The influence of probiotic administration on selected leukocyte subpopulations and the serum amyloid A concentration in the peripheral blood of dairy cows during different lactation periods

Fig. 1.

List of primary antibodies and secondary conjugates used in this study

Antibodies, dye	Cell type	Clone	Isotype
Mouse anti bovine CD4, PE	T helper cells	CC8	IgG2a
Mouse anti bovine CD8, FITC	T cytotoxic/suppressor cells	CC63	IgG2a
Mouse anti bovine CD11b, FITC	αM-integrin receptor subunit	CC126	IgG2b
Mouse anti-dog CD18, purified	β2-integrin receptor subunit	CA1.4E9	IgG1
Goat anti-mouse IgG (Fc), FITC	secondary antibody	polyclonal	polyclonal IgG
Mouse anti-bovine CD21, FITC	B lymphocytes	CC21	IgG1
Mouse anti-bovine CD25, RPE	Interleukin-2 receptor alpha chain	IL-A111	IgG1
Mouse anti-bovine Foxp3+, FITC	T regulatory cells	FJK-16s	IgG2a, kappa

Phenotyping of leukocytes in the peripheral blood of experimental (E) and control (C) cows during the experiment

Leukocyte percentage
Subpopulation	Group	7 DBD	14 DBP	7 DPP	21 DPP	60 DPP	90 DPP
CD18+	E	6.0 ± 0.6a	5.1** ± 0.6b	7.22** ± 2.9a	4.6** ± 1.5b	4.7** ± 1.2b	4.2* ± 0.9b
lymphocytes	C	6.6 ± 0.9a	3.2 ± 0.6b	2.8 ± 0.5b	2.6 ± 0.8b	2.8 ± 0.6b	3.3 ± 0.3b
CD18+	E	12.5 ± 3.9a	11.7** ± 3.2a	6.8* ± 1.6b	11.0** ± 2.3a	23.0** ± 1.6c	24.7** ± 2.5c
granulocytes	C	14.8 ± 3.0a	6.2 ± 1.7b	4.9 ± 1.7b	4.4 ± 1.5b	11.6 ± 2.6c	18.3 ± 4.6a
CD18+	E	11.7** ± 1.4a	4.5 ± 1.5b	8.6* ± 2.9c	9.5** ± 1.8c	18.0** ± 1.4d	16.1* ± 1.8d
monocytes	C	5.8 ± 1.4a	5.2 ± 0.9a	6.3 ± 0.6a	4.6 ± 0.8a	10.9 ± 1.4b	13.4 ± 1.4c
CD11b+	E	23.1* ± 4.4a	23.7** ± 4.0a	14.2 ± 1.2b	15.5 ± 2.2b	21.8** ± 2.1a	30.4** ± 3.3c
lymphocytes	C	19.5 ± 2.6a	16.0 ± 2.1b	13.2 ± 2.8b	14.1 ± 2.0b	17.0 ± 3.0ab	25.2 ± 3.1c
CD11b+	E	98.5* ± 0.9	98.9 ± 0.9	99.7 ± 0.1	99.7 ± 0.2	99.9 ± 0.0	99.6 ± 0.1
granulocytes	C	99.5 ± 0.3a	98.1 ± 0.9b	99.8 ± 0.1a	99.7 ± 0.1a	99.8 ± 0.1a	99.9 ± 0.1a
CD11b+	E	76.1 ± 4.2a	78.1** ± 4.1a	74.4 ± 5.8a	61.8 ± 5.0b	70.3** ± 4.2ab	69.3 ± 4.1ab
monocytes	C	73.3 ± 4.9a	69.8 ± 7.1a	71.2 ± 6.6a	61.0 ± 8.9b	56.0 ± 8.7b	65.9 ± 4.1b
CD21+	E	23.4 ± 3.78a	21.14* ± 4.56a	24.72 ± 3.86a	24.74 ± 4.62a	22.28** ± 4.38a	33.32** ± 2.76b
lymphocytes	C	24.88 ± 1.68	25.40 ± 3.02	25.62 ± 1.32	25.56 ± 3.32	25.50 ± 1.38	25.50 ± 1.64
CD25+	E	10.76 ± 2.28a	8.13** ± 0.6a	14.9** ± 2.19b	20.4** ± 2.62c	17.86** ± 3.0bc	23.22** ± 2.94c
lymphocytes	C	9.26 ± 1.28a	6.51 ± 1.45b	6.63 ± 1.66b	10.75 ± 2.28a	11.78 ± 1.18a	15.8 ± 2.77c
CD4+	E	28.96 ± 2.12a	32.80 ± 2.66b	34.04 ± 2.92bc	35.72* ± 2.32bc	36.62** ± 1.32c	34.94** ± 1.66bc
lymphocytes	C	26.64 ± 2.52a	33.40 ± 1.72b	33.72 ± 0.32b	33.90 ± 0.52b	32.62 ± 2.32bc	30.72 ± 0.96c
CD8+	E	10.56 ± 1.60	11.55* ± 1.12	11.96 ± 0.97	10.47* ± 1.69	10.15 ± 1.84	11.47** ± 1.48
lymphocytes	C	9.69 ± 1.11a	13.18 ± 1.56b	10.87 ± 0.78a	13.04 ± 2.27b	10.94 ± 2.12a	16.56 ± 0.46c
Foxp3+	E	42.54 ± 4.44a	44.34 ± 3.06a	49.44** ± 4.18b	42.56 ± 4.88a	47.68** ± 3.84ab	46.22** ± 4.42ab
lymphocytes	C	44.08 ± 3.52ab	43.98 ± 1.44ab	40.44 ± 4.18a	41.10 ± 2.64a	45.78 ± 3.26b	33.92 ± 2.12c

Composition of the total mixed ration and dry mass (DM) daily feed ration for lactating cows (kilograms/cow/day)

Feed component	Amount per cow, daily
	Kg	Kg DM
Maize silage	25.0	8.8
Haylage	8.0	3.2
Ensiled brewery spent grain	8.0	2.7
Wheat straw	0.8	0.7
Ensiled maize grain	2.5	1.7
Ground barley grain	1.5	1.3
Ground triticale grain	1.5	1.3
Ground rapeseeds	2.7	2.2
Extracted soyabean meal	2.0	1.7
Glycerine	0.3	0.24
Vitamin and mineral mixture	0.2	0.18
Sodium bicarbonate	0.2	0.2
Calcium carbonate	0.05	0.05
Total	52.75	24.27