M-Mode Echocardiographic Changes in Growing Beagles

Abstract

Heart growth in 6 female beagle dogs was measured by using M-mode echocardiography at 4, 7, 10, 13, 17, and 21 mo of age. The same 6 dogs were evaluated throughout the study to establish when cardiac development ends in this breed. The following parameters were measured during systole and diastole: left ventricle posterior wall thickness, interventricular septal thickness, left ventricular internal dimension, left atrial dimension during ventricular systole, aortic root dimension at end diastole, E-point to septal separation, left ventricular preejection period, ejection time of the left ventricular outflow, and time between the cessation and onset of the mitral inflow intervals. The percentage of the left ventricle posterior wall thickening, fractional shortening, ejection fraction, left ventricular end systolic and end-diastolic volumes, ratio of the left atrial dimension to aortic root dimension, and the Tei index of myocardial performance were calculated. The heart rate was measured by cardiac auscultation. The influence of ageing on each echocardiographic parameter and relationships with body weight and surface were studied. Results show that cardiac development in female beagles can be considered finished by the age of 1 y, perhaps as soon as 7 mo. The cardiac indexes studied were unaffected by the age and corporal dimensions, confirming the usefulness of these parameters for evaluating cardiac functionality alterations independent of a dog's age and body weight or surface area.

Because beagles are used in cardiovascular research as laboratory animals, knowledge about their echocardiographic reference values is necessary to evaluate possible changes in cardiac function. Several studies have established reference values in healthy beagles with specific ages, including 7-, 13-, and 20-mo-old dogs.^6,10 However, only 1 study addresses the cardiac changes evaluated by ultrasonography during the first growth period of this particular breed (from 1 to 3 mo of age).¹

Although changes in the linear dimensions of the heart during the growth period in English pointer dogs have been evaluated to establish their relationship with body weight,²¹ no similar information is available regarding age-related changes in the cardiac dimensions and function of beagles, particularly the age at which cardiac development concludes.

Many studies have been developed to establish the relationship between cardiac dimensions and body weight. Body weight, breed, somatotype, and even strain are factors that can affect cardiac dimensions and function.^{7,10,14,16,17,24} In some studies, the sex of the animal has no significant effect on cardiac dimensions,²⁴ whereas others reported significant sex-associated differences in various cardiac dimensions in several breeds,^11,13,15 including beagles.^6,10 Bearing this information in mind, we have carried out work in the beagle, studying only female dogs to avoid interferences caused by possible effects of sex-associated differences and paying special attention to the effect of age of the animals and growth indexes, specifically body weight and surface area.

The aims of this work were to study the changes in various echocardiographic measurements that take place during growth and to establish the moment at which the cardiac development was finished, from an echocardiographic point of view, in female beagles. To this end, the same group of animals has been examined throughout the growth period.

Materials and Methods

Animals.

We studied 6 female beagle dogs that were 4 mo of age at the beginning of the examination period. The animals were bred in the facilities at the Department of Medicine, Surgery, and Anatomy (University of León, León, Spain). The breeding dogs were acquired from the Laboratory Animal Facility of the Department of Animal Physiology (University Complutense, Madrid, Spain; registry no. EX0114). All animal care and husbandry procedures were in accordance with the European Council Directive and Spanish Real Decreto,^5,19 both of which regulate the protection of animals used for experimental and other scientific purposes. The experimental protocol received approval from the Ethical Committee of the University of León.

Husbandry during experiment.

After weaning, the animals were accommodated in a conventional kennel unit; immunized against parvovirus, distemper, hepatitis, and leptospirosis (Tetradog, Merial Laboratorios, Barcelona, Spain); and treated prophylactically with antihelminthics (Milbemax, Novartis Sanidad Animal, Barcelona, Spain). The animals were socialized before the beginning of the experimental procedures by means of positive contact with the investigators and positive restraint in the exploration room.

Feeding.

Dogs received a commercial feed for puppies until they were 12 mo old and then a commercial feed for adult dogs (Medium Junior and Medium Adult, Royal Canine, Aimargues, France) and water ad libitum.

Experimental procedures.

The animals underwent a complete physical examination before beginning the study and when echocardiographic examinations were performed. We paid particular attention to the cardiovascular and respiratory systems. To verify the absence of cardiac murmurs or abnormal rhythms, cardiac auscultation was performed after the dogs remained in a quiet room for 5 min. Thoracic radiographs in lateral and dorsoventral recumbent positions, resting electrocardiography, and complete echocardiography (B- and M-modes) were performed, and no cardiovascular anomalies were detected.

Echocardiographic examinations of the dogs were performed at 4, 7, 10, 13, 17, and 21 mo of age. The animals were sedated with 0.03 mg/kg acepromazine (CalmoNeosan, Pfizer, Madrid, Spain) and 0.0075 mg/kg buprenorphine (Buprex, Shearing Plough, Madrid, Spain) administered intramuscularly. All examinations (Logic 500 MD, General Electric, Chicago, IL) were performed in a dark room by the same operator, using a 5-MHz microconvex probe and continuous electrocardiographic monitoring. Examinations were performed in the right lateral recumbent position on an echocardiography table, and ultrasonic gel was used to improve the contact of the probe with the skin. Heart rate was measured by cardiac auscultation after echocardiographic examinations.

Motion (M)-mode echocardiographic measurements were recorded simultaneously with a bidimensional (B)-mode real-time display and in the right parasternal view according to the recommendations of the American Society of Echocardiography.^20,23 For each parameter, 3 measurements were made during 3 consecutive heart cycles, and the data were averaged. The following measurements were obtained from M-mode imaging: left ventricular internal diameter at end-diastole and end-systole (LVIDd and LVIDs); interventricular septum thickness at end-diastole and end-systole (IVSd and IVSs); left ventricle posterior wall thickness at end-diastole and end-systole (LVPWd and LVPWs); left atrial dimension at its maximal anterior excursion near the end of systole (LA); aortic root dimension at end diastole (Ao); E-point to septal separation (EPSS); left ventricular preejection period (PEP); ejection time of the left ventricular outflow (ET); and time between the cessation and onset of the mitral inflow interval (A). The percentage of left ventricle posterior wall thickening (PWT), fractional shortening of the left ventricle (FS), left atrial dimension to aortic root ratio (LA:Ao), ejection fraction, left ventricular preejection period to the ejection time ratio (PEP:ET); Tei index of myocardial performance; and left ventricular end-diastolic and end-systolic volumes (LVEDV, LVESV) were calculated as follows:

Statistical analysis.

Data were analyzed by using a commercial software package (Statistica 7.0, Statsoft, Tulsa, OK). One-way ANOVA was used to determine the effect of age. To determine the age at which the cardiac measurements became stable, the Newman–Keuls test was applied to parameters that were significantly affected by age. Analysis of linear regression was used to assess the relationship between each echocardiographic parameter and body weight (body weight) and surface area (body surface area). Statistical significance was defined as a P value of less than 0.05.

Results

For each cardiac parameter measured echocardiographically in female beagles, the mean and standard deviation obtained for each age group are presented in Table 1. The results of the Newman–Keuls tests for parameters showing significant (P < 0.05) age-associated effects are described in Table 1. The mean, standard deviation, minimal and maximal values, and significance level (P value) of the effect of age group obtained by 1-way ANOVA for each parameter are shown in Table 2.

Table 1.

Cardiac parameters (mean ± 1 SD) of growing female beagles

	Age (mo)
	4	7	10	13	17	21
Body weight (kg)	7.2 ± 1.5a	9.3 ± 0.9b	11.1 ± 0.8c	11.1 ± 1.0c	11.1 ± 1.2c	10.9 ± 0.8c
Body surface area (m2)	0.38 ± 0.05a	0.45 ± 0.03b	0.51 ± 0.02c	0.51 ± 0.03c	0.51 ± 0.04c	0.50 ± 0.02c
LVIDd (mm)	21.2 ± 1.6a	25.5 ± 3.7a,b	25.4 ± 2.0b	27.6 ± 3.8b	26.9 ± 4.9b	27.6 ± 2.4b
LVIDs (mm)	12.2 ± 1.8a	15.6 ± 2.0b	15.7 ± 1.8b	14.8 ± 2.4a,b	16.1 ± 1.6b	16.0 ± 3.1b
IVSd (mm)	7.0 ± 1.3	7.0 ± 1.1	8.4 ± 1.7	7.4 ± 1.9	8.7 ± 0.6	9.1 ± 1.4
IVSs (mm)	10.9 ± 1.9	11.0 ± 2.2	11.6 ± 1.9	12.2 ± 2.1	13.5 ± 1.9	13.5 ± 2.1
LVPWd (mm)	6.4 ± 1.4a	8.5 ± 1.7a,b	8.8 ± 1.6a,b	8.2 ± 1.6b	9.2 ± 1.6b	8.5 ± 0.7b
LVPWs (mm)	10.4 ± 1.5a	11.5 ± 1.1a,b	12.8 ± 1.9b	13.3 ± 1.2b	13.4 ± 1.5b	12.5 ± 1.3b
LVEDV (mm3)	2826 ± 454a	4252 ± 1342a,b	4144 ± 683a,b	4995 ± 1409b	4794 ± 1797b	4951 ± 895b
LVESV (mm3)	891 ± 283	1502 ± 411	1512 ± 355	1347 ± 475	1594 ± 333	1607 ± 677
LA (mm)	13.2 ± 1.9a	15.1 ± 2.3a,b	17.6 ± 2.2b	17.7 ± 3.8b	16.8 ± 1.4b	17.6 ± 2.6b
Ao (mm)	14.1 ± 1.8a	15.6 ± 1.6a,b	16.7 ± 2.4b	17.3 ± 1.6b	17.2 ± 1.5b	17.9 ± 1.8b
EPSS (mm)	1.0 ± 0.7	1.2 ± 1.0	1.5 ± 1.1	2.0 ± 1.5	2.4 ± 1.7	2.0 ± 1.4
PEP (ms)	33 ± 5	42 ± 4	35 ± 5	37 ± 5	40 ± 6	41 ± 9
ET (ms)	153 ± 5	163 ± 8	165 ± 20	162 ± 13	167 ± 24	176 ± 13
A (ms)	220 ± 12	225 ± 16	233 ± 36	235 ± 22	245 ± 30	261 ± 27
PWT (%)	39 ± 11	26 ± 9.0	31 ± 10	38 ± 10	31 ± 10	32 ± 5.0
FS (%)	42 ± 6	38 ± 6	38 ± 4	47 ± 2	39 ± 13	43 ± 8
Ejection fraction (%)	0.69 ± 0.07	0.64 ± 0.07	0.64 ± 0.06	0.73 ± 0.03	0.63 ± 0.17	0.68 ± 0.10
LA:Ao	0.94 ± 0.04	0.97 ± 0.16	1.07 ± 0.20	1.02 ± 0.16	0.98 ± 0.09	0.99 ± 0.11
PEP:ET	0.21 ± 0.04	0.26 ± 0.03	0.21 ± 0.04	0.23 ± 0.05	0.25 ± 0.06	0.24 ± 0.05
Tei index	0.41 ± 0.04	0.38 ± 0.11	0.48 ± 0.11	0.46 ± 0.11	0.48 ± 0.15	0.49 ± 0.12
Heart rate (bpm)	133 ± 33a	123 ± 18a,b	102 ± 11a,b	97 ± 14a,b	91 ± 28b	91 ± 15b

For variables that are affected by age (as determined by 1-way ANOVA), different superscript letters indicate significant differences (P < 0.05; Newman–Keuls test) between age groups.

Table 2.

Mean, standard deviation, and minimal (min) and maximal (max) values for each variable and the significance (P, 1-way ANOVA) of the age-associated effect

	Mean	1 SD	Min	Max	P
LVIDd (mm)	25.7	3.7	20	34.4	0.013240
LVIDs (mm)	15.2	2.5	10.3	20.4	0.039509
IVSd (mm)	7.9	1.6	5.3	10.6	0.036598
IVSs (mm)	12.2	2.2	7.8	15.8	0.087861
LVPWd (mm)	8.3	1.6	4.4	11.7	0.044345
LVPWs (mm)	12.3	1.7	8.0	15.4	0.008434
LVEDV (mm3)	4335	1313	2500	7743	0.027920
LVESV (mm3)	1431	491	602	2606	0.081046
LA (mm)	16.5	2.9	10.6	23.4	0.016823
Ao (mm)	16.5	2.1	11.3	20.3	0.011213
EPSS (mm)	1.8	1.3	0.0	4.2	0.462808
PEP (ms)	39	6.8	30	60	0.124607
ET (ms)	166	15.9	130	200	0.316158
A (ms)	239	28	180	300	0.087417
PWT (%)	33	10	10.74	54.63	0.185436
FS (%)	41	8	18.14	49.80	0.319117
LA:Ao	1.0	0.14	0.68	1.39	0.656201
ejection fraction (%)	0.66	0.10	0.35	0.77	0.354965
PEP/ET	0.24	0.05	0.17	0.33	0.604534
Tei index	0.45	0.11	0.21	0.63	0.523572
heart rate (beat/min)	101	23	64	170	0.030171

Both body weight and surface area increased to the extent that significant (P < 0.05) differences were established between 4 and 7 mo and between 7 and 10 mo. Body weight and surface area stabilized after 10 mo.

Despite a significant (ANOVA, P < 0.05) increase in IVSd with age, the Newman–Keuls test did not confirm differences between age groups. With regard to the rest of the parameters that showed significant age-associated effects (LVIDd, LVIDs, LVPWd, LVPWs, Ao, LA, heart rate, and LVEDV), Newman–Keuls tests showed that all age groups were homogeneous, starting with the second age group (7 mo), and that there were no statistical differences between any groups. Compared with the initial set of measurements, the age at which differences started to be significant varied depending on the parameter: 7 mo for LVIDs; 10 mo for LVIDd, LVPWs, Ao, and LA; 13 mo for LVPWd and LVEDV; and 17 mo for heart rate.

The age of the animals did not have significant effect on IVSs, EPSS, ET, and A. Although LVESV seemed to increase markedly between 4 and 7 mo of age, it only gradually increased thereafter. Both PEP and the cardiac function indexes obtained from the measurements (PWT, FS, LA:Ao, ejection fraction, PEP:ET, and Tei index) remained relatively constant throughout the follow-up period.

Significant correlations between various cardiac parameters studied and heart rate, body weight, and body surface area are presented in Table 3. High correlation was observed for LA (Figure 1), Ao (Figure 2), and heart rate (Figure 3) with both indexes of corporal growth.

Table 3.

Statistically significant (P < 0.05) results of the correlation matrix (r) for each parameter with body weight, body surface, and heart rate

	r
	LVIDd	LVIDs	LVPWd	LVPWs	IVSd	IVSs	LVESV	LVEDV	Ao	LA	EPSS	ET	A	LA:Ao
body weight	0.58	0.59	0.45	0.53	0.41	0.40	0.56	0.54	0.71	0.78	0.57			0.36
body surface area	0.58	0.59	0.46	0.54	0.42	0.41	0.56	0.54	0.72	0.78	0.56			0.34
heart rate			−0.48	−0.36		−0.46			−0.59	−0.43	−0.52	−0.51	−0.39

Empty cells indicate nonsignificant data.

Figure 1.

The correlation between body weight (BW) and left atrial dimensions (LA).

Figure 2.

The correlation between body weight (body weight) and aortic root dimension at end diastole (Ao).

Figure 3.

The correlation between body weight (BW) and heart rate.

Discussion

Here we found that echocardiographic parameters associated with heart size changed at different rates in female beagles, and these changes do not necessarily correspond with those in body weight and surface area. In fact, corporal growth typically ended around 9 mo of age, after which point both body weight and surface area remained fairly constant.

Despite the great variability in time needed for complete corporal development in breeds of very different sizes, the pace of cardiac development seems to be similar. The most rapid growth in the female beagle heart took place before 7 mo of age, as has also been observed in a giant breed, the Spanish mastiff,² and a medium breed, the English pointer.²¹

In our beagles, the dimensions of the left ventricle, particularly the internal dimensions and the left ventricular wall during diastole, showed the biggest increases before 7 mo. Modest cardiac growth may continue past this age, and there is some interindividual variability, as has been observed in the Irish wolfhound.²⁴

The dimensions of the cavity and wall of the left ventricle in our beagles at 4 mo were larger than those obtained at 3 mo.¹ The rapid pace of cardiac development during the first months of life,¹ which we found extends until 7 mo of age, accounts for this difference. The left ventricular values we obtained from older animals (10 to 21 mo of age), which are considered to be adults, were comparable to averages previously obtained from other female adult beagles,^6,10 although they were slightly lower than those obtained from female beagles of a different strain.¹⁰

The LA and Ao dimensions presented similar developmental evolutions. After 10 mo of age, the dimensions stabilized. In fact, they showed strong positive correlations with body weight and surface area, as has been noted for growing English pointers, Spanish mastiffs, adult Irish wolfhounds,^2,21,24 German shepherds, English bull terriers, and other dogs.^4,11,16

In humans, EPSS has been used as a practical clinical index of left ventricular function. Values obtained from beagles and German shepherds did not differ significantly and ranged from 1 to 6 mm, with values less than 6 mm considered normal.¹² However other authors noted significant correlation between EPSS and body weight when working with different sized adult dog breeds.⁶ Moreover, where reference values for adult animals of different breeds have been established, higher average EPSS values have been appreciated in the bigger breeds.^2,13,15 In a previous study,² Spanish mastiffs (a giant breed) showed increases in EPSS associated with growth and positive correlation with body weight, increasing from an average of 1.79 mm at 1 mo of age to an average of 6.71 mm in 2- to 4-y-old dogs. In the present study, age had no significant effect on EPSS, which showed positive correlation with both body weight and surface area.

The cardiac indexes obtained from measured parameters (LA:Ao, PWT, FS, and ejection fraction) were influenced neither by age nor body weight or body surface area in beagle females during growth. This fact reinforces the usefulness of these indexes for determining cardiac function independently of other parameters, such as age or corporal dimensions. However, FS and ejection fraction can present a negative correlation with body weight in breeds with the same conformation but different size.⁷ This fact again supports the need to have reference values for different breeds with different sizes and corporal conformations.

Age, body weight, and body surface area do not affect systolic time intervals in female beagles. As previously described for beagles,¹⁸ PEP remained constant during the follow-up period, regardless of changes in heart rate. Ejection time of the left ventricular outflow showed negative correlation with heart rate; the value we obtained was slightly higher than that previously noted in adult female beagle dogs²² but not as high as that observed in the same breed after induction of changes in heart rate by atropine administration.¹⁸ The pharmacologic effects of atropine on heart rate might explain the correlation between the PEP:ET ratio and heart rate observed in a previous canine study¹⁸ but not in humans²⁵ or the present study in dogs. Therefore, we consider the average reference values obtained in the present study as nonspecific indicators of the global systolic performance of the left ventricle in female beagle dogs from 4 mo of age.

In the current study, neither age, body weight or surface area, or heart rate influenced the Tei index of myocardial performance. In humans, this index is relatively independent of heart rate and age in growing patients.^3,8,9 However, ageing and the associated alteration of diastolic function can increase the Tei index, as has been reported for dogs.²² The average values we obtained from female beagles are slightly higher than those obtained from dogs of different breeds,²² suggesting the possible existence of breed differences, a possibility that should be confirmed.

Our current study has some important limitations. Cardiac dimensions were obtained by M-mode imaging, although LA body dimensions are thought to be better obtained by B-mode echocardiographic imaging because the M-mode measurement angle usually transects left auricle.²¹ In addition, our study had a small sample size and evaluated only female dogs, so our results cannot be extrapolated to male dogs of similar ages.

In conclusion, cardiac growth in female beagles can be considered complete at 13 mo of age, bearing in mind that rapid growth takes place before 7 mo, typically with only slight modification thereafter. The exception is heart rate, which does not become stable until 17 mo of age. The cardiac indexes and systolic time intervals do not show significant age-associated changes. Therefore these parameters are useful for evaluating alterations in cardiac function starting at 4 mo of age, independent of age, weight, and body weight or surface area of the animal.