CN113694182A - Application of abdominal GnIH and its homolog RFRP-3 in improving animal growth speed and quality - Google Patents
Application of abdominal GnIH and its homolog RFRP-3 in improving animal growth speed and quality Download PDFInfo
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Abstract
The invention provides an application of an abdominal cavity GnIH and a homolog RFRP-3 thereof in improving the growth speed of animals, wherein the feed intake of the animals is increased, the feed intake frequency of the animals is increased, the feed intake time of the animals is increased, the feed intake interval of the animals is shortened, and the average daily gain of the animals is increased; the invention also provides an application of the abdominal cavity GnIH and the homolog RFRP-3 thereof in improving the growth quality of animals, wherein the feed-meat ratio of the animals is reduced, the obesity index of the animals is increased, and the carcass quality of the animals is increased. The application of the abdominal cavity GnIH and the homologue RFRP-3 thereof in improving the growth speed and the growth quality of animals can be widely applied to livestock breeding in animal husbandry, improve the production efficiency and the production quality of livestock, and have wide application prospect.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to application of abdominal cavity GnIH and homologue RFRP-3 thereof in improving the growth speed and the growth quality of animals.
Background
GnIH is the first neuropeptide found in the hypothalamus of vertebrates that is capable of inhibiting gonadotropin release. After more than ten years of research, the existence of GnIH is proved in birds, poultry, mammals, even amphibians and fishes, and the GnIH participates in the regulation of reproduction among various species and becomes a key neuroendocrine factor in the regulation of animal reproduction together with GnRH. Later studies showed that GnIH is a homologue to RFRP-3. The physiological functions of neuropeptides are closely related to distribution and positioning of neuropeptides in organisms, RFRP-3 is distributed and expressed in central and peripheral tissues and organs of Luchuan pigs to different degrees, but the expression abundance of RFRP-3 in the central nervous system is higher than that of the peripheral tissues and organs. In addition, RFRP-3mRNA also has different degrees of expression in mouse central and peripheral tissues and organs, and the expression level is higher in the central nervous system. The GnIH precursor peptide is expressed at different sites in the animal body and exerts a regulatory function at that site via the corresponding receptor. For example, the action of GnIH on the pancreas may regulate insulin concentration for the treatment of diabetes.
RFRP-3 has been defined as a novel food regulatory factor that enters the field of view of researchers. Tachibana et al, first surprisingly found that injection of GnIH in the lateral ventricles of chickens resulted in a significant increase in their food intake. In addition, McConn et al found that the expression level of hypothalamus GnIH and its receptor GPR147mRNA was significantly increased in chickens after the fasting treatment, and that the expression level of hypothalamus RFRP-3 and its receptor GPR147mRNA was higher in lighter weight chickens than in heavier weight chickens, and the above results suggest that the abdominal cavity GnIH and its homologue RFRP-3 are likely to be used as a congenital feeding promoting factor to promote the feeding of animals. This shows that GnIH and its homologue RFRP-3 are closely related to regulation and control of animal growth in abdominal cavity expression.
Disclosure of Invention
Through careful study, the invention discovers that the expression of the abdominal cavity GnIH and the homolog RFRP-3 thereof in the abdominal cavity can influence the feeding behavior and the production performance of animals, and further provides the application of the abdominal cavity GnIH and the homolog RFRP-3 thereof in improving the growth speed of the animals.
In some embodiments of the invention, the growth rate is characterized by an increase in food intake of the animal.
In some embodiments of the invention, the characterization of growth rate increases the feeding frequency of the animal.
In some embodiments of the invention, the characterization of growth rate is an increase in feeding time of the animal.
In some embodiments of the invention, the characterization of growth rate is a reduction in feeding intervals for the animal.
In some embodiments of the invention, the characterization of growth rate is an increase in average daily gain of the animal.
The invention also provides application of the abdominal cavity GnIH and the homologue RFRP-3 thereof in improving the growth quality of animals.
In some embodiments of the invention, the quality of growth is characterized by a decrease in the feed-meat ratio of the animal.
In some embodiments of the invention, the quality of growth is characterized by an increase in the obesity index of the animal.
In some embodiments of the invention, the growth quality is characterized by an increase in carcass mass of the animal.
The application of the abdominal cavity GnIH and the homologue RFRP-3 thereof in improving the growth speed and the growth quality of animals can be widely applied to livestock breeding in animal husbandry, improve the production efficiency and the production quality of livestock, and have wide application prospect.
Drawings
Fig. 1 is a schematic of the feed intake of a free-feeding piglet injected intraperitoneally with GnIH;
fig. 2 is a schematic of the feeding frequency of free-feeding piglets injected intraperitoneally with GnIH;
FIG. 3 is a schematic representation of organ weights of piglets intraperitoneally injected with GnIH;
fig. 4 is a graph showing fat weight of piglets intraperitoneally injected with GnIH, wherein denotes P <0.05, P <0.01, and P < 0.001, compared to control group;
FIG. 5 is a graph showing the feed intake of the Yao chicken within 1 hour after the intraperitoneal injection of GnIH, wherein P is <0.05, P is <0.01, and P is < 0.001 in the intraperitoneal GnIH group compared with the control group; # denotes P <0.05, # denotes P <0.01, # denotes P < 0.001;
FIG. 6 is a graph showing daily feed intake of Yao chickens injected intraperitoneally with GnIH, wherein P is <0.05, P is <0.01, and P is < 0.001;
FIG. 7 is a graph showing the daily weight gain of Yao chickens upon chronic intraperitoneal injection of GnIH, wherein P is <0.05, P is <0.01, and P is < 0.001 in the abdominal GnIH group compared with the control group;
FIG. 8 is a graph showing the food intake of freely feeding rats injected intraperitoneally with RFRP-3;
FIG. 9 is a graph showing the feeding times of freely feeding rats injected with intraperitoneal RFRP-3;
FIG. 10 is a schematic representation of hypothalamic appetite-related gene expression for intraperitoneal injection of RFRP-3;
FIG. 11 is a graph showing the food intake of freely feeding male mice injected intraperitoneally with RFRP-3;
FIG. 12 is a schematic representation of the feeding times of free feeding mice injected intraperitoneally with RFRP-3;
fig. 13 is a graph of peritoneal RFRP-3 vs. mouse visceral mass morphology, where P represents P <0.05, P <0.01, and P < 0.001 in the peritoneal RFRP-3 group compared to the control group.
Detailed Description
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Many modifications may be made to the specific embodiments disclosed herein, while still obtaining the same or similar results, without departing from the spirit or scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference. The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the inventive materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical stores.
The invention researches four animals, namely pigs, Yao chickens, rats and mice, injects GnIH congeneric substance RFRP-3 to the abdominal cavity of the rat mice to research the influence on the growth speed and the growth quality, and injects GnIH to the pigs and the kiln chickens to research the influence on the growth speed and the growth quality.
The indexes for representing the growth speed of the pigs adopted by the invention comprise the feed intake, the feed intake time, the feed intake rate, the feed intake interval, the satiety rate, the average daily gain and the like after a period of fasting;
the indexes used for characterizing the growth quality of the pigs comprise feed-meat ratio, obesity index, carcass quality, slaughter rate, backfat thickness and the like.
First, test materials
The animal model three-way hybrid piglet is 14 heads, 6 weeks old and 14.5 +/-0.2 kg in weight. The groups were randomized into three groups, control (saline 1 mL/dose), GnIH low dose (0.1mg/1 mLGnIH/dose), and high dose (1mg/1 mLGnIH/dose). The test was conducted by feeding in groups and the temperature (25. + -. 2 ℃ C.), humidity (55. + -. 5%) and illumination (12L: 12D, 7: 00AM on a daily basis) of the feeding environment were strictly controlled. The animal-related tests of the test all comply with the standards of the animal administration and ethical committee of the Guangxi university experiment.
Second, the experimental instrument
The injection protocol of the test is divided into acute injection and chronic injection: acute injection is defined as the injection of the dose GnIH only once after adaptive feeding and grouping; whereas chronic injection is defined as the intraperitoneal injection of the above dose GnIH twice daily (7: 00AM and 7: 00PM) for 14 consecutive days after acclimatization and grouping.
Third, test reagent
Pig GnIH (048-46) was purchased from Phoenix Pharmaceuticals, USA; infrared night vision cameras (DS-IPC-B12-I) available from HaekWindow; 1mL syringes were purchased from Shanghai Polymu medical devices, Inc.; the full-automatic biochemical analyzer (URIT-8021AVeT) was purchased from Guilin ulite medical electronics, Inc.
1.1 Effect of intraperitoneal GnIH on the 1 st feeding behavior of fasted piglets
The test method comprises the following steps: the piglets were given a diet-deprived fasting treatment for 8 hours in the evening before the start of the experiment, the piglets were fed with feed at 7: 00AM on the next day, and were intraperitoneally injected with the above-mentioned different doses of GnIH, respectively, for each group of piglets, and their feeding behavior was recorded using a video camera, and the remaining feed was weighed, and the influence of acute intraperitoneal injection of the different doses of GnIH on their 1 st feeding behavior after the fasting treatment was analyzed, including the feed intake (kg), the feeding duration (min), and the feeding rate (feed intake/feeding time).
The test results are shown in table 1, the intraabdominal injection of the low dose and the high dose of the abdominal GnIH after 8 hours of fasting of the piglets can lead to the extremely obvious increase of the food intake and the food intake time of the prohibited piglets (P < 0.01); further analysis showed that intraperitoneal injection of a low dose of intraperitoneal GnIH significantly increased the feed rate of fasted piglets (P <0.05), while intraperitoneal injection of a high dose of intraperitoneal GnIH significantly increased the feed rate (P < 0.01). The above data suggest that intraperitoneal injection of GnIH can further enhance the hunger sensation in fasted piglets. Fig. 1 and 2 show a schematic of the feed intake and feed frequency, respectively, of a free-feeding piglet injected intraperitoneally with GnIH.
TABLE 1-1 Effect of post-fasting 1 st feeding behavior of piglets injected intraperitoneally with GnIH
Note: represents the abdominal GnIH low dose group compared with the control group, represents P <0.05, represents P <0.01, represents P < 0.001; # denotes P <0.05, # denotes P <0.01, # denotes P < 0.001 in the peritoneal GnIH high dose group compared to the control group, the same applies hereinafter.
1.2 Effect of Abdominal GnIH on the feeding behavior of free-feeding piglets
The test method comprises the following steps: in the free feeding test, GnIH is chronically intraperitoneally injected under light and dark environments, respectively, the sectional accumulated feed intake (kg), feeding times (times) and feeding time (min) under light and dark environments after injection are recorded, and the feeding behavior of rats is observed and calculated, including the feed intake rate (feed intake/feed intake time), the feeding time per meal (feed intake/feed intake time), the feeding interval [ total length of recording time-total length of feed intake time)/feeding times ], and the satiety rate (average feeding interval/average feed intake per meal).
The feeding behavior of the piglets can be changed by different stimulation in the photoperiod, so that the influence of chronic intraperitoneal injection of different dosages of abdominal cavity GnIH on the feeding behavior of the piglets is researched under different environments of light and dark. The test results are shown in table 2, the piglet feed intake can be remarkably increased by injecting the abdominal cavity GnIH with low dose and high dose in the dark environment, and the piglet feed intake can be remarkably increased by injecting the abdominal cavity GnIH with high dose only in the abdominal cavity in the light environment (P is less than 0.01); the abdominal cavity GnIH with low dose and high dose injected in the abdominal cavity under the light and dark environment can lead the piglet to remarkably increase the feeding frequency and the feeding time (P is less than 0.01); further analysis shows that the abdominal cavity GnIH injected with low dose and high dose can both remarkably shorten the feeding interval of piglets (P is less than 0.01); in addition, analysis on the satiety rate shows that the abdominal cavity injected with the low-dose abdominal cavity GnIH can obviously reduce the satiety rate in a light environment (P < 0.05); and the satiety rate is reduced very significantly in dark environment (P <0.01), the abdominal injection of high-dose abdominal GnIH can cause the satiety rate to be reduced very significantly in light and dark environment (P <0.01), and in conclusion, the increase of piglet feed intake caused by the intraperitoneal injection of GnIH is probably caused by the increase of feed intake frequency, the reduction of feed interval and the reduction of satiety rate.
TABLE 1-2 Effect of intraperitoneal GnIH injection on free-feeding piglets
1.3 Effect of Abdominal GnIH on piglet feeding behavior at different periods
The test method comprises the following steps: in the free feeding test, GnIH was chronically intraperitoneally injected under light and dark environments, respectively, the sectional cumulative food intake (kg) within 1 hour, 2 hours, and 3-12 hours after injection was recorded, and the number of times of food intake was counted by an infrared night vision camera.
The test result of 1.2 shows that different light stimuli may affect the feeding behavior change caused by the abdominal cavity GnIH, but the specific time period caused by the feeding behavior change is not clear. Therefore, the present invention further analyzed the change of piglet feeding behavior in different time periods under light and dark environments, as shown in fig. 1, the increase of piglet feeding amount had a very significant difference (P < 0.001) between 1 hour after the abdominal cavity injecting high dose abdominal cavity GnIH in light environment and 3-12 hours in the dark environment; a very significant increase (P < 0.001) only in the 3-12 hours of the dark environment after intraperitoneal injection of a low dose of intraperitoneal GnIH; as shown in FIG. 2, in the statistics of feeding frequency, there was no significant difference (P >0.05) at 2 hours after the intraperitoneal injection of the low-dose abdominal cavity GnIH in the dark environment, while the intraperitoneal injection of the low-dose abdominal cavity GnIH and the high-dose abdominal cavity GnIH in the rest of the time period can induce a very significant increase in feeding frequency (P < 0.001).
1.4 Effect of Abdominal GnIH on average daily gain, average daily feed intake and feed conversion ratio of piglets
The test method comprises the following steps: weight and feed intake were measured for each group of piglets before the start of the chronic intraperitoneal GnIH test and at the end of the test, and used to calculate feed-meat ratio [ total feed consumed (kg)/total weight gain (kg) ] during the test period
As shown in Table 3, the average daily gain of piglets (P <0.01) can be remarkably increased by injecting the low-dose and high-dose abdominal GnIH into the abdominal cavity; the average daily food intake is obviously increased (P is less than 0.05) after the abdominal cavity GnIH is injected into the abdominal cavity with low dose, and the average daily food intake of piglets can be obviously increased (P is less than 0.01) after the abdominal cavity GnIH is injected into the abdominal cavity with high dose; furthermore, the feed meat of piglets during the invention was significantly reduced (P <0.05) compared to the low dose abdominal GnIH injected in the abdominal cavity, while the high dose was very significantly reduced (P < 0.01).
TABLE 1-3 average daily gain, average daily feed intake and feed-meat ratio of piglets
1.5 Effect of Abdominal GnIH on piglet obesity index
The test method comprises the following steps: pig Obesity Index (POI) index is the most effective and intuitive indicator in evaluating obesity degree of adult animals, and for this purpose, the change of obesity index of female piglets during the test was analyzed to evaluate the effect of female piglet obesity by abdominal cavity injection of GnIH.
As shown in table 4, intraperitoneal injection of both low and high dose intraperitoneal GnIH resulted in a significant increase in the obesity index (P <0.05) in female piglets during the period of invention. The above test results suggest that intraperitoneal injection of GnIH can cause the occurrence of piglet obesity. The pig body is considered to be a truncated cone with the base represented by the abdomen (a), the top by the neck (N) and the length represented by the Body Shape (BS). POI is defined as (L/cm): where BS is the body type, A and N are the radii of the abdomen (A) and neck (N). The index in litres/centimetre can then be determined: POI 1000 pi (BS/3) (a2+ N2+ a × N)/BS.
TABLE 1-4 Change in piglet obesity index
1.6 Effect of intraperitoneal GnIH on piglet carcass weight, slaughter rate and backfat thickness
The test method comprises the following steps: after the chronic injection test is finished, the carcass mass of the piglet is weighed by removing internal organs and blood, the dressing percentage (carcass mass (kg)/total weight (kg)) is calculated, and the backfat thickness is measured at a point P2 (the point P2 is located at the circumscribed cross section of the last rib and is 6.5 cm away from the back midline, and symmetrical points on two sides of the back midline are P2 points). The weight of the pig carcass and the thickness of the backfat show the growth and development state of the pig, and the dressing percentage means the carcass weight which can be sold by the live pig and reflects the direct economic value of the live pig. For this reason, the present invention analyzes the ketone body weight and backfat thickness of piglets during the period of the invention to evaluate the influence of the growth performance of piglets injected with the intraperitoneal GnIH.
As shown in Table 5, intraperitoneal injection of both low and high dose intraperitoneal GnIH resulted in significant increases in piglet carcass weight, slaughter rate and backfat thickness (P <0.05) during the period of invention. The test results indicate that the abdominal cavity injection of GnIH can improve the nutrition level of piglets and is beneficial to development and growth. Fig. 4 shows the intraperitoneal injection of GnIH versus piglet fat weight.
TABLE 1-5 piglet Ketone body Mass, slaughter Rate and Back Scale thickness Change
1.7 Effect of Abdominal GnIH on piglet blood indices
The test method comprises the following steps: after the test, the piglets were fixed and subjected to the blood collection in the anterior vena cava, the collected blood was left to stand at 37 ℃ for 4 hours, centrifuged at low speed 2000 Xg for 20min, the upper serum was separated, and the concentrations of Triglyceride (TG), low-density lipoprotein (LDL _ C), Glucose (GLU), Total Cholesterol (TC), high-density lipoprotein (HDL _ C), alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH) and AST/ALT in the serum were measured using a hemobiochemicals.
The blood biochemical analyzer detects the change conditions of related indexes of liver damage, blood fat and blood sugar, and the detection results are shown in tables 6 and 7, and acute and chronic intraperitoneal injection of GnIH can enable the Triglyceride (TG), low-density lipoprotein (LDL _ C) and Glucose (GLU) levels in serum to be remarkably improved (P is less than 0.05) or extremely remarkably improved (P is less than 0.01), while the levels of Total Cholesterol (TC) and high-density lipoprotein (HDL _ C) are not obvious. And GnIH injected into the abdominal cavity both acutely and chronically has no influence on liver injury indexes such as alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH), AST/ALT and the like (P is more than 0.05). In addition, we also observed that chronic intraperitoneal GnIH (tables 1-6) had higher blood glucose and lipid levels than acute intraperitoneal piglets (tables 1-7), as evidenced by a very significant increase in Triglycerides (TG), low density lipoprotein (LDL _ C) and Glucose (GLU) compared to the blank group (P < 0.01). The piglet blood fat and blood sugar levels are obviously increased after the injection of the GnIH into the abdominal cavity, and liver damage is not caused.
Tables 1-6 Biochemical analysis results of acute intraperitoneal GnIH blood
Tables 1-7 Biochemical analysis results of GnIH blood by chronic intraperitoneal injection
1.8 Effect of Abdominal GnIH on the tissue and organ composition of piglets
The test method comprises the following steps: the piglets injected with the GnIH in the chronic abdominal cavity are sacrificed when the experiment is finished, the tissues and organs such as liver, pancreas, spleen, kidney, fat and the like are carefully separated according to the anatomical atlas of the piglets, the positions of the samples are ensured to be consistent, the samples are taken and weighed, then the samples are put into liquid nitrogen for quick freezing treatment, and then the samples are transferred to a refrigerator at the temperature of minus 80 ℃ for storage for later use. To examine what causes the weight gain of chronic peritoneal GnIH infused piglets, we weighed the adipose tissue, organs, as shown in fig. 3 and 4. Significant increases in liver, inguinal fat mass (P <0.05), with a very significant increase in pancreatic mass (P <0.01) were found following chronic intraperitoneal injections of low and high dose intraperitoneal GnIH. Although the fat mass around spleen, kidney and ovary of the piglet is not changed (P > 0.05). Fig. 3 shows organ weight of piglets injected intraperitoneally with GnIH.
The feed intake, daily gain and the like of the Yao chicken within one hour after GnIH is injected into the index packet for representing the growth speed of the Yao chicken;
the indexes for representing the growth quality of the Yao chicken comprise blood indexes and the like.
First, experimental material
The animal model of the test is a clean-grade Nandan Yao chicken, which is 6 weeks old, and has the weight of 850g +/-40 g and 30 chickens, each half of which is male and female. The animal-related tests of the test all obeyed the standards of animal administration and ethics committee of the university of Guangxi (approval No.: 2019-.
Second, main reagent
Chicken GnIH (048-46) was purchased from Phoenix Pharmaceuticals, USA, and prepared with 0.9% by weight of physiological saline. The formulation concentrations of this example include 0.9nmol, 2.6 nmol. Sutai (Zoletil50) was purchased from vicker, france; the test adopts group breeding, and strictly controls temperature (25 + -2 deg.C), humidity (55 + -5%) and illumination (12L: 12D, 7: 00AM on a day) of breeding environment. The animal-related tests of the test all comply with the standards of the animal administration and ethical committee of the Guangxi university experiment.
Third, test method
The injection protocol of the test is divided into acute injection and chronic injection: acute injection is defined as the injection of the dose GnIH only once after adaptive feeding and grouping; whereas chronic injection is defined as the intraperitoneal injection of the above dose GnIH twice daily (7: 00AM and 7: 00PM) for 14 consecutive days after acclimatization and grouping.
Body weight and feeding were recorded daily. Fixing Yao chicken at the end of the test, collecting blood by infrawing vein, standing the collected blood at 37 deg.C for 4h, centrifuging at low speed of 2000 Xg for 20min, separating upper layer serum, and detecting the concentration of Triglyceride (TG), low density lipoprotein (LDL _ C), Glucose (GLU), Total Cholesterol (TC), high density lipoprotein (HDL _ C), alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH) and AST/ALT in the serum by using a blood biochemical analyzer.
And (3) killing the Yao chicken at the end of the test, carefully separating tissues and organs such as liver, pancreas, spleen, kidney, fat, gastrointestinal tract and the like according to the anatomical atlas of the Yao chicken, ensuring the positions of the sampled samples to be consistent, taking a picture, weighing, putting the sampled samples into liquid nitrogen for quick freezing, and transferring the samples to a refrigerator at the temperature of-80 ℃ for storage for later use.
2.1 feed intake of Yao chicken within one hour after chronic intraperitoneal injection of GnIH
As shown in fig. 5, the food intake of the high dose group was significantly different from that of the control group within 14 days of the chronic intraperitoneal injection of GnIH and within 1 hour after the daily injection. On days 4, 7, 8, 9, 12, 13, and 14, the food intake in the high dose group was significantly different from the food intake in the low dose group. Figure 5 shows the feed intake of the Yao chicken within 1 hour after the intraperitoneal injection of GnIH.
2.2 influence of daily feed intake of Yao chicken injected with GnIH in chronic abdominal cavity
As shown in fig. 6, within 14 days of chronic intraperitoneal injection of GnIH, there was a significant difference between the daily food intake of the high dose group and the daily food intake of the control group from day 12 onward. Fig. 6 shows daily food intake of chronic intraperitoneal injection GIH p-Yao chicken.
2.3 influence of daily gain of Yao chicken injected with GnIH in chronic abdominal cavity
As shown in fig. 7, within 14 days of chronic intraperitoneal GnIH injection, the daily gain of the high dose group was significantly different from that of the control group from day 13. Figure 7 shows the daily gain of Yao chicken by chronic intraperitoneal injection of GnIH.
2.4 influence of blood index of Yao chicken injected with GnIH in chronic abdominal cavity
The chronic intraperitoneal injection of GnIH can obviously (P <0.05) or extremely obviously improve (P <0.01) the level of Triglyceride (TG), low-density lipoprotein (LDL _ C) and Glucose (GLU) in serum, and the level of Total Cholesterol (TC) and high-density lipoprotein (HDL _ C) is not obvious.
TABLE 2-1 Biochemical analysis results of GnIH blood of chronic intraperitoneal injection
The index for representing the growth rate of the rat adopted by the invention comprises the feed intake, the feed intake time, the feed intake rate, the feed intake interval, the satiety rate, the average daily gain and the like after a period of fasting;
the indices characterizing the growth quality of rats employed in the present invention include obesity index, triglyceride and total cholesterol concentrations, hypothalamic appetite-related factor expression, and the like.
First, experimental material
The animal model for this experiment was a clean grade SD (Sprague-Dawley) rat, 5 weeks old, weighing 180 + -10 g, 30 animals, each half male and female, purchased from the Experimental animals center of Guangxi medical university. The animal-related tests of the test all obeyed the standards of animal administration and ethics committee of the university of Guangxi (approval No.: 2019-.
Second, main reagent
Rat RFRP-3(048-46) was purchased from Phoenix Pharmaceuticals, USA, and prepared with 0.9% physiological saline by weight. The concentration of the formulation in this example includes 1. mu.g/100. mu.L and 10. mu.g/100. mu.L. Sutai (Zoletil50) was purchased from Fangguke.
Three, main instrument
Infrared night vision cameras (DS-IPC-B12-I) available from HaekWindow; 1mL syringes were purchased from Shanghai Polymu medical devices, Inc.; the full-automatic biochemical analyzer (URIT-8021AVeT) is purchased from Guilin ulite medical electronics, Inc.;
fourth, test method
Feeding and injection protocols
SD rats were housed in acclimatization for 7 days after being purchased and randomly divided into 3 groups (10 animals per group, half of males and females) of a control group (saline 200. mu.L/time), RFRP-3 low dose (1. mu.g/100. mu.LRFRP-3, 200. mu.L/time) and high dose (10. mu.g/100. mu.LRFRP-3, 200. mu.L/time) respectively.
The test adopts single cage breeding, and strictly controls temperature (25 + -2 deg.C), humidity (55 + -5%) and illumination (12L: 12D, 7: 00AM on-lamp every day).
The injection protocol of the test is divided into acute injection and chronic injection: the acute injection is defined as that after the adaptive feeding and grouping, the RFRP-3 dose is injected once; whereas chronic injection was defined as an intraperitoneal injection of RFRP-3 (7: 00AM and 7: 00PM) twice per day for 14 consecutive days after acclimatization and grouping.
(II) recording of ingestion behavior
The effect of intraperitoneal injection of RFRP-3 with different doses on the feeding behavior of rats in a fasting state and a free feeding state is observed and recorded in the experiment respectively, and the specific scheme is as follows.
Rats were given a diet fasting treatment for 8 hours a night before the start of the experiment, fed with feed at a rate of 7: 00AM on the next day, and were intraperitoneally injected with the different doses of RFRP-3, respectively, to each group of rats, and their feeding behavior was recorded using a video camera, and the remaining material was weighed, and the influence of acute intraperitoneal injection of the different doses of RFRP-3 on the 1 st feeding behavior of the rats after the fasting treatment, including the feed intake (g), the feed intake duration (min), and the feed intake rate (feed intake/feed intake time) was analyzed.
In the free feeding experiment, chronic intraperitoneal injection of RFRP-3 is performed in the light and dark environments respectively, the feeding intake (g), the feeding times (times) and the feeding time (min) which are accumulated in a segmented manner within 1 hour, 2 hours and 3-12 hours after injection are recorded, and the feeding behaviors of rats are observed and calculated, wherein the feeding behaviors comprise the feeding rate (feeding intake/feeding time), the feeding time (feeding time/feeding times) of each meal, the feeding interval (total length of recording time-total length of feeding time)/feeding times) and the satiety rate (average feeding interval/average feeding intake) of the rats. Each meal is defined as: the food intake of each meal is more than 0.02g, the food intake time is more than 5s, and the food intake interval is more than 5 min.
(III) determination of obesity and blood lipid indices
Measuring the weight (g) and the nose-anus distance (cm) of each group of rats after anaesthetizing each group of rats before the start and at the end of the chronic intraperitoneal injection RFRP-3 test, and calculating the feed-meat ratio (total feed consumption (g)/total weight gain (g)) and the obesity index (the obesity index is the change of the weight (g) ^ 1/3 multiplied by 1000/nose-anus distance (cm)) during the test; at the end of the test, rats were opened in the chest and subjected to cardiac blood collection, the collected blood was allowed to stand at 37 ℃ for 4 hours, centrifuged at low speed 2000 Xg for 20min, the upper serum was separated, and the concentrations of serum Triglyceride (TG) and Cholesterol (CHOL) were measured using a blood biochemical analyzer.
3.1 Effect of Abdominal RFRP-3 on the 1 st feeding behavior of fasted rats
As shown in table 9, intraperitoneal injection of low-dose and high-dose intraperitoneal RFRP-3 after 8-hour fasting of rats resulted in very significant increases in food intake and food intake time of the fasted rats (P < 0.01); further analysis showed that intraperitoneal injection of low dose intraperitoneal RFRP-3 significantly increased the feeding rate in fasted rats (P <0.05), while intraperitoneal injection of high dose intraperitoneal RFRP-3 very significantly increased the feeding rate (P < 0.01). The above data suggest that intraperitoneal injection of RFRP-3 can further enhance the hunger sensation of the rats in a fasting state. Figure 9 shows the number of feedings of free-feeding rats by intraperitoneal injection of RFRP-3.
TABLE 3-1 Effect of 1 st feeding behavior after fasting of rats intraperitoneally injected with RFRP-3
Note: p <0.05, P <0.01, P < 0.001 in the low dose group of peritoneal RFRP-3 compared to the control group; # denotes P <0.05, # denotes P <0.01, # denotes P < 0.001 in the peritoneal RFRP-3 high dose group compared with the control group, the same applies hereinafter.
3.2 Effect of Abdominal RFRP-3 on feeding behavior of freely fed rats
The feeding behavior of the rat may be changed by different stimulation in the photoperiod, so the invention researches the influence of chronic intraperitoneal injection of different doses of abdominal cavity RFRP-3 on the feeding behavior of the rat in different environments of light and dark respectively. As shown in Table 10, the abdominal cavity injection of high dose of abdominal RFRP-3 only in the light environment resulted in a very significant increase in the feed intake of rats (P < 0.01); intraperitoneal injection of low-dose and high-dose abdominal RFRP-3 in the light and dark environments can cause the feeding frequency and the feeding time of rats to be remarkably increased (P is less than 0.01); further analysis shows that abdominal cavity RFRP-3 with low dose and high dose can both remarkably shorten the feeding interval of rats (P is less than 0.01); in addition, analysis on the satiety rate shows that the abdominal cavity injected with low-dose abdominal cavity RFRP-3 can obviously reduce the satiety rate in a light environment (P < 0.05); while the satiety rate is reduced very significantly in dark environment (P <0.01), the intraperitoneal injection of high dose of intraperitoneal RFRP-3 can cause very significant reduction in satiety rate in light and dark environment (P <0.01), and in conclusion, the increase in the feed intake of rats caused by the intraperitoneal injection of RFRP-3 is likely to be caused by the increase in the feeding frequency, the reduction in the feeding interval and the reduction in satiety rate. FIG. 8 shows the abdominal injection of RFRP-3 for food intake in freely fed rats.
TABLE 3-2 Effect of intraperitoneal injection of RFRP-3 in freely fed rats
3.3 Effect of Abdominal RFRP-3 on rat feeding behavior at different time periods
The above test results 3.2 show that different light stimuli may affect the feeding behavior change caused by the abdominal cavity RFRP-3, but the specific time interval causing the feeding behavior change is not clear. Therefore, the present invention further analyzed the change of feeding behavior of rats at different time periods in light and dark environment, as shown in FIG. 8, the increase of feeding amount of rats was very significant (P < 0.001) only at 3-12 hours after intraperitoneal injection of RFRP-3 in light environment; as shown in FIG. 9, in the statistics of ingestion frequency, there was no significant difference (P >0.05) between 3 and 12 hours after the intraperitoneal injection of the low-dose abdominal cavity RFRP-3 in the light environment, while the intraperitoneal injection of the low-dose abdominal cavity RFRP-3 and the high-dose abdominal cavity RFRP-3 in the rest time periods can induce a very significant increase in ingestion frequency (P < 0.001).
3.4 Effect of Abdominal RFRP-3 on average daily weight gain, average daily feed intake and feed-meat ratio in rats
As shown in Table 11, intraperitoneal injection of both low-dose and high-dose intraperitoneal RFRP-3 significantly increased the average daily gain (P <0.01) of rats; the average daily food intake is increased after the intraperitoneal injection of the low-dose abdominal cavity RFRP-3, but the statistical significance is not generated (P is more than 0.05), but the average daily food intake of the rat can be remarkably increased (P is less than 0.01) by the intraperitoneal injection of the high-dose abdominal cavity RFRP-3; furthermore, the feed meat of the rats during the invention period was significantly reduced (P <0.05) compared to the low dose intraperitoneal RFRP-3 injection, while the high dose was very significantly reduced (P < 0.01).
TABLE 3-3 variation in average daily gain, average daily feed intake and feed-meat ratio in rats
3.5 Effect of Abdominal RFRP-3 on the obesity index of rats
The obesity index is the most effective and intuitive index in evaluating the obesity degree of adult rats, and therefore, the invention analyzes the change of the obesity index of the rats during the invention period to evaluate the influence of the obesity of the rats injected with the RFRP-3 intraperitoneally. As shown in Table 12, intraperitoneal injection of both low and high dose intraperitoneal RFRP-3 significantly increased the obesity index (P <0.05) in rats during the period of invention. The above test results suggest that intraperitoneal injection of RFRP-3 can cause obesity in rats.
TABLE 3-4 changes in the obesity index of rats
3.6 Effect of Abdominal RFRP-3 on the concentration of Triglycerides and Total Cholesterol in rat serum
The results of the biochemical blood indicators are shown in tables 2-6, and chronic intraperitoneal injection of low-dose and high-dose intraperitoneal RFRP-3 can cause the Triglyceride (TG) concentration and the Total Cholesterol (TCHO) concentration in the serum of rats to be remarkably increased (P is less than 0.01).
Tables 3-5 changes in triglyceride and Total Cholesterol concentrations in rat sera
3.7 Effect of Abdominal RFRP-3 on rat hypothalamic appetite-related factor expression
As shown in FIG. 10, intraperitoneal injection of both low and high doses of intraperitoneal RFRP-3 resulted in a significant increase in the abundance of NPYmRNA, a factor associated with appetite promotion in the hypothalamus of rats (P < 0.05); in contrast, the expression abundance of the appetite suppression related factor POMCmRNA was significantly reduced (P < 0.05). The test result indicates that the intraperitoneal injection of RFRP-3 can cause the expression of the rat hypothalamus appetite-promoting related factor to be increased, and inhibit the expression of the appetite-inhibiting related factor, thereby causing the change of the feeding behavior of the rat such as the feed intake, the feeding frequency and the like and the occurrence of obesity. FIG. 10 shows peritoneal RFRP-3 on hypothalamic appetite-related gene expression.
The index for representing the growth rate of the rat adopted by the invention comprises the feed intake, the feed intake time, the feed intake rate, the feed intake interval, the average daily gain and the like after a period of fasting;
the index for characterizing the growth quality of the rat adopted by the invention comprises feed-meat ratio, obesity index, blood index, visceral organ weight and the like.
First, experimental material
The animal model of the test is a clean KM (Kunming) mouse, 8 weeks old, 38g +/-1 g, 20 mice, half male and half female, and is purchased from the experimental animal center of Guangxi medical university. The animal-related tests of the test all comply with the standards of the animal administration and ethical committee of the Guangxi university experiment.
Second, main reagent
Mouse RFRP-3(048-46) was purchased from Phoenix Pharmaceuticals, USA, and prepared with 0.9% physiological saline by weight. The formulation concentration of this example included 10. mu.g/100. mu.L. Sutai (Zoletil50) was purchased from Vickers, France.
Three, main instrument
Infrared night vision cameras (DS-IPC-B12-I) available from HaekWindow; 1mL syringes were purchased from Shanghai Polymu medical devices, Inc.; the full-automatic biochemical analyzer (URIT-8021AVeT) is purchased from Guilin ulite medical electronics, Inc.;
fourth, test method
Feeding and injection protocols
KM mice were bred adaptively for 7 days after being purchased and randomly divided into 2 groups (10 mice per group, half of males and females), a control group (saline 200. mu.L/mouse) and a high dose (10. mu.g/100. mu.L LRFRP-3, 200. mu.L/mouse), respectively.
The test adopts single cage breeding, and strictly controls temperature (25 + -2 deg.C), humidity (55 + -5%) and illumination (12L: 12D, 7: 00AM on-lamp every day).
The injection protocol of the test is divided into acute injection and chronic injection: the acute injection is defined as that after the adaptive feeding and grouping, the RFRP-3 dose is injected once; whereas chronic injection was defined as an intraperitoneal injection of RFRP-3 (7: 00AM and 7: 00PM) twice per day for 14 consecutive days after acclimatization and grouping.
(II) recording of ingestion behavior
The experiment respectively observes and records the influence of intraperitoneal injection of RFRP-3 with different doses on the feeding behavior of mice in a fasting state and a free feeding state, and the specific scheme is as follows.
The mice were given a diet-deprived fasting treatment for 8 hours in the evening before the start of the experiment, the mice were fed with feed at a ratio of 7: 00AM on the next day, and the mice in each group were intraperitoneally injected with the different doses of RFRP-3, respectively, and the feeding behavior was recorded by using a video camera, and the remaining material was weighed, and the influence of acute intraperitoneal injection of the different doses of RFRP-3 on the 1 st feeding behavior of the mice after the fasting treatment, including the feeding amount (g), the feeding duration (min) and the feeding rate (feeding amount/feeding time), was analyzed.
In the free feeding experiment, chronic intraperitoneal injection of RFRP-3 is performed in the light and dark environments respectively, the feeding intake (g), the feeding times (times) and the feeding time (min) which are accumulated in a segmented manner within 1 hour, 2 hours and 3-12 hours after injection are recorded, and the feeding behaviors of the mice are observed and calculated, wherein the feeding behaviors comprise the feeding rate (feeding intake/feeding time), the feeding time (feeding time/feeding times) of each meal, the feeding interval (total length of recording time-total length of feeding time)/feeding times) and the satiety rate (average feeding interval/average feeding intake) of the mice. Each meal is defined as: the food intake of each meal is more than 0.02g, the food intake time is more than 5s, and the food intake interval is more than 5 min.
(III) determination of obesity and blood lipid indices
Measuring the weight (g) and the nose-anus distance (cm) of each group of mice after anesthetizing before the start and at the end of a chronic intraperitoneal injection RFRP-3 test, and calculating the feed-meat ratio (total feed consumption (g)/total weight gain (g)) and the obesity index (the obesity index is the change of the weight (g) ^ (1/3) × 1000/nose-anus distance (cm)) during the test; at the end of the test, the thoracic cavity of the mouse was opened and the heart was sampled, the sampled blood was left standing at 37 ℃ for 4 hours, centrifuged at low speed 2000 Xg for 20min, the upper serum was separated, and the concentrations of serum Triglyceride (TG) and Cholesterol (CHOL) were measured using a blood biochemical analyzer.
4.1 Effect of intraperitoneal RFRP-3 on the 1 st feeding behavior of fasted mice
As shown in table 1, intraperitoneal injection of RFRP-3(20 μ g/100 μ L) after 8-hour fasting of mice resulted in a very significant increase in food intake and food intake time of fasted mice (P < 0.01); further analysis showed that intraperitoneal injection of RFRP-3 (20. mu.g/100. mu.L) significantly increased the mouse feeding rate (P < 0.01). Figure 12 shows the number of times the mice fed free feeding were given intraperitoneal injections of RFRP-3.
TABLE 4-1 Effect of 1 st feeding behavior after fasting of mice injected intraperitoneally with RFRP-3
Note: p <0.05, P <0.01, P < 0.001 in the peritoneal RFRP-3 group compared to the control group;
4.2 Effect of Abdominal RFRP-3 on the feeding behavior of mice at different time periods
There is literature indicating that mice prefer feeding activities at night. Therefore, different lighting stimuli may affect the feeding behavior change caused by the abdominal cavity RFRP-3, but the specific time period caused by the feeding behavior change is not clear. Therefore, the invention further analyzes the change of the feeding behavior of the mice in different time periods under the light and dark environments. As shown in FIG. 11, the increase in feed intake of mice was significantly different only at 2 hours and 3-12 hours after intraperitoneal injection of RFRP-3 in a dark environment (P < 0.05); as shown in FIG. 12, in the statistics of ingestion frequency, the ingestion frequency can be induced to be increased significantly (P <0.05) in 24 hours all day after the intraperitoneal injection of RFRP-3. Likewise, it was found when the 24-hour feeding behavior was counted. Intraperitoneal injection of RFRP-3(20 mug/100 muL) can significantly improve the total food intake and the times of food intake of the mice in 24 hours all day. FIG. 11 shows the food intake of free-feeding male mice injected intraperitoneally with RFRP-3.
4.3 Effect of Abdominal RFRP-3 on average daily gain, average daily feed intake and feed-meat ratio of mice
As shown in tables 1-4, the average daily gain of mice (P <0.01) can be remarkably increased by injecting RFRP-3(20 mug/100 muL) into the abdominal cavity; the injection of RFRP-3(20 mug/100 muL) into the abdominal cavity can also remarkably increase the average daily food intake of the mice (P is less than 0.01); in addition, the feed meat of the mice was very significantly reduced during the invention period compared to the intraperitoneal injection of the high dose (P < 0.01).
TABLE 4-2 changes in average daily gain, average daily feed intake and feed-meat ratio in mice
Note: indicates abdominal cavity GnIH and its homologue RFRP-3 group, P <0.05, P <0.01, P < 0.001, compared to control group.
4.4 Effect of Abdominal RFRP-3 on the mouse obesity index
The obesity index is the most effective and intuitive index in evaluating the obesity degree of adult mice, and therefore, the invention analyzes the change of the obesity index of the mice during the invention period so as to evaluate the influence of the obesity of the mice injected with the RFRP-3 in the abdominal cavity. As shown in tables 1-7, intraperitoneal injection of RFRP-3 (20. mu.g/100. mu.L) resulted in a significant increase in the obesity index (P <0.05) in mice during the period of the invention. The above test results suggest that intraperitoneal injection of RFRP-3 can cause obesity in rats.
TABLE 4-3 changes in the obesity index of mice
Note: p <0.05, P <0.01, P < 0.001 in the peritoneal RFRP-3 group compared to the control group.
4.5 Effect of Abdominal RFRP-3 on blood indices of mice
Acute and chronic intraperitoneal injection of RFRP-3 can ensure that the levels of Lactate Dehydrogenase (LDH), low-density lipoprotein (LDL _ C) and Glucose (GLU) in serum are remarkably increased (P is less than 0.05) or extremely remarkably increased (P is less than 0.01), and the levels of Total Cholesterol (TC) and high-density lipoprotein (HDL _ C) are not obvious. No obvious liver damage. Table 17 shows the results of biochemical blood analysis of GnIH and its homologue RFRP-3
TABLE 4-4 Biochemical analysis results of chronic intraperitoneal injection abdominal RFRP-3 blood
4.6 Effect of Abdominal RFRP-3 on visceral organs of mice
After sacrifice, internal organs were dissected and weighed. As can be seen from FIG. 13, intraperitoneal injection of RFRP-3 (20. mu.g/100. mu.L) resulted in a significant increase in the weight of mouse liver and reproductive fat, while a significant decrease in the weight of testis (P < 0.05). Among them, spleen and groin fat weight increased and brown fat weight decreased, but the results were not statistically significant. Figure 13 shows abdominal cavity RFRP-3 versus mouse visceral mass morphology.
The technical features of the above-mentioned embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above-mentioned embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the spirit of the invention, which falls within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. An application of abdominal cavity GnIH and its homologue RFRP-3 in increasing animal growth speed is provided.
2. The use of the intraperitoneal GnIH and its homologue RFRP-3 according to claim 1, for increasing the growth rate of an animal, wherein said growth rate is characterized by an increased feed intake of the animal.
3. The use of the intraperitoneal GnIH and its homologue RFRP-3 according to claim 1, to increase the growth rate of an animal, wherein said growth rate is characterized by an increased feeding frequency of the animal.
4. The use of the intraperitoneal GnIH and its homologue RFRP-3 according to claim 1, to increase the growth rate of an animal, wherein said growth rate is characterized by an increased feeding time of the animal.
5. The use of the intraperitoneal GnIH and its homologue RFRP-3 according to claim 1, to increase the growth rate of an animal, wherein said growth rate is characterized by a shortened feeding interval of the animal.
6. The use of the intraperitoneal GnIH and its homologue RFRP-3 according to claim 1, for improving the growth rate of an animal, wherein said growth rate is characterized by an increase in the average daily gain of the animal.
7. An application of abdominal cavity GnIH and its homologue RFRP-3 in improving animal growth quality is provided.
8. The use of the intraperitoneal GnIH and its homologue RFRP-3 according to claim 7, for improving the growth quality of an animal, wherein said growth quality is a decrease in the feed-meat ratio of the animal.
9. The use of the intraperitoneal GnIH and its homologue RFRP-3 according to claim 7, for improving the growth quality of an animal, wherein said growth quality is an increase in the animal's obesity index.
10. The use of the intraperitoneal GnIH and its homologue RFRP-3 according to claim 7, for improving the growth quality of an animal, wherein said growth quality is an increase in carcass mass of the animal.
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| Title |
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| BETTY MCCONN等: "Gonadotropin-inhibitory hormone-stimulation of food intake is mediated by hypothalamic effects in chicks" * |
| TETSUYA TACHIBANA等: "Gonadotropin-inhibiting hormone stimulates feeding behavior in chicks" * |
| 霍孔林等: "RFRP-3 对大鼠采食行为和生长性能的影响" * |
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