Sage Journals: Discover world-class research

Abstract

Changes in clinical pathology parameters, particularly those related to blood coagulation, were examined throughout the gestation period in New Zealand White rabbits. As compared with the non-pregnant group, the following major changes were observed in the pregnant group. For blood coagulation-related parameters, platelets increased progressively and fibrinogen increased slightly from organogenesis, prothrombin time was significantly prolonged during organogenesis and shortened in the late fetal growth stage, activated partial thromboplastin time was significantly prolonged during the fetal growth stage, and antithrombin III increased during and after late organogenesis. Such changes in blood coagulation-related parameters during the later stages of gestation seem to be physiological responses in preparation for protecting against excessive haemorrhage or haemostasis at parturition. For the other haematological and blood chemical parameters as well as progesterone, red blood cell counts, haemoglobin and haematocrit began to decrease during organogenesis and continued to decrease thereafter. Reticulocyte counts significantly increased during organogenesis and decreased thereafter. White blood cell parameters, except for neutrophils, showed significant decreases during the fetal growth stage. Serum progesterone concentration reached its highest level early in organogenesis and decreased thereafter. Total protein, albumin, glucose, cholesterol, calcium, blood urea nitrogen and creatinine decreased significantly during the middle and/or late periods of gestation. In conclusion, the data obtained from the present study can be used as background data for effective evaluation of reproductive toxicology in rabbits, and pregnant rabbits may serve as models of pregnant women in research pertaining to clinical pathology and gestation.

Keywords

Pregnant rabbits clinical pathology parameters blood coagulation

It is generally acknowledged that gestation induces progressive alterations in several haematology and blood biochemistry parameters in laboratory animals. Therefore meaningful interpretation of data from toxicology studies using pregnant animals becomes difficult without the benefit of historical control data. In addition, to precisely assess the fetotoxicity of the compound administered, it is necessary to take the secondary effects of maternal toxicity on the fetus into consideration.

Pregnant rabbits of the New Zealand White strain (Kbl:NZW) are frequently and widely used in embryo-fetal development toxicity (teratology) studies. However, there is a paucity of data pertaining to haematology and blood biochemistry parameters in pregnant rabbits.^1,2 Particularly, there are few detailed reference studies on the changes in blood coagulation-related parameters during gestation. This is important as it has been reported that disorders of the blood coagulation system tend to occur in women in the late stage of pregnancy.^3–7

This study was performed to clarify how haematology and blood biochemistry parameters, especially those related to blood coagulation, change during the course of gestation in the rabbit and how well pregnant rabbits may serve as models of pregnant women in research pertaining to clinical pathology and gestation.

Materials and Methods

Animals

Thirty-five female rabbits of the New Zealand White strain (Kbl:NZW; Kitayama Labs, Co, Ltd, Nagano, Japan) at five or seven months of age were used in this study. The animals were maintained individually in aluminium cages in an animal room under controlled conditions (temperature: 22.5 ± 3.5°C; relative humidity: 50 ± 20%; air ventilation: 10-15 times per hour; artificial lighting: 12 h per day), and were allowed free access to diet (RC4; Oriental Yeast Co, Ltd, Tokyo, Japan) and to water via an automatic water supply system (Fuji Water Union, Shizuoka, Japan). After a one-week acclimatization period, 19 females that were judged to be in oestrus were housed together with male rabbits from the same colony in circles (650 mm in diameter × 500 mm in height) for mating on a 1:1 basis. The day when copulation was confirmed was regarded as gestational day 0 (GD 0). The 19 copulated animals were assigned to the pregnant (P) group and the remaining 16 animals that were not subjected to mating were assigned to the non-pregnant (NP) group. Furthermore, from the standpoint of animal welfare, the P and NP groups were subdivided into two subgroups each (Figure 1) in order to reduce stress by blood sampling. As a result of statistical analysis performed on all parameters between the two subgroups in each P group and NP group on the first blood sampling day (GD 0 [mating day] versus GD 6 [one day before implantation]), there were significant but slight differences detected sporadically in several parameters but there were no significant differences in blood coagulation-related parameters, which are the main concern of this study. Therefore, the two subgroups were merged and treated as one group.

Figure 1

Experimental design, ↑ or ↓: blood sampling point; GD: gestational day; organogenesis: the period of organogenesis; fetal growth: the period of fetal growth

All animals were euthanized as scheduled on GD 28 by exsanguination from the abdominal aorta under pentobarbital anaesthesia.

The experimental procedures were conducted according to the Animal Welfare Guidelines of Bozo Research Center Inc, the Standards of the Institutional Laboratory Animal Resources Guide and the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes.

Body weight and food consumption

During the experimental period, all animals were checked for general condition every day, body weight on GDs 0, 3, 6, 8, 10, 12, 16, 18, 19, 22, 24, 26 and 28, and food consumption from GD 1 to GD 28.

Haematological and blood chemical examinations

Blood sampling was done twice each during the following gestation periods (Figure 1): (1) the period between fertilization of the ovum or ova and implantation (GDs 0 and 6); (2) the period of organogenesis (GDs 13 and 18); and (3) the period of fetal growth (GDs 25 and 28). Blood samples were collected from the auricle artery under sedation using the restrainer.

As shown in Figure 1, to reduce the stress on the animals, blood sampling was done in 10 animals of the P group and eight animals of the NP group on GDs 0, 13 and 25, and in the remaining nine animals of the P group and eight animals of the NP group on GDs 6, 18 and 28.

At each sampling timepoint, 1 mL blood was collected into collecting tubes containing ethylenediaminetetraacetic acid -2K (SB-41: Sysmex Corporation) and subjected to measurement or calculation of haematological parameters (red blood cells [RBC], haemoglobin [Hb], haematocrit [Hct], mean cell volume [MCV], mean cell haemoglobin [MCH], mean corpuscular haemoglobin concentration [MCHC], reticulocyte, platelets, white blood cells [WBC], differential leukocyte count and absolute value) using an ADVIA^® 120 Haematology System (Siemens Healthcare Diagnosis, NY, USA). In addition, a blood sample (1.8 mL) collected into tubes containing 3.8 w/v% sodium citrate (Venoject^® 2, VP-C052K, Terumo Corp Co, Ltd, Tokyo, Japan) was centrifuged at approximately 1600 × g for 10 min, and the plasma sample obtained was examined for prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen using a Coagulometer ACL 100 (Instrumentation Laboratory Co, Lexington, KY, USA) and for antithrombin time III (ATIII) using a Biochemical Analyzer TBA-120FR (Toshiba Medical Systems Corp, Otawara, Tochigi, Japan). Further blood samples collected in plain tubes (Venoject^® 2, VP-P070K, Terumo Corp) at the same time as for haematology were kept at roomtemperature for about 30 min and centrifuged at approximately 1600 × g for 10 min, and the serum samples obtained were examined for the following parameters (blood urea-N [BUN], creatinine, total protein, albumin, alkaline phosphatase [ALP], total cholesterol, triglyceride, glucose, sodium, potassium, calcium, phosphorus and chloride). Approximately 2 mL of blood collected in a heparinized tube (Venoject^® 2, VP-H050K, Terumo Corp) was centrifuged at approximately 1600 × g for 10min, and plasma obtainedwas examined for the remaining parameters (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LD] and creatine kinase [CK]) using the Biochemical Analyzer TBA-120FR (ToshibaMedical Systems Corp). Serum progesterone concentrations were measured by Chemiluminescent Enzyme Immunoassay using IMMULYZE (Diagnostic Products Corp, Los Angeles, CA, USA).

Statistical analysis

The means±standard deviations were calculated for the parameters examined in each group, and were first analysed for homogeneity of variance by the F-test between the NP and P groups. The homogeneous data were analysed by Student's t-test and the heterogeneous data by Aspin-Welch's t-test. A two-way analysis of variance was performed (Group × Day) and there were significant differences for the Group × Day interaction in many parameters, including blood coagulation values, so further analysis was performed on the data from each day.

Results

Changes in body weight and food consumption

There were no abnormal clinical signs observed in any animal throughout the gestation period. Body weight was significantly higher in the P group than in the NP group on GDs 6, 12, 18, 24 and 28 (Table 1). Although food consumption in the P group was higher than that in the NP group during early pregnancy, it was significantly lower on GDs 25 and 28 (Table 1). In comparison with that of the NP group, food consumption of the P group was similar until GD 18, but showed a 30% decrease on GD 28.

Table 1.

Changes in body weights and daily food consumptions in pregnant (P) and non-pregnant (NP) rabbits

Day	n	Body weight (kg)	Food consumption (g)
NP
0	16	3.68 ± 0.26	-
1	16	-	112 ± 35
6	16	3.69 ± 0.29	110 ± 38
12	16	3.71 ± 0.30	-
13	16	-	115 ± 31
18	16	3.74 ± 0.31	117 ± 30
24	16	3.74 ± 0.35	-
25	16	-	104 ± 33
28	16	3.77 ± 0.37	107 ± 33
P
0	19	3.88 ± 0.35	-
1	19	-	124 ± 20
6	19	3.97 ± 0.31^**	134 ± 23^#
12	19	3.99 ± 0.30^*	-
13	19	-	97 ± 38
18	19	4.07 ± 0.30^**	122 ± 37
24	19	4.07 ± 0.30^**	-
25	19	-	73 ± 29^**
28	19	4.13 ± 0.28^**	87 ± 27^*

Day: gestational day in pregnant rabbits or day after sampling point in non-pregnant rabbits; -: not measured

P < 0.05,

P < 0.01, significantly different from the NP group (Student's t-test);

P < 0.05, significantly different from the NP group (Aspin-Welch t-test)

Changes in blood coagulation-related parameters

Changes in blood coagulation-related parameters are shown in Table 2 and Figure 2. In the P group, prolongation of APTT was observed on and after GD 18 and it was remarkable on GDs 25 and 28. PT prolonged significantly on GDs 13 and 18, but it shortened on GD 28. ATIII showed significantly higher values on and after GD 18. Platelets showed significantly higher values on GDs 18 and 25. Fibrinogen showed slightly higher values in the P group than in the NP group on and after GD 13, although there were no significant differences between the two groups.

Table 2.

Changes in blood coagulation-related parameters in pregnant (P) and non-pregnant (NP) rabbits

Day	n	Fibrinogen (g/L)	Platelets (310⁹/L)	APTT (s)	PT (s)	ATIII (%)
NP
0	8	2.79 ± 0.46	3.15 ± 0.88	22.7 ± 1.7	6.8 ± 0.2	97 ± 9
6	8	2.66 ± 0.44	2.65 ± 0.76	23.0 ± 2.5	6.6 ± 0.1	99 ± 11
13	8	2.76 ± 0.36	3.12 ± 0.99	21.7 ± 2.0	6.4 ± 0.1	100 ± 7
18	8	2.70 ± 0.33	2.46 ± 0.66	23.4 ± 5.5	6.5 ± 0.2	103 ± 7
25	8	2.60 ± 0.44	3.19 ± 1.14	22.2 ± 1.5	6.5 ± 0.2	100 ± 6
28	8	2.50 ± 0.12	2.42 ± 0.57	23.9 ± 2.6	6.7 ± 0.1	98 ± 5
P
0	9	2.75 ± 0.33	2.90 ± 0.71	20.8 ± 1.9	6.8 ± 0.2	96 ± 8
6	10	2.63 ± 0.24	3.13 ± 0.71	20.2 ± 3.1	6.8 ± 0.3	94 ± 8
13	9	2.93 ± 0.32	3.88 ± 0.78	20.0 ± 2.4	6.7 ± 0.3^#	97 ± 9
18	10	2.74 ± 0.39	4.13 ± 1.26^##	25.2 ± 5.0	6.7 ± 0.2^*	110 ± 6^*
25	9	2.69 ± 0.45	3.29 ± 0.59	31.8 ± 4.6^##	6.4 ± 0.2	128 ± 6^*
28	10	2.65 ± 0.43	3.96 ± 0.83^*	34.2 ± 5.6^##	6.4 ± 0.1^*	130 ± 7^*

APTT: activated partial thromboplastin time; PT: prothrombin time; ATIII: antithrombin time III; Day: gestational day in pregnant rabbits or day after sampling point in non-pregnant rabbits.

P < 0.05,

P < 0.01, significantly different from the NP group (Student's t-test)

P < 0.05

P < 0.01, significantly different from the NP group (Aspin-Welch t-test)

Figure 2

Changes in blood coagulation-related parameters in pregnant and non-pregnant rabbits. Day: gestational day in pregnant rabbits or day after sampling point in non-pregnant rabbits. Pregnant group (•); Non-pregnant group (○). *P < 0.05, **P < 0.01, significantly different from the non-pregnant group (Student's t-test); ^#P < 0.05, ^##P < 0.01, significantly different from the non-pregnant group (Aspin-Welch t-test). APTT: activated partial thromboplastin time; PT: prothrombin time; ATIII: antithrombin time III

Serum progesterone concentration

Serum progesterone concentration was markedly higher in the P group than in the NP group throughout the gestation period (Table 3). The concentration in the P group reached its highest level on GD 13, decreased on GD 18 and maintained a similar level until GD 28, although there were no significant differences observed among all pregnant groups.

Table 3.

Changes in serum progesterone concentrations in pregnant (P) and non-pregnant (NP) rabbits

Day	n	Concentration (ng/mL)
NP
-	4	0.2 ± 0.2
P
6	4	8.6 ± 1.6
13	4	15.0 ± 5.1
18	4	9.8 ± 3.6
25	4	11.7 ± 3.0
28	4	10.7 ± 2.4

Day: gestational day

Haematological findings

Sequential changes of RBC parameters are shown in Table 4a. In the P group, RBC, Hb and Hct decreased from GD 18, and they were significantly lower than those in the NP group. On the other hand, reticulocytes in the P group increased about 50% from GD 13 to GD 18, being significantly higher than those in the NP group. However, they became significantly lower than those in the NP group on GD 28. MCV, MCH and MCHC showed almost no significant changes throughout the gestation period.

Table 4a.

Changes in red blood cell (RBC) parameters in pregnant (P) and non-pregnant (NP) rabbits

Day	n	RBC (×10¹²/L)	Hb (g/L)	Hct (L/L)	MCV (fL)	MCH (pg)	MCHC (%)	Reticulocyte (×10⁹/L)
NP
0	8	6.37 ± 0.42	141 ± 9	0.39 ± 0.03	61 ± 2	22.2 ± 0.7	36.3 ± 1.0	1.37 ± 0.26
6	8	6.68 ± 0.47	148 ± 5	0.42 ± 0.02	62 ± 2	22.2 ± 1.1	35.6 ± 1.0	1.27 ± 0.48
13	8	6.16 ± 0.47	137 ± 8	0.38 ± 0.03	62 ± 2	22.2 ± 0.7	36.0 ± 1.1	1.52 ± 0.36
18	8	6.48 ± 0.29	144 ± 3	0.40 ± 0.01	62 ± 3	22.2 ± 0.8	36.1 ± 0.8	1.83 ± 0.62
25	8	6.19 ± 0.41	137 ± 6	0.39 ± 0.02	63 ± 2	22.2 ± 0.7	35.6 ± 1.0	1.55 ± 0.33
28	8	6.44 ± 0.41	142 ± 3	0.40 ± 0.01	62 ± 3	22.1 ± 1.2	35.5 ± 0.4	1.34 ± 0.26
P
0	9	6.78 ± 0.28^*	149 ± 4^#	0.42 ± 0.02^*	62 ± 3	22.0 ± 0.7	35.4 ± 0.9	1.43 ± 0.48
6	10	6.36 ± 0.29	143 ± 8	0.40 ± 0.03	63 ± 3	22.5 ± 0.7	35.9 ± 0.9	0.96 ± 0.26
13	9	6.57 ± 0.31	145 ± 7^*	0.41 ± 0.03	62 ± 2	22.1 ± 0.6	35.4 ± 0.9	2.33 ± 0.48^*
18	10	5.84 ± 0.34^*	134 ± 7^##	0.37 ± 0.02^*	64 ± 2	23.0 ± 0.7^*	36.1 ± 0.4	3.51 ± 0.63^##
25	9	5.99 ± 0.37	134 ± 5	0.38 ± 0.02	64 ± 2	22.3 ± 0.8	35.1 ± 0.7	1.77 ± 0.45
28	10	5.55 ± 0.37^*	124 ± 8^##	0.36 ± 0.02^*	65 ± 1	22.4 ± 0.5	34.7 ± 0.7^*	0.71 ± 0.32^##

Hb: haemoglobin; HCT: haematocrit; MCV: mean cell volume; MCH: mean cell haemoglobin; MCHC: mean corpuscular haemoglobin concentration; Day: gestational day in pregnant rabbits or day after sampling point in non-pregnant rabbits.

P < 0.05,

P < 0.01, significantly different from the NP group (Student's t-test)

P < 0.05

P < 0.01, significantly different from the NP group (Aspin-Welch t-test)

Sequential changes of WBC parameters are shown in Table 4b. The parameters, except for neutrophils, showed significantly lower values in the P group than in the NP group on GDs 25 and/or 28. There were no significant differences in neutrophils between the P and NP groups throughout the gestation period. Lymphocytes were significantly higher in the P group than in the NP group on GD 13.

Table 4b.

Changes in white blood cell (WBC) parameters in pregnant (P) and non-pregnant (NP) rabbits

Day	n	WBC (×10⁹/L)	Neutrophils (×10⁹/L)	Lymphocytes (×10⁹/L)	Eosinophils (×10⁹/L)	Basophil (×10⁹/L)	Monocytes (×10⁹/L)
NP
0	8	6.82 ± 1.79	1.78 ± 1.27	43.7 ± 9.1	0.11 ± 0.05	0.28 ± 0.09	0.24 ± 0.07
6	8	8.24 ± 2.34	1.36 ± 0.24	60.0 ± 21.3	0.11 ± 0.04	0.39 ± 0.19	0.32 ± 0.15
13	8	6.77 ± 1.60	1.39 ± 0.32	46.8 ± 12.5	0.10 ± 0.05	0.31 ± 0.10	0.24 ± 0.14
18	8	7.07 ± 2.03	1.24 ± 0.36	50.5 ± 17.9	0.12 ± 0.06	0.34 ± 0.15	0.25 ± 0.10
25	8	6.85 ± 1.25	1.24 ± 0.27	49.6 ± 10.1	0.10 ± 0.04	0.29 ± 0.07	0.18 ± 0.07
28	8	7.13 ± 1.82	1.22 ± 0.29	50.8 ± 14.8	0.13 ± 0.04	0.38 ± 0.19	0.28 ± 0.11
P
0	9	6.06 ± 0.97	1.85 ± 0.38	36.7 ± 8.4	0.07 ± 0.04	0.23 ± 0.03	0.15 ± 0.06^*
6	10	9.10 ± 2.32	1.66 ± 1.09	64.7 ± 19.2	0.12 ± 0.04	0.46 ± 0.19	0.29 ± 0.10
13	9	8.28 ± 1.46	1.17 ± 0.29	62.8 ± 14.7^*	0.10 ± 0.04	0.31 ± 0.09	0.36 ± 0.13
18	10	6.38 ± 1.66	0.95 ± 0.38	47.6 ± 13.1	0.08 ± 0.02	0.23 ± 0.13	0.30 ± 0.14
25	9	5.56 ± 0.96^*	1.19 ± 0.23	39.4 ± 9.5^*	0.06 ± 0.02^#	0.14 ± 0.04^*	0.18 ± 0.11
28	10	4.82 ± 1.01^*	1.26 ± 0.29	32.2 ± 8.6^*	0.04 ± 0.01^##	0.11 ± 0.05^##	0.15 ± 0.05^#

Day: gestational day in pregnant rabbits or day after sampling point in non-pregnant rabbits.

P < 0.05,

P < 0.01, significantly different from the NP group (Student's t-test);

P < 0.05,

P < 0.01, significantly different from the NP group (Aspin-Welch t-test)

Blood biochemical findings

Sequential changes in blood chemistry are shown in Tables 5a and 5b. As compared with the NP group, total protein and albumin were significantly lower in the P group on and after GD 18. Similar sequential changes were observed in glucose and cholesterol. On the other hand, in the P group, triglyceride began to increase significantly on GD 13, reached the highest value on GD 18, and decreased markedly on GDs 25 and 28. As for electrolytes, the P group showed significantly lower calcium values on GDs 25 and 28 and significantly higher phosphorus values on GDs 13 and 25. In addition, as compared with the NP group, ALP and ALT activities in the P group were significantly lower on and after GD 18 and on GD 28, respectively. BUN and creatinine showed similar sequential changes to that of ALP activity. AST and LD activities in the P group were significantly higher on GD 28.

Table 5a.

Changes in blood biochemical parameters in pregnant (P) and non-pregnant (NP) rabbits

Day	n	TP (g/L)	Albumin (g/L)	ALP (IU/L)	AST (IU/L)	ALT (IU/L)	LD (IU/L)	CK (IU/L)	Cholesterol (g/L)	Triglycerides (g/L)
NP
0	8	56 ± 4	41 ± 3	127 ± 31	24 ± 12	61 ± 34	122 ± 34	583 ± 300	0.53 ± 0.15	0.41 ± 0.11
6	8	56 ± 2	40 ± 2	181 ± 90	22 ± 6	63 ± 23	86 ± 15	325 ± 55	0.52 ± 0.16	0.65 ± 0.27
13	8	56 ± 4	40 ± 3	116 ± 34	25 ± 14	60 ± 39	120 ± 49	452 ± 152	0.49 ± 0.14	0.45 ± 0.16
18	8	56 ± 4	40 ± 3	165 ± 81	33 ± 16	62 ± 26	116 ± 20	403 ± 101	0.44 ± 0.13	0.84 ± 0.60
25	8	57 ± 3	41 ± 2	116 ± 31	24 ± 13	57 ± 32	74 ± 17	324 ± 127	0.51 ± 0.14	0.46 ± 0.13
28	8	56 ± 2	39 ± 2	153 ± 66	21 ± 8	66 ± 29	47 ± 13	203 ± 50	0.44 ± 0.11	0.53 ± 0.17
P
0	9	57 ± 2	42 ± 2	150 ± 61	17 ± 3	44 ± 22	133 ± 25	582 ± 93	0.54 ± 0.12	0.45 ± 0.13
6	10	55 ± 4	39 ± 3	157 ± 82	23 ± 8	52 ± 22	76 ± 13	298 ± 76	0.44 ± 0.15	0.62 ± 0.23
13	9	53 ± 2	38 ± 2	123 ± 36	18 ± 6	45 ± 25	124 ± 23	297 ± 66^#	0.38 ± 0.12	0.67 ± 0.19^*
18	10	51 ± 3^*	37 ± 2^*	88 ± 42^#	36 ± 17	53 ± 27	132 ± 27	339 ± 112	0.22 ± 0.07^*	1.53 ± 0.63^*
25	9	48 ± 4^*	35 ± 3^*	59 ± 18^*	33 ± 46	50 ± 51	67 ± 29	235 ± 83	0.08 ± 0.03^##	0.31 ± 0.24
28	10	43 ± 3^*	30 ± 2^*	61 ± 17^##	58 ± 36^#	39 ± 21^*	76 ± 15^*	292 ± 143	0.10 ± 0.02^##	0.24 ± 0.06^##

TP: total protein; ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; LD: lactate dehydrogenase; CK: creatine kinase; Day: gestational day in pregnant rabbits or day after sampling point in non-pregnant rabbits.

P < 0.05,

P < 0.01, significantly different from the NP group (Student's t-test);

P < 0.05,

P < 0.01, significantly different from the NP group (Aspin-Welch t-test)

Table 5b.

Changes in blood biochemistry parameters in pregnant (P) and non-pregnant (NP) rabbits (cont'd

Day	n	Glucose (mmol/L)	Na (mmol/L)	K (mmol/L)	Ca (mmol/L)	P (mmol/L)	Cl (mmol/L)	Urea-N (mmol/L)	Creatinine (mmol/L)
NP
0	8	7.07 ± 0.59	143 ± 2	4.3 ± 0.5	3.56 ± 0.10	1.09 ± 0.16	110 ± 3	4.50 ± 0.45	0.11 ± 0.02
6	8	7.49 ± 1.22	145 ± 2	4.3 ± 0.2	3.52 ± 0.14	1.19 ± 0.20	109 ± 2	4.63 ± 0.42	0.10 ± 0.01
13	8	6.88 ± 0.76	144 ± 1	4.3 ± 0.5	3.53 ± 0.05	1.02 ± 0.09	108 ± 3	4.40 ± 0.39	0.09 ± 0.02
18	8	6.94 ± 0.55	142 ± 3	4.1 ± 0.4	3.45 ± 0.14	1.12 ± 0.14	107 ± 2	4.31 ± 0.55	0.10 ± 0.01
25	8	7.00 ± 0.75	142 ± 1	4.3 ± 0.2	3.30 ± 0.12	1.00 ± 0.10	107 ± 2	4.50 ± 0.35	0.10 ± 0.02
28	8	7.01 ± 0.14	143 ± 2	4.1 ± 0.5	3.19 ± 0.17	1.10 ± 0.11	108 ± 1	4.40 ± 0.59	0.10 ± 0.02
P
0	9	7.65 ± 0.70	144 ± 2	4.2 ± 0.4	3.50 ± 0.14	1.20 ± 0.14	111 ± 2	4.69 ± 0.45	0.10 ± 0.01
6	10	6.95 ± 0.91	143 ± 3	4.3 ± 0.5	3.62 ± 0.10	1.12 ± 0.20	109 ± 2	4.80 ± 0.65	0.09 ± 0.01
13	9	6.82 ± 0.69	142 ± 2	4.0 ± 0.3	3.56 ± 0.10	1.14 ± 0.13^*	106 ± 2	4.48 ± 0.45	0.09 ± 0.01
18	10	6.62 ± 0.52	141 ± 2	4.0 ± 0.4	3.61 ± 0.18	1.21 ± 0.15	107 ± 3	3.52 ± 0.68^*	0.08 ± 0.01^*
25	9	6.26 ± 0.67^*	142 ± 1	4.0 ± 0.2^*	3.58 ± 0.11^*	1.13 ± 0.11^*	107 ± 2	3.37 ± 0.69^##	0.08 ± 0.01^*
28	10	6.20 ± 0.73^##	143 ± 2	4.3 ± 0.4	3.55 ± 0.13^*	1.11 ± 0.14	109 ± 2	3.25 ± 0.63^*	0.08 ± 0.01^#

Day: gestational day in pregnant rabbits or day after sampling point in non-pregnant rabbits.

P < 0.05,

P < 0.01, significantly different from the NP group (Student's t-test);

P < 0.05,

P < 0.01, significantly different from the NP group (Aspin-Welch t-test)

Discussion

In the present study, changes in haematology and blood biochemistry parameters, particularly those related to blood coagulation, were examined throughout the course of gestation in New Zealand White rabbits.

As mentioned in the introduction, it is reported that disorders in the blood coagulation system tend to occur during the late period of pregnancy in women. However, there are only a small number of reports of detailed changes in blood coagulation-related parameters in either pregnant women or rabbits during pregnancy. For example, an increase in fibrinogen,^6,7 variable changes in platelets^5,7,8 and no changes in ATIII⁶ have been reported in women during pregnancy. In pregnant rabbits, Wells et al.² reported a decrease in platelets due to haemodilution.

The present data confirmed that there were significant changes in many blood coagulation-related parameters in the late period of pregnancy in rabbits. Namely, platelets increased progressively from organogenesis through the stage of fetal growth stage, as they do in pregnant rats.⁹

As in the case of pregnant women, fibrinogen showed slightly higher values (but not significant) in pregnant than in non-pregnant rabbits from early organogenesis through the fetal growth stage, while fibrinogen showed significantly higher values in pregnant than in non-pregnant rats.⁹ This suggests that there is a species difference in the effect of pregnancy on fibrinogen production.

In addition, PT prolonged during organogenesis and shortened in the last stage of fetal growth in pregnant rabbits while it prolonged throughout the gestation period and recovered to normal value on GD 20 in pregnant rats.⁹ PT was slightly shortened during the late period of pregnancy. A similar change was observed in rats and was considered to be one of the physiological responses to protect prolonged bleeding at delivery.¹⁰ Furthermore, the prolongation of APTT was observed during the fetal growth stage in pregnant rabbits, the same as in pregnant rats.⁹ ATIII apparently increased from late organogenesis through the fetal growth stage in pregnant rabbits. Prolongation of APTT and a high value of ATIII during fetal organogenesis were observed in rats.¹⁰ Therefore, these changes in blood coagulation-related parameters during the late period of pregnancy seem to be physiological responses in preparation for protecting against excessive haemorrhage or blood coagulation at parturition, or a mechanism to prevent the development of deep tissue thrombosis in dams.

In women under normal pregnancy, increases in fibrinogen and platelets through the gestation period, an increase in prothrombin volume in the first trimester, and a decrease or steady status in prothrombin volume in the third trimester have been reported.^4–7 In addition, increases in such factors as VII, VIII, IX, X and XII and a decrease in XI factor have been observed during gestation.

In the present study on pregnant rabbits, as in the case of pregnant women, a slight increase in fibrinogen was observed from organogenesis to fetal growth.

In addition, prolongation of PT in organogenesis as well as shortening of PT and an increase in platelets in late gestation were observed. However, this study did not examine coagulation factors, for which changes were reported in pregnant women as mentioned above, and therefore total similarity in pregnancy-related changes in the blood coagulation system of women and rabbits could not be confirmed. Further studies are needed to clarify this point.

RBC, Hct and Hb began to decrease during organogenesis and decreased progressively thereafter as reported by Wells et al.² In addition to the changes in red blood parameters, decreases in total protein, creatinine, BUN and serum calcium² as well as albumin were observed in the P group. Therefore, these changes were considered to be secondary to haemodilution.^11,12 The change in ALT activity observed in the present study was different from that reported by Wells et al.,² but the cause of such difference is obscure. Haemodilution in both rabbits and humans is considered to begin around mid-organogenesis, and haemodilution in pregnant rabbits was reported to be lower in magnitude than that observed in pregnant women.¹³ The increase in plasma volume in the second trimester in women is thought to be the cause of ‘physiological anaemia of pregnancy’, i.e. the progressive decrease in Hb and Hct concentrations that occurs until approximately week 30 of gestation.¹⁴

In the present study, reticulocytes drastically increased during organogenesis (GD 13 to GD 18) probably because of the increased demand for RBC during this stage,¹² and they decreased thereafter as previously reported.^2,15 In addition, the present data demonstrated that serum progesterone concentration reached its highest level on GD 13, suggesting that the increase of serum progesterone concentration might initiate the enhancement of erythropoiesis. ^12,16 There are, however, conflicting reports of reticulocyte changes in pregnant women.^17,18

WBC parameters, except for neutrophils, showed significant decreases during the late stage of pregnancy in the present study, and this is thought to be brought about by haemodilution. ² However, it was reported that WBC parameters increased during the whole period of gestation in pregnant women^19–21 and rats,¹² and the reason for such a discrepancy between species remains obscure.

Total protein and albumin decreased at the same level as previously reported in pregnant rabbits,² rats²² and women,²³ and this is thought to reflect the increased blood volume. As mentioned above, these changes were thought to be mainly brought about by haemodilution. In addition, glucose and total cholesterol decreased markedly from late organogenesis through the fetal growth stage.² Wells et al.² suggested that the decrease in serum concentration of glucose with the progression of pregnancy might be due to the increased demand for fetal growth. Although decreases in all kinds of lipids throughout the pregnancy were reported in rats,²⁴ TG increased significantly during organogenesis and then decreased during the fetal growth period in the present pregnant rabbit. Otherwise, food consumption on GD 28 showed about a 30% decrease in comparison with that on GD 18. This suggests that the decreases of cholesterol, ALP and TG on GD 28 may reflect the decreased food consumption. In women, conflicting reports exist for serum lipid levels during pregnancy.^25,26

Serum calcium, BUN and creatinine decreased significantly during the fetal growth stage in the present study. It has been reported that women exhibited steady decreases in electrolytes because of electrolyte demands by the fetus during the fetal growth stage.²⁷

In the present study, ALP activity, one of the indicators of liver function, decreased from the end stage of organogenesis, and a striking decline in ALP activity was observed during the fetal growth stage. Wells et al.² reported that a decrease in ALP activity might reflect increased oestrogen levels or an increase in the ratio of serum oestrogen to progesterone. It has been reported that, in pregnant rabbits, oestrogen concentration increased from GD 7 and thereafter it remained relatively stable until the last two or three days before parturition,^1,28 and that progesterone concentration decreased from GD 15 to the last stage of gestation.²⁸ In the present study, serum progesterone concentration reached its highest level on GD 13, decreased on GD 18 and was at a similar level until GD 28, although plasma oestrogen concentration was not examined.

In conclusion, the data obtained from present study can be used as background data for effective evaluation of reproductive toxicology in rabbits. In addition, pregnant rabbits may serve as models of pregnant women in research pertaining to clinical pathology and gestation, although further studies are needed.

Footnotes

Acknowledgement

The authors would like to thank Dr Kunio Doi, Emeritus Professor of the University of Tokyo, for reviewing of this manuscript.

References

Mariscal

, Sanchez

, Melo

, Beyer

, Rosenblatt

. Maternal behavior in New Zealand white rabbits: quantification of somatic events, motor patterns, and steroid plasma levels. Physiol Behav 1994; 55: 1081–9

Wells

, Decobecq

, Decouvelaere

, Justice

, Guttin

. Changes in clinical pathology parameters during gestation in the New Zealand white rabbit. Toxicol Pathol 1999; 27: 370–9

Bremme

. Haemostatic changes in pregnancy. Best Pract Res Clin Haematol 2003; 16: 153–68

Clark

. Changes of hemostasis variables during pregnancy. Semin Vasc Med 2003; 3: 13–24

Brenner

. Haemostatic changes in pregnancy. Thromb Res 2004; 114: 409–14

Holmes

, Wallace

JMW

. Haemostasis in normal pregnancy: a balancing act? Biochem Soc Trans 2005; 33: 428–32

Franchini

. Haemostasis and pregnancy. Thromb Haemost 2006; 95: 401–13

Fenton

, Saunders

, Cavill

. The platelet count in pregnancy. J Clin Pathol 1977; 30: 68–9

Papworth

, Clubb

. Clinical pathology in the female rat during the pre- and postnatal period. Comp Haematol Int 1995; 5: 13–24

10.

Urasoko

, Jun

, Ebata

. Changes in blood parameters and coagulation-related gene expression in pregnant rats. J Am Assoc Lab Anim Sci 2009; 48: 272–8

11.

Burtis

, Ashwood

. Clinical chemistry of pregnancy. In: Burtis

, Ashwood

, eds. Tietz Textbook Clinical Chemistry. 2nd edn. Philadelphia: WB Saunders, 1994: 2107–48

12.

DeRijk

, Esch

, Flik

. Pregnancy dating in the rat: placental morphology and maternal blood parameters. Toxicol Pathol 2002; 30: 271–82

13.

Horger

, Zarrow

. Certain endocrine aspects of the anemia of pregnancy in the rabbit. Am J Physiol 1957; 189: 407–11

14.

Stock

, Metcalfe

. Maternal physiology during gestation. In: Knobil

, Neil

, eds. The Physiology of Reproduction. Vol 2, 2nd edn, New York: Raven Press, 1994: 947–83

15.

Zarrow

, Zarrow

. Anemia in the rabbit during pregnancy and following treatment with water soluble ovarian extracts. Endocrinology 1953; 52: 424–35

16.

Beischer

, MacKay

. Obstetrics and the Newborn. East Sussex: Saunders Ltd, 1978

17.

Howells

, Jones

, Napier

, Saunders

, Cavill

. Erythropoiesis in pregnancy. Br J Haematol 1986; 64: 595–9

18.

Mercelina-Roumans

, Ubachs

, Wersch

. The reticulocyte count and its subfractions in smoking and non-smoking women. Eur J Clin Chem Clin Biochem 1995; 33: 263–5

19.

Peck

, Arias

. Hematologic changes associated with pregnancy. Clin Obstet Gynecol 1979; 22: 785–98

20.

Mercelina-Roumans

, Ubachs

, Wersch

. Leucocyte count and leucocyte differential in smoking and non-smoking females during pregnancy. Eur J Obstet Gynecol Reprod Biol 1994; 55: 169–73

21.

Cincotta

, Balloch

, Metz

, Layton

, Leischke

. Physiological neutrophilia of pregnancy is not associated with a rise in plasma granulocyte colony-stimulating factor (G-CSF). Am J Hematol 1995; 48: 288

22.

Naismith

, Richardson

, Ritchie

. Dietary protein and energy as determinants of foetal growth in the rat [proceedings]. Proc Nutr Soc 1976; 35: 124A–125A

23.

Lind

. Clinical chemistry of pregnancy. In: Latner

, Schwartz

, eds. Advances in Clinical Chemistry. Vol 21. London: Academic Press, 1980: 1–24

24.

LaBorde

, Wall

, Bolon

. Haematology and serum chemistry parameters of the pregnant rat. Lab Anim 1999; 33: 275–87

25.

Bacq

, Zarka

. La foie au cours de la grossesse normale. Gastroenterol Clin Biol 1994; 18: 767–4

26.

Chiang

, Yang

, Hung

, Chou

, Shyn

, Ng

. Alterations of serum lipid levels and their biological relevances during and after pregnancy. Life Sci 1995; 56: 2367–5

27.

Watney

, Rudd

. Calcium metabolism in pregnancy and in the newborn. J Obstet Gynaecol Br Commonw 1974; 81: 210–19

28.

Challis

JRG

, Davies

, Ryan

. The concentrations of progesterone, estrone and estradiol-17β in the plasma of pregnant rabbits. Endocrinology 1973; 93: 971–6

Changes in blood parameters in New Zealand White rabbits during pregnancy

Abstract

Keywords

Materials and Methods

Animals

Body weight and food consumption

Haematological and blood chemical examinations

Statistical analysis

Results

Changes in body weight and food consumption

Changes in blood coagulation-related parameters

Serum progesterone concentration

Haematological findings

Blood biochemical findings

Discussion

Footnotes

Acknowledgement

References