CCK 胆囊收缩素

（5）Statistical analyses ）
• Data were expressed as means±Standard Error of Mean (SEM). • Comparisons were made using ANOVA and the Tukey multiple comparisons test for unequal N at the 0.05 significance level .
RIA-CCK
• Quantification of CCK in the extracts was determined by competitive RIA using RIACCK kits . CCK levels were expressed in fmol ind−1 . • The RIA-CCK kit used in this study is based on human CCK-8. Cloning of CCK in herring has demonstrated an identical amino acid sequence between human and herring CCK-8 .
1. Introduction
• CCK alimentary roles involve the stimulation of secretion of pancreatic enzymes, gallbladder contraction, regulation of intestinal motility, and delay of gastric emptying . • In mammals, CCK is secreted in response to the presence of digestive products (fats, proteins and amino acids) in the upper intestine and exerts its endocrine effects on the pancreas and bile.
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2.2 Methods
（2）Series 2: CCK diurnal variation in fed ） and fasted larvae. • Fasting group: ca 1000 larvae aged 23 DAH with continuous seawater exchange and no prey items being offered. • Fed group: similar conditions, but was offered food .
3. Results
3.1. Changes in CCK during ontogeny
3. Results
3.2. CCK diurnal variation in fed and fasted larvae
Fig. 2. Diurnal changes in larval CCK level of fed and fasted.Arrows indicate the time of food addition to the larval tanks.
2.2 Methods
（1）Series 1: changes in CCK levels during ） ontogeny. • Collected 20 larvae from 0 to 40 DAH ,and kepted them overnight for gut evacuation. • After central nervous system (CNS) lesion, they were quickly dissected on ice to separate the gut, head, and carcass. • Samples were placed in EP tubes and flash frozen by liquid N2. All samples were stored at −70 °C.
Comparative Biochemistry and Physiology
Cholecystokinin (CCK) in Atlantic herring (Clupea harengus L.) — Ontogeny and effects of feeding and diurnal rhythms
2. Materials and methods
2.1. Materials • Series 1
• Herring eggs were collected during spring 2003 and larvae were reared up to 40 days after hatching (DAH) to study ontogeny.
• The present study has shown that CCK peptide level in whole herring larvae increases during larval development (particularly from 14 to 20 DAH). • The neural pool (head) of CCK was quantitatively dominant while the digestive tract pool was lower in the larval stages.
• larvae were fed daily collected natural zooplankton . • Daylight conditions with light 08:00–17:00 h including 30min twilight . • Temperature was set at 6℃ from 0 to 20 ℃ DAH and slowly increased to 8 ° from 21 C to 40 DAH. • Salinity averaged 33.9 g· L−1 during the experiment.
4.Conclusions
• During feeding periods (daylight), herring larvae may have high rates of CCK biosynthesis to sustain long hours of food digestion. • The significant decrease in CCK gut level in fed larvae following food being offered to the larvae, support the participation of CCK in the regulation of digestive processes in herring larvae.
（4）Sample preparation and CCK ） radioimmunoassay (RIA-CCK)
Samples thawed → homogenized in cold distilled water →added methanol → incubated during 1 h for extraction→ centrifuged → transferred the methanol supernatant to a clean tube →evaporated using a SpeedVac→ The dried extracts were stored at −20℃ . ℃
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• Series 2
• A separate group was reared in autumn 2003 and used to study diurnal variations and postprandial effects in larvae from 23 to 26 DAH.
Conditions
（3）Experimental plan ）
• Larvae from the feeding and fasting tanks were sampled during 3 consecutive days at 1-h intervals from 08:00 to 11:00 h and after this point every 3 h until 08:00 h. • Lights started to be turned on gradually at 08:00 h and were turned off completely at 17:00 h. • Zooplankton, rotifers, and algae were added to the fed group tank between 10:15 and 10:45 h.
3. Results
3. Results
3.3. Postprandial CCK changes
No statistically significant differences were found over this time period (p<0.05).
3. Results
4.Conclusions
4.Conclusions
• No marked pattern of diurnal variation was noted in CCK level related to daylight, while CCK production and/or release appeared to be affected by feeding. • Fed larvae had in general higher alimentary CCK levels compared to fasted control.