No mortality was found in any groups treated with marine collagen peptides preparation (MCP) for 12 months. In the 24-month study, the survival rates of 0%, 2.25%, 4.5%, 9% and 18% MCP-treated groups were 55%, 70%, 55%, 60% and 60% for females, and 45%, 65%, 65%, 70% and 60% for males, respectively. There was no statistically significant difference noted in the survival analysis between the control group and the MCP-treated groups (P > 0.05) (Figure 1).

There was no remarkable difference in general conditions between the vehicle-treated and MCP-treated groups. As shown in Figure 2, there was an age-related tendency to increase bodyweight of the same group. Meanwhile, the bodyweight gain significantly decreased after 18-month MCP treatment. For food consumption, there was a remarkable age-related decreasing trend in each group; while no dose-related change in food consumption was found in either sex when the animals were treated with MCP for 12 months or 24 months.

Moreover, the food efficiency in the same group significantly decreased with age (Table 1). Throughout the experimental period, since no statistical difference or dose-dependent tendency of body weight and food efficiency was found between the control and MCP-treated groups in either sex, the actual daily intake of the test substance showed a good correlation with its dietary concentrations in different groups. During the 24 months treatment, MCP intake of 2.25%, 4.5%, 9% and 18% MCP-treated groups were estimated to be 1.063, 2.216, 4.609 and 8.586 g/kg·bw/day for females, and 0.907, 1.798, 3.418, 6.658 g/kg·bw/day for males, respectively (Table 2).

As shown in Table 3, no dose-related change or statistical difference was revealed from the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total protein (TP) and blood urea nitrogen (BUN) after 12-month and 24-month treatment. When animals were treated with MCP for 24 months, the levels of creatinine (CR) non-significantly increased in 9% and 18% male groups as compared with the control group (P > 0.05). Additionally, a significant increase in serum TP level was observed in 9% MCP-treated male group (P < 0.05), whereas no significant increase of serum TP level was found in female MCP-treated groups versus the control group. While the increase of serum TP level in 9% MCP-treated male group was considered to be incidental and not considered treatment-related for the absence of significant dose dependence.

After 12 months treatment, the significantly decreased serum triglyceride (TG) levels were observed in 2.25% and 4.5% MCP-treated female groups as compared with the female controls (P < 0.05), whereas the serum TG levels of male groups did not differ significantly between the control and MCP-treated groups (P > 0.05). Moreover, there was no MCP-related effect on the serum levels of total cholesterol (TCHOL) in both sexes (P > 0.05). After 24 months of MCP treatment, significantly lower serum levels of TG were observed in 4.5% and 9% MCP-treated female groups as well as in all MCP-treated male groups when compared with the control group (P < 0.05). Similarly, the serum levels of TCHOL in 4.5%, 9% and 18% MCP-treated male groups markedly decreased as compared with the control male group (P < 0.05), while no significant inter-group difference was observed in the female groups (P > 0.05). In addition, no significant difference in the levels of serum glucose (GLU) between the control and MCP-treated groups was revealed from the 12 months and 24 months evaluation data (Table 3).

No MCP-related effect was evident in urinalysis parameters and microscopic examination of urine sediment. Urine volume, pH and specific gravity were all within normal limits (data not shown).

The variability in the organ weights was due to the presence of gross findings such as mass, atrophy, swollen, fibrosis, enlarged and hyperemia area [13]. After 24 months of MCP administration, no significant MCP-related difference was indicated from the relative organ weights of brain, heart, lungs, liver, spleen, kidneys, adrenal glands, testis and ovaries (Table 4).

Upon histological examination, data of non-neoplastic lesions are presented in Table 5. Most of the lesions related to inflammatory changes and age-related degenerations (fibrosis, emphysema, atrophy and hyperplasia) were found in all groups. Compared with the control group, the incidence of hepatocyte fatty vacuolation decreased markedly in MCP-treated groups of both sexes, while no significance was indicated (P > 0.05). No other significantly increased incidence of pathologic change or lesion was observed in MCP-treated groups as compared with the control group (P > 0.05).

Marine collagen peptides preparation (MCP) provided by CF Haishi Biotechnology Ltd. Co. (Beijing, China) was prepared from the skin of wild-caught Chum Salmon (O. keta) (from the East China Sea, average body weight, 1.47 kg). The procedure of MCP preparation and identification was according to the method described in our previous research [23]. Briefly, the fish skin was cleaned, scaled, cut into small pieces, defatted and homogenized. After being homogenized and emulsified in distilled water, the material was enzymatically hydrolyzed by complex protease (3000 U/g protein), which including 7% of trypsin, 65% of papain and 28% of alkaline proteinase, at 40 °C and pH 8 for 3 h. The resultant hydrolysate was re-extracted by centrifugation and subsequently separated through ceramic membrane (200 µm). After being purified through a procedure of nanofiltration, condensed by cryoconcentration under vacuum, decolorized with active carbon, filtrated and dried by spray drying, MCP powder used in the following investigations was obtained.

The MCP sample contains about 90% hydrolyzed protein, 6.0% ash, 1.4% carbohydrate, 2.5% water and 0.1% fat. Then the molecular weight distribution of the samples were analyzed by high-performance liquid chromatography (HPLC, Waters Corp., Milford, MA, USA) and ascertained by LDI-1700 matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Liner Scientific Inc., Reno, NV, USA).The amino acid composition was analyzed with an amino acid analyzer (H835-50; Hitachi, Tokyo, Japan). In addition, the amino acid composition was further analyzed by an H835-50 automatic amino acid analyzer (Hitachi, Tokyo, Japan). The analysis of MCP indicated 86.5% of the molecular weights distributed between 130 and 1000 Da. The results of amino acid composition in Table 6 show that MCP is rich in Glycine > Glutamic acid > Proline > Hydroxyproline > Aspartic acid > Alanine > Arginine.

Young weaning S-D rats, four-week-old, from both sexes, weighing 80~90 g, were acquired from the Animal Service of Health Science Center, Peking University. Rats were housed two per plastic cages with free access to chow and tap water, and maintained in a filter-protected air-conditioned room with controlled temperature (25 ± 2 °C), relative air humidity (60 ± 5%) and 12 h light/dark cycles (light on 07:30–19:30 h). All animals were handled in accordance with the guidelines of the Principle of Laboratory Animal Care [24] and the guidelines of the Peking University Animal Research Committee.

After a one-week acclimation period, 200 rats were randomly assigned to five groups (20 animals/sex/group), i.e., vehicle control group and four experimental groups. Control rats were fed with the basal rodent diet (GB14924.3-2001, Vital River Limited Company, Beijing, China). The basal diet contained about 25% protein, 4% fat, 5% fiber and 60% carbohydrate. Rats in the experimental groups were fed with 2.25%, 4.5%, 9% and 18% (wt/wt) MCP in the control diet, respectively. Here, compared with the control diet, 2.25%, 4.5%, 9% and 18% (wt/wt) crude protein in the MCP-added diets of four experimental groups were respectively reduced to keep the constant dietary protein.

Clinical investigations included observations of daily general condition, bodyweight, food consumption, food efficiency, clinical biochemical examinations and urinalysis.

All animals were observed three times daily, at 08:00, 14:00 and 20:00 h, respectively, for mortality and moribundity. General condition observations included changes in the skin, fur, eyes, somatomotor activity and behavior.

Animals were single-housed. Specific amount of fresh diet was provided to each animal weekly. The remainder diet and spilled diet were measured weekly to calculate the actual food intakes of individual animal. The food consumption value (g/kg·bw/day) is the ratio of the mean daily food intake of individual animal and mean bodyweight in the individual period of observation. The food efficiency (g/100 g) of individual animals for individual intervals was the average value of bodyweight gain from the per 100 g food intake. The actual food intake and bodyweight of individual animal were recorded weekly in the first six months and every two weeks thereafter until sacrifice. Food consumption and food efficiency calculations for individual intervals and individual animals were summed consecutively to calculate overall food consumption and food efficiency.

Blood samples for serum chemistry evaluations were collected from the lateral tail vein of the ten survival animals/group/sex every three months during the study. To avoid of selection bias, the first ten animals were selected according to their serial numbers in the order from small to large. The animals were fasted overnight prior to the collection of blood samples. The clinical biochemical parameters included alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CR), triglyceride (TG), total cholesterol (TCHOL) and glucose (GLU), which were determined using an automatic biochemistry analyzer of Olympus AU400 (Olympus Optical Co., Ltd., Tokyo, Japan).

Urine routine examinations were conducted when animals were treated with MCP for 12 months and 24 months. The first ten survival animals of each group were placed in metabolic cages to collect the urine of 24 h. Urine was examined for volume, color, pH and specific gravity, as well as for levels of protein, ketone, urobilirubin, urobilinogen, glycosuria, blood and nitrite. Microscopic examination of the urine sediment was conducted to assess possible changes in the amount of epithelial cells, leukocytes, erythrocytes, cylindrical and crystalline precipitates.

All animals in moribund status during the treatment period were immediately sacrificed by exsanguination and subjected to full necropsies. The skin, all the internal organs, and the cranial, thoracic, abdominal and pelvic cavities were examined to detect any lesion. There were no interim scheduled autopsies. Treated with MCP for 24 months, the remaining surviving animals were all subjected to complete gross necropsy.

At the time of necropsy, the following tissues and organs were collected and fixed in 10% buffered formalin solution: brain, pituitary, heart, lungs, liver, spleen, kidneys, adrenal glands, stomach, intestine, pancreas, urinary bladder, skin, mammary gland, trachea, esophagus, thyroid gland, femoral muscle, epididymis, seminal vesicles, prostate gland, testis, uterus, ovary, vagina and eyes. Then the fixed tissue samples were embedded in paraffin and sectioned at 5 µm. After being stained with hematoxylin and eosin, the slides were examined with an Olympus BH2 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) to detect any lesions. The organs, i.e., brain, heart, lungs, liver, spleen, kidneys, adrenal glands, testis and ovaries of the first ten survival animals/group/sex were trimmed of extraneous fat and weighed to calculate the relative organ weights (the organ to final body weight ratio). Paired organs were weighed together.

Statistical analyses were performed using SPSS (Version 13.0, SPSS Inc.). Variances in the measurement data for bodyweights, food efficiency, food consumption, clinical biochemistry parameters and organ weights were checked for homogeneity by the Bartlett’s test. When the variance data were homogeneous, the one-way analysis of variance (ANOVA) test and multiple comparison of Dunnett’s t-test were used. The heterogeneous cases were analyzed with Kruskal-Wallis rank sum test. The results of incidences of pathologic changes or lesions were compared using Chi-square test and the Fisher’s exact probability test. The survival data were analyzed using Kaplan-Meier survival method. The dose-dependent tendency was analyzed using Spearman rank correlation test. A p value < 0.05 was considered significant.