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Abstract

Free-roaming cats in New York, NY, USA, that presented to a trap–neuter–return program were surveyed for biochemical data. One hundred and one cats had blood collected for a plasma biochemistry panel after the induction of surgical anesthesia. Reference intervals for 18 analytes were generated for the sample population, along with age-specific reference intervals when statistically appropriate. Age groups (juveniles and adults) differed in 10 of the 18 analytes measured, including protein levels and albumin/globulin ratio, aspartate aminotransferase, alkaline phosphatase, creatine kinase, creatinine, phosphorus, calcium and potassium. No differences were found between males and females. This is the first report of biochemical reference intervals for a group of free-roaming cats within the USA.

Introduction

Free-roaming cats are found worldwide; however, the exact number of free-roaming cats in the USA is unknown. The free-roaming cat population includes unsocialized or feral cats, semi-feral cats, loosely owned cats, lost or abandoned pet cats, and currently owned cats that are allowed outside.¹ The vast majority of such cats are not spayed/neutered.² The well-being of these cats, as well as the potential overgrowth of their numbers, has become of increasing concern over the past few decades. To this extent, trap–neuter–return (TNR) programs provide a means of free-roaming cat population control that does not involve mass euthanasia.³

TNR programs focus on humane sterilization of free-roaming cats with subsequent return to their original location outdoors.³ Cats of various age groups and from multiple colonies are often presented to these programs, but the same or similar anesthetic protocols are typically utilized for all cats within an individual TNR. Although previous research has gathered information regarding the health of free-roaming cats that undergo sterilization,² the lack of regularly scheduled veterinary care and comprehensive diagnostic testing limits our knowledge regarding the overall health and disease status of cats within these populations.

As TNR programs become increasingly popular throughout the USA, more and more cats are brought to these subsidized clinics. Such programs typically allocate their limited resources toward sterilization as opposed to collecting a minimum database before surgery. Additionally, the temperament of feral cats often necessitates anesthesia prior to being handled, which does not allow for collection of pre-induction blood samples.

While the mortality rate of free-roaming cats undergoing surgical sterilization is low (0.4%),² factors that contribute to this rate are still unknown and only a few studies have thoroughly investigated the biochemical and/or hematological variables of free-roaming cats.^4–8 The purpose of this study was to assess the biochemistry variables from a large group of free-roaming cats who presented to a spay/neuter program in New York, NY, USA, determine age- and gender-specific reference intervals when appropriate, and identify any trends within this population that may be of clinical importance.

Materials and methods

Cats

The study population included 104 free-roaming cats that presented to a once-monthly TNR program in New York, NY, USA during January 2010. This TNR program historically draws participants from the five New York, NY, USA boroughs. Cats were not enrolled in the survey if they had a history of being indoors for 1 month or more.

Following standard protocol of the spay/neuter program, caregivers gave consent for surgical sterilization. Consent was also required for the collection and processing of blood. Cats were anesthetized and then received a physical examination followed by a spay or neuter. The age of cats was based on examination of teeth and body condition. Hydration status was assessed by evaluation of skin turgor and mucous membranes.

Return of cats to their caregivers occurred after anesthetic recovery on the same day of surgery. Instructions were provided regarding monitoring and care during the postoperative period.

Anesthesia and blood collection

All cats were anesthetized with a combination of tiletamine/zolazepam (Telazol; Pfizer) at 3 mg/kg, butorphanol (Torbugesic; Fort-Dodge) at 0.15 mg/kg, and dexmedetomidine (Dexdomitor; Pfizer) at 6 μg/kg. These drugs were combined into a single cocktail with the cocktail administered intramuscularly (IM) at approximately 0.03 ml/kg.⁹ Meloxicam (Metacam; BIVI) at approximately 0.1 mg/kg was also given subcutaneously to each cat during anesthesia. Cats that did not awake quickly after surgery were administered 0.1 mg of atipamezole (Antisedan; Pfizer) IM.

Approximately 1.0–1.5 ml of blood was drawn from an accessible vein in each cat after being anesthetized, but prior to surgery. The blood samples were split equally between sodium or lithium heparin and ethylenediamine tetra-acetic acid microtubes for biochemical and hematological analysis, respectively. Heparin samples were stored and submitted for processing unspun. All samples were refrigerated within 5 h of collection and kept refrigerated or within a cooler until they were processed by the receiving laboratory (Antech Diagnostics) within 24 h of collection. Complete blood count data were excluded owing to the lack of manual leukocyte differentials and other analytical errors; glucose data were excluded owing to pre-analytical error (ie, delayed processing).

Biochemical analysis

Biochemical analysis was performed using heparinized plasma on an Olympus AU5431 (Olympus America). Measured biochemical variables included total protein, albumin, globulin, albumin/globulin (A/G) ratio, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), total bilirubin, blood urea nitrogen (BUN), creatinine, inorganic phosphorus, total calcium, sodium, potassium, chloride, cholesterol and amylase (Table 1).

Table 1

Methodology used for biochemical variable measurement in free-roaming cats (Felis catus)

Analyte*	Method
Total protein	Biuret
Albumin	Bromocresol green
AST	Modified IFCC method using oxaloacetate and malate dehydrogenase
ALT	Modified IFCC method using pyruvate and lactate dehydrogenase
ALP	IFCC method using p-nitrophenyl phosphate at pH 10.4
CK	Modified IFCC method using hexokinase and glucose-6-phosphate dehydrogenase
Total bilirubin	Diazo/caffeine/surfactant
BUN	Urease/glutamate dehydrogenase
Creatinine	Kinetic modification of Jaffe reaction using alkaline picrate
Phosphorus (inorganic)	Phosphomolybdate
Calcium	Arsenazo III
Sodium	Ion-selective electrode, indirect
Potassium	Ion-selective electrode, indirect
Chloride	Ion-selective electrode, indirect
Cholesterol	Cholesterol esterase/cholesterol oxidase/trinder endpoint
Amylase	2-chloro-4-nitrophenyl-α-D-maltotrioside

AST = aspartate aminotransferase; ALT = alanine aminotransferase; ALP = alkaline phosphatase; CK = creatine kinase; BUN = blood urea nitrogen; IFCC = International Federation of Clinical Chemistry and Laboratory Medicine

Globulin and albumin/globulin ratio are not included as they are calculated values

Statistical analysis

Reference intervals were determined using the freeware Reference Value Advisor v2.1.^10,11 Variables were analyzed using histograms and box-plots for visual inspection of outliers, and also mathematically using Dixon’s outlier range statistic or Tukey’s interquartile fences for non-Gaussian or Gaussian distributions, respectively. As recommended by the American Society of Veterinary Clinical Pathology, robust and non-parametric methods for reference interval calculation were employed based on sample size, distribution of data (Anderson–Darling test), symmetry of data for robust methods and clinical relevance of the generated reference interval. The non-parametric bootstrap method was used for calculation of 90% confidence intervals of the lower and upper limits of the reference intervals.^10–12 Differences between age and gender groups were analyzed with SPSS 22.0 (SPSS Inc). Variables were compared between age groups (juveniles classified as cats ⩽1 year and adults classified as cats >1 year) and gender (male and female) using the independent sample t-test or the Mann–Whitney U-test based on Gaussian or non-Gaussian data distribution as determined by the Shapiro–Wilk test, respectively. For all calculations, the P value was set at 0.05 for significance.

Results

Cats

Caregivers of 104 cats agreed to participate in the study. Of the original 104 cats, biochemistry data were obtained on 101 cats. One cat’s results were not reported for unknown reasons, and the results of two additional cats were also excluded secondary to sample mislabeling. Fifty-seven males and 42 females composed 99 of the 101 cats sampled; the remaining two did not have a sex recorded. Age varied, with 20 cats estimated at >3 to ⩽6 months, 19 at >6 to ⩽12 months, 46 at >1 to ⩽3 years and 10 at >3 years old. Six cats did not have an age recorded. All cats had uneventful anesthetic recoveries.

Common physical examination findings of the 101 cats included waxy ears/possible ear mites (25), dehydration (13), ocular discharge (12), small abrasions/cuts (7), conjunctivitis (6), nasal discharge (5), fleas/flea dirt (3), sneezing (4), corneal opacity/ulcer (3), perianal tapeworm proglottids (2), lice (2) and abscesses (2). Wheezing, fever (39.9°C), purulent vaginal discharge, hematuria, suspected eosinophilic granuloma and observation of roundworms in the stool were noted in one cat each.

Biochemistry

Forty-five cats had varying levels of hemolysis within their plasma sample and thus several analytes (AST, ALT, total bilirubin, potassium and cholesterol) have less than the maximum number of data points owing to exclusion for possible analytical interference. For outliers, a total of nine data points were rejected, as these were highly suspected to be aberrant observations: two cats each for globulin (12.4 and 19.4 g/dl) along with the corresponding total protein and A/G ratios; one cat for calcium (2.4 g/dl); one cat for cholesterol (1316 mg/dl); and one cat for CK (7183 U/l). The rejected data points were excluded from reference interval calculations and group comparisons.

Significant differences in total protein, globulin, A/G ratio, AST, ALP, CK, creatinine, phosphorus, calcium and potassium were observed between adults and juveniles, and age-specific intervals were calculated for these analytes and reported along with the population reference interval for comparison. All other analytes are reported with a population reference interval only (Table 2). No statistical differences were observed between genders.

Table 2

Biochemistry reference intervals for free-roaming cats (Felis catus)

Analyte	Unit	Group*	n^†	Minimum, maximum(median)^‡	Reference interval(90% CI)^§	Method
Total protein	g/dl	All cats	99	6, 11	5.8–9.4 (5.4–6.1, 9.0–9.7)	RUD
		Adults	56	6.6, 10.5	6.1–9.6(5.8–6.5, 9.1–10.0)	RUD
		Juveniles	37	6, 11(7.3)	5.3–9.1(4.5–6.1, 8.4–9.9)	RUD
Albumin (A)	g/dl	All cats	101	2.2, 4.2	2.7–4.3(2.6–2.9, 4.1–4.4)	RUD
Globulin (G)	g/dl	All cats	99	2.6, 7.8	1.8–6.3(1.4–2.3, 5.8–6.7)	RUD
		Adults	56	3.0, 7.8	2.1–6.5(1.6–2.7, 5.9–7.1)	RUD
		Juveniles	37	2.6, 7.7(3.7)	1.4–5.9(0.6–2.4, 4.9–6.8)	RUD
A/G ratio	–	All cats	99	0.3, 1.4	0.4–1.4(0.3–0.5, 1.3–1.4)	RUD
		Adults	56	0.3, 1.3	0.3–1.3(0.3–0.4, 1.2–1.4)	RUD
		Juveniles	37	0.3, 1.4(1.0)	0.5–1.5(0.3–0.6, 1.4–1.6)	RUD
AST	U/l	All cats	56	13, 90	16–79(14–19, 68–91)	RTD
		Adults	31	13, 72(34)	13–64(0–17, 55–74)	RTD
		Juveniles	21	19, 90(39)	17–95(14–23, 72–118)	RTD
ALT	U/l	All cats	56	1, 127	6–96(2–13, 81–111)	RTD
ALP	U/l	All cats	101	3, 158	5–149(3–7, 131–168)	RTD
		Adults	56	3, 82	5–87(3–8, 70–102)	RTD
		Juveniles	39	19, 158(87)	17–162(7–33, 146–178)	RTD
CK	U/l	All cats	100	62, 2909	96–1537(87–109, 1054–2382)	RTD
		Adults	56	99, 1264	0–631(0, 467–789)	RTD
		Juveniles	38	92, 2909(359)	116–3188(100–142, 1448–7641)	RTD
Total bilirubin	mg/dl	All cats	80	0.1, 0.3	0.1–0.3(0.1–0.1, 0.2–0.3)	NP
BUN	mg/dl	All cats	101	13, 39	13–32(11–15, 31–34)	RUD
Creatinine	mg/dl	All cats	101	0.2, 1.2	0.4–1.3(0.4–0.5, 1.2–1.3)	RUD
		Adults	56	0.4, 1.2	0.6–1.4(0.5–0.7, 1.3–1.4)	RUD
		Juveniles	39	0.2, 1.1(0.7)	0.4–1.1(0.3–0.5, 1.0–1.2)	RUD
Phosphorus	mg/dl	All cats	101	3.3, 10.4	3.7–10.7(3.2–4.1, 10.3–11.2)	RUD
		Adults	56	3.7, 9.7	3.2–8.9(2.6–3.7, 8.3–9.6)	RUD
		Juveniles	39	4.7, 10.4(8.7)	6.6–10.9(6.0–7.3, 10.2–11.6)	RUD
Calcium	mg/dl	All cats	100	8.2, 11.3	8.5–11.3(8.4–8.7, 11.2–11.5)	RUD
		Adults	56	8.2, 11.1	8.3–11.0(8.2–8.6, 10.7–11.3)	RUD
		Juveniles	38	8.6, 11.3(10.35)	9.2–11.7(8.8–9.5, 11.4–12.0)	RUD
Sodium	mEq/l	All cats	101	145, 165	145–158(143–146, 156–159)	RUD
Potassium	mEq/l	All cats	80	3.4, 5.7	3.4–5.5(3.3–3.6, 5.3–5.6)	RUD
		Adults	44	3.4, 5.1	3.3–5.3(3.1–3.5, 4.9–5.4)	RUD
		Juveniles	30	4.0, 5.7(4.7)	3.8–5.7(3.6–4.0, 5.4–5.9)	RUD
Chloride	mEq/l	All cats	101	109, 127	110–123(109–111, 121–124)	RUD
Cholesterol	mg/dl	All cats	98	53, 209	65–180(57–74, 171–188)	RUD
Amylase	U/l	All cats	101	597, 1872	470–1424(399–549, 1336–1508)	RUD

CI = confidence interval; AST = aspartate aminotransferase; ALT = alanine aminotransferase; ALP = alkaline phosphatase; CK = creatine kinase; BUN = blood urea nitrogen; RUD = robust untransformed data; RTD = robust transformed data; NP = non-parametric

All cats = all cats sampled regardless of age or gender; adults = cats aged >1 year old; juveniles = cats aged ⩽ 1 year old

†

n = number of cats. Six cats did not have an age recorded and were not included in the age-specific reference intervals. Various analytes have less than the initial number of cats sampled owing to exclusion of suspected outliers and rejection of certain data points due to possible interference from hemolysis (AST, ALT, total bilirubin, potassium and cholesterol)

‡

Median given for groups with n <40

When a statistically significant difference between age groups was found for an analyte, age-specific reference intervals were calculated and included. CI determined by non-parametric bootstrap method

Discussion

Comprehensive reports of biochemical data in free-roaming cats are lacking within the current literature and, to our knowledge, no other studies of this regard have been published from this population of cats within the USA. In this study, a large group of free-roaming cats was surveyed for biochemical data, allowing for the calculation of reference intervals for the sample population, as well as age-specific reference intervals when statistically warranted.

Adult and juvenile free-roaming cats significantly differed in 10 of the 18 variables measured. Higher reference intervals for ALP, phosphorus and calcium in the juvenile cats are consistent with bone growth, while the lower creatinine reference interval likely stems from decreased muscle mass compared with adults. Interestingly, the adult cats also had higher calcium and phosphorus reference intervals than what is typically expected for pet cats (personal observation). This may signify dietary, such as a high prey-based diet, or hormonal differences in this population. The juvenile cats also had elevated CK compared with adults, which may also be related to muscle development; however, it cannot be excluded that for both juveniles and adults the CK reference intervals may have been affected by muscle trauma secondary to exertion or struggling during capture (‘capture myopathy’), or the IM injections given for anesthesia. The higher AST and potassium in the juveniles could also represent a component of muscle trauma, but age-related increases in AST are also possible. In some cases, the age-related reference intervals are similar and of low clinical significance, or have large or overlapping confidence intervals that must be considered during interpretation.

One limitation to this study is the absolute determination of health and age within this population of free-roaming cats. Although the majority of physical examination results found would be expected in a normal free-roaming cat population or unlikely to affect biochemical variables, it is possible that some cats were included that had disease(s) uncommon to typical free-roaming cats. This is an inherent limitation to determining reference intervals where the complete health of the population being investigated is unknown. As teeth and body condition were used to estimate cat age, it may be that some cats were incorrectly classified as juveniles or adults. It is also fundamental to recognize that many statistical tests were performed for all comparisons. With an a level of 5%, a type I error could have occurred, resulting in some group differences being statistically significant when no difference truly exists.

Additionally, another limitation was the inability to obtain pre-anesthetic blood samples in this population of cats. Several recent studies in cats using similar, although not identical, anesthetic protocols have shown blood work alterations when comparing pre- and post-induction samples.^13–15 However, while statistical differences in biochemical analytes were noted in these studies, they were not clinically significant.^14,15

Conclusions

This study is the first large-scale report of biochemical data in free-roaming cats from the USA. This population of free-roaming cats exhibited multiple differences between juveniles and adults, of which several can be explained by age-related physiology. This finding serves as an important reminder of using age-appropriate intervals for biochemical data interpretation, especially in TNR programs and other veterinary services that treat both young and adult animals. Interestingly, a few analytes, such as calcium and phosphorus, also appeared elevated in adult free-roaming cats compared with what is expected in pet cats. This may be related to dietary or hormonal differences, but the exact cause was not investigated in this study. Further investigation into biochemical variables of other groups of free-roaming cats is needed to determine if these trends are consistent. The effects of this particular anesthetic protocol should also be investigated to determine if any clinically significant differences can be detected between pre- and post-induction samples.

Footnotes

Acknowledgements

Special thanks to the ASPCA spay/neuter staff and volunteers who coordinated and implemented the Sunday Spay/Neuter Clinic for free-roaming cats, as well as the staff and administration of Bergh Memorial Animal Hospital, New York, NY, where the clinic was held. Thanks also to Dr Mark Mitchell for assistance with the SPSS statistical analysis.

Conflict of interest

The authors do not have any potential conflicts of interest to declare.

Funding

Biochemical testing was donated by Antech Diagnostics. The remainder of the study was funded internally by ASPCA.

Accepted: 25 November 2013

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Biochemical survey of free-roaming cats ( Felis catus ) in New York City presented to a trap–neuter–return program

Abstract

Introduction

Materials and methods

Cats

Anesthesia and blood collection

Biochemical analysis

Statistical analysis

Results

Cats

Biochemistry

Discussion

Conclusions

Footnotes

Acknowledgements

Conflict of interest

Funding

References