A total of 197 blood samples were collected from pure and mixed breed dogs (small and medium to large size). Fifteen blood samples were excluded from the biochemical analysis: nine lipemic, four hemolyzed, and two with jaundice. In addition, seven observations were excluded from the statistical analysis by subclinical disease. Table 2 shows the mean, standard deviation, median, interquartile range, and significant difference with a 95% confidence level. It also shows a lower and upper limit of the reference intervals and the limits of reference calculated with a 90% confidence interval. Data from all the dogs were divided into four age groups: 4-8, 9-24, 25-52, and > 52 weeks of age.
This study evaluated the effect of age, sex, and breed on biochemical variables serum (AST, ALT, LDH, ALP, GGT, total protein, albumin, globulins, cholesterol, triglycerides, glucose, urea, and creatinine). Additionally, those effects on body temperature (rectal thermometer), heart rate, and respiratory rate were evaluated. In some response variables, we observed statistically significant differences.
Effect of age
No significant differences were observed in the results for AST concerning age (F (3,158) = 0.59, p = 0.62). On the other hand, the enzymatic activity of ALT had a significant effect (F (3,158) = 22.11, p = 0.00). The average of ALT in puppies from 4-8 wk of age was 26 U/L (SD = 8.8), significantly lower than the enzymatic activity in dogs from 9-24 wk of age (p = 0.01), 25-52 wk of age, and >52 wk of age (p = 0.00). Moreover, the activity was significantly lower in dogs from 9-24 wk of age (M = 32 U/L, SD = 9.1) than the adult's group IV (p = 0.00). Furthermore, ALT in young dogs of 25-52 wk of age (M = 37 U/L, SD = 7.6) was significantly similar to the enzymatic activity in adult dogs > 52 wk of age (M = 44 U/L, SD = 12.6; p = 0.17). Therefore, the ALT levels were lower in puppies of 4-24 wk of age; from 25 wk of age, the enzymatic activity is the same as that of adults (Figure 1).
The enzymatic activity of LDH had a significant effect on age (F (3,158) = 19.44, p = 0.00). The average of LDH in puppies from 4-8 wk of age was 244 U/L (SD = 164.6), significantly higher than the enzymatic activity in groups II (M = 73 U/L, SD = 32.4), III (M = 63 U/L, SD = 32), and IV (M = 67 U/L, SD = 42) p = 0.00. Our research showed that LDH decreases as age increases, and the values stabilize at 9 wk of age (Figure 1).
The enzymatic activity of ALP had a significant effect on age (F (3,158) = 165.04, p = 0.00). The average of ALP in puppies from 4-8 wk of age was 215 U/L (SD = 71.6), significantly greater than the enzymatic activity of groups III and IV (p = 0.00). The ALP of group II (M = 193 U/L, SD = 39.2) also showed an activity significantly higher than the one of groups III and IV (p = 0.00). Finally, the ALP enzymatic activity of group III (M = 85 U/L, SD = 36.7) is significantly greater than that of group IV (M = 52 U/L, SD = 23.3; p = 0.00). These results suggest that ALP enzymatic activity decreases as the age of dogs increases. The serum ALP activity at 4-24 wk of age in puppies is four times higher than that in adults; in young dogs from 25-52 wk of age, the ALP activity is almost two times higher than the activity in adults (Figure 1). On the other hand, the enzymatic activity of the GGT did not have a significant effect on age (F (3,158) = 1.33, p = 0.26).
The concentration of total proteins showed a significant effect on age (F (3,158) = 32.21, p = 0.00). The average of serum total proteins in puppies from 4-8 wk of age (M = 4.6 g/dL, SD = 0.6) was significantly lower than the one in dogs from 9-24 wk of age (p = 0.02), 25-52 wk of age, and >52 wk of age (p = 0.00). Furthermore, that average was significant lower in dogs from 9-24 wk of age (M = 5.1 g/dL, SD = 0.7) compared to adults of group IV (p = 0.00). Finally, the concentration of group from 25-52 wk of age (M = 5.7 g/dL, SD = 0.8) was lower than in dogs of >52 wk of age (M = 6.2 g/dL, SD = 0.9; p = 0.03). Therefore, these results show that the total serum proteins in puppies from 4-8 wk of age are low while in dogs from 9-52 wk of age begin to increase until they stabilize after 52 wk of age (Figure 2).
The concentration albumin also had a significant effect on age (F (3,158) = 21.38 p = 0.00). The albumin levels in the group I (M = 2.5 g/dL, SD = 0.7) were significantly lower than the levels of groups II (M = 2.7 g/dL, SD = 0.3; p = 0.01), III (M = 3.1 g/dL, SD = 0.3), and IV (M = 3.1 g/dL, SD = 0.4) p = 0.00. From group II, puppies from 9-24 wk of age, the concentration was significantly lower than that of group IV (p = 0.00). These results show that serum albumin concentration increase as the dog's age advances. The albumin concentration is low in puppies from 4-8 wk of age while in dogs from 9 weeks of age begins to increase; however, until 25 weeks of age, the albumin concentration stabilizes at adult values (Figure 2).
Regarding the concentration of globulins, a significant effect on age was observed (F (3,158) = 12.89, p = 0.00). The average of serum globulins in puppies from 4-8 wk of age was 2.1 g/dL (SD = 0.6) and 2.3 g/dL (SD = 0.6) in puppies from 9-24 wk of age; significantly lower than that in the dogs from >52 wk of age (M = 3 g/dL, SD = 0.7; p = 0.00). The average of globulins in young dogs from 25-52 wk of age was 2.6 g/dL (SD = 0.6): the same as in groups I (p = 0.25), II (p = 0.94), and IV (p = 0.06). Therefore, these results indicate that the serum levels globulins in puppies from 4 to 24 wk of age are low whereas in dogs from 25 wk of age begin to increase the levels until they stabilize after 52 wk of age (Figure 2).
On the other hand, there were no statistically significant differences regarding age in the results of cholesterol (F (3,158) = 1.49, p = 0.22) and triglycerides (F (3,158) = 2.52, p = 0.06). Whilst, the concentration of glucose showed an effect on age (F (3,158) = 4.14, p = 0.01). This concentration was higher in puppies from 4-8 wk (M = 89 mg/dL, SD = 21, p = 0.02) and from 9-24 wk of age (M = 90 mg/dL, SD = 18, p = 0.00) than the levels observed in adults > 52 wk (M = 77 mg/dL, SD = 21). This result suggests that there is a decrease in glucose concentration as the age of the dog increases. In addition, it was established that puppies reached the glucose levels of an adult at 25 wk of age (M = 81 mg/dL, SD = 21) (Figure 2).
Our research evince an effect of age on the serum urea (F (3,158) = 14.84 p = 0.00). The urea concentration was significantly lower in puppies from 4-8 wk of age (M = 23 mg/dL, SD = 7) compared to that from group II (p = .047), III, and IV (p = 0.00). Similarly, puppies from 9-24 wk of age (M = 27 mg/dL, SD = 8.7) showed significantly lower urea levels than dogs from group IV (p = 0.00). Furthermore, urea in young dogs from 25-52 wk of age (M = 36 mg/dL, SD = 9.5) was equal to that of adult dogs > 52 wk of age (M = 34 mg/dL, SD = 10; p = 0.99). Therefore, the urea concentration was lower in puppies from 4-24 wk of age compared to adults, and it stabilized in week 25 (Figure 3).
Regarding the concentration of creatinine, it showed an effect on age (F (3,158) = 78.92 p = 0.00). In puppies from 4-8 wk of age (M = 0.45 mg/dL, SD = 0.09), the creatinine levels were lower than the concentration from groups II, III, and IV (p = 0.00). Creatinine levels in puppies from 9-24 wk of age (M = 0.59 mg/dL, SD = 0.16) were lower than those from groups III and IV (p = 0.00). Furthermore, creatinine in young dogs from 25-52 wk of age (M = 1.00 mg/dL, SD = 0.30) was significantly similar to the concentration in adult dogs > 52 wk of age (M = 1.03 mg/dL, SD = 0.25; p = 0.81) while creatinine levels in puppies from 4 -24 wk of age were lower. from 25 wk of age, the concentration is the same as that of adults (Figure 3).
On the other hand, in body temperature, a significant effect on age was observed (F (3,158) = 18.62, p = 0.00). Body temperature in puppies from 4-8 weeks of age (M = 37.9° C, SD = 0.5) is lower than the temperature obtained from groups II, III (M = 38.7° C, SD = 0.4), and IV (M = 38.8° C, SD = 0.4) p = 0.00. Furthermore, in puppies from 9-24 weeks of age (M = 38.5° C, SD = 0.6), their body temperature is lower than that of adults > 52 weeks of age (p = 0.01). These results indicate that dogs from 4 to 24 weeks of age have a lower body temperature. In dogs from 25 weeks of age, the temperature begins to increase and it is similar to that from adults > 52 weeks of age (p = 0.53) (Figure 4).
Regarding heart rate, there was a significant effect of age (F (3,158) = 5.44, p = 0.00). In puppies from 4-8 (M = 155 bpm, SD = 28.4; p = 0.00) and from 9-24 (M = 145 bpm, SD = 33; p = 0.01) weeks of age the heart rate was higher than that of adults > 52 weeks (M = 74 bpm, SD = 33). Additionally, in the group from 25 weeks of age, the heart rate was similar to that of adults (p = 0.86). Therefore, our study shows a decrease in the heart rate as the animal's age increases (Figure 4). On the other hand, there was no statistically significant difference in respiratory rate (F (3,158) = 1.21, p = 0.31).
Effect of sex
No significant effect of sex was observed on the enzymatic activity of AST (F (1,158) = 2.98, p = 0.09), ALT (F (1,158) = 1.24, p = 0.28), LDH (F (1,158) = 1.74, p = 0.19), ALP (F (1,158) = 2.75, p = 0.10), and GGT (F (1,158) = 0.12, p = 0.73). Neither was there effect of sex on the serum concentration of total proteins (F (1,158) = 0.14, p = 0.70), albumin (F (1,158) = 0.45, p = 0.50), globulins (F (1,158) = 0.62, p = 0.43), cholesterol (F (1,158) = 0.44, p = 0.51), triglycerides (F (1,158) = 1.11, p = 0.29), glucose (F (1,158) = 0.02, p = 0.90), urea (F (1,158) = 0.50, p = 0.48), and creatinine (F (1,158) = 1.40, p = 0.24). Physiological constants did not have either an effect on sex: body temperature (F (1,158) = 0.71, p = 0.40), heart rate (F (1,158) = 2.86, p = 0.09), and respiratory rate (F (1,158) = .80, p = 0.37).
Effect of breed
From all the variables evaluated, the effect of breed showed statistically noteworthy differences in creatinine concentration (F (3,158) = 4.00, p = 0.01). The average creatinine in small breed dogs was 0.73 mg/dL (SD = 0.31): significantly lower than that in medium-large breed dogs (M = 0.92, SD = 0.33, p = 0.01). Nevertheless, the other variables did not show significant effects.
No statistical major differences were observed in enzymatic activity of AST (F (3,158) = 1.34 p = 0.26), ALT (F (3,158) = 0.87, p = 0.46), LDH (F (3,158) = 2.61, p = 0.05), ALP (F (3,158) = 1.32, p = 0.27), and GGT (F (3,158) = 0.77, p = 0.51). There was also no effect of breed on the serum concentration of total proteins (F (3,158) = 0.21, p = 0.88), albumin (F (3,158) = 0.50, p = 0.68), globulins (F (3,158) = 0.59, p = 0.63), cholesterol (F (3,158) = 2.39, p = 0.07), triglycerides (F (3,158) = 1.85, p = 0.14), glucose (F (3,158) = 1.36, p = 0.26), and urea (F (3,158) = 1.36 p = 0.26). Neither did breed have an effect on Physiological constants: body temperature (F (3,158) = 0.47, p = 0.70), heart rate (F (3,158) = 1.94, p = 0.13), and respiratory rate (F (3,158) = 1.77, p = 0.15).
Age, sex and breed interaction
No statistical remarkable effects were observed in enzymatic activity of AST (F (9,158) = 0.76, p = 0.65, ALT (F (9,158) = 0.87, p = 0.55), LDH (F (9,158) = 1.41, p = 0.19), ALP (F (9,158) = 0.44, p = 0.91), and GGT (F (9,158) = 1.54, p = 0.14). There was not effect of interaction on the serum concentration of total proteins (F (9,158) = 0.81, p = 0.61), albumin (F (9,158) = 0.78, p = 0.64), globulins (F (9,158) = 0.77, p = 0.64), cholesterol (F (9,158) = 0.79, p = 0.62), triglycerides (F (9,158) = 1.29, p = 0.25), glucose (F (9,158) = 0.54, p = 0.84), urea (F (9,158) = 1.92, p = 0.05) and creatinine (F (9,158) = 0.79, p = 0.63). Body temperature (F (9,158) = 0.69, p = 0.71), heart rate (F (9,158) = 0.55, p = 0.84), and respiratory rate (F (9,158) = 0.74, p = 0.67) did not show effects either.