Funding for the production of this manuscript was provided by Nottingham Trent University. The authors wish to thank the University yard manager, Anna Gregory for the use of her horses and provision of fecal samples for use in the protocol. Thanks also to Chester Zoo Wildlife Endocrinology Laboratory for use of their facilities.

윤리 문 : 현장 샘플링과 동물 과목을 포함하는 절차는 노팅엄 트렌트 대학의 동물, 농촌 및 환경 과학 (ARES)의 학교에 의해 승인되었습니다. 대변​​ 샘플 1. 컬렉션 참고 : 분변 샘플 및 메탄올을 취급 할 때는 장갑을 착용한다. 동물은 또한 착용해야 실험실 코트와 같은 인수 공통 질병, 보호 복을 앓고 될 수 있다는 강한 의심이있는 경우. <ol><li> 배변 다음 (몇 시간에 분 이내) 가능한 한 빨리 배설물 샘플을 수집하고 샘플 가방에 배치합니다. 약 10 g의 총 중량과 어떤 똥 샘플의 3 ~ 5 부 표본을 가져 가라. 시간과 수집, 개별 ID의 날짜와 가방 레이블 및 분석 때까지 -20 ° C에서 샘플을 동결. </li><li> 모든 샘플을 동결 오차를 최소화 -20 ° C까지의 연구이다 완료 후 일괄 적으로 분석을 위해 함께 샘플을 수집합니다. &lt;/ 리&gt; </ol> 2. 대변 호르몬 추출 <ol><li> 실온에서 해동하는 배설물 샘플을 남겨주세요. </li><li> 샘플을 수동으로 자신의 샘플 가방에 각각의 샘플을 혼합하여 균질화되어 있는지 확인합니다. </li><li> 각 샘플은 무게 보트에서 마이크로 저울에 0.50 분변 물질 (g 0.003 ±)을 무게를 들어 다음 8 ㎖의 유리 병에 대변을 전송합니다. 각 샘플에 대한 새로운 무게 보트를 사용하여 각 샘플 사이에 30 % 메탄올로 핀셋을 청소하십시오. </li><li> 5 90 % 메탄올 용액으로 분뇨 0.5 g의 결합 물, 및 RT에서 오비탈 진탕 기에서 O / N을 흔들어. </li><li> 다음 날 아침, 샘플 와동 후 600g에서 20 분 동안 원심 분리. </li><li> 37 ℃의 온도로 설정된 수욕 랙에 16 × 125mm이 유리관 제자리 메탄올 분획을 경사 분리한다. 흄 찬 공기를 사용하여 건조시켜 증발. 추출 병을 씻어 폐기에 대한 임상 폐기물 가방에 남아있는 대변 펠렛을 배치합니다. </li><li> 100 %에서 1 ml의 메탄올을 대변 추출물을 다시 중단하고 12 X 75mm 2 폴리 프로필렌 튜브에 전송할 수 있습니다. 즉시 분석 할 때까지 -20 ° C에서 모자 및 저장. </li></ol> 3. 호르몬 분석 <ol><li> 클론 코르티 코스 테론의 CJM006 항혈청을 사용하여 효소 연결된 면역 측정법 (EIA)와 분석을 수행합니다. 참고 :. 두 번째 EIA는 (코르티솔 R4866 항혈청을 테스트했지만,이 (4.1 절 참조) 폴리 클로 날 코르티 코스 테론 CJM006 항혈청과 코르티솔 R4866 항혈청과 각각의 HRP의이 CJ 먼로, 캘리포니아 대학에서 제공하는 생화학 적 검증 단계를 달성하는 데 실패, 데이비스, 캘리포니아 </li><li> 1의 항혈청 희석 : 코팅 버퍼 (0.05 M을 NaHCO3, pH를 9.6)에서 15,000 반복적 인 피펫과 50 μL 피펫 팁을 사용하여 / 잘 50 μL와 96 웰 마이크로 타이 플레이트를로드합니다. 접시 실러와 함께 접시를 덮고 4 ° C에서 O / N을 품어. (N을 비 - 특이 적 결합을 나타내는 웰을 코팅되지 않은 두 개의 웰을 남겨SB). </li><li> 사용 직전 자동화 된 마이크로 타이 터 플레이트 세척기 (0.15 M의 NaCl, 0.05 % 트윈 20 세정액)를 이용하여 마이크로 타이 터 플레이트 다섯 번 세척 하였다. </li><li> 1,000 페이지 / - NSB의 잘 당 50 μL와 &#39;제로&#39;우물 [EIA 버퍼 (0.1 M 나포 4, 0.149 M의 NaCl, 0.1 % 소 혈청 알부민, pH를 7.0) 만, 표준 (코르티 코스 테론, 3.9으로 판을로드 잘) 또는 적절하게 1시 20분 EIA 버퍼에 희석 배설물 추출물 샘플 (,) 섹션 4.1을 참조하십시오. 표준과 0은 각각 세중 및 4 번씩 실행해야합니다. 희석 배설물 추출물 샘플과 NSB의 중복에서 실행해야합니다. </li><li> 70,000 : 50 ㎕를 첨가 / 웰의 1 EIA 버퍼에 희석 라벨 고추 냉이 퍼 옥시 다제 공액 ᵻ 즉시이 작업을 수행합니다. </li><li> 실온에서 2 시간 동안 어둠 속에서 마이크로 타이 플레이트를 품어. </li><li> 100 μL와 함께 어둠 속에서 실온에서 접시를 세척 용액으로 5 회 마이크로 타이 플레이트를 세척하고 품어 / 아RT 기판 [0.4mm의 2,2&#39;- 아지노 디 - (3-ethylbenzthiazoline 술폰산) 디 암모늄 염, 1.6 mM의 H 2 O 2, 0.05 M 시트 레이트, pH가 4.0]의 L. 사용하기 직전에 기판을 준비합니다. </li><li> 미크로 역가 플레이트를 읽는 405 nm의 파장에서 spectrophometer를 사용한다. 제로 웰의 광학 밀도가 0.8 이상 1.0에 도달 할 때 배양 플레이트는 완료된 것으로 간주된다. </li><li> 반복성을 보장하기 위해, 약 30 % (EIA 표준), 50 % (생물학적 샘플 예를 들어, 국내 말 배설물 추출물의 수영장) 70 % (EIA 표준)를 결합하는 EIA 버퍼에 희석 제어 [EIA 표준 또는 생물학적 샘플을 사용 ]는 각각 &lt;10 %의 변화와 &lt;15 %의 인트라 간 분석 계수를 설명합니다. </li></ol> 4. 효소 연결된 면역 : 연구 종과 섹스에 대한 검증 <ol><li> 생물 유효성 검사 (병렬) <ol><li> EIA 버퍼를 사용하여 풀 배설물 여분의 직렬 희석을 수행코네티컷, 이상적으로 여러 사람들로부터 의심 높고 낮은 호르몬 농도 샘플에서 가져온 (범위 : 깔끔한, 1 : 8192 : 2 1)과 3 장에 설명 된대로 EIA에서 실행됩니다. 주 : 분변 추출물의 계열 희석 표준 곡선의 선형 회귀 결과 변위 곡선 평행 수득 때 병렬성이 달성 된 것으로 간주된다 R2&gt; 0.90, 기울기&gt; 0.50 및 p &lt;0.05를 보여준다. 표준 곡선에 약 50 %를 결합하는 일련 묽은 배설물 추출물은 모든 배설물 추출물을 실행하는 데 이상적인 희석을 고려하여야한다 (예를 들면 1:20). 선형 회귀 결과 R2&gt; 0.90 및 p &lt;0.05을 설명 할 때 분석에서 고려 간섭이 없다. </li></ol></li><li> 생물 유효성 검사 (간섭) <ol><li> 50 %로 희석 적절하게 풀 희석 된 배설물 추출물 [200 μL가 paralleli에 의해 결정 바인딩과 (3.9 1000 NG / 잘 코르티 코스 테론 제로) 직렬 희석 기준의 200 μl를 스파이크SM (1:20)] 및 EIA 분석 플롯 다음 섹션 3과 EIA에서 실행 관측 된 농도 - 예상 농도 대 배경 (제로 샘플의 농도). 주 : 표준 희석 풀링 분변 추출물의 첨가가 유의 양 예상 선형 회귀 결과를 변경하지 않으면 R이&gt; 0.90, 1.0 및 p &lt;0.05에 가까운 기울기를 산출한다. 환경 영향 평가와 간섭을 달성 없습니다. </li></ol></li><li> 생물 유효성 검사 : 참고 : 선택한 EIA 관심 호르몬의 생물학적으로 중요한 변화를 측정 할 수있는 능력이있는 데모 [예는, 약리학 다음 글루코 코르티코이드 농도의 상승 또는 생물학적 도전 (ACTH가 종종 허가 절차) (예를 들어, 심리적 및 / 또는 환경, 기회주의 관계가없는 이벤트가 될 가능성이 더 높습니다)]. <ol><li> 선택 추출물과 글루코 코르티코이드 대사 산물 농도에 대한 분변 샘플을 분석이전과 도전 이벤트 - 3 절에서 1 설명 된 프로세스를 사용하여. 주 : 도전 이벤트가 특정 종 수 있습니다. 예시적인 방법은 국내 말은 아래 제공된다. 말에 대한 도전 이벤트의 전체 예는 관리 9 축산 절차 (16)의 사용을 포함하여 이전에 발표되었다. 도전 이벤트 다음 글루코 코르티코이드의 대사 산물 농도의 유의 한 증가가 관찰 및 통계 분석을 사용하여 확인해야합니다. 이것은 반복 측정 샘플 날짜 및 개인의 영향을 적절 포함한다. 생물학적 검증은 달성 된 것으로 간주된다. </li></ol></li></ol>

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The authors have nothing to disclose.

분변 코르티 분석 말 부신 활성의 장기 패턴을 평가하는 수단을 제공한다. 상기 방법의 비 침습적 자연 타액 플라즈마 분석 9 포함한 부신 활성을 평가하기 위해 사용되는 다른 샘플링 방법의 교란 효과를 극복한다. 또한이 기술은 무료에 이르기까지 말을 공부하는 경우 명확한 비 침습적 인 장점이 있습니다. 이 방법의 적절한 사용에 관한 논의하기 위해 몇 가지 주?...

방법 설명 목적은 부신 활성의 비 침습적 평가를 제공하기 위해서 말 대변 코르티 코스 농도를 분석한다. 시상 하부 - 뇌하수체 - 부신 (HPA) 축 활동을 측정하는 것은 포로 및 국내 종 모두에서 잠재적으로 혐오 상황에 대한 반응을 연구하는 허용 방법입니다. 참조 기술과 가장 널리 사용되는 방법은 그러나, 배설물 분석과 같은 다른 방법이없는 범위 종을 감시하는 기능을 채혈 자체에 의해 유도 된 응력을 극복 할 수 있도록하기 위해 개발 된 혈장 (1)의 사용이다. 혐오 상황시, 생리적 항상성이 파괴된다. 앞쪽에 뇌하수체에 작용하여 부 신피질 자극 호르몬 (ACTH)의 방출을 자극 방출 호르몬 (CRH)를 corticotrophin 뇌 릴리스의 시상 하부. ACTH는 혈류를 입력하고 종을 분비하는 부신 피질을 자극s의 특정 글루코 코르티코이드 (GC). 글루코 코르티코이드는 밀접하지 않고 지속적으로 그러므로 그들은 종종 다른 스트레스 링크 호르몬이 이상 환경에서 측정 된 모든 에너지를 높게 한 상태에서 생산되고보다 스트레스가 이벤트에 연결되어 있습니다. 글루코 코르티코이드는 말을 여러 적응 효과에 대한 책임이 있습니다. 에너지가 급속 지방산 및 글루코스의 형태로 본문에 저장 사이트로부터 동원되는 산소 섭취량은 감각 기능 3을 향상 혈류가 이동 4 불필요 영역 감소, 증가된다. 뿐만 아니라 대응 메커니즘으로 작용하는 글루코 코르티코이드에 의한 응력 상승은 다음 스트레스 5 동물을 제조하는 데 도움이된다. 호르몬 혈장 수준과 침을 평가하는 것은 배설물 대책 호르몬의 대사 최종 제품 대사를 측정하지만 실제 순환 호르몬을 측정하는 것을 포함한다. 순환 스테로이드는 리터에 이화된다그들은 장 트랙 6에서 세균성 식물의 효소 활성에 의해 촉진 추가 변경을 거쳐 담즙에 배설하기 전에 이베. 따라서, 혈중 글루코 코르티코이드 지향 면역은 배설물 글루코 코르티코이드의 대사 산물 (7)의 분석에 적합하지 않을 수 있습니다. 찌끼 모음 말 아무런 방해하여 수행 될 수있는 바와 같이, 코르티 대한 대변의 분석은 다수의 환경에서 HPA 활동을 모니터링하기 위해 광범위하게 사용되고있다. 말의 배설물에 상승 코르티 코스 테론은 수술 후 의학적 치료 8시 제한적 하우징 (9)을 포함하여 잠재적 인 혐오 상황에 대한 응답으로보고되었다. 대변 ​​샘플링은 시간이 지남에 풀링 된 글루코 코르티코이드 수준보다는 장기, 만성 또는 계절 패턴 (10)을 모니터링하기위한 적절하고 혈장과 타액에서 제공하는 시간 샘플링에있는 점을 반영한다. 때문에 비 침습적으로방법의 본질은, 샘플 캡처하거나 구속 11 없이도 개인 반복적으로 수집 될 수있다. 샘플링 프로토콜을 계획 할 때, 종 특정 장내 통과 시간이 고려되어야한다. 말에서 소화관 통과 시간 따라서, 부신 반응과 후속 코르티 대사는 HPA 축의 활성화 후 초기 언젠가 대변 검출 할 수 약 18 내지 12 시간이다. 비 침습적 면역 기술을 활용하면 조사중인 종에 대한주의 깊은 검증이 13 필수적이다. 또한, 호르몬 대사 산물의 배설에 성별 차이로 인해 신진 대사 속도와 마우스 (14), 닭 (15) 등 다양한 종으로 배설 코르티 코스 테론의 대사 산물의 종류의 차이에 아마보고되었다. 일에 자세히 설명되어 있습니다로는 분석이 남성과 여성의 국내 말 모두에 사용하기 위해 검증 된이 방법의 일환으로, 따라서 중요전자 프로토콜입니다. 남녀 간의 호르몬 대사의 차이는 해결하지 않고 분석 검증의 일부로 포함 거의되어 아직 데이터 품질에 대한 처벌을 받게됩니다. 이 비 침습적 방법은 국내 말에 부신 활동의 장기 평가를 할 수 있습니다. 프로토콜 세부 분석의 검증 및 분석 기술 자체를 모두.

<table><tbody><tr><td>Corticosterone antibody &amp;amp; HRP kit</td><td>Coralie Munro - UC Davis</td><td></td><td>No longer available through UC Davis - please see Arbor Assays</td></tr><tr><td>Cortisol antibody &amp;amp; HRP kit</td><td>Coralie Munro - UC Davis</td><td></td><td>No longer available through UC Davis - please see Arbor Assays</td></tr><tr><td>Corticosterone synthetic standard hormone</td><td>Sigma Aldrich</td><td>50-23-7</td><td>Harnful if ingested or with skin contact. Use in fume cupboard</td></tr><tr><td>Cortisol synthetic standard hormone</td><td>Sigma Aldrich</td><td>15087-01-1</td><td>Harmful if ingested or with skin contact. Use in fume cupboard</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>67-56-1</td><td>Irritant. Use in fume cupboard</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>144-55-8</td><td>Irritant</td></tr><tr><td>Sodium Carbonate Anhydrous</td><td>Sigma Aldrich</td><td>497-19-8</td><td>Irritant</td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma Aldrich</td><td>7558-79-4</td><td>Irritant</td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma Aldrich</td><td>10049-21-5</td><td>Irritant</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>9048-46-8</td><td>Irritant</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>9005-64-5</td><td>Irritant</td></tr><tr><td>Citric Acid</td><td>Sigma Aldrich</td><td>77-92-9</td><td>Irritant</td></tr><tr><td>ABTS</td><td>Sigma Aldrich</td><td>30931-67-0</td><td>Irritant</td></tr><tr><td>Hydrogen Peroxide 30%</td><td>Sigma Aldrich</td><td>7722-84-1</td><td>Irritant</td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>7647-14-5</td><td>Irritant</td></tr><tr><td>Buffer capsules - pH 4</td><td>VWR</td><td>332732B</td><td></td></tr><tr><td>Buffer capsules - pH 7</td><td>VWR</td><td>332742D</td><td></td></tr><tr><td>Buffer capsules - pH 10</td><td>VWR</td><td>332762H</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Sigma Aldrich</td><td>435570</td><td>Irritant. Use in fume cupboard</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>Irritant</td></tr><tr><td>Analytical balance</td><td>Fisher Scientific</td><td>BFS-525-010A</td><td></td></tr><tr><td>Air compressor</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Computer +printer</td><td></td><td></td><td></td></tr><tr><td>fridge-freezer</td><td></td><td></td><td></td></tr><tr><td>Drying apparatus</td><td></td><td></td><td></td></tr><tr><td>+tubing</td><td></td><td></td><td></td></tr><tr><td>Flammable liquid storagecabinet</td><td>VWR</td><td>649-002</td><td></td></tr><tr><td>Fume cupboard</td><td></td><td></td><td></td></tr><tr><td>Hot-plate stirrer</td><td>VWR</td><td>640-282</td><td></td></tr><tr><td>Microplate reader</td><td>VWR</td><td></td><td></td></tr><tr><td>Microplate washer</td><td>VWR</td><td></td><td></td></tr><tr><td>pH meter</td><td>VWR</td><td></td><td></td></tr><tr><td>Eppendorf Research&amp;reg; pipettes - multipack option 2</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 1,000 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 200 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 20 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Repeater pipette</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette filler</td><td>VWR</td><td></td><td></td></tr><tr><td>Orbital shaker</td><td>Progen Scientific</td><td></td><td></td></tr><tr><td>Sonicator</td><td>Hilsonic</td><td></td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td></td><td></td></tr><tr><td>Warm water bath</td><td></td><td></td><td></td></tr><tr><td>Water purification system</td><td>Millipore</td><td></td><td></td></tr></tbody></table>

fecal glucocorticoid analysis: non-invasive adrenal monitoring in equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

국내 말 (N = 16, 8 암말, 8 geldings) 십오년 (± 3)의 평균 연령은 성별과 사회적 고립 (N = 4 말 / 치료)의 증가 수준 4 개의 주택 디자인 대상에 따라 분류 하였다. 밀접하게 그들의 자연 서식지를 시뮬레이션, 무리 환경에서 생활 주택 1 관련된 말. 실내 헛간에 쌍에 살고있는 주택이 포함 된 말. 하우징 (3) 참여 말은 마구간에 있지만, 다른 말과 말의 하우징 (4) 관련 총 격리 시각?...

Watch this Scientific Journal Video about Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids at JoVE.com

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

fecal-glucocorticoid-analysis-non-invasive-adrenal-monitoring

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.6K 조회수.  Nottingham Trent University. Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

비디오: Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Cortisol Measurement in Koala Phascolarctos cinereus Fur

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

코알라의 코티솔 측정(파스콜아크토시네레우스)모피

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

연구

생화학

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

철갑상어 지느러미와 턱뼈 매트릭스에서 코티솔 추출

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

환경과학

Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report formats, a growing number of studies now incorporate biological stress-related hormones and catalysts, such as cortisol and &#945;-amylase, to measure levels of stress. Presented here is a protocol to examine the effects on levels of biophysical and psychological stress following visitation to three different locations with differing levels of nature. Biophysical and self-reported psychological stress levels are measured immediately upon entering the selected locations and just prior to the visitors leaving the site. Using a &#34;drool&#34;&#160;method, the biophysical measure consists of 1-2 mL samples of saliva provided by study subjects upon entry to one of three study locations. As prescribed by extant literature, the saliva is collected within a 45 minute time frame following the end of the visitor&#8217;s engagement at the location. Following saliva collection, the samples are labeled and transported to a biological lab. Cortisol is the biophysical variable of interest in this study and measured using an ELISA process with a TECAN plate reader. To measure self-reported stress, the Perceived Stress Questionnaire (PSQ), which reports levels of worry, tension, joy, and perceived demands. Data are collected at all three sites in the late afternoon through early evening. When compared across all three settings, stress levels, as measured by both the biological markers and self-reports, are significantly lower after visitation to the most natural setting.

The purpose of this paper is to identify changes in stress levels after visitation to three different settings and to describe the methods used in identifying stress levels based on measures of salivary cortisol, &#945;-amylase, and a psychological self-report instrument.&#160;

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report ...

행동분석학

Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a substantial pressure coming from customers, legislators and other stakeholders, to make management changes to improve the well-being of these animals. Several studies have demonstrated that providing environmental enrichment to pigs allows them to express their natural behavior, such as rooting and exploring, as well as nesting prior to farrowing, and is associated with reduced stress and improved production and welfare. However, many considerations should be taken into account when providing environmental enrichment, such as the type of floor, drainage and sewage systems, the pigs&#39; stage in life, the material, as well as its hanging method, height and location within the pen. The objectives of this paper are (1) to give methodologic information on how to provide a relatively simple and practical meaningful environmental enrichment for pigs which are housed on slatted floors during the different stages of their life, and (2) to demonstrate how to collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress. Protocols include information regarding the use of jute, cotton ropes, straw in racks, as well as chewable silicone sticks devices as environmental enrichment in pens of farrowing and lactation, weaners and finishers. In addition, the use of cotton rope for a non-invasive saliva samples collection for cortisol concentrations analysis is detailed. The protocols provided are relevant for professionals aiming to improve and monitor animal welfare, in both research and industrial swine farming.

This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

슬레이트 바닥에 보관된 돼지에서 의미 있는 환경 농축 및 타액 코티솔 측정

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a ...

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

열 화상 비 침습적 무제한 조류의 스트레스를 연구

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

신경과학

A Novel Method: Super-selective Adrenal Venous Sampling

Primary aldosteronism (PA) and subclinical Cushing&#39;s syndrome (SCS) are conditions in which the adrenal glands autonomously produce excessive amounts of aldosterone and cortisol, respectively. The conventional adrenal venous sampling (cAVS) method collects blood samples from both adrenal central veins and is useful for identifying the laterality of excess hormone production in a unilateral lesion(s), as documented in PA cases. In cAVS, plasma cortisol concentrations (PCCs) are used to normalize plasma aldosterone concentrations (PACs). A novel &#34;super-selective&#34; adrenal venous sampling (ssAVS) method was developed using a micro-catheter, which collects blood samples from adrenal tributary veins (TVs). PACs in ssAVS samples do not require PCC normalization because samples contain a limited amount of systemic venous blood, if any. The ssAVS method enabled segmental lesion(s) to be detected in both adrenal glands, which may be treated by bilateral adrenalectomy, thereby sparing lesion-free segment(s). Right and left adrenals typically have three TVs each, i.e., the superior, lateral, and inferior TVs in the right adrenal as well as the superior-median, superior-lateral, and lateral TVs in the left adrenal. In the ssAVS method, specific parent catheters and a technique to handle them are required, and have been described herein. Furthermore, ssAVS results from three cases of PA are presented: bilateral aldosterone-producing adenoma (APA) (Case #1), left APA and right possible cortisol-producing adenoma causing SCS (Case #2), and idiopathic hyperaldosteronism in which bilateral adrenal segments produced excessive amounts of aldosterone (Case #3). The ssAVS method is not difficult for expert angiographers, and, thus, is recommended worldwide to treat PA cases for which cAVS does not represent a viable surgical treatment option.

&#39;Super-selective&#39; adrenal venous sampling (ssAVS, also called segmental adrenal venous sampling: sAVS)&#160;was performed using a micro-catheter to identify adrenal segment(s) that produce excessive amounts of hormones. The ssAVS technique was described, cases in which adrenal segmental lesion(s) were identified by ssAVS were presented, and the usefulness of ssAVS in future adrenal research was discussed.

새로운 방법: 최고 선택적 부 신 정 맥 샘플링

Primary aldosteronism (PA) and subclinical Cushing&#39;s syndrome (SCS) are conditions in which the adrenal glands autonomously produce excessive amounts ...

의학

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Zebrafish의 유충 루시 페라 제 리포터 시스템과 글루코 코르티코이드 신호에 대한 화학적 스크리닝 절차

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

생물학

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

추출 및 인간과 원숭이의 머리에서 코르티솔 분석

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

부신 크로마핀 세포에서 융합 기공 역학의 세 가지 모드를 측정하는 공초점 현미경 검사

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

쥐 Adrenocortical 셀 및 Steroidogenic 기능의 분석 실험의 기본 문화

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

대변 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

요약

더 많은 비디오 탐색

이 비디오의 챕터

코알라의 코티솔 측정(파스콜아크토시네레우스)모피

철갑상어 지느러미와 턱뼈 매트릭스에서 코티솔 추출

Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature

슬레이트 바닥에 보관된 돼지에서 의미 있는 환경 농축 및 타액 코티솔 측정

열 화상 비 침습적 무제한 조류의 스트레스를 연구

새로운 방법: 최고 선택적 부 신 정 맥 샘플링

Zebrafish의 유충 루시 페라 제 리포터 시스템과 글루코 코르티코이드 신호에 대한 화학적 스크리닝 절차

추출 및 인간과 원숭이의 머리에서 코르티솔 분석

부신 크로마핀 세포에서 융합 기공 역학의 세 가지 모드를 측정하는 공초점 현미경 검사

쥐 Adrenocortical 셀 및 Steroidogenic 기능의 분석 실험의 기본 문화

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

요약

더 많은 비디오 탐색

이 비디오의 챕터

코알라의 코티솔 측정(파스콜아크토시네레우스)모피

철갑상어 지느러미와 턱뼈 매트릭스에서 코티솔 추출

Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature

슬레이트 바닥에 보관된 돼지에서 의미 있는 환경 농축 및 타액 코티솔 측정

열 화상 비 침습적 무제한 조류의 스트레스를 연구

새로운 방법: 최고 선택적 부 신 정 맥 샘플링

Zebrafish의 유충 루시 페라 제 리포터 시스템과 글루코 코르티코이드 신호에 대한 화학적 스크리닝 절차

추출 및 인간과 원숭이의 머리에서 코르티솔 분석

부신 크로마핀 세포에서 융합 기공 역학의 세 가지 모드를 측정하는 공초점 현미경 검사

쥐 Adrenocortical 셀 및 Steroidogenic 기능의 분석 실험의 기본 문화

대변 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링