Age- and sex-associated differences in hematology and biochemistry parameters of Dunkin Hartley guinea pigs (Cavia porcellus)

The Dunkin Hartley is the most common guinea pig strain used in biomedical research, particularly for studies of asthma, allergy, infectious disease, reproduction, and osteoarthritis. Minimally invasive blood tests, such as complete blood counts and serum biochemistry profiles, are often collected for diagnostics and laboratory analyses. However, reference intervals for these assays have not yet been well-documented in this strain. The purpose of this study was to establish reference intervals for hematologic and biochemical parameters of Dunkin Hartley guinea pigs and determine age- and sex-related differences. Hematologic and biochemical parameters were retrospectively obtained from 145 male and 68 female guinea pigs between 2 and 15 months of age. All blood parameters were analyzed by a veterinary clinical pathology laboratory. Reference intervals were established according to the American Society for Veterinary Clinical Pathology guidelines. Age- and sex-related differences were determined using unpaired t-tests or nonparametric Mann-Whitney tests. Hematocrit, red blood cell distribution width, mean platelet volume, white blood cell count, heterophils, monocytes, eosinophils, glucose, blood urea nitrogen, creatinine, calcium, magnesium, total protein, albumin, globulin, cholesterol, aspartate aminotransferase, gamma glutamyl transferase, and bicarbonate increased with age. Mean corpuscular hemoglobin concentration, cellular hemoglobin concentration mean, platelets, lymphocytes, phosphorus, albumin/globulin ratio, alkaline phosphatase, anion gap, and calculated osmolality decreased with age. Males had higher hemoglobin, hematocrit, red blood cell count, mean corpuscular hemoglobin concentration, white blood cell count, heterophils, Foa-Kurloff cells, alanine aminotransferase, and bicarbonate and lower mean corpuscular volume, red blood cell distribution width, platelets, mean platelet volume, eosinophils, total protein, albumin, globulin, cholesterol, potassium, anion gap, calculated osmolality, and iron compared to females. Establishing age and sex differences in hematologic and biochemical parameters of Dunkin Hartley guinea pigs provides valuable insight into their physiology to better evaluate diagnostics and experimental results.

Introduction Due to their docile nature, small size, and biological similarities to humans, guinea pigs (Cavia porcellus) have been a mainstay of biomedical research for hundreds of years. They are most commonly used in allergy, immunology, infectious disease, nutritional, auditory, and osteoarthritis studies, among others [1,2]. The standard laboratory guinea pig for research is the Dunkin Hartley, an outbred, smooth-coated, albino strain. This strain was first developed by Dunkin and Hartley in 1926 and is commercially available from several laboratory breeders [1].
Complete blood counts (CBC) and serum biochemistries are routine blood tests performed to screen health status in both animals and humans. Reference intervals, defined as the set of values comprising 95% of the healthy reference population [3], are essential for laboratory diagnostic testing, as well as clinical decision-making. The American Society for Veterinary Clinical Pathology (ASVCP) has set forth guidelines for determining reference intervals for veterinary species [3]. Despite their widespread use in research, there are few publications reporting hematology and clinical chemistry reference intervals in clinically healthy guinea pigs, particularly of the Dunkin Hartley strain. Waner et al. compared hematologic and clinical chemistry parameters of 1 month old, male haired (n = 10) and hairless (n = 12) Dunkin Hartley guinea pigs [4], but did not include a high enough sample size to establish reference intervals as put forth by the ASVCP guidelines [3]. Prior studies have determined reference intervals in Weiser-Maples [5] and strain 13 [6] guinea pigs. However, there are likely differences in hematology and biochemistry parameters between strains of guinea pigs, similar to other laboratory rodents [7][8][9]. These studies in guinea pigs [5,6] and other laboratory rodents [8][9][10][11][12] have also demonstrated age and sex to be important factors impacting hematologic and biochemical parameters.
The purpose of this study was to develop CBC and serum biochemistry reference intervals for the Dunkin Hartley strain and determine age-and sex-related differences. To accomplish this, we accumulated historical CBC and serum biochemistry data from healthy control animals used in our laboratory's previous studies that represent both males and females of a large age range.

Animals
Retrospective CBC and serum biochemistry data from a total of 145 male (age, 2-15 mo) and 68 female (age, 2-12 mo) Dunkin Hartley guinea pigs were included in this study. At the time of blood collection, male guinea pigs weighed (mean ± SD) 939.29 ± 214.06 g, and female guinea pigs weighed 839.46 ± 200.45 g. All guinea pigs were purchased from Charles River Laboratories (Wilmington, MA) and housed in Fort Collins, Colorado. All animals were singly-housed in 30.80 cm × 59.37 cm × 22.86 cm isolator cages (Maxi-Miser Interchangeable IVC Caging, Thoren, Hazleton, PA) with 0.125-in corn cob bedding (Harlan, Madison, WI) and a red hut (BioServe, French Town, NJ). Caging was changed 2-3 times weekly. Teklad Global Guinea Pig Diet 2040 (Envigo, Madison, WI) and filter-sterilized water were provided ad libitum. Hay cubes (PMI Nutrition International LLC, Brentwood, MO) were provided daily. Animal rooms were maintained at a 12:12 h light:dark cycle, 20-26˚C temperature, and 30-70% humidity. As per the vendor, all animals were free of Sendai virus, lymphocytic choriomeningitis virus, pneumonia virus of mice, guinea pig adenovirus, guinea pig reovirus, Helicobacter spp., Mycoplasma pulmonis, and ectoparasites. All original experiments were performed in accordance with The Guide for the Care and Use of Laboratory Animals and approved by the Colorado State University Institutional Animal Care and Use Committee.

Blood collection and analysis
Blood was collected from anesthetized guinea pigs (isoflurane 3-5% in oxygen) from either the cranial vena cava with a 25-gauge needle and 1-mL syringe (n = 40 males; 8 females) or at study harvest via direct cardiac puncture with a 20-gauge butterfly catheter (95 males; 60 females). In a small subset of adult male animals, blood was also collected from an implanted jugular vein catheter while awake (n = 10). Collected blood was placed into 0.5 mL ethylenediaminetetraacetic acid (EDTA) microtubes and red top serum collection tubes. After allowing samples to clot for 20-30 minutes at room temperature, red top tubes were placed into a centrifuge at 3000 x g for 15 minutes at 4˚C for serum collection. EDTA microtubes and serum aliquots were maintained at 4˚C and submitted to the Colorado State University Clinical Pathology Laboratory within 4 hours of collection. CBCs were performed using the Advia 120 hematology analyzer (Siemens, Munich, Germany). Automated parameters included: hemoglobin (Hgb) as measured spectrophotometrically, Hgb (cell) as measured optically, hematocrit (Hct), red blood cell count (RBC), red blood cell distribution width (RDW), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), cell hemoglobin concentration mean (CHCM), platelets, mean platelet volume (MPV), and white blood cell count (WBC). Manual blood film differentials were performed by trained laboratory staff with experience in guinea pig hematology and identified heterophil, lymphocyte, Foa-Kurloff, monocyte, eosinophil, and basophil percentages and absolute counts. The Roche Cobas 6000 (Basel, Switzerland) was used to measure the following parameters in serum: glucose, blood urea nitrogen (BUN), creatinine, phosphorus, calcium, magnesium, total protein, albumin, globulin, albumin/globulin ratio (A/G), cholesterol, creatine kinase (CK), total bilirubin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), sodium, potassium, chloride, bicarbonate, anion gap, and iron. All analyzers were operating within laboratory established quality assurance protocols that incorporated ASVCP established total allowable error estimates [13,14].

Statistical analyses
Animals were partitioned into groups based on age. Guinea pigs less than 5 months old were classified as juveniles, and guinea pigs 5 months of age or older were classified as adults. Adult animals were further partitioned into subgroups by sex. Due to low numbers of juvenile females, juveniles were unable to be partitioned by sex.
Following guidelines provided by the ASVCP [3], descriptive statistics (sample size, mean, SD, median, and minimum and maximum values) and reference intervals with confidence intervals were determined for each group using Reference Value Advisor v2.1 [15]. Histograms were evaluated to assess distribution and outliers. Outliers identified by the Tukey test were removed. After outlier removal, normality was assessed using the Anderson-Darling test with P value < 0.05 considered statistically significant. For parameters with � 40 reference samples, the nonparametric method was performed to determine the 2.5 th and 97.5 th percentile of each parameter to serve as the lower and upper limits of the reference interval, respectively. The 90% confidence intervals of the lower and upper limits of the reference interval were then determined using the bootstrap method. For parameters with < 40 reference samples, reference intervals with 90% confidence intervals of the reference limits were calculated by parametric or robust methods. If the distribution was non-Gaussian, the data were transformed using the Box-Cox method and rechecked for distribution.
Data were analyzed using Prism (version 8.4.0, GraphPad Software, La Jolla, CA). Normality was assessed using the D'Agostino-Pearson normality test. An unpaired t-test for normally distributed data or a nonparametric Mann-Whitney test for non-normally distributed data was used to determine age-and sex-associated differences. Age correlation was determined using the Spearman coefficient. Results were considered statistically significant with a P value < 0.05.

Discussion
As comprehensive reference intervals for blood parameters have not previously been published for the Dunkin Hartley guinea pig, the purpose of this study was to establish reference intervals for hematologic and serum biochemical parameters of this strain according to the ASVCP   guidelines. Age-and sex-associated differences were also determined. These results provide the foundation for interpreting hematology and serum biochemistry values of the Dunkin Hartley guinea pig. There were several age-and sex-related changes in hematology and biochemical parameters in the Dunkin Hartley strain. However, it is important to use clinical judgement when evaluating these differences, as a statistically significant difference does not always correspond to a clinically significant difference. For example, Hct was positively correlated with age in males, leading to significantly higher levels in adults compared to juveniles. However, the median difference between juveniles and adults was 1%, and the upper limit of the reference interval was only 1% higher in adults compared to juveniles. This data will prove useful in study design and analysis when the primary endpoint is statistical detection of differences and likely will not have a significant impact on clinical interpretation of the data from a single individual.
Of the RBC parameters, males had significantly higher Hgb, Hct, RBC, and MCHC compared to females. Similar findings have been reported in numerous animal species, including other rodents [6,10,11] as well as humans [16]. This may be due to the varying effects of estrogen and testosterone on erythropoietin production [16]. These sex differences were small and unlikely to affect clinical interpretation. Although RDW was higher in females, there was a weak correlation with age in males. MCV was strongly correlated with age in females, leading to higher levels in females compared to males. Juveniles had significantly higher numbers of platelets, but lower MPV, than adults. While MPV was strongly correlated with age in both males and females, the number of platelets moderately decreased with age in males. Both platelets and MPV were significantly higher in females compared to males. In contrast, platelets were positively correlated with age in male Weiser-Maples guinea pigs [5] and C57BL/6J mice [9], and no sex differences were observed in platelets of Strain 13 guinea pigs [6]. Platelets in Sprague-Dawley rats were shown to markedly decrease with age, and later increase in old age [12]. Additionally, platelet numbers were higher in male 129SV/EV and C3H/HeJ, but not C57BL/6J, mice compared to females [9]. Clinical relevance of this variability in platelet numbers among different sexes and strains of rodents is unknown and may be worthy of additional research. Similar to other strains of guinea pigs [5,6], juvenile guinea pigs had higher numbers of heterophils and lower numbers of lymphocytes compared to adults. In both sexes, heterophils markedly increased with age, while lymphocytes decreased with age. Additionally, total WBC, Foa-Kurloff cells, monocytes, and eosinophils mildly increased with age and Foa-Kurloff cells markedly increased with age in males, but not females. Although females had more eosinophils, males had significantly higher WBC counts, due to higher numbers of heterophils and Foa-Kurloff cells. Foa-Kurloff cells are a type of estradiol-dependent white blood cell unique to guinea pigs that contain a large granular intracytoplasmic inclusion. Although the function of these cells is unknown, they are thought to have natural killer cell activity [17] and protect the fetus during pregnancy. Foa-Kurloff cells are commonly associated with pregnancy in older females and are reported to rarely be seen in young animals or males [18]. In contrast, higher numbers of Foa-Kurloff cells were seen in males compared to females in the current study. Additionally, higher numbers were reported in male strain 13 guinea pigs than females [6].
Other studies did not report numbers of Foa-Kurloff cells [4,5]. As this cell type is not recognized in automated hematology analyzers, manual differential leukocyte counts are particularly important for accurate white blood cell counts in guinea pigs. Glucose was positively correlated with age in male Dunkin Hartley guinea pigs, but negatively correlated in strain 13 males. Additionally, our reference intervals for glucose were much higher than those reported for strain 13 and Weiser-Maples guinea pigs [5,6], which may be due to variations in diet or fasting status. Animals were not fasted prior to blood collection in the current study. As fasting has been shown to affect numerous clinical pathology parameters in rats [19], it may also influence similar parameters in guinea pigs.
Like other guinea pig strains, BUN, creatinine, and calcium were positively correlated with age [5,6]. An increase in these parameters may be associated with the development of renal disease. Spontaneous renal lesions, such as nephrosclerosis, are a common incidental finding in guinea pigs that may result in renal insufficiency. Total protein, albumin, and globulin were also significantly increased with age in both sexes, with females having higher levels compared to males. A similar age-related increase in total protein was observed in Weiser-Maples guinea pigs [5], but not strain 13 guinea pigs [6]. Although total bilirubin measured 0 mg/dL in all guinea pigs included in this study, this value is consistent with reference intervals of other guinea pigs [18].

PLOS ONE
ALP and phosphorus significantly decreased with age, likely due to the decline in bone growth as animals reached skeletal maturity. In the guinea pig, bone growth is purported to cease by 4 months of age [20]. Liver enzymes ALT and GGT markedly increased with age in males, and AST increased with age in both sexes. However, these enzymes were not correlated with age in other strains [5,6]. Male Hartleys had significantly higher levels of ALT than females, whereas strain 13 males had significantly higher levels of ALT, AST, and GGT than females. Increased levels of these enzymes may be indicative of hepatocellular and/or biliary disease.
Consistent with Weiser-Maples guinea pigs [5] and Sprague Dawley rats [10,12], cholesterol increased with age in males, but females had higher overall levels compared to males [5]. Dunkin Hartley females also had an age-associated increase in iron, with significantly higher levels compared to males. In contrast, iron decreased with age in both male and female mice [9]. Magnesium was positively correlated with age in males, leading to significantly higher levels in adults than juveniles. Male guinea pigs had higher levels of bicarbonate, whereas females had higher potassium, anion gap, and calculated osmolality. Additionally, bicarbonate was positively correlated with age and potassium was negatively correlated with age in males, which led to a negative age correlation in anion gap. Similar to Weiser-Maples guinea pigs [5], sodium was positively correlated with age in female Dunkin Hartley guinea pigs. Iron, magnesium, bicarbonate, and calculated osmolality were not evaluated in other guinea pig studies. The reason for these age-and sexrelated differences in electrolytes is unknown and warrants further investigation.
In addition to strain, age, and sex, blood parameters may be affected by other factors that should be considered when applying these reference intervals to other animals. For example, the majority of guinea pigs included in this study were anesthetized with isoflurane for blood collection. As blood collection is challenging in this species, anesthesia is often necessary to collect large blood volumes to minimize trauma and stressful handling. Isoflurane has been shown to increase white blood cells and liver enzymes and decrease red blood cells and plasma proteins in Dunkin Hartley guinea pigs [21]. Additionally, this study included data from blood collected from the cranial vena cava and jugular vein, as well as directly from the heart. In rodents, hematologic and biochemical parameters can vary when blood is collected from different anatomical locations [22][23][24][25][26][27]. It should also be noted that all guinea pigs in this study were housed in Colorado at an elevation of approximately 5,000 ft, which may impact certain RBC parameters. For example, humans at high altitude have increased erythropoietin and hemoglobin levels, as well as increased red blood cell volume [28]. Future work would assess a comparison of subjects at high altitude versus sea level.
In conclusion, this study found several important differences in hematologic and biochemical parameters of Dunkin Hartley guinea pigs based on age and sex. Additionally, our results showed many differences between Dunkin Hartley and other strains of guinea pigs, which further emphasizes the need for strain-specific reference intervals. Establishing these differences provides valuable insight into their physiology to better evaluate diagnostics and experimental results.