Evaluation of pregnancy associated glycoproteins assays for on farm determination of pregnancy status in beef cattle

Adalaide C Kline; Saulo Menegatti Zoca; Kaitlin M Epperson; Lacey K Quail; Jaclyn N Ketchum; Taylor N Andrews; Jerica J J Rich; Jim R Rhoades; Julie A Walker; George A Perry

doi:10.1371/journal.pone.0306325

. 2024 Jul 25;19(7):e0306325. doi: 10.1371/journal.pone.0306325

Evaluation of pregnancy associated glycoproteins assays for on farm determination of pregnancy status in beef cattle

Adalaide C Kline ^1,^#, Saulo Menegatti Zoca ^1,^‡, Kaitlin M Epperson ^2,^3,^‡, Lacey K Quail ^2,^3,^‡, Jaclyn N Ketchum ^2,^3,^‡, Taylor N Andrews ^4,^‡, Jerica J J Rich ^5,^‡, Jim R Rhoades ^6,^‡, Julie A Walker ^1,^‡, George A Perry ^3,^*,^#

Editor: Angel Abuelo⁷

¹Department of Animal Science, South Dakota State University, Brookings, South Dakota, United States of America

²Department of Animal Science, Texas A&M University, College Station, Texas, United States of America

³Texas A&M AgriLife Research, Overton, Texas, United States of America

⁴Department of Animal Science, New Mexico State University, Las Cruces, New Mexico, United States of America

⁵Department of Animal Science, Arkansas State University, Jonesboro, Arkansas, United States of America

⁶IDEXX Laboratories, Westbrook, Maine, United States of America

⁷Michigan State University, UNITED STATES

Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Jim Rhoades is employed by IDEXX.

‡ SMZ, KME, LKQ, JNK, TNA, JJJR, JRR and JAW also contributed equally to this work.

^✉

* E-mail: george.perry@ag.tamu.edu

Contributed equally.

Roles

Adalaide C Kline: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Saulo Menegatti Zoca: Data curation, Investigation, Visualization, Writing – original draft, Writing – review & editing

Kaitlin M Epperson: Investigation, Writing – review & editing

Lacey K Quail: Investigation, Writing – review & editing

Jaclyn N Ketchum: Investigation, Writing – review & editing

Taylor N Andrews: Investigation, Writing – review & editing

Jerica J J Rich: Investigation, Writing – review & editing

Jim R Rhoades: Funding acquisition, Resources, Writing – review & editing

Julie A Walker: Funding acquisition, Resources, Writing – review & editing

George A Perry: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Angel Abuelo: Editor

PMCID: PMC11271854 PMID: 39052584

Abstract

Transrectal ultrasonography is known as the gold standard for pregnancy detection, but requires costly equipment and technical skills; therefore, access to an inexpensive and more user-friendly method with similar accuracy could benefit cattle producers. Detection of pregnancy-associated glycoproteins can accurately determine pregnancy in ruminants; however, usually requires specialized equipment for the assay. Thus, the objectives of these studies were to 1) validate the IDEXX Alertys OnFarm Pregnancy Test (lateral flow) and compare the accuracy of all three commercial PAG assays to transrectal ultrasonography and 2) to determine the postpartum interval necessary for clearance of pregnancy-associated glycoproteins from the previous pregnancy to avoid false positives. In study 1, blood samples from previously identified pregnant Bos taurus females from six different herds (nulliparous n = 1,205 and multiparous n = 1,539; samples collected between d 27 to 285 of gestation over a three-year period) were utilized. In study 2, postpartum females (primiparous n = 48 and multiparous n = 66) from one herd were utilized: (n = 1,066; samples collected weekly for up to 12 weeks postpartum). In study 1, level of agreement between different methods of pregnancy detection was determined by Pearson’s correlation and Kappa scores. In study 2, data were analyzed as a repeated measure using the MIXED procedure of SAS with main effects of parity, days postpartum (dpp), and parity by days postpartum, then data were analyzed further using the REG procedure of SAS. In study 1, transrectal ultrasonography and lateral flow were positively correlated (r = 0.77; P <0.01), with 92.4% agreement. In study 2, the abundance of absorbance of PAGs rapidly decreased from 0 to 50 days postpartum, then continued to gradually decrease (P <0.01; r = 0.90). Prior to 42 days postpartum, PAG concentrations were sufficiently elevated resulting in false positive readings in all assays. In conclusion, there is very good agreement between transrectal ultrasonography and PAG assays, but likelihood of false positive results are highif assays are performed fewer than 42 days postpartum.

Introduction

Pregnancy diagnosis within an operation is not only important, but necessary to increase profitability and have a complete and successful reproductive management program [1]. Fertilization occurs greater than 90% of the time following insemination of beef cows that have been detected in estrus, but calving rates to a single insemination are usually only about 55% [2]. According to recent USDA NAHMS data, only 31.6% of all cow-calf operations use a method of pregnancy determination [3], and given the discrepancy between fertilization rate and calving rate, an accurate pregnancy detection method is critical to maximizing herd profitability and production efficiency. In order to increase the percentage of operations that utilize a pregnancy detection method; however, it must be accurate and easy to use.

Transrectal ultrasonography is considered the gold standard for pregnancy detection, but it is costly and requires a skilled technician [4]. An alternative method to determine pregnancy is by detecting pregnancy-associated glycoproteins (PAGs) in circulation [5–8]. Blood pregnancy tests are also increasing in popularity due to ease of use and the unique feature of not requiring costly equipment or special training. Pregnancy-associated glycoproteins are synthesized by trophoblast giant cells (TGCs) of the trophectoderm in the ruminant placenta [9]. Binucleate giant cells then migrate to fuse with maternal uterine epithelial cells where the granular content within the TGC is released into the maternal circulation. Once the granular content is released to maternal circulation, PAGs can be measured in either milk or blood samples to determine pregnancy status [10–12]. These glycoproteins can be detected in the maternal bloodstream as early d 22 of gestation [13, 14].

Pregnancy-associated glycoproteins concentrations steadily increase in the maternal bloodstream throughout gestation, are elevated at time of parturition, and then decrease after parturition [5, 6, 15]. They also have a long half-life in the blood of postpartum females, ranging from 80 to 100 days postpartum (dpp) [7, 16]. Because PAGs peak at parturition and have a long half-life, residual concentrations can still exist in both primiparous and multiparous animals at the start of the subsequent breeding season. Thus, when trying to use PAG concentrations as a marker for early pregnancy diagnosis, these residual concentrations may impact the result. Therefore, the objectives of these studies were to 1) validate the IDEXX Alertys OnFarm Pregnancy Test (lateral flow) and compare the accuracy of all three commercial PAG assays to transrectal ultrasonography and 2) to determine postpartum interval necessary for clearance of PAGs from the previous pregnancy to avoid false positives.

Materials and methods

All procedures were approved by the South Dakota State University Institutional Animal Care and Use Committee (IACUC number17-046A, 18-014E and 1910-061E).

Experimental design

In study 1, blood samples (nulliparous n = 1,205 and multiparous n = 1,539) from six different Bos taurus herds in the state of South Dakota were utilized. Blood samples were collected over a three-year period (2018, 2019, and 2020) from d 27 to 285 of gestation. Pregnancy detection was performed by transrectal ultrasonography between d 30 and 80 post-insemination in all animals.

In study 2, blood samples from Angus and Angus-cross postpartum females (primiparous n = 48 and multiparous n = 66) from one herd in South Dakota were utilized. Blood samples were collected once a week for up to 12 weeks postpartum (n = 1,066 samples; range of first and last sample was 1–7 to 85–91 dpp).

Blood sampling

Serum

Blood samples were collected by venipuncture of either the coccygeal or jugular vein into 10-mL Vacutainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) and stored at room temperature (20°C) for approximately 2 h until centrifuged. Samples were centrifuged at 2,000 x g for 30 min, serum was collected and stored at -20°C until PAG assays were conducted.

Plasma

Blood samples were by venipuncture of either the coccygeal or jugular vein into 10-mL EDTA Vacutainer tubes containing (Becton, Dickinson and Company, Franklin Lakes, NJ) and were immediately placed on ice. Samples were centrifuged (1200 × g for 20 min at 4 °C) within 2 h of collection, and plasma was aspirated and stored at -20°C until PAG assays were conducted.

Transrectal ultrasonography

All animals were evaluated for pregnancy by transrectal ultrasonography by a trained technician with an Ibex EVO ultrasound and 5 MHz linear array probe on d 28 following their first insemination. Pregnancy diagnosis was based on the visualization of an embryo or absence of one. A final pregnancy diagnosis occurred between d 30 and 80 following the end of the breeding season to determine if early fetal loss occurred.

IDEXX Alertys Rapid Visual Pregnancy Test (RVPT)

Pregnancy was determined in whole blood and serum samples utilizing the commercially available blood pregnancy tests, IDEXX Alertys Rapid Visual Pregnancy Test (IDEXX, Westbrook, ME) according to the manufacturer’s directions. Briefly, positive/negative controls, and samples were pipetted into coated plates, and plates were washed and treated with reagents according to the manufacturer’s instructions. Visual evaluation of the plates based on a numerical scale, established by color intensity were made upon completion of the procedure by one technician. Color intensity evaluation and description were described by Northrop et al. [17]. The scoring system included a yes/no assignment and numerical value from 0–3 based on color intensity in comparison to the positive and negative control wells, where a score of 0 had the same or less color than the negative control, a score of 1 had slightly more color than the negative control, a score of 2 had slightly less color than the positive control, and a score of 3 had the same or more color than the positive control. A numerical score of 0 or 1 would result in “no” meaning the female is not pregnant, while a numerical score of 2 or 3 would result in “yes” meaning the female is pregnant [17]. This scoring system is not provided by the manufacture with the kit, but instead it was an internal lab assessment.

IDEXX Alertys Ruminant Pregnancy Test (RPT)

Pregnancy was determined in samples using the commercially available blood pregnancy tests, IDEXX Alertys Ruminant Pregnancy Test (RPT; IDEXX, Westbrook, ME) according to the manufacturer’s directions. Briefly, controls and serum samples were pipetted in duplicate into wells of the coated plates, and plates were washed and treated with reagents according to the manufacturer’s instructions. The results from the RPT were analyzed on a Molecular Devices SpectraMax 190 microtiter plate reader that measures the optical density of the wells (San Jose, California). The interassay CV was 3.8% and 3.7% for plasma and serum respectively. The intraassay CV was 2.82%, and the cutoff for pregnancy was a S-N value of ≥0.300.

IDEXX Alertys OnFarm Pregnancy Test (lateral flow)

Pregnancy was determined using commercially available blood pregnancy tests, IDEXX Alertys OnFarm Pregnancy Test (IDEXX, Westbrook, ME) according to the manufacturer’s directions. Inside an IDEXX Alertys OnFarm Pregnancy Test kit includes a lateral flow test, pipette, and a dropper with chase buffer. Briefly, 150 μL of serum or plasma was pipetted into the well of the lateral flow test followed with 175 μL of chase buffer. After a 20 min incubation, the tests were scored and evaluated by two technicians that were blind to ultrasonography pregnancy status. A third technician was utilized to break any discrepencies. Interpretation of IDEXX Alertys OnFarm Pregnancy Test with just the internal positive control, “C”, visible line indicates the female was not pregnant, while visibility of the test sample, “T”, line means the female was pregnant at time of blood sample (S1 Fig).

Statistical analysis

In study 1, blood samples were analyzed to determine pregnancy status agreement between tests (Ultrasonography, RVPT, RPT and lateral flow) using the CORR procedure of SAS (9.4). Since the correlation between tests was significant, further analysis were performed using the FREQ procedure of SAS to evaluate the frequency of pregnant and open between each test comparatively to each other. Further analysis include measurements of specificity, sensitivity, positive predicitive value, negative predictive value, and percent correct between the pregnancy detection methods with herd as the random effect. The Kappa scoring scale utilized is as follows: 0.80–1.00 = Very good, 0.60–0.80 = Good, 0.40–0.60 = Moderate, 0.20–0.40 = Fair, and < 0.20 = Poor. Statistical significance was considered at P ≤ 0.05, and a tendency at 0.05 < P ≤ 0.10.

In study 2, samples were grouped by postpartum week of collection. Pregnancy status/optical density readings were analyzed as a repeated measure using the MIXED procedure of SAS (9.4) using the compound semetry covariant structure. The statistical model consisted of time postpartum, parity, and their interactions. The effect of parity was analyzed using animal within treatment as the error term, and effects of time and the interaction were analyzed using the residual as the error term. The effect of dpp on PAG concentrations (pregnancy status/opticial density) using the REG procedure of SAS. Statistical significance was determined at P ≤ 0.05, and a tendency at 0.05 < P ≤ 0.10. All data are reported as (LSmeans ± SEM).

Results

Study 1

Agreement based on Kappa scores was very good among all tests in the study (Table 1). Additionally, there was a positive correlation (P < 0.05) among all tests (Ultrasonography:RVPT r² = 0.81311, Ultrasonography:RPT r² = 0.83856, Ultrasonography:lateral flow r² = 0.84126, RVPT:RPT r² = 0.94739, RVPT: lateral flow r² = 0.90138, RPT:lateral flow r² = 0.91257; Table 2). Of the 1,096 animals that were diagnosed nonpregnant by transrectal ultrasonography, less than 7% were diagnosed pregnant by any of the PAG assays. Of the 1,648 animals diagnosed pregnant by transrectal ultrasonography, less than 3% were diagnosed nonpregnant by any of the PAG assays. Thus, a greater than 90% agreement occurred between transrectal ultrasonography and all of the PAG assays (Table 3). Comparisons were also made between the three PAG assays, and a greater than 95% agreement occurred between all assays (Table 3).

Table 1. Agreement between pregnancy tests to determine accuracy of pregnancy detection.

Test	Ultrasonography ¹	RVPT ²	RPT ³	Lateral Flow ⁴
Ultrasonography ¹		0.8108	0.8344	0.8388
RVPT ²	very good		0.9472	0.9005
RPT ³	very good	very good		0.9122
Lateral Flow ⁴	very good	very good	very good

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Values depicted above the diagonal line are the Kappa scores, while values below the diagonal are the overall agreement of the tests based on the Kappa score. The Kappa scoring scale is: 0.80–1.00 = Very good, 0.60–0.80 = Good, 0.40–0.60 = Moderate, 0.20–0.40 = Fair, and < 0.20 = Poor.

¹Ultrasonography = transrectal ultrasonography

² RVPT = IDEXX Alertys Rapid Visual Test

³ RPT = IDEXX Alertys Ruminant Pregnancy Test

⁴ Lateral Flow = IDEXX Alertys OnFarm Pregnancy Test

Table 2. Correlation between pregnancy tests to determine accuracy of pregnancy detection.

Test	Ultrasonography ¹	RVPT ²	RPT ³	Lateral Flow ⁴
Ultrasonography ¹		0.81311	0.83856	0.84126
RVPT ²	< 0.0001		0.94739	0.90138
RPT ³	< 0.0001	< 0.0001		0.91257
Lateral Flow ⁴	< 0.0001	< 0.0001	< 0.0001

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Values depicted above the diagonal line are the correlation coefficients, r², of the tests, while values below the diagonal are the P-values of all the tests. A positive correlation and significant difference were found among the tests in comparison to each other.

¹Ultrasonography = transrectal ultrasonography

² RVPT = IDEXX Alertys Rapid Visual Test

³ RPT = IDEXX Alertys Ruminant Pregnancy Test

⁴ Lateral Flow = IDEXX Alertys OnFarm Pregnancy Test

Table 3. Agreement between pregnancy detection methods to determine accuracy among methods.

Test¹	Agreement, %	False Positive², %	False Negative³, %	Sensitivity, %⁴	Specificity, %⁵	Samples, n
Ultrasonography ⁶ :Lateral Flow ⁷	92.38	5.61	2.00	97.2	85.9	2,744
Ultrasonography ⁶ :RVPT ⁸	90.73	6.46	2.80	93.1	89.1	1,533
Ultraound ⁶ :RPT ⁹	92.61	5.91	1.48	97.7	83.4	2,436
RVPT ⁸ :Lateral Flow ⁷	95.07	3.56	1.37	96.9	92.9	1,460
RPT ⁹ :Lateral Flow ⁷	96.22	1.31	2.46	96.5	95.7	2,360
RPVT ⁸ :RPT ⁹	97.36	1.80	0.83	98.2	96.6	1,443

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¹Comparison between tests first:second

² False Positive = a result that shows a female is pregnant when she is actually non-pregnant

³False Negative = a result that shows a female is non-pregnant when she is actually pregnant

⁴Sensitivity = number of true positives divided by number of true positives plus number of false negatives

⁵Specificity = number of true negatives divided by number of true negatives plus number of false positives

Ultrasonography = transrectal ultrasonography

⁵Lateral Flow = IDEXX Alertys OnFarm Pregnancy Test

⁶RVPT = IDEXX Alertys Rapid Visual Test

⁷RPT = IDEXX Alertys Ruminant Pregnancy Test

Study 2

PAG clearance

When using the RPT assay, there were no differences detected in PAG concentrations between primiparous and multiparous females (parity; P = 0.73) and parity by dpp (P = 0.55); however, there was a significant effect of dpp group on PAG concentrations in postpartum females (P < 0.01). A linear decrease from 0 to 50 dpp occurred, and then PAG concentrations reached a sustained nadir from 50 to 84 dpp. There was a strong correlation between dpp group and PAG concentrations, accounting for 67.48% of the variance. Since PAGs reached a sustained nadir after 50 dpp, statistical analysis was performed to determine the clearance from 0 (calving) to 50 dpp. Elimination of the samples when they first reached a sustained nadir allowed for a stronger correlation between PAG concentrations and dpp, accounting for 80.83% of the variance (P < 0.01). When determining average PAG concentrations of samples by dpp group broken down into 7 d intervals through d 84, clearance below pregnancy threshold of the RPT assay occurred by 42 dpp (optical density (od) = 0.26 ± 0.036, Fig 1A). Animals were considered pregnant when od were ≥ 0.3. There was no significant difference in determining the clearance of PAGs with the IDEXX Alertys Ruminant Pregnancy Test between both multiparous and primiparous females (P = 0.55; Fig 1B).

Fig 1 — Mean (± SEM) serum pregnancy-associated glycoprotein (PAG) concentration levels among postpartum beef females (A) for the Ruminant Pregnancy Test. PAG concentration levels were below the detectable range by 42 dpp (optical density (od) = 0.2636; A). Values not sharing the same superscripts (a-h) differ (P < 0.01; A). Superscript (*) represents values that tended to differ (P = 0.08; A). There was no significant difference in clearance patterns among postpartum multiparous and primiparous beef females (P = 0.55; B).

For postpartum samples analyized by the RVPT, there was a significant difference (P = 0.04) in PAGs between parity, an effect of dpp (P < 0.01), and a dpp by parity interaction (P = 0.03). All animals regardless of parity were still considered pregnant from the previous pregnancy through 21 dpp (98.63 ± 2.62%), whereas by 28 dpp, 88.36 ± 2.58% were considered pregnant (Fig 2A). The percentage of females that received a false positive pregnancy diagnosis rapidly decreased as dpp increased. By 49 dpp, 11.82 ± 2.64% of the females were considered positive for pregnancy, and at 56 dpp, there were 1.98 ± 2.70% positive for pregnancy (Fig 2A). The detection of false positives rapidly decreased from 21 to 56 dpp, then sustained nadir from 56 to 84 dpp (Fig 2A). There was a significant difference in the clearance of PAGs considering the parity by dpp interaction (P = 0.03; Fig 2B). Between 35 to 49 dpp, there was a greater decrease in false positives among primiparous compared to multiparous animals (at 49 dpp 5.01 ± 4.02% and 18.63 ± 3.43%; respectively). At 84 dpp 7.85 ± 6.52% of primiparous and 1.77 ± 6.14% of multiparous females were still considered pregnant (Fig 2).

Fig 2 — Postpartum samples were analyzed by the Rapid Visual Pregnancy Test (RVPT) to determine an accurate timeframe to test pregnant females without getting a false positive test from the residual pregnancy-associated glycoproteins (PAGs). There was a significant effect of days postpartum (dpp; P < 0.01; A). All animals were still considered pregnant from the previous pregnancy on 21 dpp (98.63%; A). There was a significant difference of dpp by parity (P = 0.03; B). # values between multiparous and primiparous between 35 to 49 dpp differed P < 0.05.

For postpartum samples analyzed by the lateral flow test, there was no difference (P = 0.21) of parity, but there was an effect of dpp (P < 0.01) and a tendency for a dpp by parity interaction (P = 0.06). All animals regardless of parity were still considered pregnant from the previous pregnancy through 35 dpp (100 ± 2.58%), whereas by 42 dpp, 98.16 ± 2.55% were considered pregnant (Fig 3A). The percentage of females that received a false positive pregnancy diagnosis declined as dpp increased. By 77 dpp, there were 13.72 ± 3.16% of the females positive for pregnancy, and at 84 dpp, there were 4.11 ± 4.39% positive for pregnancy (Fig 3A). The detection of false positives rapidly decreased between 42 and 70 dpp group, then slowly decreased from 70 to 84 dpp group (Fig 3A). There was a tendency for a parity by dpp interaction (P = 0.06; Fig 3B). Between 63 to 77 dpp group there was a greater decrease in false positives among primiparous compared to multiparous animals (at 77 dpp 5.12 ± 4.26% and 22.31 ± 4.67%; respectively). At 84 dpp 3.56 ± 6.38% of primiparous and 4.66 ± 6.03% of multiparous females were still considered pregnant (Fig 3).

Fig 3 — Postpartum samples were analyzed by the OnFarm Pregnancy Test (lateral flow) to determine an accurate timeframe to test pregnant females without getting a false positive test from the residual pregnancy-associated glycoproteins (PAGs) from the previous pregnancy. There was a significant effect of dpp (P < 0.01; A). All animals were still considered pregnant from the previous pregnancy on 35 dpp (100%; A). Days postpartum by parity tended to be different (P = 0.06; B). a-f values not sharing the same superscripts differed P < 0.05. *† values not sharing the same superscripts tended to differ P ≤ 0.08. ‡§ values not sharing the same superscripts tended to differ P ≤ 0.07. # values between multiparous and primiparous between 63 to 77 dpp differed P < 0.05.

Discussion

Currently, there is no pregnancy detection method that is 100% accurate for pregnancy determination without being invasive (i.e. harvesting the reproductive tract), which makes it difficult to evaluate the accuracy of a new pregnancy detection method [18]. The most accurate, minimally-invasive (i.e. without harvesting the reproductive tract) method currently known as the gold standard, is transrectal ultrasonography. Efforts have been ongoing to develop a method which accurately determines pregnancy status and is also producer friendly and quick to use. IDEXX Laboratories have created a series of blood pregnancy tests, (Alertys Rapid Visual; Alertys Ruminant Pregnancy Test; and Alertys OnFarm Pregnancy Test), to help producers determine pregnancy status of females within their herd. The RVPT and RPT utilize polyclonal antibodies, allowing for the detection of various members of the PAG family that are secreted, some at different times throughout gestation, and found in the maternal bloodstream to determine pregnancy status. The lateral flow test utilizes a monoclonal antibody, meaning the antibody used binds to specific members of the PAGs family (proprietary information) that is secreted in order to determine the pregnancy status of the female.

In order for a pregnancy detection test to be beneficial, it must be sensitive, meaning it is accurate in identifying pregnant females, and specific, meaning it also accurately identifies females who are nonpregnant. Transrectal ultrasonography was used as a comparative measure to determine the accuracy of all the IDEXX PAG pregnancy tests as it is considered the gold standard.

Epperson et al. [19] conducted a comparison between RVPT, ultrasonography, RPT and resynchronization pregnancy diagnosis. Blood samples were taken on d 28 and the final pregnancy diagnosis was made 31 to 80 d post-second artificial insemination (AI2). The kappa statistic scores among the comparisons between RVPT, ultrasonography, and RPT against the final resynchronization pregnancy diagnosis were very good; 0.90, 0.82, and 0.90, respectively. In a study completed by Silva et al. [20], the accuracy of PAG ELISA to transrectal ultrasonography on d 27 post-AI, d 39 post-AI2, and d 39 post-AI3 (post-third artificial insemination) was compared utilizing dairy cows. These comparisons found the PAG blood pregnancy test to have a kappa statistic score of 0.87 to 0.90, which is very good, when compared to ultrasonography. Similarly, a study by Romano and Larson [18] compared pregnancy specific protein B (PSPB; PAG-1 subgroup) ELISA to transrectal ultrasonography on d 28, 30, and 35 post-AI. Romano and Larson [18] found between d 28 to d 35 PSPB compared to transrectal ultrasonography had a very good kappa statistic score of 0.93 for accurately detecting pregnancy status. Piechotta et al. [21] utilizing dairy cows, compared two ELISA blood pregnancy tests for PSPB) against transrectal ultrasonography between d 26 to 58 post-AI. Between the two tests there was no significant difference found in comparison to transrectal ultrasonography. Northrop et al. [17] also reported similar results when comparing the RPT and RVPT to transrectal ultrasonography in beef cows between d 28 to 40 post-AI, and found them to have very good agreement, 0.86 and 0.85 respectively. The current study found similar results when comparing RVPT and RPT to ultrasonography with very good kappa scores (0.81 and 0.83, respectively) and agreement (81.3% and 83.9%, respectively). Similarly, validating the IDEXX Alertys OnFarm Pregnancy Test to ultrasonography, RVPT, and RPT, there was very good kappa scores (0.84, 0.90, 0.91, respectively) and agreement (84.1%, 90.1%, and 91.3%, respectively). The results from the current study along with previous research highlight that blood pregnancy tests are highly accurate compared to transrectal ultrasonography. Considering the lateral flow test had a very good kappa score and agreement among all three tests used in the present study, the lateral flow test would make a great pregnancy detection alternative to the costly ultrasound equipment or laboratory equipment to run the RPT. The costs associated with the blood test methods range from $4.50 to $8 per head while the cost for ultrasound is going to depend on the veterinarian’s rate to perform the test and their speed since many veterinarians charge an hourly rate. A skilled ultrasound technician can also provide information about the pregnancy (viability, stage of gestation, fetal sex, etc) that is not provided by a blood test. In areas where skilled ultrasound technitions are not readally available; however, or when animals numbers do not make them cost effective the use of a cow side test will allow producers to benefit from pregnancy diagnosis and identification of non-pregnant cows which will help their bottom line.

Caution should be used when implementing the lateral flow test to minimize false positives from performing the test too early in gestation, unlike the RVPT or RPT. It would be advised to perform the lateral flow test on d 40 of gestation or later to receive the most accurate results, thus, not keeping or rebreeding a female in the herd who happens to be not pregnant. Commercial PAG tests have a 1 to 5% false positive rate compared to transrectal ultrasonography [22]. These false positives could be due to residual PAG concentrations from the previous pregnancy or from the current embryo being lost.

Due to residual concentrations of PAGs after parturition, blood pregnancy tests need to be conducted at the appropriate time postpartum to accurately determine pregnancy status and avoid false positives. Previous research suggested that PAG pregnancy blood tests should not be utilized until 91 to 120 dpp [7, 8]. Specifically, Zoli et al. [7] used boPAG_{67 kDa}, which is part of the boPAG-1 group that is detectable in the maternal bloodstream through 100 dpp [10]. Others found that PAG pregnancy tests should not be performed until 70 to 90 dpp or later [16, 23, 24]. However, after 80 dpp, Kiracofe et al. [16], determined PAG concentrations were <1 ng/mL, which indicates PAGs from the previous pregnancy would not produce a false positive result. The differences reported between authors could be due to the different PAGs that were detected in each study.

The RPT resulted in fewer false positives at 42 dpp, thus allowing for earlier testing in gestation compared to the RVPT and lateral flow test evaluated in our laboratory. The utilization of the RPT can be implemented as early as d 28 of gestation, so the RPT could be conducted at 58 dpp or 28 d post-AI to allow for uterine involution to occur [25] and improve accuracy of results. Clearance of residual PAGs from the previous pregnancy determined by the RVPT test took longer than the clearance within the RPT. Also, the clearance of residual PAGs in the lateral flow test surpassed the length of time needed for uterine involution to occur (30 dpp) as well as the manufacturer’s recommended utilization day, d 28 post-AI. For the most accurate results (less than 20% false positive) an allotment of 49 dpp or 19 d post-AI and 70 dpp or 40 d post-AI should be made before performing the RVPT and lateral flow test, respectively. Between the RPT and lateral flow, to decrease the amount of false pregnancy diagnoses there is at least a 12 dpp difference if the RPT is utilized on d 28 and the lateral flow is utilized on d 40 of gestation. The RPT uses a polyclonal antibody against several PAGs (e.g. PAGs 4, 6, 9, 16, 18, 19; described in US Patent no. 7,604,950B2; [26]). Specifically, PAGs 4, 9, and 6 are secreted from d 25 (PAG 4 and 9) and d 45 (PAG 6) through d 250 of gestation [27]. The lateral flow test is a monoclonal antibody test where the PAGs identified is not stated (proprietary information). Thus, the difference in detection of PAGs between the two tests may be due to the influence in clearance of different PAGs from circulation.

Performing the lateral flow later in gestation when there would be fewer false positives would effectively detect embryonic loss that occurs through d 45 [28], and may improve management decisions and reproductive opportunities. Implementation of the lateral flow test on postpartum females, may potentially increase the calving interval compared to the RPT test since pregnancy determination would occur later in gestation, causing the possibility of rebreeding, if utilizing AI, to be later as well. In nulliparous females, implementation of the lateral flow test can occur on d 28 of gestation as the manufacturer’s instructions state, like the two other tests (RPT and RVPT), since there are no residual PAGs from a previous pregnancy.

For practical and research use in the cattle industry, any female that is diagnosed by a pregnancy detection method as nonpregnant, the female may receive an injection of prostaglandin-F_2α. Receiving an injectionof prostaglandin-F_2α would cause luteal regression decreasing progesterone concentrations and allow for the dominant follicle to increase estradiol production and estrus to be initiated [29]. Females falsely classified as nonpregnant by a pregnancy detection method may potentially receive a treatment with prostaglandin-F_2α, which could cause abortion. Therefore, producers with unintentionally aborted females due to pregnancy misdiagnosis would experience experience an economic loss of $550 to $800 for the loss of a live calf, resynchronization drugs, time, and labor [18, 30]. Silva et al. [20] found the RPT to have a great false negative predicted value from the three different AI days, d 27 post-AI, d 39 post-AI2, and d 39 post-AI3 (97.1%, 96.9%, 97.7% respectively). This study also found a significant negative predictive value between ultrasonography:lateral flow, ultrasonography:RVPT, and ultrasonography:RPT (98.0%, 97.2%, and 98.5%, respectively). Having a greater negative predictive value decreases the likelihood of giving prostaglandin-F_2α to a female who is truly pregnant. On the other hand, early and accurate pregnancy detection allows for more management decisions to be made by identifying females who are nonpregnant post-breeding. Animals that are accurately detected as nonpregnant can be rebred or culled earlier to reduce the number of days a nonpregnant female is cared for which ultimately would result in maximizing an operation’s economic gains and minimize reproduction associated financial losses [31].

Conclusion

The utilization of the IDEXX Alertys OnFarm Pregnancy Test (lateral flow) is a competitive alternative in pregnancy detection compared to the gold standard transrectal ultrasonography with an 92.38% agreement comparison in postpartum females. Of the three IDEXX Laboratories tests available, the lateral flow test is the most user-friendly method. Due to the additional time required for diagnosis of pregnancy with blood based tests; however, management decisions may be delayed compared to ultrasonography and may result in additional labor to sort open females once test results are available.

Concentrations of PAGs decreased rapidly for the first 3 weeks after parturition, and after 42 dpp PAG concentrations fell below the concentrations for pregnancy detection using the RPT assay. Thus, there is more confidence gained by the results received from a PAG blood pregnancy test when it is performed at least 42 dpp. More caution should be used with the utilization of the RVPT and lateral flow test postpartum in primiparous and multiparous females until at least 49 or 70 dpp, respectively, due to residual PAGs. Based on the present study, to decrease the likelihood of false positives, pregnancy detection by the lateral flow test should be preformed at d 40 of gestation on beef cattle who were bred at or after 30 dpp, which is later than the mansufacture’s recommended day, d 28. Based on results found with the current study utilizing the RVPT and RPT for pregnancy detection, there can be confidence gained on the manufacture’s recommended day of use, d 28, with decreased false positive results.

Supporting information

S1 Fig. Comparison of a pregnant and not pregnant IDEXX Alertys OnFarm Pregnancy Test, lateral flow.

The test on the left indicates that particular female at the timepoint the test was taken is pregnant due to the visibility of the test sample, “T”, line. The test on the right indicates that particular female is not pregnant due to there only being one line visible and that is the internal positive control, “C”, line. If the C line does not show up at all the test is invalid and the sample should be reran on a different test.

(DOCX)

pone.0306325.s001.docx^{(180KB, docx)}

S1 File. Data file 1.

This is the S1 data file.

(DOCX)

pone.0306325.s002.docx^{(261.4KB, docx)}

S2 File. Data file 2.

This is the S2 data file.

(DOCX)

pone.0306325.s003.docx^{(153.8KB, docx)}

Acknowledgments

The authors would like to thank IDEXX Laboratories for the donation of the IDEXX Alertys Ruminant Pregnancy Test and IDEXX Alertys OnFarm Pregnancy Test, as well as the cooperator herds for the use of their cattle, and zoetis for synchronization products.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This project was funded in part by South Dakota Hatch Funds and Texas A&M AgriLife Multistate Hatch project 9835. Products were contributed by IDEXX and Zoetis.

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PLoS One. doi: 10.1371/journal.pone.0306325.r001

Decision Letter 0

Angel Abuelo

18 Feb 2024

PONE-D-24-00715Evaluation of pregnancy associated glycoproteins assays for on farm determination of pregnancy status in beef cattlePLOS ONE

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Reviewer #1: Overall, the manuscript is straightforward and easy to read. It provides valuable insight on a newly available method of pregnancy diagnosis in ruminants. Additional comments and suggestions are in the attached document.

Reviewer #2: Specific Comments:

Title and Abstract

Appropriate title.

In abstract should probably be inexpensive rather than cheap? Line 27

.. likelihood of false positive results are high if assays are performed fewer than 42 days pp. Might be less confusing (Lines 49-50.)

Introduction

Line 72; only 22days necessary when describing as early as…

Line 73: probably best to describe where the PAG’s are increasing- circulation?

Aims of study clearly stated.

M&M’s

IACUC stated- ethical research.

Study 1

It would be useful to know if those that were diagnosed pregnant via ultrasonography also calved, illustrating no pregnancy loss occurred to validate the PAG assay results. Otherwise the results would need to be assessed for the possibility of pregnancy loss and inherent PAG decreases.

Study 2

Good array of time points.

Ultrasonography

Please state transducer frequency. Line 112

Please also state either conceptus was detected; or an embryo or fetus were detected. The embryo becomes a fetus at between 40-45 days depending on publications and interpretations. Similarly embryonic loss can only occur up to day 45; maybe best to describe as late embryonic or early fetal loss occurred? Lines 114-115.

Lateral Flow

Please update the Figure descriptions, in line 127 the relevant figure is not numbered, and might not be featured in the SI?

RVPT

The scoring for the RVPT appears to be a bit confusing, and should probably be a discussion point?

Statistical analysis

Please use ultrasonography rather than ultrasound. It might be better to compare the tests in the same order as they are introduced: ultrasonography, lateral flow, RVPT and RPT, to make it easier for the reader.

Line 166

My understanding is the ‘tendencies’ should not be part of statistical analysis.

Results

Table 1 useful.

For table 2 and for all others please alter ultrasound for ultrasonography.

Please ensure all the figures are correctly documented in the article, and correlate with the relevant illustrated figures.

Line 282; not sure that tendency should be recorded; maybe in the discussion. Similarly for line 290, and for all mentions of a tendency.

Discussion and conclusion

Line 323; remove ‘a’ or member to singular.

Lines 357-359: It is difficult to interpret the data without knowing the costs of the tests relative to the costs of ultrasonography. And, recognising the skill required to take the correct blood samples. If this could be addressed please.

Lines 360-364:

Please discuss the relevance of a false positive compared with a false negative in relation to the managers requirements- feeding of a non-pregnant animal, or culling of a pregnant animal.

Discussion of relevance of DPP PAG results in relation to the likelihood of pregnancy if insemination is unlikely? And, importance of multiparity cf primiparity esp 35-49d pp. (Why?) (Lines 376-390)

Line 388: Probably does not need: ‘with that said’; and to explain the differences RPT and Lateral Flow options more succinctly.

Line 408: A ‘shot’ although a common colloquialism, should probably be best described scientifically as some sort of drug administration/injection?

I think there needs to be some discussion as to the ‘immediacy’ of the gold standard ultrasonography compared with the relative delays associated with the tests described in the article. Particularly in relation to the handling and management of the animals in relation to the animals that are not pregnant.

Some mention of the likelihood of a bovid in most management systems being inseminated within 50 days, hence the likelihood of being pregnant should be discussed in relation to the levels of PAG in post-partum cows. This in relation to the points mentioned in lines 442-446. (Uterine involution and voluntary waiting periods are typically more than 40 days in most dairy animals, and the effects of the described lactational anoestrus in beef animals typically resulting in breeding not occurring prior to 50days PP.)

Competing Interests

I think it is difficult to believe that no competing interests were declared when Idexx and Zoetis supported the study to a degree, and at least one author is an employee of Idexx.

The Funding declaration in lines 452-453 appears to be at odds with that in the Financial disclosures section of the pre-manuscript section.

Recommendation:

This is useful information that should be published, with minor revisions suggested.

**********

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Reviewer #2: No

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pone.0306325.s004.docx^{(14.6KB, docx)}

PLoS One. 2024 Jul 25;19(7):e0306325. doi: 10.1371/journal.pone.0306325.r002

Author response to Decision Letter 0

29 Apr 2024

Dr. Abuelo,

We greatly appreciate the effort you and the reviewers have made in reviewing this manuscript. We have incorporated almost all of the suggested comments, and our individual responses to each suggestion is listed below.

Overall, the manuscript is well written and easy to read. It details an evaluation of a newly marketed cow-side pregnancy test. Evaluation of such new tools is important for the beef and dairy industry including veterinarians and producers. Understanding test function, limitations, and accuracy is critically important for those making decisions on whether to use such tools for their herd. There is certainly value in reporting the results of this study. Below are some items to consider to strengthen the manuscript as currently written.

Lines 59-61 – Beef NAHMS 2017 survey from USDA has data on the percentage of beef herds that use ultrasound for pregnancy diagnosis or perform any pregnancy diagnosis at all. According to this data set, only 31.6% of beef operations surveyed used either blood based, ultrasound, or palpation pregnancy diagnosis. Including this data could strengthen your argument for use of a rapid, cow-side test for pregnancy and the impacts on production efficiency and profitability.

Reference to the NAHMS data has been included.

Line 71 – Roberts et al., Reprod Domest Anim 2015; 50(4):651-8 investigated the use of a milk-based ELISA in beef cows. This could be included here to indicate it’s not only been investigated in dairy cows.

This reference has been added.

The ultimate gold standard for comparison of any pregnancy diagnosis method is confirmed delivery of a fetus. Since this study spanned 3 years, do you have calving data for which you would be able to compare and calculate sensitivity and specificity of the pregnancy diagnosis method used relative to calving? This would also allow for some discussion of rates of loss of pregnancy in beef cows which would be valuable to practitioners and producers.

The authors do have this data on a subset of animals; however, loss among this group was only so low that that analysis of this data would not be valid.

Reporting the loss rates between the 28d ultrasound examination and the recheck, as well as how this compared to the results of the assays would be useful and practical information for users of these tests.

Only 68 lost a pregnancy between the first and final pregnancy diagnosis. With these low numbers it is not possible to evaluate the data as a predictive test. When a retrospective analysis is done values will appear different, but as a predictive value there is too much variation in PAG concentrations to predict which animals might lose a pregnancy.

Lines 145-147 – We intra- and inter-assay coefficients of variation calculated for the OD readings on the RPT? What were the cutoffs used for pregnant, non-pregnant, and inconclusive/recheck?

The inter and intra assay CVs have been included “The interassay CV was 3.8% and 3.7% for plasma and serum respectively. The intraassay CV was 2.82%, and the cutoff for pregnancy was a S-N value of ≥0.300.”

There are no sensitivities or specificities calculated in this study. You have only reported false negative and false positive results with NPV and PPV which can be impacted by the population of this one study (ie prevalence of open or pregnant cows in your study population). The sensitivity and specificity, along with confidence intervals, should be included, as this reflects the expected performance of the test. Previous studies comparing PAG tests to a gold standard, routinely report sensitivity and specificity in addition to test agreement and NPV/PPV. Please incorporate this into the manuscript.

Sensitivity and specificity have been added to Table 3.

Figures 1 and 2 do not, in this reviewer’s opinion, add additional insight into the data. While it shows a linear relationship of PAG OD to dpp for each parity group or dpp group, the following Figure 3 is a clearer depiction of the relationship of dpp OD, and assay cut off. It is this reviewer’s recommendation to remove Figures 1 and 2, keeping figure 3.

Figures 1 and 2 have been removed from the manuscript.

There are multiple mentions of the PAG levels “plateauing” during the postpartum period. Plateau typically indicates that something has reached its highest point and stays there but in this case, the PAGs have reached a low point. From a clarity standpoint for the reader, consider using different terminology such as “sustained nadir” or “baseline” to indicate the PAG levels have reached their lowest point and stayed there.

This has been revised to sustained nadir.

Lines 330-359 – There is extensive discussion of the previous studies and days at which the performance of the test was evaluated. Despite referencing Ricci, et al (Ref #10), early in the manuscript, there is no mention here of their finding that the IDEXX RPT has a period where PAG OD may drop into the recheck or inconclusive range (d 46-67 post AI) and was more pronounced in multiparous cows. Roberts et al 2015 also found this to be a gestational window where false negatives were possible in beef cows. This is of particular importance for beef producers using this test and understanding its limitations when the population they are testing may have a wide range of days of gestation depending on breeding strategy. These limitations identified by Ricci and discussed in both the Ricci and Roberts studies are hypothesized to be due to varying expression of PAG throughout early gestation. Given that the specific PAG in the lateral flow is unknown, is it possible that there could also be a window of gestation with this test that yields equivocal results or contributes to some of the false negatives depending on the expression pattern of the monoclonal PAG antibody used?

The authors agree with the reviewer that this is an important consideration; however, in the present study only 78 samples were collected from pregnant animals during that time point and of those samples all were above the 0.3 cutoff used to determine pregnancy. So this is something that cannot be evaluated with this data set.

Lines 378-384 – It is unclear what the process of uterine involution has to do with the use of a test to detect PAGs. Presumably, the authors are inferring that uterine involution is a critical step in return to fertility in the postpartum beef cow prior to the next conception. However, the way it is worded in this section is confusing. Please rework this paragraph to clarify the relationship you are making between uterine involution and the use of the blood-based pregnancy testing methods for the next pregnancy.

This sentence has been removed to prevent confusion.

Reviewer #2: Specific Comments:

Title and Abstract

Appropriate title.

In abstract should probably be inexpensive rather than cheap? Line 27

This correction was made.

.. likelihood of false positive results are high if assays are performed fewer than 42 days pp. Might be less confusing (Lines 49-50.)

This correction was made.

Introduction

Line 72; only 22days necessary when describing as early as…

This correction was made.

Line 73: probably best to describe where the PAG’s are increasing- circulation?

This has been revised to read Pregnancy-associated glycoproteins steadily increase “in the maternal bloodstream” throughout gestation, are elevated at time of parturition, and then decrease after parturition [4,6,13].

Aims of study clearly stated.

M&M’s

IACUC stated- ethical research.

Study 1

That is not what this study was designed to evaluate. The objectives were to 1) validate the IDEXX Alertys OnFarm Pregnancy Test (lateral flow) and compare the accuracy of all three commercial PAG assays to transrectal ultrasonography and 2) to determine the postpartum interval necessary for clearance of pregnancy-associated glycoproteins from the previous pregnancy to avoid false positives. So, of the animals that were tracked to calving only 68 lost a pregnancy between first and final pregnancy diagnosis, and very few (<10) lost a pregnancy between final pregnancy diagnosis and calving. With these low numbers it is not possible to evaluate the data as a predictive test.

Study 2

Good array of time points.

Ultrasonography

Please state transducer frequency. Line 112

“5 MHz linear array probe” has been added to line 113.

This has been revised to read Pregnancy diagnosis was based on the visualization of “an embryo” or absence of one. A final pregnancy diagnosis occurred between d 30 and 80 following the end of the breeding season to determine if “early fetal” loss occurred.

Lateral Flow

Please update the Figure descriptions, in line 127 the relevant figure is not numbered, and might not be featured in the SI?

There is an existing number, “1”, S1 Fig.

RVPT

The scoring for the RVPT appears to be a bit confusing, and should probably be a discussion point?

The authors Added the following: “, where a score of 0 had the same or less color than the negative control, a score of 1 had slightly more color than the negative control, a score of 2 had slightly less color than the positive control, and a score of 3 had the same or more color than the positive control. A numerical score of 0 or 1 would result in “no” meaning the female is not pregnant, while a numerical score of 2 or 3 would result in “yes” meaning the female is pregnant.”

Statistical analysis

Line 166

This has been revised in the materials and methods section, so it follows the order of the statistical analysis and results section.

My understanding is the ‘tendencies’ should not be part of statistical analysis.

The definition of what level was considered a tendency is stated in the statistical section.

Results

Table 1 useful.

For table 2 and for all others please alter ultrasound for ultrasonography.

Revised as suggested.

Please ensure all the figures are correctly documented in the article, and correlate with the relevant illustrated figures.

This has been revised

Line 282; not sure that tendency should be recorded; maybe in the discussion. Similarly for line 290, and for all mentions of a tendency.

Tendencies were left in to allow readers to evaluate their own cutoff level, and the actual p-values are included with all statements.

Discussion and conclusion

Line 323; remove ‘a’ or member to singular.

Revised as suggested.

Additional information has been added to address this concern.

Lines 360-364:

Please discuss the relevance of a false positive compared with a false negative in relation to the managers requirements- feeding of a non-pregnant animal, or culling of a pregnant animal.

Additional information has been added to address this concern

Discussion of relevance of DPP PAG results in relation to the likelihood of pregnancy if insemination is unlikely? And, importance of multiparity cf primiparity esp 35-49d pp. (Why?) (Lines 376-390)

This sentence has been removed

Line 388: Probably does not need: ‘with that said’ change made.; and to explain the differences RPT and Lateral Flow options more succinctly. I believe it already is.

Revised as suggested.

Line 408: A ‘shot’ although a common colloquialism, should probably be best described scientifically as some sort of drug administration/injection?

Revised to injection.

Additional information has been added to this point

Competing Interests

I think it is difficult to believe that no competing interests were declared when Idexx and Zoetis supported the study to a degree, and at least one author is an employee of Idexx.

This statement has been changed to list Dr. Jim Rhoades as an employee of IDEXX.

The Funding declaration in lines 452-453 appears to be at odds with that in the Financial disclosures section of the pre-manuscript section.

The financial statement has been added to and now reads. “This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. This project was partially funded by donations from IDEXX and Zoetis.”

Attachment

Submitted filename: Response to Reviewers (003).docx

pone.0306325.s005.docx^{(22.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0306325.r003

Decision Letter 1

Angel Abuelo

24 May 2024

PONE-D-24-00715R1Evaluation of pregnancy associated glycoproteins assays for on farm determination of pregnancy status in beef cattlePLOS ONE

Dear Dr. Perry,

There are a remaining few minor items that would need to be addressed - see comments from reviewer #1 below.

Please submit your revised manuscript by Jul 08 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Angel Abuelo, DVM, MRes, MSc, PhD, DABVP (Dairy), DECBHM

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Thank you for the time and effort you have put into revising this manuscript. The revised version is much closer to completion and reads more easily than the first. However, there are a few additional minor revisions this reviewer would suggest prior to final acceptance.

Line numbers correspond to the marked up version of the manuscript

Line 63-65 This section, starting with “In order…” is a sentence fragment. Please revise so it is more clear and complete.

Line 237 – In table and in legend, please correct spelling of “sensitivity”

Lines 393-394 – In this sentence, it would be appropriate to use “ultrasound equipment” rather than “equipment of ultrasonography”

Lines 394-397 – A better way to discuss the costs would be to state that the blood test methods range from $4.50 to $8 per head while the cost for ultrasound is going to depend on the veterinarian’s rate to perform the test and their speed. Many veterinarians, working with an efficient crew, can run 30-45 cows through the chute in an hour (or more) likely making the cost of all methods relatively similar and maybe even less per head for ultrasonography since veterinarians typically charge an hourly rate for these services (rather than per head). It also needs to be mentioned that a skilled ultrasonographer can provide valuable information about the pregnancy (viability, stage of gestation, fetal sex, etc) that is not provided by a blood test. Because of the extreme variability in rates charged by practicing veterinarians providing ultrasonography services across the country, I would suggest not including that dollar figure. Additionally, the most logical argument for the use of a cow side test is in rural areas where producers are unable to find veterinarians to perform pregnancy diagnosis. This tool allows those producers to benefit from pregnancy diagnosis and identification of non-pregnant cows which will help their bottom line even when professional services are unavailable.

Lines 479-481 – The added sentences do not flow with the rest of the paragraph content. Please consider revising so they complete the paragraph. One suggested revision would be to word as follows: “Due to the additional time required for diagnosis of pregnancy with blood based tests, management decisions may be delayed compared to ultrasonography and may result in additional labor to sort open females once test results are available.”

Reviewer #2: Thank you for addressing the review comments; hopefully it has been beneficial to the publication to have done so.

I have two concerns, both of which I will leave to the editor/s to address:

I am not convinced that funding acquisition only is sufficient grounds for scientific authorship, although I am sure that J Rhodes did more than obtain funding, and would have been involved in the study concepts and editing the submitted document, thereby making an intellectual contribution to the research and it's output.

I think that 'tendency' should be left for the reader to determine, and not be suggested by the authors.

I am happy for the amended article to be published.

**********

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Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2024 Jul 25;19(7):e0306325. doi: 10.1371/journal.pone.0306325.r004

Author response to Decision Letter 1

13 Jun 2024

Line 63-65 This section, starting with “In order…” is a sentence fragment. Please revise so it is more clear and complete.

This has been revised to read “Pregnancy diagnosis within an operation is not only important, but necessary to increase profitability and have a complete and successful reproductive management program”

Line 237 – In table and in legend, please correct spelling of “sensitivity”

Revised as suggested

Lines 393-394 – In this sentence, it would be appropriate to use “ultrasound equipment” rather than “equipment of ultrasonography”

Revised as suggested

This has been revised as suggested.

Attachment

Submitted filename: response to review.docx

pone.0306325.s006.docx^{(15KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0306325.r005

Decision Letter 2

Angel Abuelo

16 Jun 2024

Evaluation of pregnancy associated glycoproteins assays for on farm determination of pregnancy status in beef cattle

PONE-D-24-00715R2

Dear Dr. Perry,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Angel Abuelo, DVM, MRes, MSc, PhD, DABVP (Dairy), DECBHM

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0306325.r006

Acceptance letter

Angel Abuelo

21 Jun 2024

PONE-D-24-00715R2

PLOS ONE

Dear Dr. Perry,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

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on behalf of

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PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Comparison of a pregnant and not pregnant IDEXX Alertys OnFarm Pregnancy Test, lateral flow.

(DOCX)

pone.0306325.s001.docx^{(180KB, docx)}

S1 File. Data file 1.

This is the S1 data file.

(DOCX)

pone.0306325.s002.docx^{(261.4KB, docx)}

S2 File. Data file 2.

This is the S2 data file.

(DOCX)

pone.0306325.s003.docx^{(153.8KB, docx)}

Attachment

Submitted filename: PONE-D-24-00715_reviewer comments.docx

pone.0306325.s004.docx^{(14.6KB, docx)}

Attachment

Submitted filename: Response to Reviewers (003).docx

pone.0306325.s005.docx^{(22.2KB, docx)}

Attachment

Submitted filename: response to review.docx

pone.0306325.s006.docx^{(15KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Evaluation of pregnancy associated glycoproteins assays for on farm determination of pregnancy status in beef cattle

Adalaide C Kline

Saulo Menegatti Zoca

Kaitlin M Epperson

Lacey K Quail

Jaclyn N Ketchum

Taylor N Andrews

Jerica J J Rich

Jim R Rhoades

Julie A Walker

George A Perry

Roles

Abstract

Introduction

Materials and methods

Experimental design

Blood sampling

Serum

Plasma

Transrectal ultrasonography

IDEXX Alertys Rapid Visual Pregnancy Test (RVPT)

IDEXX Alertys Ruminant Pregnancy Test (RPT)

IDEXX Alertys OnFarm Pregnancy Test (lateral flow)

Statistical analysis

Results

Study 1

Table 1. Agreement between pregnancy tests to determine accuracy of pregnancy detection.

Table 2. Correlation between pregnancy tests to determine accuracy of pregnancy detection.

Table 3. Agreement between pregnancy detection methods to determine accuracy among methods.

Study 2

PAG clearance

Fig 1. Clearance of PAG concentrations in postpartum beef cattle (A) and overall comparison between parity (B).

Fig 2. False-positive percentage by days postpartum (A) and parity (B) utilizing the Rapid Visual Pregnancy Test.

Fig 3. False positive percentage by days postpartum (A) and parity (B) utilizing the OnFarm Pregnancy Test.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Angel Abuelo

Roles

Author response to Decision Letter 0

Decision Letter 1

Angel Abuelo

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