López-Arjona et al. BMC Veterinary Research (2021) 17:140 https://doi.org/10.1186/s12917-021-02838-5 RESEARCH ARTICLE Open Access Oxytocin in bovine saliva: validation of two assays and changes in parturition and at weaning Marina López-Arjona1, Eva Mainau2, Elena Navarro2, María Dolores Contreras-Aguilar1, Damián Escribano1, Sandra V. Mateo1, Xavier Manteca2, José Joaquín Cerón1* and Silvia Martínez-Subiela1 Abstract Background: The possible use of oxytocin in saliva as an indicator of positive emotions in bovine species has been poorly investigated. In the present study, two new assays (one using a monoclonal antibody and the other using a polyclonal antibody) for the measurement of oxytocin in bovine saliva were developed and validated. Also, the changes in oxytocin in saliva were explored in two different situations. One was around parturition, and for this purpose, saliva samples from 13 cows were collected at three different times: 7 days before the parturition, the day of parturition and 7 days after the parturition. The second situation was weaning and grouping of calves, and for this purpose, saliva from 25 calves was collected at three different times: 1 day before weaning, 2 days after weaning or milk withdrawal and 4 days after grouping calves. Results: In cows, oxytocin concentrations showed an increase on the day of parturition with both assays, while in calves, oxytocin concentrations showed a decrease 4 days after the grouping. Conclusions: The assays validated in this report could be used for the measurement of oxytocin in bovine saliva and detect changes in this analyte that can occur in different physiological or productive situations such as parturition and weaning. Keywords: Calves, Cow, Oxytocin, Saliva Background Saliva is gaining importance as a sample for oxytocin Oxytocin is a hormone released by neurons from the measurements because it has the advantage, compared neurohypophysis, which is involved in milk ejection with serum or cerebrospinal fluid, of being obtained and uterine contractions [1]. In addition to these through a non-invasive way and does not cause any stress physiological functions, oxytocin has a role in mater- to the animal [8]. Recently, two new accurate and pre- nal behavior [2] and it is involved in positive social cise assays have been developed for oxytocin measure- interactions [3, 4] and positive emotions in humans ment in the saliva of pigs. One uses a monoclonal [5] and animals [6, 7]. antibody, and it can measure the oxytocin liberated from proteins after a reduction-alkylation (R/A) procedure, and the other assay uses a polyclonal antibody and it can * Correspondence: jjceron@um.es measure oxytocin linked to proteins [9]. 1 Interdisciplinary Laboratory of Clinical Analysis, Regional Campus of Some studies have evaluated oxytocin in cows in International Excellence ’Campus Mare Nostrum’, University of Murcia different physiological situations. Suckling increases oxy- (Interlab-UMU), University of Murcia, Campus de Espinardo s/n, Espinardo, 30100 Murcia, Spain tocin in serum in both dams and calves, being these Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. López-Arjona et al. BMC Veterinary Research (2021) 17:140 Page 2 of 8 increases related to positive animal welfare [6, 10]. However, in saliva, Geburt et al. [11] indicated that oxy- tocin is not a valid biomarker for maternal behavior in cattle and Lurzel et al. [12] found that salivary oxytocin concentrations in cattle do not change consistently after interaction of 10 min with a familiar or unfamiliar human. Cows and calves face stressful situations during their production cycle. The occurrence of stress and reduced animal welfare can cause decreases in production, poor reproductive performance, or an increase in disease sus- ceptibility [13, 14]. Parturition in cows is recognized as a painful and stressful process [15], resulting in hyperadre- nocortical activity [16], which increases plasma gluco- corticoid [17]. Regarding the separation of calves from their mothers, Pérez et al. [18] found that it had a stress- ful effect on both the mothers and the calves, showing some behaviors associated with stress, like vocalizations and locomotor activity, and also increase in cortisol con- centration and loss of weight. Moreover, the calves also Fig. 1 AlphaLISA monoclonal protocol for oxytocin measurement in need to adapt to new social companions, and the bovine saliva regrouping and mixing of unfamiliar animals might lead to an increase in agonistic interactions [19]. To the author`s knowledge, in cows, there are no stud- The polyclonal method showed intra-assay CVs of ies reported about different forms of oxytocin in saliva, 5.1–15.1 % and inter-assay CVs of 9.3–16.2 %. Dilution and about changes in oxytocin concentrations after some of saliva samples resulted in linear regression equations stressful situations, such as parturition and the separ- with a correlation coefficient between 0.98 and 0.99. The ation of calves from their mothers. The purposes of this results of the recovery obtained were between 99 and study were: (a) to optimize and validate two assays, one 120 %. The assay LD and LOQ were 4.7 and 2.4 ng/mL, using a monoclonal antibody and the other using a poly- respectively. clonal antibody for oxytocin, which can measure differ- ent oxytocin components in the saliva of other species Evaluation of the R/A effect such as pigs, for oxytocin measurements in bovine saliva. Oxytocin concentrations with the monoclonal method (b) To evaluate if oxytocin measured by these two assays showed no significant differences (P = 0.6237) between can change in two situations in which variations in oxy- samples with the R/A procedure (358.6 pg/mL; 25-75th tocin concentrations would be expected, one affecting percentile: 56.8-567.4 pg/mL) and without the R/A pro- cows such as parturition, and one affecting calves such cedure (337.0 pg/mL; 25-75th percentile: 22.6-860.1 pg/ as weaning and grouping. mL). In the case of the polyclonal method, oxytocin con- centrations showed no differences (P = 0.0707) between samples with the R/A procedure (75.8 ng/mL; 25-75th Results percentile: 59.1–91.9 ng/mL) and without the R/A proced- Optimization of monoclonal and polyclonal assay ure (62.9 ng/mL; 25-75th percentile: 46.4–76.5 ng/mL). protocol for oxytocin measurement in bovine saliva These results are shown in Fig. 3. The protocols that resulted after optimization for mono- When buffers were analyzed as samples, they gave a clonal and polyclonal assays are shown in Figs. 1 and 2. result under the detection limit before and after the R/A procedure in the two assays. Monoclonal and polyclonal assay validation The correlations between assays and with or without The monoclonal method showed intra-assay CVs of R/A procedure are shown in Table 1. The monoclonal 2.3–8.1 % and inter-assay CVs of 10.7–13.4 %. Dilu- method showed a significant positive correlation be- tion of saliva samples resulted in linear regression tween values with and without R/A procedure (r = 0.873; equations with a correlation coefficient between 0.98 P< 0.0001), while the polyclonal method showed a no and 0.99. The results of the recovery obtained were significant correlation (r = 0.162; P = 0.495) with and between 93 and 120 %. The assay LD and LOQ were without R/A procedure. The monoclonal method with- 7.1 and 56.8 pg/mL, respectively. out R/A procedure showed a significant positive López-Arjona et al. BMC Veterinary Research (2021) 17:140 Page 3 of 8 Fig. 2 AlphaLISA polyclonal protocol for oxytocin measurement in bovine saliva correlation with polyclonal method without R/A proced- 18.3–41.6 ng/mL) (P = 0.0189). T0 did not show sig- ure (r = 0.565; P = 0.009) and a no significant correlation nificant changes when compared with T-7 (30.4 ng/ with polyclonal method with R/A procedure (r = 0.056; mL; 25-75th percentile: 24.7–51.7 ng/mL) (P = 0.1821) P = 0.821). (Fig. 4). Oxytocin concentrations in saliva of cows around Oxytocin concentrations in saliva of calves after weaning parturition and grouping When the monoclonal method was used in saliva sam- When the monoclonal method was used, there were ples, a significant increase was observed in T0 (1105.0 higher values for oxytocin before weaning (780.0 pg/mL; pg/mL; 25-75th percentile: 286.0-1643.0 pg/mL) com- 25-75th percentile, 591.3-955.4 pg/mL) and after wean- pared with T-7 (69.1 pg/mL; 25-75th percentile: 7.13– ing time (795.0 pg/mL; 25-75th percentile, 657.3-952.3 401.5 pg/mL) and T7 (7.1 pg/mL; 25-75th percentile: pg/mL) than after grouping time (18.6 pg/mL; 25-75th 7.1–1028.0 pg/mL) (P = 0.0375 and P = 0.0306, respect- percentile, 7.1-299.1 pg/mL) (P < 0.0001). ively). When the polyclonal method was used in saliva With the polyclonal method, there were higher values samples, a significant increase was observed in T0 for oxytocin before weaning (107.2 ng/mL; 25-75th per- (40.5 ng/mL; 25-75th percentile: 28.4–64.3 ng/mL) centile, 81.9-130.7 ng/mL) and after weaning time (88.8 compared with T7 (30.1 ng/mL; 25-75th percentile: ng/mL; 25-75th percentile, 70.5-122.2 ng/mL) than after López-Arjona et al. BMC Veterinary Research (2021) 17:140 Page 4 of 8 a b Fig. 3 Results for oxytocin concentrations in saliva of cows before (No R/A) and after (R/A) the R/A treatment to the saliva samples with AlphaLISA monoclonal (a) and polyclonal (b) method. The plots show median (line within box), 25th and 75th percentiles (box) and minimum and maximum values (whiskers) grouping time (64.6 ng/mL; 25-75th percentile, 37.9– since no significant changes were detected before and 85.5 ng/mL) (P = 0.0016 and P = 0.0068, respectively). after the R/A procedure, while in pig saliva, the concen- Oxytocin concentrations at different times of sampling trations were significantly lower after the R/A procedure with the monoclonal and polyclonal methods are shown [9]. Therefore, according to these results, it could be in Fig. 5. postulated that the monoclonal method could evaluate the oxytocin that would be liberated after the R/A pro- Discussion cedure without the need for this procedure in saliva Our findings indicate that oxytocin is present in bovine samples [9]. However, more studies should be under- saliva as was reported in previous studies [11, 12] and that taken to determine which form of oxytocin is measured the assays used in our research can measure oxytocin in by the polyclonal assay, since contrarily to that reported saliva samples with high precision and accuracy. These as- in other species such as pig, in cows, this assay seems says are based on the alphaLISA technology, which offers not to measure the oxytocin which is bound to proteins some advantages compared to ELISA (enzime-linked im- and it is not separated with the R/A procedure [9]. The munosorbent assay) kits, such as a lower sample volume high correlation found in alphaLISA monoclonal method and the no need for any washing step [20]. between the values obtained before and after the R/A Regarding the effect of the R/A procedure with the procedure leads us to use the samples without R/A monoclonal method, the results were similar to previous treatment in the experiments made to evaluate the studies made with the same antibody in the saliva of pigs changes in this indicator in parturition and weaning. [9], with no significant changes and similar values of The oxytocin concentrations detected by our poly- oxytocin concentrations before and after the R/A pro- clonal method were much higher than those for the cedure. However, the results with the polyclonal method monoclonal method. This could indicate that the poly- were not similar to previous studies in other species, clonal assay measures other different forms of oxytocin. Table 1 Table 1 Spearman correlation coefficients between the two methods without (no R/A) and with R/A treatment (R/A). Asterisks indicate statistical significance (*P < 0.05; **P < 0.01; ****P < 0.0001) AlphaLISA monoclonal no AlphaLISA monoclonal R/ AlphaLISA polyclonal no R/ AlphaLISA polyclonal R/ R/A A A A AlphaLISA monoclonal no 0.873**** 0.565** 0.056 R/A AlphaLISA monoclonal R/A 0.873**** 0.523* 0.112 AlphaLISA polyclonal no R/A 0.565** 0.523* 0.162 AlphaLISA polyclonal R/A 0.056 0.112 0.162 López-Arjona et al. BMC Veterinary Research (2021) 17:140 Page 5 of 8 a b Fig. 4 Results for oxytocin concentrations in saliva of cows at 7 days before the parturition (T-7), the day of parturition (T0) and 7 days after the parturition (T7) with AlphaLISA monoclonal (a) and polyclonal (b) methods. Asterisks indicate significant differences (*P < 0.05). The plots show median (line within box), 25th and 75th percentiles (box) and minimum and maximum values (whiskers) Our monoclonal method yielded a similar range of method (16 fold) compared with the polyclonal method values to a recent study in the saliva of cattle that used (1.3 fold). This increase could be because of oxytocin is an ELISA kit, in which the saliva samples were extracted involved in the mechanism of parturition [21, 22]. with solid-phase extraction [12]. Our However, our assay Although the parturition is a painful process for the would have the advantage of not needing solid-phase cows [15] and would potentially decrease oxytocin levels, extraction and would be faster and easier to perform. the high values obtained on the day of parturition would In our experimental conditions, we found an increase indicate that the role of oxytocin in the mechanisms of in salivary oxytocin at the day of parturition, being the parturition predominates at this stage. In future studies, magnitude of increase higher with the monoclonal it would be interesting to evaluate oxytocin changes in a b Fig. 5 Oxytocin concentrations measured with monoclonal (a) and polyclonal (b) method in saliva of calves at 1 day before weaning (BW), 2 days after weaning or milk withdrawal (AW) and 4 days after grouping calves (AG) Asterisks indicate significant differences (**P < 0.01; ****P < 0.0001). Plots show median (line within box), 25th and 75th percentiles (box) and minimum and maximum values (whiskers) López-Arjona et al. BMC Veterinary Research (2021) 17:140 Page 6 of 8 dystocia or in other disorders related to parturition and For the polyclonal assay, a polyclonal antibody previ- explore if the concentrations would decrease as a conse- ously described was used (López-Arjona et al., 2020). An quence of the pain. The decrease in oxytocin found in indirect competition assay was developed, in which our study in lactation would be in line with other studies Acceptor beads are coated to protein G (PerkinElmer, that indicate that oxytocin secreted at nursing was Inc., USA). In the optimization of the assay, different lower that at parturition [23]. concentrations of biotinylated oxytocin (0–6 nM) and The decrease found in oxytocin concentrations in sal- polyclonal anti-oxytocin antibody (10–20 nM) were iva in calves after grouping would indicate that the tested. Different concentrations of both Donor (Perki- calves face a stressful situation at these times, with nega- nElmer, Inc., USA) and Acceptor beads (with protein G) tive welfare implications [19, 24]. This data would sug- between 20 and 40 µg/mL were evaluated. gest that oxytocin in saliva can reflect situations of negative emotions and be a marker of welfare in cows. This would be in contrast with other reports that found Monoclonal and polyclonal assay validation that oxytocin in saliva did not change in situations re- Oxytocin standards were prepared by diluting conju- lated to maternal behavior or interaction with humans gated oxytocin to bovine serum albumin (oxytocin-BSA, [11, 12]. The different physiological situations evaluated Cusabio) in AlphaLISA Universal buffer. The standard and/or the assays used could be involved in these curve was generated using eight standards at concentra- divergences. tions of 2400, 1200, 600, 300, 150, 75, 37.5 and 0 pg/mL There is some controversy about using oxytocin in in case of alphaLISA monoclonal method and 160, 80, serum as an indicator of stress or welfare due to its short 40, 20, 10, 5, 2.5 and 0 ng/mL in case of alphaLISA poly- half-life. However, in previous studies in humans, oxyto- clonal method. cin persisted elevated during hours in saliva after its in- For analytical validation of the assays, the inter- and tranasal administration [25, 26], so oxytocin could have intra-assay coefficients of variations (CVs) were calcu- enough half-life in saliva to act as an indicator of stress lated for imprecision. Five replicates of each pooled sam- or welfare. ple with high, medium and low oxytocin concentrations were analyzed at the same time to determine the intra- assay precision. Five aliquots of each pool were stored in Conclusions plastic vials at -80 ºC until analysis. These samples were In conclusion, two new assays have been analytically val- thawed at room temperature and measured in duplicate idated for oxytocin measurement in bovine saliva. five times over five different days for inter-assay These assays could be used to measure oxytocin in bo- precision. vine saliva and detect the changes that can occur in dif- The accuracy was evaluated by an assessment of lin- ferent physiological or productive situations such as earity under dilution and recovery experiments. For the parturition and weaning. In our particular conditions, linearity evaluation, two samples were serially diluted the assay that uses a monoclonal antibody showed from 1:2 to 1:128 with AlphaLISA Universal buffer. For changes of higher magnitude and significance, and this the recovery test, different amounts (4000, 2000, 1000, would indicate that different results in oxytocin mea- 500 and 250 pg/mL in case of monoclonal method and surements in saliva of cows can be obtained depending 32, 16, 8, 4 and 2 ng/mL in case of polyclonal method) of the assay used. of commercially available conjugated oxytocin (oxytocin- BSA) were added to saliva samples with known oxytocin Methods concentrations. The percentages of the measured to the Optimization of monoclonal and polyclonal assay expected oxytocin concentrations were calculated. protocol for oxytocin measurement in cow saliva The limit of detection (LD) was obtained by the The monoclonal antibody used for assay develop- mean + two standard deviations (SD) of 12 replicated ment was previously described in a report about oxytocin measurements of the AlphaLISA Universal buffer for measurement in pigs [27]. The monoclonal assay, is a monoclonal and polyclonal method. For the limit of direct competition assay based on AlphaLisa (PerkinEl- quantification (LOQ), the lowest oxytocin concentra- mer, (MA, USA)) technology in which Acceptor beads tions that could be measured with < 20 % imprecision coated to the monoclonal anti oxytocin antibody was calculated, so the saliva samples with a known oxy- were used. In the assay optimization, different concen- tocin concentration (1300 pg/mL and 90 ng/mL for trations of biotinylated oxytocin (0–6 nM), acceptor monoclonal and polyclonal method, respectively) were beads (10–30 µg/mL) and streptavidin donor beads (with serially diluted from 1:2 to 1:128 with AlphaLISA uni- protein G) were tested (20–40 µg/mL) until an optimal versal buffer and analyzed five times at the same run for concentration was found. each dilution. López-Arjona et al. BMC Veterinary Research (2021) 17:140 Page 7 of 8 Evaluation of R/A procedure placed into the Salivette tubes that were immediately For evaluation of the R/A procedure in the saliva of centrifuged at 4500 rpm for 10 min at 4ºC. Saliva was cows, saliva samples were obtained from 20 cows then transferred into 1.5 mL eppendorf and stored at (Holstein breed) from a farm of Murcia, Spain. The cows -80ºC until analysis. were all healthy and the saliva samples were obtained The saliva samples of these experiments were analyzed between 9.00 and 12.00 h. Each cow was sampled one by the monoclonal and polyclonal assays without R/A. time during the study. These saliva samples were mea- sured by both assays with and without application of the Study of oxytocin changes at weaning and grouping R/A procedure. The samples used in this study were obtained from 25 The R/A procedure was carried out according to calves (newborn female Friesian calves born from multipar- Brandtzaeg et al. [28] and 20 saliva samples from cows ous and primiparous dams) from a commercial farm. The were used. At first, 100 µL of each sample was diluted study was performed from October 2018 to June 2019. The into 200 µL 50 mM Tris-HCl (pH 8.0). Then 5 µL of calves were sampled three times during the study: 1 day be- 0.5 M DTT (dithiothreitol) (GE Healthcare Life Sciences, fore weaning (BW), 2 days after weaning or milk with- Marlborough, MA, USA) were added to 100 µL of the drawal (AW) and 4 days after grouping calves (AG). diluted saliva sample, mixed for 30 s and incubated at Saliva samples were obtained from all the calves 37ºC for 45 min, followed by cooling to room included in the study. Saliva samples were collected from temperature (22ºC). After that, 15 µL of 0.5 M IAM the calves using Salivette tubes (Sarstedt, Aktienge- (iodoacetamide) (GE Healthcare Life Sciences, Marlbor- sellschaft & Co. Nümbrecht, Germany). Each tube con- ough, MA, USA) were added to each solution, mixed for tained a cotton swab, that was clipped through a Kocher 30 s and incubated at 22ºC in the dark for 20 min. Then, clip, and calves were allowed to chew it during 1 min. ice-cold 80 % ACN (acetonitrile) (Scharlab, S.L., Barce- Then, the cotton swab was placed in the tube and centri- lona, Spain) in ultrapure water (Millipore) (v/v) was fuged at 6000 rpm for 12 min. Saliva samples (approxi- added to the solution, mixed for 30 s and centrifuged for mately 1–2mL per cotton swab) were stored in Eppendorf 15 min at 14,000 rpm in a Sorvall ST 8R Centrifuge tubes and frozen at − 80 °C until analysis. (Thermo Fisher Scientific, Waltham, MA, USA). After The saliva samples of these experiments were analyzed centrifugation, the supernatant was pipetted into a new by the monoclonal and polyclonal assays without R/A. Eppendorf and evaporated to dryness in a speed vac The study was carried out in compliance with the concentrator (Eppendorf Concentrator 5301) followed ARRIVE guidelines [30]. by reconstitution in 200 µL of PBS (phosphate-buffered saline). The treated samples were diluted 1:2 with buffer Statistical analysis corresponding to each assay. Medians and 25th-75th percentile were calculated by To evaluate if the R/A procedure could affect the assays use of routine descriptive statistical procedures and used in our study independently from the effect that it has computer software (Excel 2016, Microsoft Corporation, on bound oxytocin, the Universal assay buffer used for Redmond, Washington, USA). Statistical analyses were alphaLISA methods was processed using the R/A protocol performed using Graph Pad Software Inc (GraphPad and assayed as an unknown sample in each of the assays. Prism, version 6 for Windows, Graph Pad Software Inc, San Diego, USA). The distribution of the studied vari- Study of oxytocin changes around parturition ables was checked by using the Kolmogorov-Smirnov Saliva samples were obtained from 13 cows (between 3 and test and did not follow a normal distribution. Mann- 8 years old, Holsteins) from a commercial farm. The cows Whitney test was used for evaluation of differences be- were all healthy and with a body condition score between fore and after the R/A procedure with each method, and 2.5 and 4. The saliva samples were obtained between 9.00 the Spearman correlation coefficients were calculated to and 12.00 h. Each cow was sampled three times during the evaluate the correlation between the two methods before study: 7 days before the parturition (T-7), the day of partur- and after R/A procedure. A one-way Repeated Measures ition (T0) and 7 days after the parturition (T7). The calves Analysis of Variance (ANOVA) followed by uncorrected were separated from the cows the same day of parturition. Fisher’s Least Significant Difference (LSD) test was used, Saliva samples were collected using Salivette tubes after log-transformation of data, to compare oxytocin (Sarstedt, Aktiengesellschaft & Co. Nümbrecht, values obtained in cows at different times (T-7, T0 and Germany) containing a sponge (Esponja Marina, La T7) and to determine if there were any significant differ- Griega E. Koronis, Madrid, Spain) as previously ences in the values obtained in calves at different times described by López-Arjona et al. [29]. The cows were (BW, AW and AG). Results were reported as median allowed to chew the sponge, which was clipped to a flex- and 25th-75th percentile (in the text) and line-box plots ible thin metal rod for 1 min. Then, the sponges were (in Figures) and a P < 0.05 was considered significant. López-Arjona et al. BMC Veterinary Research (2021) 17:140 Page 8 of 8 Acknowledgements cholecystokinin, somatostatin and insulin in dairy cows and their calves. J Not applicable. 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