Determination of natural progesterone levels in bovine serum using gas chromatography coupled with mass spectrometry

Parameter	Spiking level (μg L⁻¹)
Mean concentration (μg L⁻¹) / apparent recovery (%)	0.500	0.433 / 86.5
	1.000	0.893 / 89.3
	2.000	1.778 / 88.9
Repeatability (s_r, μg L⁻¹) / RSD (%)	0.500	0.056 / 12.8
	1.000	0.111 / 12.4
	2.000	0.333 / 18.7
Within-lab reproducibility (s_R, μg L⁻¹) / RSD (%)	0.500	0.066 / 15.2
	1.000	0.193 / 21.6
	2.000	0.366 / 20.6
Decision limit (CCa, μg L⁻¹)		0.058
Detection capability (CCβ, μg L⁻¹)		0.100
Expanded measurement uncertainty (U, k = 2, %)	0.500	47
	1.000	50
	2.000	55
Standard calibration curve
Slope ± s_b		3.2134 ± 0.7344
y-intercept ± s_a		0.1394 ± 0.0642
Correlation coefficient		0.9999
Standard error		0.1169
Matrix-matched calibration curve
Slope ± s_b		2.8158 ± 0.0242
y-intercept ± s_a		-0.0952 ± 0.7603
Correlation coefficient		0.9999
Standard error		0.0951

1 RSD – relative standard deviation; s_r – standard deviation under repeatability conditions; s_R – standard deviation under reproducibility conditions; s_a – standard deviation of slope; s_b – standard deviation of intercept

Table 2.

Detection of progesterone (P4) by cattle sex and age in the earlier part of the investigation

		Age (months)	Year of sampling
		Age (months)	2017	2018	2019	2020	2021
Male cattle	Sample size		122	124	117	59	52
	Number tested / with detected P4	>12	22 / 7	35 / 26	26 / 26	6 / 4	7 / 2
		<12 to ≤24	94 / 36	78/ 67	69 / 60	42 / 24	38 / 19
		>24	5 / 4	8 / 7	15 / 13	10 / 7	7 / 4
		not specified	1 / 1	3 / 1	7 / 0	1 / 0	0 / 0
Female cattle	Sample size		132	146	140	165	130
	Number tested / with detected P4	≤6	20 / 2	37 / 25	15 / 14	8 / 6	8 / 4
		>6 to ≤12	12 / 6	11 / 10	17 / 17	9 / 5	4 / 1
		≤12	32 / 8	48 / 35	32 / 31	17 /11	12 / 5
		>12 to ≤24	64 / 32	60 / 58	41 / 40	60 / 48	38 / 28
		>2	35 / 21	36 / 36	67 / 65	88 / 78	80 / 65
		not specified	1 / 0	2 / 2	0 / 0	0 / 0	0 / 0
Male and female cattle	Combined all-age sample size		254	270	257	224	182
Male cattle	All-age number tested with detected P4		48	101	99	35	25
Male cattle	Percentage		39	81	85	59	48
Female cattle	All-age number tested with detected P4		61	131	136	137	98
Female cattle	Percentage		46	90	97	83	75
Male and female cattle	Combined all-age number tested with detected P4		109	232	235	172	123
	Percentage		43	86	91	77	68

Table 3.

Detection of progesterone (P4) by cattle sex and age in the later part of the investigation and a summary of the entire research period

		Age (months)	Year of sampling
		Age (months)	2022	2023	2024	2025	2017–2021
Male cattle	Sample size		79	34	43	12	642
	Number tested / with detected P4	≤12	6 / 0	4 / 1	3 / 2	0 / 0	109 / 68
		>12 to ≤24	61 / 20	21/ 5	28 / 10	7 / 6	438 / 247
		>24	12 / 6	8 / 3	10 / 7	5 / 5	80 / 56
		not specified	0 / 0	1 / 1	2 / 1	0 / 0	15 / 4
Female cattle	Sample size		95	55	27	5	895
	Number tested / with detected P4	≤6	0 / 0	2 / 0	1 / 1	1 / 1	92 / 53
		>6 to ≤12	3 / 0	4 / 0	2 / 0	0 / 0	62 / 39
		≤12	3 / 0	6 / 0	3 / 1	1 / 1	154 / 92
		>12 to ≤24	38 / 22	24 / 17	8 / 6	3 / 3	336 / 254
		>24 months	54 / 31	25 / 21	14 / 11	1 / 1	400 / 329
		not specified	0 / 0	0 / 0	2 / 2	0 / 0	5 / 4
Male and female cattle	Combined all-age sample size		174	89	70	17	1,537
Male cattle	All-age number tested with detected P4		26	10	20	11	375
Male cattle	Percentage		33	29	47	92	57
Female cattle	All-age number tested with detected P4		53	38	20	5	679
Female cattle	Percentage		56	69	74	100	77
Male and female cattle	Combined all-age number tested with detected P4		79	48	40	16	1,054
	Percentage		45	54	57	94	68

Histogram of progesterone concentrations in male cattle

Histogram of progesterone concentrations in female cattle. The charts on the left present concentration data for specific age groups (including the entire cows population); the histograms on the right side show progesterone concentrations that may indicate the current oestrous cycle phase

Histogram of progesterone concentrations in female cattle. The charts on the left present concentration data for specific age groups; the histograms on the right side show progesterone concentrations that may indicate the current oestrous cycle phase (continuation of Fig. 2)

Table 4.

Serum progesterone (P4) concentration levels in the studied cattle population, and levels in females that may indicate the current oestrous cycle phase

	Age (months)		Concentration (μg L⁻¹)							Number of results below decision limit	Percentage of results below decision limit
	Age (months)		Minimum	Maximum	Mean	Median	Standard deviation	95^thCentile	99^thCentile	Number of results below decision limit	Percentage of results below decision limit
Male cattle	≤12		0.001	31.792	1.170	0.174	4.187	4.038	19.227	16	24
	>12 to ≤24		0.002	4.154	0.385	0.138	0.598	1.572	3.044	65	26
	>24		0.019	3.905	0.727	0.172	1.042	3.138	3.804	7	13
	All ages combined		0.001	31.792	0.575	0.155	1.905	2.169	4.310	90	24
Female cattle	≤6		0.010	61.842	2.541	0.216	10.615	3.640	54.755	10	19
	>6 to ≤12		0.001	11.503	0.791	0.206	1.895	2.038	8.442	5	13
	≤12		0.001	61.842	1.799	0.211	8.164	2.689	49.439	15	16
	>12 to ≤24		0.004	71.415	2.031	0.659	6.706	5.108	23.866	28	11
	>24		0.002	496.059	6.348	0.720	35.245	13.667	109.185	35	11
	All ages combined		0.001	496.059	4.083	0.622	25.130	8.542	68.112	78	11
Female cattle	≤6	P4 conc. <1 (oestrus)	0.010	0.996	0.240	0.108		0.717	0.968
		P4 conc. >1 (ovulation)	1.011	5.998	2.009	1.151		4.819	5.762
		P4 conc. ≥3 and ≤7 (midluteal period)	5.998	5.998	5.998	5.998		5.998	5.998
	>6 to ≤12	P4 conc. <1 (oestrus)	0.001	0.816	0.231	0.122		0.757	0.811
		P4 conc. >1 (ovulation)	1.036	3.449	1.742	1.604		2.979	3.355
		P4 conc. ≥3 and ≤7 (midluteal period)	3.449	3.449	3.449	3.449		3.449	3.449
	≤12	P4 conc. <1 (oestrus)	0.001	0.996	0.236	0.119		0.753	0.947
		P4 conc. >1 (ovulation)	1.011	5.998	1.875	1.446		4.341	5.666
		P4 conc. ≥3 and ≤7 (midluteal period)	3.449	5.998	4.723	4.723		5.870	5.971
	>12 to ≤24	P4 conc. <1 (oestrus)	0.004	0.997	0.347	0.250		0.875	0.980
		P4 conc. >1 (ovulation)	1.024	5.809	2.511	2.264		5.098	5.359
		P4 conc. ≥3 and ≤7 (midluteal period)	3.007	5.809	4.035	3.718		5.316	5.675
	≥24	P4 conc. <1 (oestrus)	0.002	0.999	0.343	0.299		0.859	0.981
		P4 conc. >1 (ovulation)	1.002	7.996	2.831	2.154		6.027	7.898
		P4 conc. ≥3 and ≤7 (midluteal period)	3.085	6.351	4.424	4.147		6.027	6.307
	All ages combined	P4 conc. <1 (oestrus)	0.001	0.999	0.323	0.245		0.845	0.991
		P4 conc. >1 (ovulation)	1.002	7.996	2.635	2.154		5.745	7.833
		P4 conc. ≥3 and ≤7 (midluteal period)	3.007	6.351	4.264	4.037		5.960	6.267

Chromatograms of A – a blank bovine serum sample; B – a serum sample spiked with progesterone (P4) at 0.500 μg L⁻¹; C – a serum sample from a male bovine with P4 detected at the concentration of 1.336 μg L⁻¹; D – a serum sample from a female bovine with P4 detected at the concentration of 13.548 μg L⁻¹ and diagrams of structure of P4 and its heptafluorobutyric anhydride (HFBA) derivative); E – a serum sample from female bovine with P4 detected at the concentration of 4.734 μg L⁻¹; F – a serum sample from female bovine with P4 detected at the concentration of 0.362 μg L⁻¹. 17β-T-D2 – deuterated analogue of 17β-testosterone used as internal standard

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DOI: https://doi.org/10.2478/jvetres-2025-0071 | Journal eISSN: 2450-8608 (formerly 2300-3235)

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Language: English

Page range: 561 - 573

Submitted on: Aug 18, 2025

Accepted on: Dec 10, 2025

Published on: Dec 16, 2025

Published by: National Veterinary Research Institute in Pulawy

In partnership with: Paradigm Publishing Services

Keywords:

GC-MS,

Related subjects:

Microbiology and virology,

Life sciences, other,

Medicine,

Veterinary medicine

© 2025 Iwona Matraszek-Żuchowska, Alicja Kłopot, Paulina Zdonek, Beata Korycińska, Justyna Grzelak, published by National Veterinary Research Institute in Pulawy
This work is licensed under the Creative Commons Attribution 4.0 License.

Volume 69 (2025): Issue 4 (December 2025)

Determination of natural progesterone levels in bovine serum using gas chromatography coupled with mass spectrometry

Figures & Tables

Table 1.

Table 2.

Table 3.

Fig. 1.

Fig. 2.

Fig. 3.

Table 4.

Fig. 4.

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