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In This Article

  • Erratum Notice
  • Summary
  • Abstract
  • Introduction
  • Protocol
  • תוצאות
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Erratum
  • Reprints and Permissions

Erratum Notice

Important: There has been an erratum issued for this article. Read More ...

Summary

שני פרוטוקולי אבלציה של דיבור עיניים (כלומר, גישות צריבה וניתוח) בוצעו על נקבות סרטנים מורדמות. אבלציה של סרטני בוץ זירזה את הבשלת השחלות מבלי להפחית את שיעור ההישרדות.

Abstract

סרטני בוץ (Scylla spp.) הם מיני סרטנים בעלי חשיבות מסחרית שניתן למצוא בכל אזור הודו-מערב האוקיינוס השקט. במהלך התרבות, השראת הבשלת השחלות חשובה כדי לענות על הביקוש הצרכני לסרטני בוץ בוגרים ולהאיץ את ייצור הזרעים. אבלציה של Eyestalk היא כלי יעיל לשיפור התבגרות השחלות בסרטני בוץ. עם זאת, אין פרוטוקול סטנדרטי לאבלציה של סרטני בוץ. במחקר זה מתוארות שתי טכניקות אבלציה של דיבור עיניים: צריבה (שימוש במתכת חמה כדי לעבד את העיניים של סרטן מורדם) וניתוח (הסרת העיניים באמצעות מספריים כירורגיים). לפני אבלציה של דיבור עיניים, נקבות בוגרות מינית (CW > 86 מ"מ) הורדמו באמצעות שקית קרח (-20 מעלות צלזיוס) עם מי ים. כאשר טמפרטורת המים הגיעה ל -4 מעלות צלזיוס, שקית הקרח הוסרה מהמים. מי ים זורמים (טמפרטורת סביבה: 28 מעלות צלזיוס) שימשו להתאוששות מההרדמה מיד לאחר אבלציה של דיבור עיניים. התמותה לא התרחשה במהלך או אחרי תהליך אבלציה של דיבור עיניים. פרוטוקול האבלציה של שיחות העיניים שהוצג כאן האיץ את ההבשלה השחלתית של סרטני הבוץ.

Introduction

כל ארבעת מיני סרטני הבוץ השייכים לסוג Scylla הם מיני סרטנים בעלי חשיבות מסחרית בחקלאות ימית 1,2. גדילתם של סרטנים, כולל סרטני בוץ, והפיכתם משלב הפג (תת-בוגר או התבגרות מינית) לשלב הבוגר מינית (בוגר) מתרחשים בתהליך התכה הכולל נשירה תקופתית של שלד חיצוני ישן וקטן יותר. רוחב קרפצ'ה (CW), כלפידים ומורפולוגיה של דש הבטן נמצאים בשימוש נרחב כדי לקבוע את הבגרות המינית של Scylla spp. 3,4,5. תהליך ההתכה מוסדר על ידי פעולה של הורמונים שונים ודורש כמות עצומה של אנרגיה6. בנוסף לתהליך ההתכה הרגיל, אובדן הגפיים, בין אם מרצון או על ידי גורמים חיצוניים, מזרז את ההתכה של סרטנים מבלי להשפיע על שיעור ההישרדות שלהם 7,8,9. לכן, אוטוטומיה של הגפיים משמשת בדרך כלל להשראת מותך בתעשיית גידול סרטני הבוץ הרכים 7,9.

אבלציה חד צדדית או דו-צדדית פופולרית בעיקר בסרטני מים מתוקים ושרימפס ימיים להבשלת גונאד וייצור זרעים10,11,12,13. טכניקות אבלציה נפוצות בסרטנים כוללות את הדברים הבאים: (i) קשירה בבסיס העין באמצעות מחרוזת14,15; (2) צריבת העיניים באמצעות מלקחיים חמים או מכשירים חשמליים16; (3) הסרה או צביטה ישירה של העיניים כדי להשאיר פצע פתוח12; ו-(4) הסרת תוכן העיניים דרך חתך לאחר חיתוך החלק הדיסטלי של העין בתער17. איברי X הם איברים אנדוקריניים חשובים בסרטנים, שכן הם מווסתים הורמונים היפרגליקמיים סרטניים (CHH), הורמונים מעכבי מותך (MIH) והורמונים מעכבי ויטלוגנזה (VIH)6,18,19,20,21,22. איברי X של Eyestalk (או קומפלקס בלוטת הסינוס) מסנתזים ומשחררים הורמונים מעכבי גונאד (GIH), הידועים גם בשם הורמונים מעכבי ויטלוגנזה (VIH), השייכים למשפחת ההורמונים הנוירופפטידים6. אבלציה חד צדדית או דו-צדדית מפחיתה את סינתזת GIH, וכתוצאה מכך דומיננטיות של הורמונים מעוררים (כלומר, הורמונים מעוררי גונאד, GSH) והאצת תהליך ההתבגרות השחלתית בסרטנים23,24,25,26. ללא השפעת GIH לאחר אבלציה של דיבור עיניים, נקבות סרטנים מקדישות את האנרגיה שלהן להתפתחות השחלה27. נמצא כי אבלציה חד צדדית בעיניים מספיקה להשראת הבשלה שחלתית בסרטנים11 וכי העיניים האבלטיות של שרימפס וסרטנים יכולות להתחדש לאחר מספר התכות28. ישנם ארבעה שלבי התפתחות שחלות שתועדו ב- Scylla spp.: i) לא בשלים (שלב 1), ii) הבשלה מוקדמת (שלב -2), iii) טרום הבשלה (שלב -3), ו- iv) הבשלה מלאה (שלב 4)29,30. השלב השחלתי הלא בשל נמצא אצל נקבות לא בוגרות. לאחר ההתכה המינית וההזדווגות, השחלה הלא בשלה מתחילה להתפתח ולבסוף מבשילה (שלב 4) לפני ההשרצה31.

פרוטוקול אבלציה של Eyestalk חיוני להתפתחות סרטני בוץ ולייצור זרעים. בשוק המזון העולמי, סרטני בוץ בוגרים עם שחלות בשלות לחלוטין (שלב 4) על פני סרטנים עם תכולת שרירים גבוהה יותר מועדפים על ידי הצרכנים, ולכן יש להם ערך מסחרי גבוה יותר, אפילו גבוה יותר מאשר זכרים גדולים. אין פרוטוקול מלא לאבלציה של סרטני בוץ בעיניים. פרוטוקול אבלציה של דיבור עיניים בעבודה זו ממזער מתח על ידי שימוש בסרטנים מורדמים לחלוטין וממזער את הפגיעה הפיזית באנשי צוות מעקיצות סרטנים. פרוטוקול זה קל וחסכוני. כאן, אנו מציגים פרוטוקול עבור אבלציה עיניים של Scylla spp. שיכול לגרום להבשלה של הגונדה. נבדקו שתי טכניקות של אבלציה של דיבור עיניים (צריבה וניתוח) ויעילותן הושוותה בהתבסס על קצב התפתחות הגונדל של נקבות סרטני הבוץ.

Protocol

פרוטוקול זה עוקב אחר קוד העיסוק המלזי לטיפול ושימוש בבעלי חיים למטרות מדעיות שנקבע על ידי האגודה למדעי חיות מעבדה של מלזיה. הקרבת דגימות הניסוי נעשתה על פי מדריך המכונים הלאומיים לבריאות לטיפול ושימוש בחיות מעבדה (פרסומי NIH מס '8023, מתוקן 1978). סרטני בוץ נקביים פגים מינית (סרטן בוץ כתום S. olivacea) נאספו מהשוק המקומי (5°66′62′′N, 102°72′33′′E) בביצות סטיו במלזיה. מין סרטני הבוץ זוהה על סמך מאפיינים מורפולוגיים1.

1. איסוף דגימות וחיטוי

  1. אספו סרטני בוץ בריאים, פעילים ופגים (איור 1).
    הערה: לנקבות סרטנים פגות יש דשי בטן משולשים ובהירים יחד עם טווח CW של 80-85 מ"מ.
  2. שטפו את הסרטנים במי ברז עם כלור (מים מתוקים) כדי להסיר פסולת וטפילים אוסמופיליים.
  3. משרים את הסרטנים בפורמלדהיד של 150 ppm עם מליחות של 20 ppt למשך 30 דקות.
  4. יש לשמור על אוורור רציף ועדין עם אבני אוויר במהלך הטיפול בפורמלדהיד. מקור האוורור יכול להיות מקו אוורור מרכזי או משאבת אוורור אקווריום.
  5. שטפו את הסרטנים במי ים זורמים כדי להסיר שאריות פורמלדהיד.

figure-protocol-1182
איור 1: מורפולוגיה בטנית של נקבות סרטני בוץ המשמשת לזיהוי שלבי ההתבגרות המינית. אנא לחץ כאן כדי להציג גרסה גדולה יותר של איור זה.

2. התאקלמות

  1. מעבירים כל נקבה שעברה חיטוי למיכל עגול נפרד בנפח 32 ליטר.
  2. מגדלים את הנקבות במשך 3 ימים במליחות של 20 ppt וממשיכות להאכיל פעמיים ביום (בוקר 09:00 בבוקר וערב 20:00) עם דגי ים קצוצים בכ-4%-5% ממשקל הגוף של הסרטנים.
  3. הסר מזון עודף ולא נאכל על ידי siphoning לפני האכלת הבוקר.
  4. החליפו 10% ממי הים המגדלים סרטנים (20 ppt) מדי יום.

3. השראת התכה לבגרות מינית

  1. חותכים את כל הרגליים למעט רגלי השחייה באמצעות מספריים מעוקרים.
    1. תפסו את הסרטן עם רשת סקופ, והחזיקו את הסרטן בזהירות. חותכים תחילה את שתי הרגליים ההולכות במפרק השני באמצעות מספריים. הסרטן יבצע אוטוטומיזציה אוטומטית של התוספתן הפגוע. הרדמה אינה נדרשת עבור אוטוטומיה של הגפיים.
  2. לשטוף את הסרטן במים מתוקים מיד לאחר אוטוטומיה הגפיים.
  3. מעבירים בנפרד את הסרטנים האוטוטומיים לגפיים לסלי פלסטיק מחוררים (רוחב 28 ס"מ x 22 ס"מ רוחב x 7 ס"מ גובה), ומניחים אותם במיכל פיברגלס (אורך 305 ס"מ x רוחב 120 ס"מ x גובה 60 ס"מ).
    הערה: ניתן לקשור ולחתוך שני סלים זה לזה. הסל העליון משמש ככיסוי כך שהסרטן לא יכול לברוח מהסל.
  4. השתמש במערכת חקלאות ימית מחזורית (RAS) עם מליחות של 20 ppt ועומק מים של לפחות 10 ס"מ כדי להבטיח שכל סל הפלסטיק שקוע.
  5. המשיכו להאכיל את נקבת הסרטן בדג ימי קצוץ פעמיים ביום ב-5%-7% ממשקל גופו של הסרטן.
  6. מגדלים את הסרטנים עד לבגרות מינית באמצעות התכה (35 יום).
    הערה: ניתן לדלג על התכה מושרית לצורך הבשלה מסחרית של השחלות וייצור זרעים עם נקבות סרטני בוץ בוגרות בטבע. נקבות בוגרות שנקטפו מהטבע חייבות להתאקלם ולהיות חשופות ישירות להלם קר ולהלציה של דיבור עיניים לאחר מכן.

4. הרדמה

  1. בחרו נקבות בוגרות מינית עם דש בטן בצורת אליפסה בצבע כהה עם CW >86 מ"מ (איור 1).
  2. תפסו את הסרטנים עם רשת סקופ, ושמרו אותם בנפרד באקווריומים קטנים להרדמה.
  3. לאחר 5 דקות של תקופת התאקלמות, הוסף 2-פנוקסיאתנול (2-PE) ב 2 מ"ל / ליטר לכל אקווריום ולאפשר 15 דקות של טיפול הרדמה.
  4. ודא שהסרטנים מורדמים לחלוטין על ידי חוסר תנועה ספונטנית.

5. אבלציה של דיבור עיניים

  1. טכניקת צריבה
    1. בצע את כל ההליכים על גבי שולחן ובשטח פתוח.
    2. קחו מוט מתכת מפלדת ניקל עם ראש שטוח (למשל, מברג) עם ידית עץ או פלסטיק, וכסו את הידית במגבת כותנה רטובה.
    3. לעקר שני מלקחיים כירורגיים מנירוסטה באוטוקלאב.
    4. הכינו אתנול 70% בבקבוק תרסיס והרחיקו אותו מכל מקור הקשור לאש, כגון לפיד ניפוח ומברג חם אדום. הכינו נייר טישו לשימוש.
      הערה: אתנול דליק מאוד. יש לשמור על מרחק בטוח ממקורות האש.
    5. חבר פנס נשיפה לבלון גז (בוטאן) בצורה מאובטחת.
      התראה: בצע את ההוראות המופיעות על לפיד הנשיפה ובלון הגז. ודא כי הלפיד כבוי בעת חיבור עם בלון הגז. קרא ופעל לפי כל אמצעי בטיחות האש המוזכרים על בלון הגז.
    6. לבשו כפפות כותנה עבות כדי למנוע פציעה מחפצים חמים.
    7. יש להכפיף את קצה מוט המתכת לאש הלפיד עד שמוט המתכת יהיה אדום בוהק.
    8. מכסים את הסרטן המורדם במגבת כותנה רטובה.
      הערה: כסו את האנטנות של הסרטן כדי למנוע נזק מיותר.
    9. החזיקו עין אחת של הסרטן במלקחיים מעוקרות.
      הערה: יש לעקר את המלקחיים באוטוקלאבה לשימוש ראשון, ולחטא באמצעות 70% אתנול לשימוש עתידי על סרטנים אחרים.
    10. החזיקו את קצה המתכת האדום-חם על עין הסרטן ולחצו מעט במשך כ-10-15 שניות עד שצבע העיניים יהפוך לכתום או אדמדם-כתום. היזהר בעת ביצוע צעד זה כדי למנוע נזק למבנים סמוכים.
      הערה: שני אנשים נדרשים לבצע אבלציה של דיבור עיניים בשיטת הצריבה: אחד להחזיק את הסרטן והשני לבצע את הליך האבלציה.
    11. יש לחטא את המלקחיים בתרסיס אתנול 70% כדי להבטיח שאין זיהום צולב בין סרטנים.
      הערה: בצע שלב זה רק לפחות 5 דקות לאחר הליך אבלציה של דיבור עיניים כדי להבטיח קירור המלקחיים לפני החיטוי באמצעות אתנול 70% כדי למנוע סיכוני אש פוטנציאליים.
    12. לאחר ביצוע אבלציה של דיבור העיניים על כל הסרטנים, טבלו את מוט המתכת החם מפלדת ניקל (מברג) במי ברז.
    13. יש לחטא את המגבת לפני שימוש חוזר. ניתן להשתמש במספר מגבות כדי לחסוך זמן.
      הערה: שטפו את המגבת במי ברז וטבלו אותה במי כלור של 30 ppm למשך 5 דקות. לאחר מכן, שטפו שוב את המגבת במי ברז וטבלו אותה בתמיסת נתרן תיוסולפט 1 גרם / ליטר.
    14. יש לשמור את הפנס במקום בטוח לאחר כיבויו, ולהמתין עד שהוא יחזור לטמפרטורת הסביבה (כ-30 דקות) לפני הניתוק.
  2. טכניקת ניתוח
    1. בצע את ההליך באזור מאוורר היטב.
    2. לעקר שני מספריים כירורגיים ומלקחיים באוטוקלאב.
    3. יוצקים 50 מ"ל של 70% אתנול לתוך זכוכית 100 מ"ל.
    4. יש ללבוש כפפות כותנה עבות.
    5. החזיקו את הסרטן המורדם, וכסו אותו במגבת כותנה רטובה.
    6. החזיקו עין אחת של הסרטן במלקחיים מעוקרות.
    7. חתכו במהירות את העיניים באמצעות מספריים כירורגיים מעוקרים.
      הערה: המולימפה עלולה ללכת לאיבוד מהחלק הפצוע של הסרטן.
    8. טובלים את המספריים והמלקחיים באתנול 70% לאחר כל שימוש, ומייבשים אותם באמצעות נייר טישו לפני שימוש חוזר.

6. טיפול לאחר הרדמה

  1. הכינו מי ים מסוננים של 20 ppt, ושמרו במיכל עילי עם אוורור רציף.
  2. חבר צינור גמיש עם המיכל העילי לזרימת מים כבידתית.
  3. מיד לאחר אבלציה של דיבור עיניים, הכניסו את הסרטן לסלסלה, וחשפו את הסרטן למי ים זורמים (טמפרטורת הסביבה: 28 מעלות צלזיוס) מהמיכל שמעליו.
  4. שמרו על זרימת מי הים, ועקבו אחר הסרטן עד שהוא יכול לנוע באופן ספונטני, מה שמעיד על התאוששות מההרדמה.
    הערה: ניתן להכין מי ים במיכל קרקע, וניתן להשתמש במשאבת מים טבולים לזרימת המים.
  5. שמור את הסרטנים בנפרד במי ים של 20 ppt עם אוורור באקווריום למשך 30 דקות לתצפית נוספת.
    הערה: הסרטנים שהתאוששו יתרבו בנפרד בתהליך תרבית הברודסטוק הבא.

7. תצפית על התבגרות השחלות

  1. גידול ברודסטוק
    1. מעבירים את הסרטנים הבוגרים למיכלים עגולים בודדים של 32 ליטר.
    2. המשיכו להאכיל עם דגים ימיים קצוצים (קפואים בטמפרטורה של 20°C-) פעמיים ביום (בבוקר 09:00 בבוקר ובערב 20:00 בערב), והוציאו מזון שלא נאכל לפני האכלת הבוקר.
    3. מחזירים את הברודסטוק בנפרד למשך 30 יום במליחות של 20 ppt.
    4. יש להסיר צואה ולהחליף 10% ממי הים (20 ppt) מדי יום.
  2. דיסקציה
    1. נקו מגש ניתוח, מספריים ומלקחיים עם 70% אתנול.
    2. מרדימים את הנקבות בנפרד בשיטת הרדמה טבילה 2-PE.
    3. בחרו באופן אקראי נקבות חדשות (לאחר התכה של נקבות פגות) שלא עברו אבלציה של דיבור עיניים כדי לאשר את שלבי הגונדל שלהן.
    4. הקריבו את כל נקבות הניסוי בעיניים בנפרד, וזהו את שלבי ההבשלה של הגונד. להרוס את גרעיני החזה של הסרטן באמצעות awl סטרילי חד. הסר תחילה את השריון העליון ולאחר מכן את hepatopancreas כדי להפוך את השחלה גלוי. התבוננו בצבע השחלה וזהו את שלב ההבשלה השחלתית (איור 2).
  3. זיהוי שלבי התבגרות השחלות
    1. שימו לב לצבע השחלה בעין בלתי או מתחת לסטריאומיקרוסקופ.
    2. זהה את שלבי ההבשלה השחלות על סמך צבע30: הלא בוגר (שלב -1) מראה צבע לבן שקוף או שמנת; ההתבגרות המוקדמת (שלב 2) מראה צבע צהבהב חיוור עד בהיר; (iii) טרום ההתבגרות (שלב 3) מראה צבע צהוב עד כתום בהיר; ו-(iv) הבשלה מלאה (שלב 4) מראה צבע כתום כהה עד אדמדם.

תוצאות

התבגרות גונאד
רקמות שחלה לבנות וקרמיות (שחלות לא בשלות, שלב 1) נמצאו ב-100% מהנקבות שנותחו (n = 6) לפני ביצוע אבלציה של דיבור עיניים (איור 2). שיעור ההבשלה של נקבות סרטנים עם דיבור עיניים (n = 63; 31 נקבות עם טכניקת הצריבה ו-32 נקבות עם טכניקת הניתוח) היה גבוה יותר בהשוואה לנקב?...

Discussion

פרוטוקול זה פותח עבור אבלציה של סרטן הבוץ, Scylla spp., וניתן ליישם אותו כשיטה יעילה לגרימת התבגרות הגונד. פרוטוקול זה ניתן לשכפול בקלות להבשלה מסחרית של סרטני בוץ וניתן ליישם אותו כדי להפחית את התקופה הסמויה (הזמן מהשרצה אחת לאחרת) בייצור זרעי סרטני בוץ.

אבלציה של סרטנים (כל?...

Disclosures

לאף אחד מהכותבים אין ניגודי עניינים.

Acknowledgements

מחקר זה נתמך על ידי משרד החינוך, מלזיה, במסגרת תוכנית מרכז המצוינות של המוסד הגבוה (HICoE), מלזיה, מוכר למכון לחקלאות ימית טרופית ודיג, אוניברסיטת מלזיה טרנגאנו (הצבעה מס '63933 והצבעה מס '56048). אנו מכירים בתמיכתם של Universiti Malaysia Terengganu ו- Sayap Jaya Sdn. Bhd. באמצעות מענק המחקר לשותפות פרטית (הצבעה מס '55377). כמו כן מוכרת משרת עמית אקדמי נספח מאוניברסיטת סאיינס מלזיה לח'ור וואיהו וחנפיה פאז'אן.

Materials

NameCompanyCatalog NumberComments
Aeration tube Ming Yu ThreeN/Aaquarium and pet shop
AirstoneMing Yu ThreeN/Aaquarium and pet shop
Autoclave machineHIRAYAMA MANUFACTURING CORPORATIONN/AMADE IN JAPAN
Bleaching powder (Hi-Chlon 70%)Nippon Soda Co.Ltd,JapanN/AN/A
Blow torch MR D.I.Y. Group BerhadN/AN/A
Circular tank (32L)BEST PLASTIC INDUSTRY SDN. BHD. N/AN/A
Cotton hand gloves (thick) MR D.I.Y. Group BerhadN/AN/A
Cotton towelMR D.I.Y. Group BerhadN/AN/A
Digital thermometerHanna InstrumentHI9814Hanna Instruments GroLine Hydroponics Waterproof pH / EC / TDS / Temp. Portable Meter HI9814
Digital Vernier CaliperINSIZE Co., Ltd.N/A
Dissecting trayHatcheri AKUATROP N/AResearch Center of Universiti Malaysia Terengganu
Dropper bottle/Plastic Pipettes DropperShopee MalaysiaN/AN/A
Ethanol 70%Thermo Scientific Chemicals033361.M1Diluted to 70% using double distilled water
Fiberglass tank (1 ton)Hatcheri AKUATROP N/AResearch Center of Universiti Malaysia Terengganu
Fine sandN/AN/Acollected from Sea beach of Universiti Malaysia Terengganu
First Aid KitsWatsons MalaysiaN/AN/A
Flat head nickel steel metal rod (Screw driver)MR D.I.Y. Group BerhadN/AN/A
FormaldehydeThermo Scientific Chemicals119690010
Gas cylinder (butane gas) for blow  torchMR D.I.Y. Group BerhadN/AN/A
Gas lighter gun (long head)MR D.I.Y. Group BerhadN/AN/A
Glass beaker (100 mL))Corning Life Sciences1000-100
Ice bag Watsons MalaysiaN/AN/A
Perforated plastic baskets Eco-Shop Marketing Sdn. Bhd.N/AN/A
PVC pipe 15mmBina Plastic Industries Sdn Bhd (HQ)N/AN/A
RefractometerATAGO CO.,LTD.
RefrigeratorSharp Corporation JapanN/AChest Freezer SHARP 110L - SJC 118
Scoop netMR D.I.Y. Group BerhadN/A
SeawaterHatcheri AKUATROP N/AResearch Center of Universiti Malaysia Terengganu
Siphoning pipeMR D.I.Y. Group BerhadN/AN/A
Spray bottleMr. DIY Sdn BhdN/AN/A
Stainless surgical forceps N/AN/AN/A
Stainless surgical scissors N/AN/AN/A
Submersible water pump ASN/Amodel: Astro 4000
Tincture of iodine solution  (Povidone Iodine)Farmasi Fajr Sdn BhdN/AN/A
Tissue paper N/AN/A
Transparent plastic aquariumMing Yu ThreeN/Aaquarium and pet shop
Waterproof tableHatcheri AKUATROP N/AResearch Center of Universiti Malaysia Terengganu

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Erratum


Formal Correction: Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs
Posted by JoVE Editors on 5/26/2023. Citeable Link.

An erratum was issued for: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs. The Introduction, Protocol, Discussion and References were updated.

The forth sentence in the third paragraph of the Introduction has been updated from:

The eyestalk ablation protocol in this work minimizes stress by using fully sedated crabs and minimizes physical injury to personnel from crab bites. 

to:

The eyestalk ablation protocol in this work minimizes stress by using fully anesthetized crabs and minimizes physical injury to personnel from crab bites. 

The start of the Protocol has been updated from:

This protocol follows the Malaysian Code of Practice for the Care and Use of Animals for Scientific Purposes outlined by the Laboratory Animal Science Association of Malaysia. The sacrifice of the experimental samples was done according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Sexually pre-mature female mud crabs (orange mud crab S. olivacea) were collected from the local market (5°66′62′′N, 102°72′33′′E) at the Setiu Wetlands in Malaysia. The mud crab species was identified based on morphological characteristics1.

to:

This protocol follows the Malaysian Code of Practice for the Care and Use of Animals for Scientific Purposes outlined by the Laboratory Animal Science Association of Malaysia and was approved by the Universiti Malaysia Terengganu's Research Ethics Committee (Animal ethics approval number: UMT/JKEPHMK/2023/96). The sacrifice of the experimental samples was done according to the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition. Sexually pre-mature female mud crabs (orange mud crab Scylla olivacea) were collected from the local market (5°66′62′′N, 102°72′33′′E) at the Setiu Wetlands in Malaysia. The mud crab species was identified based on morphological characteristics1.

Section 4 of the Protocol has been updated from:

4. Cold-shock anesthesia

  1. Select sexually mature females with a dark-colored oval-shaped abdominal flap with a CW >86 mm (Figure 1).
  2. Catch the crabs with a scoop net, and keep them individually in small aquariums for cold shock anesthesia.
  3. Prepare 2 L of 4 °C to 1 °C seawater (20 ppt) in a transparent plastic aquarium. Maintain the temperature using (−20 °C) ice bags for cold shock anesthesia.
    NOTE: Check the temperature with a digital thermometer.
  4. Immerse the crab in the 4 °C seawater until sedated (about 3−5 min).
  5. Ensure the crabs are fully anesthetized by the lack of spontaneous movement. The legs and chelipeds joints will still show minor movements when touched with forceps.

to:

4. Anesthesia

  1. Select sexually mature females with a dark-colored oval-shaped abdominal flap with a CW >86 mm (Figure 1).
  2. Catch the crabs with a scoop net, and keep them individually in small aquariums for anesthesia.
  3. After 5 min of acclimatization period, add 2-phenoxyethanol (2-PE) at 2 mL/L into each aquarium and allow 15 min of anesthesia treatment.
  4. Ensure the crabs are fully anesthetized by the lack of spontaneous movement.

Section 5 of the Protocol has been updated from:

5. Eyestalk ablation

  1. Cauterization technique
    1. Perform all procedures on top of a table and in an open area.
    2. Take a flat head nickel-steel metal rod (e.g., a screwdriver) with a wooden or plastic handle, and cover the handle with a wet cotton towel.
    3. Sterilize two stainless surgical forceps in an autoclave.
    4. Prepare 70% ethanol in a spray bottle. Have tissue paper ready for use.
      NOTE: Ethanol is highly flammable. Maintain a safe distance from fire sources.
    5. Connect a blowtorch to a gas cylinder (butane) securely.
      CAUTION: Follow the instructions on the blowtorch and gas cylinder. Make sure that the blowtorch is switched off when connecting with the gas cylinder. Read and follow all the fire safety precautions mentioned on the gas cylinder.
    6. Wear thick cotton gloves to avoid injury from hot objects.
    7. Subject the tip of the metal rod to the fire of the blowtorch until the metal rod is bright red.
    8. Cover the anesthetized (sedated) crab with a wet cotton towel.
      NOTE: Cover all the tentacles of the crab to avoid unnecessary damage.
    9. Hold one eye of the crab with sterilized forceps.
      NOTE: Sterilize the forceps in an autoclave for first-time use, and disinfect using 70% ethanol for subsequent use on other crabs.
    10. Hold the red-hot metal flat tip onto the eye of the crab and press slightly for about 10−15 s until the eyestalk turns an orange or reddish-orange color.
      NOTE: Two people are needed to execute eyestalk ablation following the cauterization method: one to hold the crab and another to perform the ablation procedure.
    11. Disinfect the forceps with 70% ethanol spray to ensure no cross-contamination between crabs.
    12. After performing the eyestalk ablation on all crabs, dip the hot nickel steel metal rod (screwdriver) into tap water.
    13. Disinfect the towel before reuse. Multiple towels can be used to save time.
      NOTE: Wash the towel with tap water, and dip it into 30 ppm chlorinated water for 5 min. Then, wash the towel with tap water again, and dip it in a 1 g/L sodium thiosulphate solution.
    14. Keep the blowtorch in a safe place after turning it off, and wait until it returns to environmental temperature (about 30 min) before disconnecting.
  2. Surgery technique
    1. Perform the procedure in a well-ventilated area.
    2. Sterilize two surgical scissors and forceps in an autoclave.
    3. Pour 50 mL of 70% ethanol into a 100 mL glass beaker.
    4. Prepare the tincture of iodine solution in a dropper bottle.
      NOTE: Tincture of iodine (iodine tincture or weak iodine solution) is made up of 2%-7% elemental iodine and potassium iodide, or sodium iodide, dissolved in ethanol and water.
    5. Wear thick cotton gloves.
    6. Hold the sedated crab, and cover it with a wet cotton towel.
    7. Hold one eye of the crab with sterilized forceps.
    8. Swiftly cut off the eyestalk using sterilized surgical scissors.
      NOTE: Hemolymph may be lost from the wounded part of the crab.
    9. Dip the scissors and forceps in 70% ethanol after every use, and dry them using tissue paper before reuse.
    10. Apply two to three drops of iodine tincture to the wounded part of the eyestalk immediately after cutting it off.
      NOTE: Tincture of iodine is used for healing and to prevent infection.

to:

5. Eyestalk ablation

  1. Cauterization technique
    1. Perform all procedures on top of a table and in an open area.
    2. Take a flat head nickel-steel metal rod (e.g., a screwdriver) with a wooden or plastic handle, and cover the handle with a wet cotton towel.
    3. Sterilize two stainless surgical forceps in an autoclave.
    4. Prepare 70% ethanol in a spray bottle and keep it away from any fire-related sources, such as blow torch and red hot screwdriver. Have tissue paper ready for use.
      NOTE: Ethanol is highly flammable. Maintain a safe distance from fire sources.
    5. Connect a blowtorch to a gas cylinder (butane) securely.
      CAUTION: Follow the instructions on the blowtorch and gas cylinder. Make sure that the blowtorch is switched off when connecting with the gas cylinder. Read and follow all the fire safety precautions mentioned on the gas cylinder.
    6. Wear thick cotton gloves to avoid injury from hot objects.
    7. Subject the tip of the metal rod to the fire of the blowtorch until the metal rod is bright red.
    8. Cover the anesthetized crab with a wet cotton towel.
      NOTE: Cover the antennae of the crab to avoid unnecessary damage.
    9. Hold one eye of the crab with sterilized forceps.
      NOTE: Sterilize the forceps in an autoclave for first-time use, and disinfect using 70% ethanol for subsequent use on other crabs.
    10. Hold the red-hot metal flat tip onto the eye of the crab and press slightly for about 10−15 s until the eyestalk turns an orange or reddish-orange color. Be careful when conducting this step to avoid damage to adjacent structures. 
      NOTE: Two people are needed to execute eyestalk ablation following the cauterization method: one to hold the crab and another to perform the ablation procedure.
    11. Disinfect the forceps with 70% ethanol spray to ensure no cross-contamination between crabs.
      NOTE: Only perform this step at least waiting for 5 min after the eyestalk ablation procedure to ensure the forceps are cooled down before disinfection using 70% ethanol to prevent potential fire hazards.
    12. After performing the eyestalk ablation on all crabs, dip the hot nickel steel metal rod (screwdriver) into tap water.
    13. Disinfect the towel before reuse. Multiple towels can be used to save time.
      NOTE: Wash the towel with tap water, and dip it into 30 ppm chlorinated water for 5 min. Then, wash the towel with tap water again, and dip it in a 1 g/L sodium thiosulphate solution.
    14. Keep the blowtorch in a safe place after turning it off, and wait until it returns to environmental temperature (about 30 min) before disconnecting.
  2. Surgery technique
    1. Perform the procedure in a well-ventilated area.
    2. Sterilize two surgical scissors and forceps in an autoclave.
    3. Pour 50 mL of 70% ethanol into a 100 mL glass beaker.
    4. Wear thick cotton gloves.
    5. Hold the anesthetized crab, and cover it with a wet cotton towel.
    6. Hold one eye of the crab with sterilized forceps.
    7. Swiftly cut off the eyestalk using sterilized surgical scissors.
      NOTE: Hemolymph may be lost from the wounded part of the crab.
    8. Dip the scissors and forceps in 70% ethanol after every use, and dry them using tissue paper before reuse.

Step 7.2.2 of the Protocol has been updated from:

Sedate the females individually with the cold shock anesthesia method.

to:

Anesthetize the females individually with the 2-PE immersion anesthesia method.

The Discussion has been updated from:

This protocol was developed for the eyestalk ablation of the mud crab, Scylla spp., and can be applied as an efficient method to induce gonad maturation. This protocol can be easily replicated for the commercial ovary maturation of mud crabs and can be implemented to reduce the latent period (time from one spawning to another) in mud crab seed production.

The eyestalk ablation of crustaceans (i.e., freshwater prawn, marine shrimp) is typically done to induce gonad maturation and out-of-season spawning11,12,13. Eyestalk ablation in brachyuran crabs has also been done to study molting25,32,33, hormonal regulation18, gonad maturation34, and induced breeding and reproductive performance35,36,37,38,39. Unilateral or bilateral eyestalk ablation influences the physiology of the crustacean. Eyestalk ablation following the protocol stated in this study also influences the ovarian maturation rate of mud crabs. In the control treatment (without eyestalk ablation), 43.33% ± 5.77% of female crabs had an immature ovary (stage-1). However, in the same rearing period (30 days), eyestalk-ablated female crabs had pre-maturing ovaries (stage-3; 56.67% ± 11.55% and 53.33% ± 15.28% with the cauterization and surgery techniques, respectively), which shows that eyestalk ablation can increase the gonad maturation of mud crabs. Previous studies have also reported that the ovarian development of intact crabs (without eyestalk ablation) is slower than that of eyestalk-ablated crabs25,31. Due to the slower gonadal development in intact crustaceans, eyestalk ablation is widely done in commercial prawn and shrimp hatcheries. In this protocol, the eyestalk-ablated female crabs achieved higher percentages of ovarian maturation compared to the female crabs without the eyestalk ablation treatment (Figure 3).

The gonad maturation of the mud crab is regulated by hormones21,40,41. The eyestalk contains important endocrine glands (i.e., the X-organ-sinus gland complex) that play vital roles in the gonadal maturation process of mud crabs18,21. Unilateral eyestalk ablation, either by cauterization or surgery, damages one of the major endocrine glands that is involved in the synthesis and release of inhibiting hormones (e.g., VIH), thereby resulting in a higher level of gonad-stimulating hormones (i.e., VSH).

The ovarian maturation stages of Scylla spp. can be differentiated by observing the ovarian tissue coloration with the naked eye29,30,42. Translucent or creamy white ovarian tissues are indications of immature ovaries29,30,42,43. In this study, immature ovaries (stage-1) were still found in the group of female crabs without eyestalk ablation due to the slower ovarian maturation process. However, the crabs in the eyestalk-ablated groups (both by the cauterization and surgery techniques) mostly showed pre-maturing ovaries (stage-3), with some individuals exhibiting fully matured ovaries (stage-4). Therefore, the protocol of eyestalk ablation described here can be used to increase ovarian maturation in female mud crabs. This protocol can also be applied directly to wild-collected mature female mud crabs to hasten their seed production. To evaluate the effectiveness of cauterization and surgery methods on mud crab gonad maturation and to ensure the accurate estimation of molting duration, sexually pre-mature crabs were used. After the (induced) molting of sexually pre-mature female crabs, we noticed that their ovaries were still in the immature or early developing stages29,44. After 30 days of rearing the newly mature female crabs (either eyestalk-ablated or without eyestalk ablation), the ovarian development stages (stage-1 to stage-4) were determined by the color of the ovarian tissues. This protocol encourages the use of the cauterization technique to perform eyestalk ablation in mud crabs to avoid any hemolymph loss and prevent infection at the ablated sites. Cauterization immediately seals the wound, whereas the surgery technique requires an additional step of disinfection using iodine. For commercial purposes, larger mature crabs, preferably at a later stage of ovarian maturation, should be selected for eyestalk ablation to shorten the time to reach the fully matured ovary stage for subsequent commerce or brood stock culture. In addition to eyestalk ablation, individual rearing with sand substrate and sufficient feeding, preferably with live feed, can increase the gonad maturation rate of mud crabs in captivity30,35,45,46.

Crustacean blood is called hemolymph and can be lost during eyestalk ablation. An excessive loss of hemolymph may lead to the death of eyestalk-ablated crabs, especially when performing surgery to remove the eyestalk. The hemolymph can coagulate in the wounded part to prevent loss. The application of a tincture of iodine can prevent infection of the wounded part. However, in comparison to the surgery technique, the cauterization technique seals the wounded part immediately, thereby preventing the loss of hemolymph and possible infection.

Mud crab mortality after unilateral eyestalk ablation with either cauterization or surgery was not found within the first 7 days. Thus, eyestalk ablation can be done with a higher survival rate. Unilateral eyestalk ablation does not hamper the survival rate of the crab33.

Stress during crab handling and eyestalk ablation may contribute to crab mortality. Proper anesthesia is needed to minimize handling stress during eyestalk ablation. In crustacean eyestalk ablation, chemical anesthetics (i.e., xylocaine, lidocaine) are used at the base of the eyestalk before eyestalk ablation14,15,17,47. However, due to the aggressive nature and large size of mud crabs, the use of anesthesia only at the base of the eyestalk is not sufficient and might result in additional stress to the animals during the injection. On the other hand, anesthesia by subjecting them to a lower water temperature is more economical and safer. The use of cold water for anesthesia in mud crabs is common and has been used in other studies due to its efficiency, simplicity, and minimal impact on recovery and survival37,48,49.

Although eyestalk ablation using both cauterization and surgery methods has a minimal effect on crab survival and enhances ovarian maturation, performing eyestalk ablation requires professional mastery of the techniques. The timing between the steps is critical as any delay between protocols adds additional stress for the crabs. Unlike the surgery technique, the cauterization technique is dangerous because it involves the use of flammable equipment (i.e., a blow torch and butane gas). Thus, extra caution is needed when performing the cauterization technique.

Crabs are cannibalistic in nature, and they are known to prey on others that have just completed their molt and are still in their soft-shell conditions7,50,51. Thus, rearing the crabs individually can avoid unnecessary mortality due to cannibalism. The use of individual rearing in mud crab culture is commonly practiced, both in high-density culture and pond culture, for fattening and soft-shell crab farming purposes8,52. This protocol also utilized individual rearing and maintenance. During the transportation of the crabs for rearing or commerce, the crab chelipeds are tied up securely (or even autotomized) to prevent fighting, unnecessary injury, and limb loss34.

The described protocol for eyestalk ablation should be performed with multiple persons. After completing the eyestalk ablation, non-disposable equipment (e.g., the aquarium, tray, towel, etc.) should be disinfected with 30 ppm chlorine. The crabs must be monitored at least twice per day. Any dead crabs, uneaten feed, ablated limbs, or molted crab shells should be swiftly disposed of (i.e., buried in soil with bleaching powder) to prevent any potential for disease spread.

to:

This protocol was developed for the eyestalk ablation of the mud crab, Scylla spp., and can be applied as an efficient method to induce gonad maturation. This protocol can be easily replicated for the commercial ovary maturation of mud crabs and can be implemented to reduce the latent period (time from one spawning to another) in mud crab seed production.

The eyestalk ablation of crustaceans (i.e., freshwater prawn, marine shrimp) is typically done to induce gonad maturation and out-of-season spawning11,12,13. Eyestalk ablation in brachyuran crabs has also been done to study molting25,32,33, hormonal regulation18, gonad maturation34, and induced breeding and reproductive performance35,36,37,38,39. Anesthesia via immersion in 2-phenoxyethanol was used as it is comparable to the use of tricaine methanesulfonate (MS-222) in arthopods but cheaper and does not require the use of additional buffer40. Unilateral or bilateral eyestalk ablation influences the physiology of the crustacean. Eyestalk ablation following the protocol stated in this study also influences the ovarian maturation rate of mud crabs. In the control treatment (without eyestalk ablation), 43.33% ± 5.77% of female crabs had an immature ovary (stage-1). However, in the same rearing period (30 days), eyestalk-ablated female crabs had pre-maturing ovaries (stage-3; 56.67% ± 11.55% and 53.33% ± 15.28% with the cauterization and surgery techniques, respectively), which shows that eyestalk ablation can increase the gonad maturation of mud crabs. Previous studies have also reported that the ovarian development of intact crabs (without eyestalk ablation) is slower than that of eyestalk-ablated crabs25,31. Due to the slower gonadal development in intact crustaceans, eyestalk ablation is widely done in commercial prawn and shrimp hatcheries. In this protocol, the eyestalk-ablated female crabs achieved higher percentages of ovarian maturation compared to the female crabs without the eyestalk ablation treatment (Figure 3).

The gonad maturation of the mud crab is regulated by hormones21,41,42. The eyestalk contains important endocrine glands (i.e., the X-organ-sinus gland complex) that play vital roles in the gonadal maturation process of mud crabs18,21. Unilateral eyestalk ablation, either by cauterization or surgery, damages one of the major endocrine glands that is involved in the synthesis and release of inhibiting hormones (e.g., VIH), thereby resulting in a higher level of gonad-stimulating hormones (i.e., VSH).

The ovarian maturation stages of Scylla spp. can be differentiated by observing the ovarian tissue coloration with the naked eye29,30,43. Translucent or creamy white ovarian tissues are indications of immature ovaries29,30,43,44. In this study, immature ovaries (stage-1) were still found in the group of female crabs without eyestalk ablation due to the slower ovarian maturation process. However, the crabs in the eyestalk-ablated groups (both by the cauterization and surgery techniques) mostly showed pre-maturing ovaries (stage-3), with some individuals exhibiting fully matured ovaries (stage-4). Therefore, the protocol of eyestalk ablation described here can be used to increase ovarian maturation in female mud crabs. This protocol can also be applied directly to wild-collected mature female mud crabs to hasten their seed production. To evaluate the effectiveness of cauterization and surgery methods on mud crab gonad maturation and to ensure the accurate estimation of molting duration, sexually pre-mature crabs were used. After the (induced) molting of sexually pre-mature female crabs, we noticed that their ovaries were still in the immature or early developing stages29,45. After 30 days of rearing the newly mature female crabs (either eyestalk-ablated or without eyestalk ablation), the ovarian development stages (stage-1 to stage-4) were determined by the color of the ovarian tissues. This protocol encourages the use of the cauterization technique to perform eyestalk ablation in mud crabs to avoid any hemolymph loss and prevent infection at the ablated sites. Cauterization immediately seals the wound, whereas the surgery technique takes time for the wound to heal and this would allow for chance of infection. For commercial purposes, larger mature crabs, preferably at a later stage of ovarian maturation, should be selected for eyestalk ablation to shorten the time to reach the fully matured ovary stage for subsequent commerce or brood stock culture. In addition to eyestalk ablation, individual rearing with sand substrate and sufficient feeding, preferably with live feed, can increase the gonad maturation rate of mud crabs in captivity30,35,46,47.

Crustacean blood is called hemolymph and can be lost during eyestalk ablation. An excessive loss of hemolymph may lead to the death of eyestalk-ablated crabs, especially when performing surgery to remove the eyestalk. The hemolymph can coagulate in the wounded part to prevent loss. However, in comparison to the surgery technique, the cauterization technique seals the wounded part immediately, thereby preventing the loss of hemolymph and possible infection.

Mud crab mortality after unilateral eyestalk ablation with either cauterization or surgery was not found within the first 7 days. Thus, eyestalk ablation can be done with a higher survival rate. Unilateral eyestalk ablation does not hamper the survival rate of the crab33.

Stress during crab handling and eyestalk ablation may contribute to crab mortality. Proper anesthesia is needed to minimize handling stress during eyestalk ablation. In crustacean eyestalk ablation, chemical anesthetics (i.e., xylocaine, lidocaine) are used at the base of the eyestalk before eyestalk ablation14,15,17,48. However, due to the aggressive nature and large size of mud crabs, the use of anesthesia only at the base of the eyestalk is not sufficient and might result in additional stress to the animals during the injection. On the other hand, anesthesia by subjecting them to a lower water temperature is more economical and safer. The use of cold water for anesthesia in mud crabs is common and has been used in other studies due to its efficiency, simplicity, and minimal impact on recovery and survival37,49,50. In addition, future research on pain assessment following eyestalk ablation on mud crabs is recommended to highlight the change in behaviours associated with pain and stress, as evident in freshwater prawn Macrobrachium americanum51.

Although eyestalk ablation using both cauterization and surgery methods has a minimal effect on crab survival and enhances ovarian maturation, performing eyestalk ablation requires professional mastery of the techniques. The timing between the steps is critical as any delay between protocols adds additional stress for the crabs. Unlike the surgery technique, the cauterization technique is dangerous because it involves the use of flammable equipment (i.e., a blow torch and butane gas). Thus, extra caution is needed when performing the cauterization technique.

Crabs are cannibalistic in nature, and they are known to prey on others that have just completed their molt and are still in their soft-shell conditions7,52,53. Thus, rearing the crabs individually can avoid unnecessary mortality due to cannibalism. The use of individual rearing in mud crab culture is commonly practiced, both in high-density culture and pond culture, for fattening and soft-shell crab farming purposes8,53. This protocol also utilized individual rearing and maintenance. During the transportation of the crabs for rearing or commerce, the crab chelipeds are tied up securely (or even autotomized) to prevent fighting, unnecessary injury, and limb loss34.

The described protocol for eyestalk ablation should be performed with multiple persons. After completing the eyestalk ablation, non-disposable equipment (e.g., the aquarium, tray, towel, etc.) should be disinfected with 30 ppm chlorine. The crabs must be monitored at least twice per day. Any dead crabs, uneaten feed, ablated limbs, or molted crab shells should be swiftly disposed of (i.e., buried in soil with bleaching powder) to prevent any potential for disease spread.

The References have been updated from:

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  2. Fazhan, H. et al. Morphological descriptions and morphometric discriminant function analysis reveal an additional four groups of Scylla spp. PeerJ. 8, e8066 (2020).
  3. Ikhwanuddin, M., Bachok, Z., Hilmi, M. G., Azmie, G., Zakaria, M. Z. Species diversity, carapace width-body weight relationship, size distribution and sex ratio of mud crab, genus Scylla from Setiu Wetlands of Terengganu coastal waters, Malaysia. Journal of Sustainability Science and Management. 5 (2), 97-109 (2010).
  4. Ikhwanuddin, M., Bachok, Z., Mohd Faizal, W. W. Y., Azmie, G., Abol-Munafi, A. B. Size of maturity of mud crab Scylla olivacea (Herbst, 1796) from mangrove areas of Terengganu coastal waters. Journal of Sustainability Science and Management. 5 (2), 134-147 (2010).
  5. Waiho, K. et al. On types of sexual maturity in brachyurans, with special reference to size at the onset of sexual maturity. Journal of Shellfish Research. 36 (3), 807-839 (2017).
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  10. Okumura, T. et al. Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 143 (2), 246-253 (2006).
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  16. Chu, K. H., Chow, W. K. Effects of unilateral versus bilateral eyestalk ablation on molting and growth of the shrimp, Penaeus chinensis (Osbeck, 1765) (Decapoda, Penaeidea). Crustaceana. 62 (3), 225-233 (1992).
  17. Taylor, J. Minimizing the effects of stress during eyestalk ablation of Litopenaeus vannamei females with topical anesthetic and a coagulating agent. Aquaculture. 233 (1-4), 173-179 (2004).
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  27. Arcos, G. F., Ibarra, A. M., Vazquez-Boucard, C., Palacios, E., Racotta, I. S. Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone. Aquaculture Research. 34 (9), 749-755 (2003).
  28. Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
  29. Wu, Q. et al. Growth performance and biochemical composition dynamics of ovary, hepatopancreas and muscle tissues at different ovarian maturation stages of female mud crab, Scylla paramamosain. Aquaculture. 515, 734560 (2020).
  30. Ghazali, A., Azra, M. N., Noordin, N. M., Abol-Munafi, A. B., Ikhwanuddin, M. Ovarian morphological development and fatty acids profile of mud crab (Scylla olivacea) fed with various diets. Aquaculture. 468 (Part 1), 45-52 (2017).
  31. Farhadi, A. et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8 (2021).
  32. Sukardi, P., Prayogo, N. A., Harisam, T., Sudaryono, A. Effect of eyestalk-ablation and differences salinity in rearing pond on molting speed of Scylla serrata. AIP Conference Proceedings. 2094, 020029 (2019).
  33. Stella, V. S., López Greco, L. S., Rodríguez, E. M. Effects of eyestalk ablation at different times of the year on molting and reproduction of the estuarine grapsid crab Chasmagnathus granulata (Decapoda, Brachyura). Journal of Crustacean Biology. 20 (2), 239-244 (2000).
  34. Jang, I. K. et al. The effects of manipulating water temperature, photoperiod, and eyestalk ablation on gonad maturation of the swimming crab, Portunus trituberculatus.Crustaceana. 83 (2), 129-141 (2010).
  35. Millamena, O. M., Quinitio, E. The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata. Aquaculture. 181 (1-2), 81-90 (2000).
  36. Zeng, C. Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development. Aquaculture Research. 38 (14), 1478-1485 (2007).
  37. Rana, S. Eye stalk ablation of freshwater crab, Barytelphusa lugubris: An alternative approach of hormonal induced breeding. International Journal of Pure and Applied Zoology. 6 (3), 30-34 (2018).
  38. Yi, S.-K., Lee, S.-G., Lee, J.-M. Preliminary study of seed production of the Micronesian mud crab Scylla serrata (Crustacea: Portunidae) in Korea. Ocean and Polar Research. 31 (3), 257-264 (2009).
  39. Azra, M. N., Abol-Munafi, A. B., Ikhwanuddin, M. A review of broodstock improvement to brachyuran crab: Reproductive performance. International Journal of Aquaculture. 5 (38), 1-10 (2016).
  40. Muhd-Farouk, H., Abol-Munafi, A. B., Jasmani, S., Ikhwanuddin, M. Effect of steroid hormones 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology. 9 (1), 23-28 (2013).
  41. Muhd-Farouk, H., Jasmani, S., Ikhwanuddin, M. Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture. 451, 78-86 (2016).
  42. Ghazali, A., Mat Noordin, N., Abol-Munafi, A. B., Azra, M. N., Ikhwanuddin, M. Ovarian maturation stages of wild and captive mud crab, Scylla olivacea fed with two diets. Sains Malaysiana. 46 (12), 2273-2280 (2017).
  43. Aaqillah-Amr, M. A., Hidir, A., Noordiyana, M. N., Ikhwanuddin, M. Morphological, biochemical and histological analysis of mud crab ovary and hepatopancreas at different stages of development. Animal Reproduction Science. 195, 274-283 (2018).
  44. Amin-Safwan, A., Muhd-Farouk, H., Mardhiyyah, M. P., Nadirah, M., Ikhwanuddin, M. Does water salinity affect the level of 17β-estradiol and ovarian physiology of orange mud crab, Scylla olivacea (Herbst, 1796) in captivity? Journal of King Saud University - Science. 31 (4), 827-835 (2019).
  45. Wu, X. et al. Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards), female broodstock. Aquaculture. 273 (4), 602-613 (2007).
  46. Azra, M. N., Ikhwanuddin, M. A review of maturation diets for mud crab genus Scylla broodstock: Present research, problems and future perspective. Saudi Journal of Biological Sciences. 23 (2), 257-267 (2016).
  47. Maschio Rodrigues, M., López Greco, L. S., de Almeida, L. C. F., Bertini, G. Reproductive performance of Macrobrachium acanthurus (Crustacea, Palaemonidae) females subjected to unilateral eyestalk ablation. Acta Zoologica. 103 (3), 326-334 (2022).
  48. Zhang, C. et al. Changes in bud morphology, growth-related genes and nutritional status during cheliped regeneration in the Chinese mitten crab, Eriocheir sinensis. PLoS One. 13 (12), e0209617 (2018).
  49. Zhang, C. et al. Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation. Fish & Shellfish Immunology. 81, 266-275 (2018).
  50. Mirera, D. O., Moksnes, P. O. Comparative performance of wild juvenile mud crab (Scylla serrata) in different culture systems in East Africa: Effect of shelter, crab size and stocking density. Aquaculture International. 23 (1), 155-173 (2015).
  51. Ut, V. N., Le Vay, L., Nghia, T. T., Hong Hanh, T. T. Development of nursery cultures for the mud crab Scylla paramamosain (Estampador). Aquaculture Research. 38 (14), 1563-1568 (2007).
  52. Fazhan, H. et al. Limb loss and feeding ability in the juvenile mud crab Scylla olivacea: Implications of limb autotomy for aquaculture practice. Applied Animal Behaviour Science. 247, 105553 (2022).

to:

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  41. Muhd-Farouk, H., Abol-Munafi, A. B., Jasmani, S., Ikhwanuddin, M. Effect of steroid hormones 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology. 9 (1), 23-28 (2013).
  42. Muhd-Farouk, H., Jasmani, S., Ikhwanuddin, M. Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture. 451, 78-86 (2016).
  43. Ghazali, A., Mat Noordin, N., Abol-Munafi, A. B., Azra, M. N., Ikhwanuddin, M. Ovarian maturation stages of wild and captive mud crab, Scylla olivacea fed with two diets. Sains Malaysiana. 46 (12), 2273-2280 (2017).
  44. Aaqillah-Amr, M. A., Hidir, A., Noordiyana, M. N., Ikhwanuddin, M. Morphological, biochemical and histological analysis of mud crab ovary and hepatopancreas at different stages of development. Animal Reproduction Science. 195, 274-283 (2018).
  45. Amin-Safwan, A., Muhd-Farouk, H., Mardhiyyah, M. P., Nadirah, M., Ikhwanuddin, M. Does water salinity affect the level of 17β-estradiol and ovarian physiology of orange mud crab, Scylla olivacea (Herbst, 1796) in captivity? Journal of King Saud University - Science. 31 (4), 827-835 (2019).
  46. Wu, X. et al. Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards), female broodstock. Aquaculture. 273 (4), 602-613 (2007).
  47. Azra, M. N., Ikhwanuddin, M. A review of maturation diets for mud crab genus Scylla broodstock: Present research, problems and future perspective. Saudi Journal of Biological Sciences. 23 (2), 257-267 (2016).
  48. Maschio Rodrigues, M., López Greco, L. S., de Almeida, L. C. F., Bertini, G. Reproductive performance of Macrobrachium acanthurus (Crustacea, Palaemonidae) females subjected to unilateral eyestalk ablation. Acta Zoologica. 103 (3), 326-334 (2022).
  49. Zhang, C. et al. Changes in bud morphology, growth-related genes and nutritional status during cheliped regeneration in the Chinese mitten crab, Eriocheir sinensis. PLoS One. 13 (12), e0209617 (2018).
  50. Zhang, C. et al. Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation. Fish & Shellfish Immunology. 81, 266-275 (2018).
  51. Diarte-Plata, G., Sainz-Hernandez, J. C., Aguiñaga-Cruz, J. A., Fierro-Coronado, J. A., Polanco-Torres, A., Puente-Palazuelos, C. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012). 
  52. Mirera, D. O., Moksnes, P. O. Comparative performance of wild juvenile mud crab (Scylla serrata) in different culture systems in East Africa: Effect of shelter, crab size and stocking density. Aquaculture International. 23 (1), 155-173 (2015).
  53. Ut, V. N., Le Vay, L., Nghia, T. T., Hong Hanh, T. T. Development of nursery cultures for the mud crab Scylla paramamosain (Estampador). Aquaculture Research. 38 (14), 1563-1568 (2007).
  54. Fazhan, H. et al. Limb loss and feeding ability in the juvenile mud crab Scylla olivacea: Implications of limb autotomy for aquaculture practice. Applied Animal Behaviour Science. 247, 105553 (2022).

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