Peritoneal fluid collection in healthy cattle and evaluation of changes in cell morphology during storage in refrigerated and non-refrigerated samples

Sales, Nathali A.A.; Flaiban, Karina K.M.C.; Fonteque, Joandes H.; Lisbôa, Júlio A.N.

doi:10.1590/1678-5150-PVB-6444

ABSTRACT:

This study aimed to evaluate the appropriate sites of abdominocentesis for peritoneal fluid collection in cattle and to investigate the time of cell viability in vitro, comparing three methods of sample conservation. Twenty-one healthy cattle (19 females and 2 males) were subjected to a laparocentesis procedure to obtain peritoneal fluid, with punctures in three defined sites: left cranial, right cranial, and right caudal. The total peritoneal fluid collected was divided into three aliquots and maintained under three preservation conditions: room temperature (26°C), refrigeration (4°C), and room temperature (26°C) with the addition of 1μL of 10% formaldehyde per 1mL of peritoneal fluid. The peritoneal fluid analysis performed immediately after collection consisted of: physical examination (color, appearance, volume, and specific gravity), biochemical measures (pH, total protein, fibrinogen, creatinine, and glucose), and cellularity (total and differential counts). The determination of proteins and the examination of cells were repeated in each separate aliquot at two, four, six, and eight hours after harvest. Data were analyzed through repeated measures ANOVA or Friedman test. The harvest was productive in 67% of cattle. The left cranial and the right cranial puncture sites were the most appropriate. Peritoneal fluid analyzed after collection, the total protein concentration ranged from 1.4 to 3.6g/dL, and number of leukocytes ranged from 54 to 1,322 cells/μL; 60 to 95% of leukocytes were lymphocytes. The protein concentration decreased, but the absolute values of leukocytes, lymphocytes, and segmented neutrophils did not change up to eight hours after collection, independent of the maintenance method. Cell lysis was delayed by cooling, and the addition of formaldehyde did not help preserve the integrity of cellular morphology. Laparocentesis is a safe and secure procedure in cattle and maybe more productive when performed in specific sites on the left or right sides of the cranial abdominal wall. Peritoneal fluid samples may be analyzed with reliable results for up to eight hours after collection when kept refrigerated and for up to six hours when kept at room temperature.

INDEX TERMS:
Peritoneal fluid; healthy cattle; cell morphology; abdominocentesis; cell viability; conservation; cattle

[1] *Corresponding author: janlisboa@uel.br

Cattle	Left cranial	Right cranial	Right caudal
1	3.5mL	>5mL	1mL
2	>5mL	>5mL	Drops
3	0	0	Drops
4	Drops of blood	0	Drops
5	Drops	1.3mL	Drops
6	Drops of blood	0	>5mL
7	0	Drops	Drops
8	0	0	Drops
9	>5mL	3mL	Drops of blood
10	0	0	Drops of blood
11	Drops	1mL	0
12	0.5mL	5mL	Not collected
13	Drops	0	Not collected
14	>5mL	>5mL	Not collected
15	5mL	>5mL	Not collected
16	Drops	5mL	Not collected
17	>5mL	>5mL	Not collected
18	0	>5mL	Not collected
19	Drops	0	Not collected
20	5mL	>5mL	Not collected
21	>5mL	>5mL	Not collected

		x	s	Md	Minimum	Maximum
Peritoneal fluid
	Color	Light yellow predominant (n = 11) and occasional orange (n = 3)
	Aspect	Slightly cloudy predominant (n = 9) and occasional cloudy (n = 5)

Specific gravity		1,014.5	3.2	1,015	1,010	1,020
pH		7.22	0.40	7.0	7.0	8.0
Total protein (g/dL)		2.1	0.6	2.0	1.4	3.6
Fibrinogen (mg/dL)		112.5	34.1	100	100	200
Glucose (mg/dL)		55.9	5.4	55.3	48.7	66.6
Creatinine (mg/dL)		1.3	0.4	1.2	0.7	2.2
Leukocytes (/μL)		1,079	977	896	54	1,322
Segmented neutrophils (%)		15.2	9.3	14.0	5	40
Lymphocytes (%)		84.7	9.4	86.0	60	95
Segmented neutrophils (/μL)		182	230	127	4	303
Lymphocytes (/μL)		921	862	767	50	1,065

Plasma
	Total protein (g/dL)	7.8	0.6	7.5	7.2	9.4
	Fibrinogen (mg/dL)	350	89,4	400	200	500
	Creatinine (mg/dL)	1.5	0.4	1.6	0.9	2.5

	0 h	2 h	4 h	6 h	8 h
Proteins (g/dL)
RT	1.95± 0.45^a	1.91 ± 0.48^ab	1.88±0.49^ab	1.86±0.49^b	1.84±0.50^b
R	1.95 ± 0.45^a	1.91 ± 0.48^ab	1.88±0.49^b	1.88±0.49^b	1.85±0.51^b
RTF*	1.95 ± 0.45^a	1.91 ± 0.48^ab	1.88±0.49^b	1.88±0.49^b	1.85±0.51^b
Leukocytes (/μL)
RT	901^a	880^a	816^a	826^a	858^a
R	901^a	880^a	805^a	1,066^a	944^a
RTF*	901^a	869^a	896^a	1,002^a	1,002^a
Segmented neutrophils (/μL)
RT	127^a	109^a	163^a	66^a	118^a
R	127^a	148^a	89^a	134^a	124^a
RTF*	127^a	151^a	128^a	118^a	118^a
Lymphocytes (/μL)
RT	856^a	674^a	591^a	715^a	670^a
R	856^a	752^a	653^a	783^a	771^a
RTF*	856^a	741^a	774^a	832^a	863^a

	0 h	2 h	4 h	6 h	8 h
Cytoplasmic membrane irregularity or rupture
RT	90	78	90	80	70
R	90	80	81	72	70
RTF*	90	77	60	50	66
Cell lysis
RT	81	88	90	70	60
R	81	80	100	90	90
RTF*	81	88	90	70	55
Cytoplasmic vacuoles
RT	100	100	100	100	100
R	100	100	100	100	100
RTF*	100	100	100	100	100
Nuclear membrane irregularity or rupture
RT	100	100	100	100	100
R	100	100	100	100	100
RTF*	100	100	100	100	100
Fragmented chromatin
RT	100	100	100	100	100
R	100	100	100	100	100
RTF*	100	100	100	100	100

Brasil

Brasil

Peritoneal fluid collection in healthy cattle and evaluation of changes in cell morphology during storage in refrigerated and non-refrigerated samples

Colheita do fluido peritoneal de bovinos sadios e verificação das alterações na morfologia celular quando mantido refrigerado ou à temperatura ambiente

ABSTRACT: