Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

  • Print ISSN 0367-6722

  • Online ISSN 0976-0555

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Reference Values for Selected Blood Parameters in Rabbits: Effects of Age and Physiological Status

M.J. Argente1,*, D.M. Abad-Salazar2, E. Bermejo-González2, M.L. García1, A. López-Palazón2
1Departamento de Tecnología Agroalimentaria, Universidad Miguel Hernández de Elche, Ctra de Beniel Km 3.2, 03312 Orihuela, Spain.
2IES Francisco de Goya, Calle Luchador nº 77, 30500 Molina de Segura, Spain.
Rabbit is widely used as an experimental animal model in infectious and non-infectious diseases. The haematologic data can be helpful in evaluating the health status of animals over time. The aim of this study was to determine the levels of red blood cells (RBC), white blood cells (WBC) and differential leukocyte counts in 5 nulliparous and 5 multiparous females, i.e. in young and older animals, at mating and at delivery. The values of RBC did not change with age, but WBC and lymphocytes decreased with age, a -33% and a -60% less in multiparous females than nulliparous ones. Monocytes count was double at delivery than at mating. In conclusion, aging on the immune system is manifested as reduction in production of mature lymphocytes and as a result, older females would not respond to immune challenge as robustly as the young ones. Physiological status is only related to production of monocytes.
Rabbits have significant economic importance, both in livestock and pet industry (DG Health and Food Safety, 2017). Moreover, this species is also used frequently as an animal model for studying infectious (Bansal et al., 2014; Peng et al., 2015) and non-infectious diseases (Anjum et al., 2019; Fan et al., 2014), due to several advantages such as easier housing and handling over larger species of animals. Blood parameters represent a great diagnostic tool for animal health status and disease evaluation (Barkakati et al., 2015; Etim et al., 2013; Kumar et al., 2010). Hence, establishing their standard reference ranges become imperative for any disease diagnosis, prevention and controlling program as it forms the very basis for clinical interpretation of laboratory data (Moore et al., 2015). It is well known in rabbits that various factors as breed (Abdel-Azeem et al., 2010; Martinec et al., 2012), gender (Ozkan et al., 2012; Tokarz-Deptuła et al., 2014), physiological condition (Archetti et al., 2008; Cetin et al., 2009) and age (Jeklova et al., 2009; Tumova et al., 2017) affect the cellular hemodynamic. However, the information is really scarce about differential leukocyte counts in adult animals, with more than one year old.

The aim of this study was to determine the levels of red blood cells (RBC), white blood cells (WBC) and differential leukocyte counts at mating and delivery in nulliparous and multiparous females, i.e. in young and older animals.
Animals
 
Ten female rabbits from a synthetic line were used in this study, which were divided according to age in young (5 nulliparous females with no parturition) and older females (5 multiparous females with more than four parturitions, i.e., being one year old). This line was selected during twelve generations for decreasing litter size variability. The animals were housed individually in breeding cages (37.5x33x90 cm) at the farm of the Universidad Miguel Hernández of Elche (Spain). They were under a constant photoperiod of 16 h continuous light: 8 h continuous darkness and controlled ventilation throughout the experiment. The animals were allowed to adapt to the feed and environment for a month before collection of the samples. The rabbits were given ad libitum commercial pelleted rabbit food (Cunilactal, Nutreco) and drinking water. Commercial pelleted food contained 16.5% CP, 15.8% fibre, 4% fat, 36% NDF, 18.5% ADF, 12% indigestible fibre and 2.400 kcal digestible energy. The female rabbits were first mated at 18 weeks of age and at 10 days after parturition thereafter. The animals were housed and handled with the agreement of the Miguel Hernández University of Elche Research Ethics Committee (Reference number 2019/VSC/PEA/0077) according to European Council Directives 98/58/EC and 2010/63/EU.

Blood sampling and analysis
 
Blood sampling in nulliparous and multiparous females was performed at 2 different time points, at mating and immediately after delivery. Blood samples were collected from the marginal ear vein to 2 ml tube of K3-EDTA (Deltalab, Madrid, Spain). The animals were restrained manually. Diurnal variations in haematological parameters were avoided by sampling the animals during the same time of the day (09:00-10:00). Blood samples were delivered to the laboratory within 0.5 h of collection and immediately analysed. The total RBC and WBC counts and the absolute number of each type of leukocytes were assessed using the Abacus Junior 5 hematology analyzer (Diatron Messtechnik, Budapest, Hungary).
 
Reproductive traits
 
At delivery, it was recorded the age of female (AF), the litter size at birth (LSB) and the kit’s individual weight at birth (IWB) in both nulliparous and multiparous females.
 
Data analysis
 
The differences between nulliparous and multiparous females and between mating and delivery status for total RBC and WBC counts and the different populations of leukocytes were analysed using the following model:
 
yijk = µ + Ti + PSj + eijk
 
Where,
yijk= The observed trait.
µ= The general mean.
Ti= The effect of female’s type (with two levels: nulliparous and multiparous female).
PSj= The effect of female’s physiological status (with two levels: at mating and at delivery).
eijk= The residual term.

The model for AF and LSB only included the effect of female’s type.
The used model for IWB was:
 
yijklm= µ + Ti + S+ SEXk +pijkl + eijklm
 
Where,
yijklm= The observed trait.
µ= The general mean.
Ti= The effect of female’s type.
Sj= The effect of suckling in the first 24 hours after delivery (with two levels: the kit sucked and no sucked).
SEXk= The effect of sex (with two levels: male and female).
pijkl= The random effect of the female.
eijklm= The residual term.
 
IWB was also analysed with LSB as covariate. All statistical analyses were performed with SAS program (SAS Institute, 2019).
Effect of age on reproductive traits
 
The minimum and maximum values, the least squared mean and its standard error of female’s age (AF), litter size at birth (LSB), kit’s individual weight at birth (IWB) and statistical comparisons between nulliparous and multiparous female rabbits are presented in Table 1. We found that the nulliparous female rabbits (young females) had their first delivery around 6 month of age and the multiparous female rabbits (older females) at more than one year old at their fifth delivery (P<0.001). Besides, litter size was greater in young females than older ones (+1.7 kits, P<0.05). However, kits of young females weighed lesser at birth compared to that of those multiparous female rabbits (-11.7 g, P<0.001). This difference disappeared when the litter size was included in the model as covariate (-0.5 g, P>0.10). Therefore, larger litters in young female are related to a lower weight of their kits (P<0.001).

Table 1: Reference intervals, least square mean (LSM) and standard error (SE) for age of female (AF), litter size at birth (LSB) and individual weight of kits birth (IWB) in nulliparous and multiparous female rabbits.



In commercial rabbit breeding, females are normally inseminated at 10-12 days post-delivery; thus, in multiparous females, there is an overlap between lactation of their current litter and gestation of their future litter. The high body energy deficit in multiparous females and the negative interactions between lactation and fertility are well known (Fortun-Lamothe et al., 1999; Theau-Clement and Roustan, 1992; Xiccato et al., 2004), which would explain a lower litter size in multiparous females than nulliparous ones. In this sense, literature in rabbits shows that reproductive performance is age dependent and the litter size at birth is increased as parity increased reaching the peak at the 3rd parity (Das and Yadav, 2007; Xiccato et al., 2004) then decreased gradually thereafter (Mahmoud, 2013). As weight of kits at birth in nulliparous females is concerned, it is well established that female’s body growth has not finished during the first pregnancy because of the feed intake is lower than thereafter (Rommers et al., 1999) and that a greater embryonic uterine overcrowding decreases available uterine surface area for the development of each placenta and nutrient supply to embryo (Argente et al., 2006 and 2008), which would explain kits born from nulliparous females are lighter than those multiparous ones.
 
Effect of age on haematological parameters
 
Even though rabbit is widely used as experimental animal model for monitoring evolution in infectious and non-infectious diseases (Fan et al., 2014; Peng et al., 2015), there is scarce information about evolution in haematological reference values with age in this species. Using appropriate reference values are vital in these studies. Table 2 shows the minimum and maximum values, the least square mean and its standard error of red blood cells (RBC), white blood cells (WBC) and differential count for lymphocytes, neutrophils, monocytes, eosinophils and basophils both in nulliparous and multiparous female rabbits, i.e. young and older animals. Nulliparous and multiparous females had similar RBC count (P>0.10). This finding showed that the values of RBC do not change with age and they are consistent with reference data of RBC in other populations (Hewitt et al., 1989; Jain 1993; Jeklova et al., 2009). In contrast to our observation for RBC, WBC and lymphocytes decrease substantial with age (10.50 109/L in nulliparous female vs 7.01 109/L in multiparous females, P≤0.05, e.g. -33% for WBC; 6.99 109/L in nulliparous females vs 2.78 109/L in multiparous females, P≤0.001, e.g. -60% for lymphocytes). Accordingly, the percentage of lymphocytes to WBC count was lesser in multiparous females than nulliparous ones (-26% for LWR, P≤0.001) and the percentage of neutrophils to WBC ratio was higher in multiparous females than in nulliparous ones (+26% for NWR, P≤0.001). A reduction in number of lymphocytes may related to higher ratios of both neutrophils to lymphocytes (NLR) and monocytes to lymphocytes (MLR) in multiparous females than in nulliparous ones (+95% for NLR and +5% for MLR, P≤0.01). For the rest of haematological parameters, there were no differences between young and older females.

Table 2: Reference intervals, least square mean (LSM) and standard error (SE) for red blood cells (RBC), white blood cells (WBC) and differential count in nulliparous and multiparous female rabbits.



This strong decrease in WBC and lymphocytes would be related to impairment of the adaptive immune function primarily because of the decline in production of naïve lymphocytes in the bone marrow and thymus as well as the expansion of incompetent memory lymphocytes (Weng, 2006). Monocyte to lymphocyte ratio (MLR) has been recently proposed to a simple biomarker of immune system; moderate and low values are related to a good combative power against disease in the host, by contrary high value is associated to impair immune system (Zelmer et al., 2018). On the other hand, neutrophil to lymphocyte ratio (NLR) has been proposed as an indirect biomarker of stress (Jain, 1993). Stress stimulates release of glucocorticoids from the adrenal glands that induces neutrophilia or lymphopenia, or both, increasing NLR (Davis et al., 2008). We stress that, in healthy rabbits, NLR increases from 1:2 at 2 months of age until 1:1 at 1 year of age (Washington and Van Hoosier, 2012), as a consequence of maturation of the hypothalamic-pituitary-adrenal axis over time (Hillmann et al., 2008). Chronic stress is associated with immune dysfunction. In this regard, the highest values in NLR and MLR of multiparous females would agree with a higher adrenal activity and in consequence an increase in the susceptibility to infections in older animals.
 
Effect of physiological status on haematological parameters
 
Table 3 shows the minimum and maximum values, the least square mean and its standard error of red blood cells (RBC), white blood cells (WBC) and differential count for lymphocytes, neutrophils, monocytes, eosinophils and basophils at mating and at delivery. There was difference only in monocytes count. In this sense, monocytes count doubled at delivery compared to mating (0.28 109/L at mating vs. 0.59 109/L at delivery, P≤0.01). Accordingly, its percentages increased in WBC count (+3%, P≤0.01) and number of lymphocytes (+3% for MWR and +5% for MLR, P≤0.01).

Table 3: Reference intervals, least square mean (LSM) and standard error (SE) for red blood cells (RBC), white blood cells (WBC) and differential count at mating and at delivery in female rabbits.

Aging on the immune system is manifested as reduction in production of mature lymphocytes and as a result, older females would not respond to immune challenge as robustly as the young ones. Physiological status is only related to production of monocytes.
This study is supported by the Spanish Ministry of Economy and Competitiveness (MINECO), project no. AGL2017-86083-C2-2-P.
The authors declare that they have no competing interests.

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