתאימה, שימוש בהרדמה אינו נחוץ. הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש 4 זכרים ונקבות עבור 6 </li><li style=";text-align:right;direction:rtl"> להרדים את הזכרים ביבר </li><li style=";text-align:right;direction:rtl"> הכן כ 5 מ&quot;ל של חלב 9% לעומת אבקת חלב </li><li style=";text-align:right;direction:rtl"> מכניסים 1.5 מ&quot;ל בצלחת פטרי קטן </li><li style=";text-align:right;direction:rtl"> חותכים את epididymus ואת הזרע הזרע </li><li style=";text-align:right;direction:rtl"> מכניסים 1.5ml חלב 9% </li><li style=";text-align:right;direction:rtl"> השימוש בשתי 18 מחטים מד, באמצעות תנועה חיתוך ארוך עם מחטים, פרוסה לפתוח את epididymus ואת הזרע הזרע לשחרר את הזרע </li><li style=";text-align:right;direction:rtl"> קח את שאריות רקמת ולבדוק הזרע במיקרוסקופ </li></ol> הזרקה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שימוש במחט קהה זווית 120 ° </li><li style=";text-align:right;direction:rtl"> החזק העכבר ביד להזריק תערובת. אם לא נוח עם אדם אחד מחזיק והזרקת, שני אנשים יכולים לשמש. הרדמה לא אמור לשמש, כמו על אחד יודע איך זה יכול להשפיע על ההליך. </li><li style=";text-align:right;direction:rtl"> השתמש 0.05ml של תערובת הזרע / חלב לכל נקבה </li></ol> נמען נקבות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> האם למרוח בנרתיק על הנקבות הנמען (בזמן הזה הנקבות הנמען צריך להיות proestrus / ייחום) </li><li style=";text-align:right;direction:rtl"> נקבות שלא הגיעו ייחום על ידי יום 5 צריכה כנראה לא בשימוש </li></ol>

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harvesting sperm and artificial insemination of mice

מכרסמים של Peromyscus הסוג (עכברים צבי) הם היונקים הנפוצים ביותר ילידי צפון אמריקה. מינים Peromyscus משמשים מגוון רחב של מחקר כולל טוקסיקולוגיה, אפידמיולוגיה, אקולוגיה, התנהגות, מחקרים גנטיים. הנה הם לספק מודל שימושי עבור הפגנות של הזרעה מלאכותית. שיטות דומות לאלה המוצגות כאן בעבר שימוש במחקרים העכבר כמה צבאים, עדיין אין פרוטוקול מפורט פורסמו. כאן אנו מדגימים את השיטה הבסיסית של הזרעה מלאכותית. שיטה זו כרוכה לחילוץ האשכים של מכרסם, ואז לבודד את הזרע מן האשך ואת הזרע הזרע. הזרע בשלה, עכשיו בתערובת החלב, ממוקמים מערכת הרבייה של הנקבה בזמן הביוץ. הפריה נחשבת 0 יום התזמון של התפתחות העובר. עוברים לאחר מכן, ניתן לאחזר את נקודת הזמן הרצוי מניפולציות. הזרעה מלאכותית ניתן להשתמש במגוון של מינים מכרסם בו העובר העיתוי המדויק הוא קריטי או קשה להשיג. טכניקה זו היא חיונית עבור מינים או זנים (כולל רוב Peromyscus) אשר לא יכול להזדווג באופן מיידי ו / או שם ההזדווגות קשה להעריך. בנוסף, הזרעה מלאכותית מספק התזמון המדויק להתפתחות העובר או מיפוי התקדמות התפתחותית ו / או עבודה מהונדס. מספר מופחת של בעלי חיים יכול לשמש מאז ההפריה מובטחת. שיטה זו הייתה חיונית לקידום מערכת Peromyscus, ויהיה בתקווה להפיק תועלת גם לאחרים.

Watch this Scientific Journal Video about Harvesting Sperm and Artificial Insemination of Mice at JoVE.com

Harvesting Sperm and Artificial Insemination of Mice

פרוטוקול זה מדגים שיטות להפקת הזרע מן האשכים של זכרים ואז כשזרעו נקבות עכברים. הליך זה שימושי כאשר זמן מדויק יש צורך במחקרים התפתחותיים כמו גם עבודה מהונדס.

קצירת הזרע והזרעה מלאכותית של עכברים

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

harvesting-sperm-and-artificial-insemination-of-mice

Research

JoVE Journal

Biology

48.6K Views.  University of California, Irvine (UCI). פרוטוקול זה מדגים שיטות להפקת הזרע מן האשכים של זכרים ואז כשזרעו נקבות עכברים. הליך זה שימושי כאשר זמן מדויק יש צורך במחקרים התפתחותיים כמו גם עבודה מהונדס.

Video: Harvesting Sperm and Artificial Insemination of Mice

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

הכנת Bilayers מלאכותית לניסויים Electrophysiology

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

טכניקות הדמיה Ca 2 + איתות זרע אנושי

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

בידוד במבחנה הפעלת Caenorhabditis elegans זרע

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

שני סוגים של מבחני עבור Chemoattraction גילוי זרע צפרדע

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

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.

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

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.

הזרע עכבר cryopreservation ושחזור באמצעות · אני 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 ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

הבדלי שומה ביכולת תחרותית בזרע<em&gt; דרוזופילה</em

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

אוסף העופות זרע על ידי עיסוי Cloacal ובידוד של ה-DNA של הזרע

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

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.

הערכת זרע דגים באמצעות תוכנה, התקני קירור

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

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

אוסף זרע באיכות דיפרנציאלית בעזרת צנטריפוגה הדרגתיות צפיפות

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Artificial Intelligence Approaches to Assessing Primary Cilia

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.

The use of artificial intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

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Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. ...