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. 2021 Jul 9;16(7):e0253794. doi: 10.1371/journal.pone.0253794

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

Alexa P Spittler 1, Maryam F Afzali 1, Sydney B Bork 1, Lindsey H Burton 1, Lauren B Radakovich 1, Cassie A Seebart 1, A Russell Moore 1, Kelly S Santangelo 1,*
Editor: Antonio Humberto Hamad Minervino2
PMCID: PMC8270176  PMID: 34242236

Abstract

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 [7–9]. These studies in guinea pigs [5,6] and other laboratory rodents [8–12] 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.

Materials & methods

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 2.5th and 97.5th 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.

Results

Hematology

Descriptive statistics and reference intervals were established for hematology parameters of 68 juvenile (52 males; 16 females) and 144 adult (93 males; 51 females) Dunkin Hartley guinea pigs (Table 1). Manual WBC differential counts were unavailable from 12 adult females. Thus, reference intervals for heterophil, lymphocyte, monocyte, eosinophil, and basophil percentages and counts were calculated from 132 adults. Compared to juveniles, adults had significantly higher Hct (median difference 1%; 95% CI 0–2; P = 0.0127), RDW (median difference 0.4%; 95% CI 0–0.7; P = 0.0482), MPV (median difference 0.7 fl; 95% CI 0.3–0.7; P < 0.0001), WBC (median difference 0.8 × 103/μL; 95% CI 0.3–1.1; P = 0.0018), heterophil % (mean difference 8%; 95% CI 5–11; P < 0.0001), heterophils (median difference 0.732 × 103/μL; 95% CI 0.359–0.896; P < 0.0001), monocytes (median difference 0.046 × 103/μL; 95% CI 0.006–0.090; P = 0.0227), eosinophil % (median difference 1%; 95% CI 0–1; P = 0.0106), and eosinophils (median difference 0.042 × 103/μL; 95% CI 0.007–0.058; P = 0.0048). Juveniles had higher MCHC (mean difference 1 g/dL; 95% CI 0–1; P = 0.0008), CHCM (mean difference 1 g/dL; 95% CI 0–1; P = 0.003), platelets (median difference 32 × 103/μL; 95% CI 15–80; P = 0.0039), lymphocyte % (mean difference 11%; 95% CI 7–14; P < 0.0001), and lymphocytes (median 0.103 × 103/μL; 95% CI 0.016–0.462; P = 0.0357) compared to adults.

Table 1. Summary data and reference intervals for hematology parameters of juvenile (< 5 mo) and adult (≥ 5 mo) Hartley guinea pigs.

Analyte Age N Mean SD Median Min Max P-value Distribution Method RI 90% CI of LRL 90% CI of URL P-value
(Juveniles vs. Adults)
Hgb (g/dL) Juveniles 67 15.2 0.7 15.1 13.4 16.7 0.371 G NP 13.8–16.6 13.4–14.2 16.3–16.7 0.2668
Adults 144 15.3 0.9 15.4 11.9 17.3 0.001 NG NP 13.4–16.7 11.9–13.6 16.5–17.3
Hgb (cell) (g/dL) Juveniles 67 15.1 0.8 15.2 13.5 16.9 0.496 G NP 13.6–16.6 13.5–13.9 16.2–16.9 0.2878
Adults 144 15.2 1.0 15.4 11.9 17.4 0.002 NG NP 12.9–17.0 11.9–13.4 16.6–17.4
Hct (%) Juveniles 67 46 2 46 41 52 0.012 NG NP 41–52 41–42 50–52 0.0127a
Adults 144 47 3 47 38 53 0.000 NG NP 41–51 38–42 51–53
RBC (× 106/μL) Juveniles 68 5.59 0.32 5.56 4.76 6.53 0.599 G NP 4.93–6.30 4.76–5.12 6.12–6.53 0.2247
Adults 144 5.62 0.37 5.66 4.16 6.42 0.028 NG NP 4.79–6.20 4.16–4.98 6.15–6.42
MCV (fl) Juveniles 68 83 3 83 77 90 0.062 G NP 78–89 77–78 87–90 0.2222
Adults 143 84 3 84 78 94 0.000 NG NP 79–89 78–80 88–94
RDW (%) Juveniles 67 13.0 1.6 12.5 11.0 19.4 0.000 NG NP 11.1–17.2 11.0–11.2 15.4–19.4 0.0482a
Adults 143 13.0 0.9 12.9 11.1 16.0 0.000 NG NP 11.5–15.3 11.1–11.7 15.0–16.0
MCHC (g/dL) Juveniles 68 33 1 33 31 35 0.000 NG NP 31–35 31–32 34–35 0.0008c
Adults 144 32 1 32 30 35 0.000 NG NP 31–34 30–31 34–35
CHCM (g/dL) Juveniles 68 33 1 33 30 35 0.000 NG NP 31–35 30–31 34–35 0.0030b
Adults 143 32 1 32 30 35 0.000 NG NP 31–34 30–31 34–35
Platelets (× 103/μL) Juveniles 68 539 113 519 297 856 0.055 G NP 332–801 297–363 735–856 0.0039b
Adults 143 489 124 487 112 943 0.002 NG NP 259–800 112–298 703–943
MPV (fl) Juveniles 68 8.0 0.7 7.9 6.6 9.2 0.040 NG NP 6.8–9.1 6.6–7.0 9.0–9.2 < 0.0001d
Adults 144 8.4 0.5 8.6 7.0 9.3 0.000 NG NP 7.3–9.2 7.0–7.5 9.1–9.3
WBC (× 103/μL) Juveniles 68 5.1 1.5 4.9 2.2 9.6 0.000 NG NP 2.6–9.4 2.2–3.2 8.7–9.6 0.0018b
Adults 144 5.7 1.8 5.7 1.9 11.7 0.008 NG NP 2.7–10.3 1.9–3.1 8.9–11.7
Heterophils (%) Juveniles 68 41 10 40 18 64 0.343 G NP 18–61 18–26 56–64 < 0.0001d
Adults 132 49 11 49 23 79 0.380 G NP 29–72 23–33 66–79
Heterophils (× 103/μL) Juveniles 68 2.134 1.002 1.888 0.396 5.248 0.000 NG NP 0.738–5.219 0.396–1.012 4.349–5.248 < 0.0001d
Adults 130 2.750 1.078 2.620 0.437 6.804 0.001 NG NP 1.054–5.403 0.437–1.333 4.902–6.804
Lymphocytes (%) Juveniles 68 52 9 51 31 71 0.761 G NP 33–71 31–38 68–71 < 0.0001d
Adults 132 41 12 41 14 70 0.243 G NP 18–63 14–26 59–70
Lymphocytes (× 103/μL) Juveniles 68 2.553 0.702 2.339 1.414 4.644 0.000 NG NP 1.521–4.387 1.414–1.702 3.866–4.644 0.0357a
Adults 132 2.308 0.827 2.236 0.476 4.816 0.087 G NP 0.775–4.177 0.476–1.140 3.577–4.816
Foa-Kurloff Cells (%) Juveniles 67 3 3 2 0 9 0.000 NG NP 0–9 0–0 8–9 0.1032
Adults 132 3 2 3 0 11 0.000 NG NP 0–9 0–0 8–11
Foa-Kurloff Cells (× 103/μL) Juveniles 68 0.150 0.151 0.126 0 0.576 0.000 NG NP 0–0.512 0–0 0.415–0.576 0.0504
Adults 132 0.192 0.163 0.156 0 0.757 0.000 NG NP 0–0.710 0–0 0.500–0.757
Monocytes (%) Juveniles 68 4 2 3 0 9 0.000 NG NP 0–9 0–1 8–9 0.3164
Adults 131 4 3 4 0 14 0.000 NG NP 0–10 0–1 8–14
Monocytes (× 103/μL) Juveniles 67 0.172 0.124 0.159 0 0.558 0.000 NG NP 0–0.503 0–0.033 0.415–0.558 0.0227a
Adults 131 0.239 0.195 0.205 0 1.020 0.000 NG NP 0–0.792 0–0.019 0.696–1.020
Eosinophils (%) Juveniles 67 2 1 1 0 5 0.000 NG NP 0–5 0–0 4–5 0.0106a
Adults 130 2 2 2 0 8 0.000 NG NP 0–7 0–0 6–8
Eosinophils (× 103/μL) Juveniles 67 0.074 0.067 0.060 0 0.265 0.000 NG NP 0–0.253 0–0 0.210–0.265 0.0048b
Adults 128 0.117 0.101 0.102 0 0.442 0.000 NG NP 0–0.363 0–0 0.330–0.442
Basophils (%) Juveniles 68 0 1 0 0 2 0.000 NG NP 0–2 0–0 1–2 0.3021
Adults 130 0 1 0 0 2 0.000 NG NP 0–2 0–0 2–2
Basophils (× 103/μL) Juveniles 68 0.012 0.027 0 0 0.112 0.000 NG NP 0–0.102 0–0 0.069–0.112 0.3041
Adults 128 0.016 0.030 0 0 0.108 0.000 NG NP 0–0.102 0–0 0.089–0.108
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aP < 0.05

bP < 0.01

cP < 0.001

dP < 0.0001 between values for juvenile and adult Hartley guinea pigs. Abbreviations: G, Gaussian; NG, non-Gaussian; NP, nonparametric; RI, reference interval.

Age correlation of hematology parameters in male and female guinea pigs is shown in Table 2. In both sexes, MPV (males: r = 0.4555; females: r = 0.5114), heterophil % (males: r = 0.3237; females: r = 0.5893) and heterophils (males: r = 0.2996; females: r = 0.5587) were positively correlated with age, and CHCM (males: r = -0.2777; females: r = -0.3660) and lymphocyte % (males: r = -0.4704; females: r = -0.5799) were negatively correlated with age. In males alone, Hct (r = 0.1942), RDW (r = 0.2108), WBC (r = 0.1802), Foa-Kurloff cell % (r = 0.3274), monocytes (r = 0.1937), eosinophils % (r = 0.1972), and eosinophils (r = 0.2401) were positively correlated with age, while MCHC (r = -0.2498), platelets (r = -0.2647), and lymphocytes (r = -0.2186) were negatively correlated. MCV (r = 0.4211) was positively correlated with age in females.

Table 2. Spearman correlation (r) of hematology parameters with age in 145 male and 67 female Dunkin Hartley guinea pigs.

Analyte Males Females
Hgb (g/dL) 0.09060 0.0848
Hgb (cell) (g/dL) 0.03184 0.0255
Hct (%) 0.1942a 0.1354
RBC (× 106/μL) 0.1360 -0.0576
MCV (fl) 0.05356 0.4211c
RDW (%) 0.2108a -0.1209
MCHC (g/dL) -0.2498b -0.2016
CHCM (g/dL -0.2777c -0.3660b
Platelets (× 103/μL) -0.2647b -0.1259
MPV (fl) 0.4555d 0.5114d
WBC (× 103/μL) 0.1802a 0.1620
Heterophils (%) 0.3237d 0.5893d
Heterophils (× 103/μL) 0.2996c 0.5587d
Lymphocytes (%) -0.4704d -0.5799d
Lymphocytes (× 103/μL) -0.2186b -0.2508
Foa-Kurloff Cells (%) 0.3274d -0.2113
Foa-Kurloff Cells (× 103/μL) 0.3193d -0.1643
Monocytes (%) 0.1311 0.1191
Monocytes (× 103/μL) 0.1937a 0.2224
Eosinophils (%) 0.1972a 0.2249
Eosinophils (× 103/μL) 0.2401b 0.2165
Basophils (%) 0.07390 0.01767
Basophils (× 103/μL) 0.06626 0.0280
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aP < 0.05

bP < 0.01

cP < 0.001

dP < 0.0001.

Hematology reference intervals for adult guinea pigs were partitioned by sex (Table 3). Since manual WBC differential counts were unavailable from 12 adult females, reference intervals for heterophil, lymphocyte, monocyte, eosinophil, and basophil percentages and counts were calculated from 39 females. Males had significantly higher Hgb (median difference 0.5 g/dL; 95% CI 0.3–0.9; P = 0.003), Hgb (cell) (mean difference 0.5 g/dL; 95% CI 0.1–0.8; P = 0.0033), Hct (median difference 2%; 95% CI 0–2; P = 0.0231), RBC (median difference 0.31 × 106/μL; 95% CI 0.21–0.43; P < 0.0001), MCHC (median difference 1 g/dL; 95% CI 0–1; P = 0.0002), WBC (median difference 0.4 × 103/μL; 0.1–1.2; P = 0.0179), heterophil % (median difference 11%; 95% CI 5–13; P < 0.0001), heterophils (median difference 0.794 × 103/μL; 95% CI 0.405–1.054; P = 0.0001), Foa-Kurloff cell % (mean difference 2%; 95% CI 0–2; P = 0.0084), and Foa-Kurloff cells (median difference 0.075 × 103/μL; 95% CI 0.010–0.120; P = 0.0095) compared to females. Females had higher MCV (median difference 3 fl; 95% CI 2–4; P < 0.0001), RDW (median difference 0.4%; 95% CI 0.3–0.8; P = 0.0002), platelets (median difference 77 × 103/μL; 95% CI 45–126; P < 0.0001), MPV (median difference 0.5 fl; 95% CI 0.3–0.6; P < 0.0001), lymphocyte % (median difference 8%; 95% CI 2–12; P = 0.0017), eosinophil % (median difference 2%; 95% CI 1–3; P < 0.0001), and eosinophils (median difference 0.071 × 103/μL; 95% CI 0.036–0.121; P = 0.0001) than males.

Table 3. Summary data and reference intervals for hematology parameters of adult male and female Hartley guinea pigs.

Analyte Sex N Mean SD Median Min Max P-value Distribution Method RI 90% CI of LRL 90% CI of URL P-value
(Males vs. Females)
Hgb (g/dL) Males 93 15.5 0.9 15.6 12.4 17.3 0.022 NG NP 13.4–16.9 12.4–13.8 16.6–17.3 0.0003c
Females 50 14.9 0.9 15.1 13.3 16.7 0.063 G NP 13.3–16.6 13.3–13.5 16.1–16.7
Hgb (cell) (g/dL) Males 93 15.4 0.9 15.5 12.6 17.4 0.106 G NP 13.2–17.3 12.6–13.9 16.8–17.4 0.0033b
Females 51 14.9 1.0 15.0 11.9 16.6 0.015 NG NP 12.1–16.5 11.9–13.2 16.1–16.6
Hct (%) Males 93 47 3 48 38 53 0.000 NG NP 41–52 38–43 51–53 0.0231a
Females 51 46 3 46 40 52 0.195 G NP 40–51 40–41 50–52
RBC (× 106/μL) Males 93 5.73 0.33 5.76 4.59 6.42 0.042 NG NP 4.83–6.24 4.59–5.17 6.17–6.42 < 0.0001d
Females 51 5.41 0.36 5.45 4.16 6.18 0.259 G NP 4.35–6.11 4.16–4.92 5.88–6.18
MCV (fl) Males 93 83 2 83 78 89 0.000 NG NP 78–88 78–80 86–89 < 0.0001d
Females 51 86 3 86 79 95 0.028 NG NP 79–95 79–81 91–95
RDW (%) Males 93 12.8 0.8 12.8 11.1 15.3 0.001 NG NP 11.4–15.0 11.1–11.6 14.2–15.3 0.0002c
Females 51 13.5 1.1 13.2 11.8 17.2 0.020 NG NP 11.8–16.8 11.8–12.0 15.4–17.2
MCHC (g/dL) Males 92 33 1 33 32 35 0.000 NG NP 32–34 32–32 34–35 0.0002c
Females 51 32 1 32 30 34 0.000 NG NP 30–34 30–31 34–34
CHCM (g/dL) Males 92 32 1 32 31 35 0.000 NG NP 31–34 31–31 33–35 0.1094
Females 51 32 1 32 30 34 0.000 NG NP 30–34 30–30 34–34
Platelets (× 103/μL) Males 93 454 99 470 112 688 0.108 G NP 197–636 112–288 604–688 < 0.0001d
Females 51 562 155 547 333 1025 0.012 NG NP 335–1000 333–352 837–1025
MPV (fl) Males 93 8.3 0.5 8.3 7.0 9.2 0.014 NG NP 7.1–9.1 7.0–7.3 9.0–9.2 < 0.0001d
Females 50 8.8 0.3 8.8 7.7 9.3 0.010 NG NP 7.7–9.3 7.7–8.3 9.3–9.3
WBC (× 103/μL) Males 93 6.0 1.8 5.8 2.0 11.7 0.041 NG NP 3.0–10.7 2.0–3.2 9.5–11.7 0.0179a
Females 51 5.3 1.5 5.4 1.9 9.1 0.388 G NP 2.1–9.0 1.9–3.0 7.3–9.1
Heterophils (%) Males 93 51 10 51 31 73 0.809 G NP 33–72 31–35 68–73 0.0001c
Females 39 43 12 40 23 79 0.027 NG TS 25–75 23–28 65–88
Heterophils (× 103/μL) Males 91 2.972 1.085 2.907 1.156 6.804 0.006 NG NP 1.324–5.463 1.156–1.426 5.157–6.804 0.0001c
Females 38 2.161 0.765 2.113 0.437 4.277 0.101 G US 0.590–3.731 0.248–0.955 3.369–4.101
Lymphocytes (%) Males 93 39 10 40 14 63 0.755 G NP 18–59 14–24 56–63 0.0017b
Females 39 46 13 48 14 70 0.099 G US 18–73 12–25 67–79
Lymphocytes (× 103/μL) Males 93 2.283 0.769 2.223 0.560 4.816 0.153 G NP 0.806–3.695 0.560–1.168 3.540–4.816 0.5240
Females 39 2.367 0.959 2.420 0.476 4.450 0.519 G US 0.402–4.333 0–0.865 3.885–4.782
Foa-Kurloff Cells (%) Males 93 4 3 4 0 11 0.008 NG NP 0–9 0–0 9–11 0.0084b
Females 39 2 2 2 0 6 0.025 NG TS 0–4 0–1 4–5
Foa-Kurloff Cells (× 103/μL) Males 93 0.215 0.174 0.183 0 0.757 0.000 NG NP 0–0.713 0–0 0.513–0.757 0.0095b
Females 39 0.137 0.119 0.108 0 0.396 0.003 NG TS 0–0.436 0–0 0.349–0.521
Monocytes (%) Males 93 4 3 4 0 11 0.001 NG NP 0–10 0–1 9–11 0.1418
Females 37 3 2 3 0 7 0.056 NG US 1–7 0–0 6–8
Monocytes (× 103/μL) Males 93 0.259 0.210 0.210 0 1.020 0.000 NG NP 0.004–0.930 0–0.034 0.710–1.020 0.2136
Females 38 0.192 0.144 0.163 0 0.574 0.074 G NP 0–0.487 0–0 0.409–0.553
Eosinophils (%) Males 93 2 1 2 0 7 0.000 NG NP 0–6 0–0 4–7 < 0.0001d
Females 38 4 3 4 0 11 0.112 G UR 0–9 0–0 8–10
Eosinophils (× 103/μL) Males 93 0.097 0.089 0.077 0 0.442 0.000 NG NP 0–0.339 0–0 0.278–0.442 0.0001c
Females 37 0.195 0.157 0.148 0 0.712 0.003 NG TS 0–0.618 0–0.019 0.480–0.783
Basophils (%) Males 93 0 1 0 0 2 0.000 NG NP 0–2 0–0 1–2 0.8752
Females 37 0 1 0 0 2 0.000 NG US 0–2 0–0 1–2
Basophils (× 103/μL) Males 91 0.015 0.027 0 0 0.103 0.000 NG NP 0–0.098 0–0 0.080–0.103 0.9139
Females 37 0.019 0.037 0 0 0.128 0.000 NG US 0–0.096 0–0 0.060–0.119
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aP < 0.05

bP < 0.01

cP < 0.001

dP < 0.0001 between values for adult male and female Hartley guinea pigs. Abbreviations: G, Gaussian; NG, non-Gaussian; NP, nonparametric; US, untransformed standard; UR, untransformed robust; TS, transformed standard; RI, reference interval.

Serum biochemistry

Descriptive statistics and reference intervals were established for serum biochemistry analytes of 49 juveniles (41 males; 8 females) and 145 adult (93 males; 52 females) Dunkin Hartley guinea pigs (Table 4). Magnesium was unavailable from 11 adult males. Iron was unavailable from 28 juvenile males and 23 adult males. Additionally, there was insufficient blood volume to complete testing of all analytes in some animals: glucose (2 juveniles; 1 adult), BUN (2 juveniles; 1 adult), creatinine (1 juvenile; 1 adult), phosphorus (2 juveniles), calcium (3 juveniles), magnesium (3 juveniles), total protein (2 juveniles), globulin (2 juveniles), A/G (2 juveniles), cholesterol (1 juvenile; 1 adult), total bilirubin (2 juveniles), ALP (1 juvenile), ALT (1 juvenile), GGT (1 juvenile), bicarbonate (2 juveniles), anion gap (2 juveniles; 1 adult), and calculated osmolality (2 juveniles; 1 adult).

Table 4. Summary data and reference intervals for serum biochemistry parameters of juvenile (< 5 mo) and adult (≥ 5 mo) Hartley guinea pigs.

Analyte Age N Mean SD Median Min Max P-value Distribution Method RI 90% CI of LRL 90% CI of URL P-value
(Juveniles vs. Adults)
Glucose (mg/dL) Juveniles 47 203 47 201 125 415 0.020 NG NP 127–384 125–153 256–415 < 0.0001d
Adults 137 235 60 227 123 454 0.000 NG NP 128–427 123–146 374–454
BUN (mg/dL) Juveniles 47 18 2 18 11 23 0.048 NG NP 11–23 11–14 21–23 < 0.0001d
Adults 143 21 3 21 13 33 0.010 NG NP 16–29 13–16 27–33
Creatinine (mg/dL) Juveniles 47 0.3 0.1 0.3 0.2 0.4 0.000 NG NP 0.2–0.4 0.2–0.2 0.3–0.4 < 0.0001d
Adults 144 0.4 0.1 0.3 0.2 0.6 0.000 NG NP 0.2–0.5 0.2–0.3 0.5–0.6
Phosphorus (mg/dL) Juveniles 47 6.1 1.1 6.1 3.5 8.6 0.165 G NP 3.6–8.5 3.5–4.5 7.9–8.6 < 0.0001d
Adults 145 5.1 0.8 5.0 3.4 7.6 0.019 NG NP 3.7–6.9 3.4–3.8 6.4–7.6
Calcium (mg/dL) Juveniles 46 10.7 0.6 10.7 8.9 11.8 0.285 G NP 9.1–11.8 8.9–9.9 11.6–11.8 < 0.0001d
Adults 145 11.3 0.5 11.3 10.0 12.9 0.007 NG NP 10.5–12.5 10.0–10.6 12.2–12.9
Magnesium (mg/dL) Juveniles 46 3.3 0.5 3.2 2.5 4.6 0.021 NG NP 2.5–4.6 2.5–2.6 4.3–4.6 < 0.0001d
Adults 134 3.6 0.5 3.6 2.2 5.1 0.174 G NP 2.7–4.6 2.2–2.9 4.4–5.1
Total Protein (g/dL) Juveniles 46 4.8 0.3 4.8 4.0 5.3 0.150 G NP 4.0–5.3 4.0–4.3 5.2–5.3 < 0.0001d
Adults 145 5.4 0.3 5.3 4.7 6.4 0.030 NG NP 4.7–6.1 4.7–4.8 5.9–6.4
Albumin (g/dL) Juveniles 47 3.0 0.1 3.0 2.7 3.3 0.000 NG NP 2.7–3.3 2.7–2.8 3.2–3.3 < 0.0001d
Adults 145 3.2 0.2 3.2 2.7 3.7 0.000 NG NP 2.8–3.6 2.7–2.8 3.5–3.7
Globulin (g/dL) Juveniles 47 1.8 0.2 1.8 1.4 2.4 0.205 G NP 1.4–3.4 1.4–1.4 2.1–2.4 < 0.0001d
Adults 145 2.2 0.2 2.2 1.7 2.9 0.000 NG NP 1.8–2.6 1.7–1.9 2.5–2.9
A/G Ratio Juveniles 47 1.66 0.19 1.65 1.40 2.10 0.015 NG NP 1.40–2.10 1.40–1.40 1.90–2.10 < 0.0001d
Adults 145 1.45 0.13 1.45 1.04 1.80 0.028 NG NP 1.20–1.75 1.04–1.27 1.70–1.80
Cholesterol (mg/dL) Juveniles 48 31 7 31 19 56 0.022 NG NP 20–54 19–23 44–56 0.0100b
Adults 140 37 14 35 12 86 0.000 NG NP 16–79 12–19 68–86
CK (IU/L) Juveniles 47 434 278 380 141 1211 0.000 NG NP 142–1200 141–161 1083–1211 0.1648
Adults 139 560 437 425 107 2269 0.000 NG NP 121–1897 107–141 1505–2269
Total Bilirubin (mg/dL) Juveniles 47 0 0 0 0 0 - - NP 0–0 0–0 0–0 > 0.9999
Adults 144 0 0 0 0 0 - - NP 0–0 0–0 0–0
ALP (IU/L) Juveniles 47 188 64 181 89 322 0.057 G NP 89–320 89–97 284–322 < 0.0001d
Adults 145 79 36 72 29 205 0.000 NG NP 34–167 29–37 159–205
ALT (IU/L) Juveniles 48 35 10 33 12 69 0.002 NG NP 15–66 12–25 52–69 0.0827
Adults 142 38 11 36 15 77 0.000 NG NP 18–70 15–23 59–77
AST (IU/L) Juveniles 48 44 20 39 22 108 0.000 NG NP 23–108 22–27 84–108 < 0.0001d
Adults 141 67 36 54 22 203 0.000 NG NP 25–171 22–29 134–203
GGT (IU/L) Juveniles 48 11 6 10 0 27 0.003 NG NP 1–27 0–5 23–27 < 0.0001d
Adults 145 18 7 17 0 37 0.004 NG NP 7–34 0–8 31–37
Sodium (mEQ/L) Juveniles 49 136 2 136 130 140 0.000 NG NP 130–140 130–131 139–140 0.8469
Adults 145 136 3 137 127 144 0.000 NG NP 129–141 127–131 140–144
Potassium (mEQ/L) Juveniles 49 5.30 1.06 5.15 3.85 9.00 0.001 NG NP 3.89–8.88 3.85–4.16 7.24–9.00 0.1433
Adults 143 5.06 0.88 4.88 3.66 7.41 0.000 NG NP 3.90–7.04 3.66–3.99 6.90–4.41
Chloride (mEQ/L) Juveniles 49 101.3 2.5 101.4 94.7 106.7 0.885 G NP 95.0–106.5 94.7–98.1 105.0–106.7 0.3990
Adults 145 100.7 3.3 101.2 88.5 107.3 0.002 NG NP 91.7–107.1 88.5–95.2 105.2–107.3
Bicarbonate (mEQ/L) Juveniles 46 21.2 3.4 21.2 14.8 28.6 0.578 G NP 15.0–28.5 14.8–16.2 26.2–28.6 < 0.0001d
Adults 144 24.4 2.5 24.6 18.5 31.3 0.677 G NP 19.4–29.4 18.5–20.1 28.4–31.3
Anion Gap (mmol/L) Juveniles 47 19 3 18 11 26 0.273 G NP 12–26 11–14 24–26 < 0.0001d
Adults 142 16 2 16 12 22 0.000 NG NP 13–21 12–13 21–22
Calculated Osmolality (mOsm/kg) Juveniles 47 281 5 281 268 289 0.090 G NP 269–289 268–271 288–289 0.0001c
Adults 144 284 5 284 269 302 0.097 G NP 273–294 269–275 292–302
Iron (μg/dL) Juveniles 21 293 29 288 232 358 0.331 G US 231–356 211–252 338–377 0.5212
Adults 122 299 41 298 186 405 0.614 G NP 215–391 186–238 367–405
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aP < 0.05

bP < 0.01

cP < 0.001

dP < 0.0001 between values for juvenile and adult Hartley guinea pigs. Abbreviations: G, Gaussian; NG, non-Gaussian; NP, nonparametric; US, untransformed standard; RI, reference interval.

Significant differences in biochemistry parameters between juveniles and adults are shown in Table 4. Glucose (median difference 26 mg/dL; 95% CI 16–44; P < 0.0001), BUN (median difference 3 mg/dL; 95% CI 2–4; P < 0.0001), creatinine (median difference 0.0 mg/dL; 95% CI 0.1–0.1; P < 0.0001), calcium (median difference 0.6 mg/dL; 95% CI 0.4–0.7; P < 0.0001), magnesium (median difference 0.4 mg/dL; 95% CI 0.2–0.5; P < 0.0001), total protein (mean difference 0.6 g/dL; 95% CI 0.5–0.7; P < 0.0001), albumin (mean difference 0.2 g/dL; 95% CI 0.1–0.3; P < 0.0001), globulin (mean difference 0.4 g/dL; 95% CI 0.3–0.5; P < 0.0001), cholesterol (median difference 4 mg/dL; 95% CI 1–7; P = 0.01), AST (median difference 15 IU/L; 95% CI 9–23; P < 0.0001), GGT (median difference 7 IU/L; 95% CI 4–9; P < 0.0001), bicarbonate (mean difference 3.2 mEQ/L; 95% CI 2.3–4.2; P < 0.0001), and calculated osmolality (mean difference 3 mOsm/kg; 95% CI 2–5; P = 0.0001) were significantly increased in adults compared to juveniles. Phosphorus (median difference 1.1 mg/dL; 95% CI 0.8–1.3; P < 0.0001), A/G (mean difference 0.21; 95% CI 0.16–0.26; P < 0.0001), ALP (median difference 109 IU/L; 95% CI 85–127; P < 0.0001), and anion gap (median difference 2 mmol/L; 95% CI 2–4; P < 0.0001) were significantly increased in juveniles compared to adults.

Age correlation of serum biochemistry parameters in males and females is presented in Table 5. In both males and females, BUN (males: r = 0.3729; females: r = 0.4789), creatinine (males: r = 0.6504; females: r = 0.2947), calcium (males: r = 0.4971; females: r = 0.3740), total protein (males: r = 0.6481; females: r = 0.5333), albumin (males: r = 0.4198; females: r = 0.3804), globulin (males: r = 0.6557; females: r = 0.5455), and AST (males: r = 0.3374; females: r = 0.3494) were positively correlated with age, and phosphorus (males: r = -0.5403; females: r = -0.2627), A/G (males: r = -0.5709; females: r = -0.3273), and ALP (males: r = -0.7074; females: r = -0.7501) were negatively correlated with age. Glucose (r = 0.4273), magnesium (r = 0.3933), cholesterol (r = 0.1776), CK (r = 0.2006), ALT (r = 0.2023), GGT (r = 0.3974), and bicarbonate (r = 0.4356) increased with age, whereas potassium (r = -0.1780) and anion gap (r = -0.4931) decreased with age in males. In females, sodium (r = 0.2650) and iron (r = 0.6484) increased with age.

Table 5. Spearman correlation (r) of serum biochemistry parameters with age in male and female Dunkin Hartley guinea pigs.

Analyte Males Females
Glucose (mg/dL) 0.4273d 0.0817
BUN (mg/dL) 0.3729d 0.4789c
Creatinine (mg/dL) 0.6504d 0.2947a
Phosphorus (mg/dL) -0.5403d -0.2627a
Calcium (mg/dL) 0.4971d 0.3740b
Magnesium (mg/dL) 0.3933d 0.0363
Total Protein (g/dL) 0.6481d 0.5333d
Albumin (g/dL) 0.4198d 0.3804b
Globulin (g/dL) 0.6557d 0.5455d
A/G Ratio -0.5709d -0.3273a
Cholesterol (mg/dL) 0.177a 0.0892
CK (IU/L) 0.2006a 0.1707
Total Bilirubin (mg/dL) - -
ALP (IU/L) -0.7074d -0.7501d
ALT (IU/L) 0.2023a 0.04274
AST (IU/L) 0.3374d 0.3494b
GGT (IU/L) 0.3974d 0.2274
Sodium (mEQ/L) 0.1533 0.2650a
Potassium (mEQ/L) -0.1780a 0.1865
Chloride (mEQ/L) 0.1291 0.2298
Bicarbonate (mEQ/L) 0.4356d -0.1111
Anion Gap (mmol/L) -0.4931d 0.0681
Calculated Osmolality (mOsm/kg) 0.3648d 0.4568c
Iron (μg/dL) 0.0623 0.6484d
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aP < 0.05

bP < 0.01

cP < 0.001

dP < 0.0001.

Adult reference intervals for serum biochemistry were partitioned by sex (Table 6). Magnesium and iron were unavailable from 11 and 23 males, respectively. Due to low blood sample volumes, the following analytes were not tested in all animals: glucose (n = 1 male), BUN (1 male), creatinine (1 male), cholesterol (1 male), anion gap (1 female), and calculated osmolality (1 female). Outliers removed from males included glucose (n = 2), BUN (1), CK (4), total bilirubin (1), ALT (2), AST (2), potassium (1), and chloride (1). Glucose (n = 2), CK (3), ALT (1), AST (2), and bicarbonate (1) outliers were removed from females.

Table 6. Summary data and reference intervals for serum biochemistry parameters of adult male and female Hartley guinea pigs.

Analyte Sex N Mean SD Median Min Max P-value Distribution Method RI 90% CI of LRL 90% CI of URL P-value
(Males vs. Females)
Glucose (mg/dL) Males 90 229 55 227 123 411 0.002 NG NP 126–392 123–140 315–411 0.6527
Females 50 260 84 232 159 475 0.000 NG NP 163–473 159–184 454–475
BUN (mg/dL) Males 91 21 3 21 16 33 0.030 NG NP 16–29 16–16 26–33 0.9909
Females 52 21 4 21 13 32 0.348 G NP 14–31 13–16 27–32
Creatinine (mg/dL) Males 92 0.4 0.1 0.4 0.2 0.6 0.000 NG NP 0.2–0.5 0.2–0.3 0.5–0.6 0.0870
Females 52 0.3 0.1 0.3 0.2 0.5 0.000 NG NP 0.2–0.5 0.2–0.3 0.4–0.5
Phosphorus (mg/dL) Males 93 5.0 0.7 5.0 3.6 6.9 0.289 G NP 3.7–6.4 3.6–3.8 6.2–6.9 0.0838
Females 52 5.2 1.0 5.1 3.4 7.6 0.237 G NP 3.5–7.5 3.4–4.0 6.9–7.6
Calcium (mg/dL) Males 93 11.3 0.4 11.3 10.4 12.5 0.118 G NP 10.5–12.4 10.4–10.6 12.0–12.5 0.4169
Females 52 11.3 0.6 11.3 10.0 12.9 0.058 G NP 10.1–12.8 10.0–10.5 12.4–12.9
Magnesium (mg/dL) Males 82 3.6 0.5 3.7 2.5 5.1 0.256 G NP 2.7–5.1 2.5–2.9 4.5–5.1 0.1709
Females 52 3.5 0.4 3.5 2.2 4.4 0.389 G NP 2.4–4.3 2.2–2.8 4.1–4.4
Total Protein (g/dL) Males 93 5.3 0.3 5.3 4.7 5.8 0.018 NG NP 4.7–5.8 4.7–4.8 5.7–5.8 0.0002c
Females 52 5.5 0.4 5.5 4.7 6.4 0.245 G NP 4.7–6.3 4.7–5.0 6.1–6.4
Albumin (g/dL) Males 93 3.1 0.2 3.1 2.7 3.4 0.000 NG NP 2.8–6.4 2.7–2.8 3.3–3.4 < 0.0001d
Females 52 3.3 0.2 3.3 2.7 3.7 0.112 G NP 2.7–3.7 2.7–2.8 3.6–3.7
Globulin (g/dL) Males 93 2.2 0.2 2.2 1.7 2.6 0.003 NG NP 1.7–2.5 1.7–1.9 2.5–2.6 0.0363a
Females 52 2.2 0.2 2.2 1.9 2.9 0.001 NG NP 1.9–2.8 1.9–2.0 2.6–2.9
A/G Ratio Males 93 1.45 0.14 1.43 1.04 1.80 0.003 NG NP 1.19–1.76 1.04–1.29 1.70–1.80 0.5888
Females 52 1.46 0.11 1.49 1.20 1.65 0.038 NG NP 1.20–1.64 1.20–1.24 1.61–1.65
Cholesterol (mg/dL) Males 92 36 13 35 17 86 0.000 NG NP 18–77 17–20 55–86 0.0317a
Females 52 46 25 38 12 119 0.000 NG NP 13–117 12–17 95–119
CK (IU/L) Males 89 561 435 444 107 1975 0.000 NG NP 116–1924 107–128 1523–1975 0.7809
Females 49 524 374 387 125 1620 0.000 NG NP 130–1591 125–200 1295–1620
Total Bilirubin (mg/dL) Males 92 0 0 0 0 0 - - NP 0–0 0–0 0–0 > 0.9999
Females 52 0 0 0 0 0 - - NP 0–0 0–0 0–0
ALP (IU/L) Males 93 74 32 68 29 170 0.000 NG NP 32–160 29–36 155–170 0.0873
Females 52 88 42 78 37 205 0.001 NG NP 37–197 37–40 163–205
ALT (IU/L) Males 91 39 10 37 17 72 0.005 NG NP 23–69 17–25 58–72 0.0196a
Females 51 35 12 34 15 77 0.008 NG NP 15–75 15–19 59–77
AST (IU/L) Males 91 62 30 53 22 162 0.000 NG NP 24–142 22–27 118–162 0.1031
Females 50 75 44 56 33 215 0.000 NG NP 33–210 33–35 169–215
GGT (IU/L) Males 93 18 8 18 6 37 0.016 NG NP 6–37 6–8 32–37 0.3739
Females 52 17 7 17 0 33 0.292 G NP 2–32 0–8 28–33
Sodium (mEQ/L) Males 93 136 3 137 127 144 0.001 NG NP 128–132 127–132 140–144 0.4988
Females 52 136 3 137 127 142 0.030 NG NP 128–141 127–131 140–142
Potassium (mEQ/L) Males 92 4.93 0.79 4.81 3.66 7.21 0.000 NG NP 3.85–6.99 3.66–3.98 6.59–7.21 0.0407a
Females 52 5.37 1.16 5.00 3.89 9.90 0.003 NG NP 3.91–9.09 3.89–4.03 6.99–9.00
Chloride (mEQ/L) Males 92 100.7 2.9 101.0 91.8 107.3 0.381 G NP 94.1–107.2 91.8–96.1 104.9–107.3 0.1745
Females 52 101.1 3.6 101.5 90.5 107.2 0.011 NG NP 90.9–107.2 90.5–93.8 105.6–107.2
Bicarbonate (mEQ/L) Males 93 25.0 2.4 25.0 19.5 31.3 0.876 G NP 19.9–29.6 19.5–21.3 28.9–31.3 0.0001c
Females 51 23.4 2.3 23.6 18.5 28.4 0.530 G NP 18.5–28.2 18.5–19.5 26.5–28.4
Anion Gap (mmol/L) Males 93 16 2 15 12 24 0.000 NG NP 12–22 12–13 21–24 0.0102a
Females 51 17 3 17 13 26 0.002 NG NP 13–25 13–14 21–26
Calculated Osmolality (mOsm/kg) Males 93 284 5 284 269 302 0.017 NG NP 272–293 269–273 292–302 0.0489a
Females 51 285 5 285 276 297 0.801 G NP 276–296 276–278 294–297
Iron (μg/dL) Males 70 285 30 288 197 350 0.128 G NP 210–345 197–236 331–350 < 0.0001d
Females 52 319 46 325 186 405 0.282 G NP 199–405 186–245 390–405
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aP < 0.05

bP < 0.01

cP < 0.001

dP < 0.0001 between values for adult male and female Hartley guinea pigs. Abbreviations: G, Gaussian; NG, non-Gaussian; NP, nonparametric; RI, reference interval.

Sex-associated differences in biochemistry parameters are shown in Table 6. Males had higher ALT (median difference 3 IU/L; 95% CI 1–8; P = 0.0196) and bicarbonate (mean difference 1.6 mEQ/L; 95% CI 0.8–2.5; P = 0.0001). Females had higher total protein (mean difference 0.2 g/dL; 95% CI 0.1–0.3; P = 0.0002), albumin (mean difference 0.2 g/dL; 95% CI 0.1–0.2; P < 0.0001), globulin (mean difference 0.0 g/dL; 95% CI 0.0–0.1; P = 0.0363), cholesterol (median difference 3 mg/dL; 95% CI 0–11; P = 0.0317), potassium (median difference 0.19 mEQ/L; 95% CI 0.02–0.63; P = 0.0407), anion gap (median difference 2 mmol/L; 95% CI 0–2; P = 0.0102), calculated osmolality (mean difference 1 mOsm/kg; 95% CI 0–4; P = 0.0489), and iron (mean difference 34 μg/dL; 95% CI 21–48; P < 0.0001).

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].

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 sex-related 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 [22–27]. 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.

Acknowledgments

The authors thank the Colorado State University Clinical Pathology Laboratory for sample analysis and Laboratory Animal Resources for their compassionate care of the animals used in this study.

Data Availability

All relevant data are within the manuscript.

Funding Statement

A portion of this study was supported by the Colorado State University Clinical Pathology Research and Development Fund. APS was supported by a COHA Translational Fellowship funded by U01 TR002953. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Associated Data

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Data Availability Statement

All relevant data are within the manuscript.

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