López-Arjona et al. BMC Veterinary Research (2023) 19:96 BMC Veterinary Research https://doi.org/10.1186/s12917-023-03661-w REVIEW Open Access Oxytocin measurements in saliva: an analytical perspective Marina López-Arjona1*, María Botía2, Silvia Martínez-Subiela2 and José Joaquín Cerón2 Abstract Oxytocin has traditionally been known for its physiological effects on muscle contraction associated with birth and lactation, but in the last years is widely used as a biomarker of “positive experiences” in psychology and behavior. Different types of samples have been used for oxytocin measurements with saliva samples having the particular advantage of an easy and non-stressful collection. However, the low concentration of oxytocin in saliva can represent a limitation for its use. For this reason, sensitive assays and even a previous sample treatment in some cases are required for saliva oxytocin quantification. In addition, the lack of standardized and generally agreed-upon approach to peripheral oxytocin measurement leads to large discrepancies between different laboratories, that use different sample treatment protocols and different assays. The main objectives of this review are to describe the current status of the use of saliva for oxytocin measurement, provide details of the different sample processing techniques that can be applied and inform about the analytical techniques and assays available in different animal species, and also in humans for comparative purposes. It is expected that this information can contribute to an increase in the knowledge about the measurements of oxytocin in saliva and to its wider use in the future. Keywords Enzyme immunoassay, Extraction, Oxytocin, Saliva, Welfare Background for the first time in 1953 [5]. Mammals produce oxyto- Structure and synthesis cin, while fish produce isotocin, and amphibians, reptiles Oxytocin is a hormone composed of nine amino acids and birds have mesotocin, which are oxytocin-like pep- (CYIQNCPLG) which in many biological samples can tides [6]. In addition, oxytocin like peptides are found in have a cyclic chemical structure by the join of disulfide a large number of invertebrate species such as mollusks, bonds between cysteine residues 1 and 6, resulting in nematodes, and arthropods with some differences in a cyclic core comprising six amino acids with a flexible their structure [7]. three-residue amidated tail [1–3] (Fig. 1). The oxytocin Oxytocin is synthesized in the supraoptic and paraven- molecule was discovered in 1950 [4] and synthesized tricular nucleus of the hypothalamus[8]. It is transported along with the neurophysins to the posterior lobe of the pituitary gland and then it is released to the blood [8, *Correspondence: 9]. From the pituitary gland, oxytocin pathways are dis- Marina López-Arjona tributed in many brain regions associated with the stress marina.lopez.arjona@uab.cat 1 response, including the bed nucleus of the stria termina- Department of Animal and Food Science, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain lis and the amygdala [10]. Moreover, oxytocin receptors 2 Interdisciplinary Laboratory of Clinical Analysis, Regional Campus of have been found in some limbic structures, including the International Excellence ‘Campus Mare Nostrum’, University of Murcia bed nucleus of the stria terminalis, central nucleus of the (Interlab-UMU), University of Murcia, Campus de Espinardo s/n, Murcia 30100, Spain amygdala, septum, and hippocampus [11]. In addition to © The Author(s) 2023. 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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 (2023) 19:96 Page 2 of 11 Several mechanisms have been postulated to explain the effects of oxytocin, such as the potentiation of neuro- nal activity in areas of the brain related to cognitive pro- cesses, and the reduction of activity in areas that control the autonomic nervous system [28] as well, the modula- tion of serotonin activity leading to a reduction of anxi- ety [29] and interactions with the dopaminergic system and endogenous opioid system [30]. Oxytocin also plays important roles in peripheral tissues, which can feed- back to the central nervous system with routes of con- nection described [31, 32]. For example, inhaled oxytocin Fig. 1 The chemical structure of oxytocin (C43H66N12O12S2) can reach the central nervous system [33], and a route for MW = 1007,19 g/mol [2] active transport from blood circulating oxytocin into the brain has recently been discovered in mice [34]. brain, oxytocin is produced in several peripheral tissues and organs, such as the uterus, ovaries, testis, vascular Analytical issues endothelium, and heart [12]. There are many studies about peripheral oxytocin mea- Oxytocin has some similarities with arginine-vasopres- surements that use blood plasma samples [3, 35, 36]. sin, another neuropeptide synthesized in the hypothala- However, saliva has gained a high interest for the mea- mus, which shares with oxytocin seven of nine amino surement of oxytocin in trials related with behavior and acid sequences, differing only in two amino acids [3]. stress, since it is an ideal sample due to its non-invasive However, several of the known effects of arginine-vaso- nature and its easy collection, although the difficulties for pressin are opposite to those of oxytocin [3] and from the assay development and its usually low concentration at analytical point of view, oxytocin assays should not have these samples can represent a limitation for its use [37]. cross-reactivity with vasopressin. In general, there is little consensus on how to manage the saliva samples, the assays that should be used to mea- Function and biological relevance sure oxytocin and how to interpret the often-discordant Traditionally, oxytocin is known for its physiologi- results of various methods [38]. In this line, an issue that cal effects on muscle contraction associated with labor it is known is a matter of controversy is what the differ- (uterine contraction) and lactation (milk ejection) [13]. ent assays are measuring. Oxytocin can be present in a In addition, some reports stated that oxytocin acts as a cyclic or linear form (Fig. 2) that can be differently rec- stress responsive hormone [14–16]. However, in the ognized by the diverse antibodies used in the assays; last years, oxytocin has been widely studied in the field and also some assays involve sample treatments, such as of psychology and behavior, being considered as a bio- a reduction-alkylation process that irreversibly breaks marker of positive emotions since it increases in calm- disulfide bridges and produces the linear form of oxyto- ing and relaxing situations [17]. This hormone plays an cin [39]. These forms can have different psychophysiolog- important role in social bonding[18] with both short- ical effects [37, 40]: the cyclic molecule may act to initiate term and long-term effects[19] and also in birth, lac- social interaction, whereas linear oxytocin and C-ter- tation, and maternal-filial relationship [20], being in minal fragments may induce relaxation and anti-stress general released during positive interaction situations effects following social interaction, and anti-inflamma- [21]. Moreover, it improves social memory, social recog- tory and antioxidant effects [40]. The linear form can also nition and attention[22] and it also has a strong anxiolytic be formed by an enzymatic degradation[41, 42]. In addi- and stress-reducing effects [23, 24]. Overall, contrarily to tion, the oxytocin can bind to proteins but also be in free most biomarkers used for evaluating welfare, such as cor- form, and there can be oxytocin metabolites that can be tisol, catecholamines or alpha-amylase that usually are measured in variable degree by the different assays [38, associated with stress and negative situations, the oxyto- 43]. cin has the particularity of being associated with positive experiences. However, while oxytocin was first described Objectives of this review as a pro-social hormone, more recent research suggests The main objectives of this review are to report the cur- that oxytocin also increases in non-pro-social behaviors rent status of the use of saliva for oxytocin measurement, [25]. In addition, oxytocin has an important role in regu- describe the options of sample management and pro- lation of the immune system[26, 27] and anti-inflamma- cessing and provide an update about the analytical tech- tory effects. niques described for the measurements of oxytocin in López-Arjona et al. BMC Veterinary Research (2023) 19:96 Page 3 of 11 Fig. 2 Oxytocin can be degraded by aminopeptidase activity from to its cyclic form (left side from the observer) into a linear form (right side) [33, 38] saliva, in different animal species and also in humans for other authors have also reported that plasma oxytocin comparative purposes. levels do not predict central concentrations [51, 52]. In addition, there is in general no high correlation Use of saliva for oxytocin measurement between plasma and saliva concentrations. Quintana et Although blood plasma is also frequently used to mea- al.[33] concluded that salivary oxytocin concentrations sure peripheral oxytocin concentrations [3, 35, 36], this do not accurately reflects plasma oxytocin concentra- review will be focused on saliva samples. The secretion of tions after intranasal or intravenous oxytocin administra- oxytocin to saliva from central nervous system could be tion in men, and Javor et al.[53] did not found correlation influenced and controlled by autonomic nerves [44] and between salivary and plasma oxytocin concentrations in the circulating molecules in blood plasma are thought healthy young men. Also, according to Martins et al. [54], to transfer to salivary glands via surrounding capillar- salivary oxytocin is a weak surrogate for plasmatic oxyto- ies [45], since lipid insoluble molecules such as oxyto- cin in humans (r = 0.10). However, also here, there is some cin, could enter into saliva mainly via the tight junctions degree of divergence between studies since authors found between acinar cells through ultrafiltration [46]. How- low to moderate correlation between salivary and plasma ever, more studies should be needed to clearly elucidate oxytocin, such as Grewen et al.[55] (r = 0.59) in mothers the mechanisms and sources of the presence of oxyto- in response to infant contact and stress and Feldman et cin in saliva. In this line, to our knowledge, there are no al.[56] (r = 0.41) in mothers and fathers interacting with studies that have evaluated if oxytocin can be synthesized their infants. Much further research is required to detail directly in salivary glands. the mechanisms through which changes in brain oxyto- Saliva has various advantages compared to other sam- cin can be reflected in various peripheral systems. ple types that have been used for oxytocin measurements Maybe one of the reasons for the divergences found such as cerebrospinal fluid[47] or serum [48–50], such as in the relation between oxytocin in cerebrospinal fluid its easy, non-invasive and quick collection. In addition, and other sample types, can be the different assays used. saliva sampling usually reduces various sources of stress As an example, plasma and cerebrospinal oxytocin con- that are likely to interfere with oxytocin system activ- centrations were not correlated (r = 0.00, P = 0.99) when ity, since it does not require medical care or a laboratory radioimmunoassay (RIA) was used but showed a sig- setting. nificant correlation (r = 0.80, P < 0.01) when an enzyme Table 1 shows different correlations between peripheral immunoassay (EIA) was used [57]. Also, these relations (blood and saliva) and central (cerebrospinal fluid) oxy- could be influenced by the concentration of oxytocin. tocin concentrations. This table shows a large divergence For example, there is evidence for a positive association of correlations between different samples depending on between central and peripheral oxytocin concentrations the study. This indicates that, in general, the relationships after intranasal oxytocin administration and after experi- between oxytocin concentrations in saliva, blood, and mental stress induction; but this association did not cerebrospinal fluid and the possibility of use of periph- appear under baseline conditions [65]. eral oxytocin quantification to evaluate cerebrospinal Initially, some authors did not recommend oxytocin fluid concentrations and central oxytocin bioavailability measurement in saliva because its concentrations were are not yet fully well-understood [33]. Martin et al. [51] very low and therefore, difficult to detect with the assays found that oxytocin in cerebrospinal fluid showed a high developed at that time [66]. The low values of oxytocin in correlation with saliva but a low correlation with plasma, saliva have been confirmed with studies reporting a range so saliva oxytocin could help to assess cerebrospinal oxy- between 2 and 10 pg/mL in human saliva without extrac- tocin levels in patients in neurocritical care. In this line, tion, that is more than 10-fold lower than the values in López-Arjona et al. BMC Veterinary Research (2023) 19:96 Page 4 of 11 Table 1 Comparison between peripheral (plasma and saliva) and central oxytocin concentrations (cerebrospinal fluid) (CSF: cerebrospinal fluid; EIA: enzyme immunoassay; RIA: radioimmunoassay) Samples compared Correlation Species Experimental setting Assay used Refer- ence Plasma and CSF -0.149 (p = 0.352) Human Basal conditions RIA [52] Plasma and CSF 0.00 (P = 0.99) (RIA) Human and non- During surgery (human) and EIA (Enzo Life Sciences) [57] 0.48 (P = 0.02) (EIA) human primates awake and anesthetized ani- RIA 0.80 (P < 0.01) (EIA) mals (non-human primates) 0.59 (P = 0.04) (EIA) Plasma, saliva and CSF Saliva-CSF: Human Patients in neurocritical care RIA [51] 0.657 (P < 0.001) Saliva-Plasma: 0.361 (P = 0.010) Plasma-CSF: 0.417 (P = 0.003) Saliva and plasma -0.26 (P = 0.43) Human After intranasal oxytocin EIA (Enzo Life Sciences) [33] administration Plasma and CSF 0.10 (P = 0.714) Human After intranasal oxytocin RIA [58] administration Plasma and CSF 0.56 (P = 0.006) Human Anxiety EIA (Enzo Life Sciences) [59] Saliva and plasma 0.59 (P = 0.022) Human Breats-feeding mothers EIA (Assay Designs) [55] Saliva and plasma 0.252 (P = 0.180) Human Healthy young men EIA (LUCIO- Medical [53] ELISA Oxytocin) Plasma and CSF 0.926 (P < 0.001) Human Headache RIA [60] Plasma, saliva and CSF Saliva-CSF: Human Postpartum depression EIA (Nanjing SenBeiJia [61] 0.624 (P < 0.001) Biologic Technology Plasma-CSF: Co.) 0.407 (P < 0.001) Saliva and plasma 0.32 (P < 0.01) Human Depressive symptomatology EIA (Assay Designs) [62] Saliva and plasma 0.41 (P < 0.001) Human Parents interaction with EIA (Assay Designs) [56] infants Plasma and CSF No significant Human Suicide attempters RIA [47] Amygdala/Hippocampus 0.771 (P = 0.072) Rat and mice After intranasal and peripheral RIA [63] and plasma 0.600 (P = 0.208) oxytocin administration -0.030 (P = 0.934) -0.029 (P = 0.957) Hypothalamic and serum 0.010 (P = 0.967) Rat Mother-pup interaction EIA (Assay Designs) [64] 0.596 (P = 0.006) 0.542 (P = 0.016) plasma (150–250 pg/mL) [3]. However, Carter et al. [3] Also, oxytocin can be measured in saliva of goats (around used an EIA and concentrated samples, and accurately 500 pg/mL) and cattle (around 200 pg/mL) [69]. In pig detected salivary oxytocin in humans, suggesting that species, salivary oxytocin concentrations showed higher measurements of biologically relevant changes in salivary values in young individuals [70]. oxytocin are possible. Some years later, other authors From the applicative point of view, changes in salivary measured oxytocin in saliva with sensitive assays [55, 62] oxytocin concentrations have been observed in both and demonstrated that oxytocin concentrations in saliva humans [71, 72] and animals [43, 67, 68] in different con- can change in stressful situations. ditions. In human species, oxytocin can increase in saliva In animals, dogs and pigs have higher values of oxy- as after intranasal oxytocin administration; although part tocin in saliva than humans, with values usually higher of this increase could result from a direct movement of than 200 pg/mL [43, 67]. However, different values can mucus or it could be a residue of the spray administered be obtained depending on the assay used. For example, in present in the pharynx and oral cavity [73, 74]. In addi- canine saliva a median value of 258 pg/mL (range = 207– tion, it can increase in saliva in an affiliation relationship 471 pg/mL) was obtained with Arbor Assays kit (K048) [56], lactating women [72], physiological relaxation and and a median value of 679 pg/mL (range = 356–1073 pg/ emotional excitation status [75], and after physical effort, mL) with Cayman Chemical kit (Item #500,440) [67], psychological stress and sexual self-stimulation [71]. while in case of an assay developed with AlphaLISA tech- Oxytocin also has described to have an anti-stress func- nology, the median value in dogs was 335 pg/mL ([68]. tion[76] showing higher values in saliva in patients with López-Arjona et al. BMC Veterinary Research (2023) 19:96 Page 5 of 11 depressive symptoms [62]. In animals, oxytocin in saliva bound to other molecules, and only “free” oxytocin is has been studied in human-animal interactions [69, 77], measured [83]. stroking in dogs[68, 78] and stress or situations of posi- There are different methods for sample oxytocin tive welfare [79, 80]. extraction. One of them is solid phase extraction (SPE) that uses different types of columns [67, 82, 88]. This pro- Sample processing cedure usually treats the sample with 0.1% trifluoroacetic It should be point out the importance of using standard- acid (TFA-H2O), and after a centrifugation the super- ized collection conditions and sampling devices. For natant is applied to the extraction column (equilibrated example, in dogs, Sarstedt Salivette® yields oxytocin con- with acetonitrile and TFA- H2O). The eluate is evapo- centrations significantly lower than a SalivaBio Children’s rated, and the samples are reconstituted with assay buffer Swab, and citric acid and eating immediately prior to the [57]. Alternatively, to SPE, a solvent extraction, using for sample tended to increase oxytocin values [67]. example acetone, can be used [89]. The type of extrac- Oxytocin measurements can be made on unprocessed tion can influence the final values, since the recovery of samples or by performing a sample extraction. In addi- oxytocin values in plasma samples using SPE is higher tion, reduction/alkylation has been applied for sam- than when using a solvent [82]. SPE columns are more ple processing. Also, a concentration can be done in efficient and accurate than acetone and are the preferred cases that the limit of the assay detection is higher than method for extracting oxytocin from serum or plasma the oxytocin levels of the sample to be measured [81]. samples [88]. Table 2 shows examples of the different sample treatment There is controvery in the literature over whether oxy- applied. Overall, there is a huge variation in the methods tocin extraction is or not necessary. Some authors such as for sample treatment that researchers have used. McCollough et al. [50], Leng and Sabatier[87] and Chris- tensen et al.[90] recommended extraction, because non- Sample extraction extracted samples contain substances that are tagged The purpose of sample extraction is to obtain a higher as oxytocin in assays but are not oxytocin [87]. Also, it purity of the analyte of interest from the sample prior is described a high risk of matrix interference if samples to analysis with the elimination of potentially interfer- are not extracted [90]. Considering these issues, analyses ing molecules and the reduction of sample matrix effects of unextracted plasma have been described as “no more [87]. In the particular case of oxytocin, the sample extrac- than a random number generator” [87]. On the other tion removes the oxytocin bound to proteins and other hand, some studies report direct relationship between molecules. Therefore, with this procedure the “free frac- oxytocin concentrations determined in extracted and tion” of oxytocin is measured. In general, lower values of non-extracted samples, suggesting the no need of extrac- oxytocin are obtained after this process, possibly due to tion. In rhesus monkeys, there was a high correlation the elimination of interfering molecules, that could artifi- (r = 0.89) between oxytocin levels in non-extracted and cially increase oxytocin values, and the matrix effect [87] extracted serum samples [91]. Also, in some species and/or because extraction procedure eliminates oxytocin such as dog, the high correlation (Arbor assays: r = 0.80, Table 2 Oxytocin values in different species in saliva (in case of various references, the values in the table indicate the lower and upper limits described in the references). Species Salivary concentration Sample processing Refer- ences Human 1.80–7.60 pg/ml Extracted and concentrated [55, 74, 82] 1.5-51.05 pg/ml Non-extracted and concentrated [3, 35, 36, 71–73, 82] 500–1200 pg/ml Reduction-alkylation [83] Dog 27–418 pg/ml Extracted [77, 81] 100–5000 pg/ml Non-extracted [68, 77, 78, 84] 100–400 pg/ml Reduction-alkylation [68] Cattle 0-3000 pg/ml Non-extracted [85] 0-700 pg/ml Reduction-alkykation 20–550 pg/ml Extracted [69] Pig 129.6-814.2 pg/ml Extracted [69, 86] 718.4–2140.0 pg/ml Non-extracted [79, 80] 0-2500 pg/ml Reduction-alkylation [43] Goat 100–1000 pg/ml Non-extracted [69] López-Arjona et al. BMC Veterinary Research (2023) 19:96 Page 6 of 11 Cayman kit: r = 0.61) between the extracted and non- López-Arjona et al.[43] developed two immunoassays extracted saliva samples and the high spiking recoveries with polyclonal and monoclonal antibodies for salivary found in non-extracted saliva samples, make the extrac- oxytocin measurement. When they tested the assays after tion procedure not necessary [67]. Similarly, this has the reduction/alkylation treatment to saliva samples, the been recently described in saliva of pigs [86]. This could monoclonal antibody showed a high affinity by the total indicate that probably if the assay is adequate, sample oxytocin (without significant differences before and after extraction is not necessary, and this would avoid some the treatment) and the polyclonal antibody showed a high risks and limitations associated to the extraction proce- affinity by bound oxytocin and other forms or metabo- dure such as: lites of oxytocin. These results were obtained in saliva of •  The lower oxytocin values that are obtained with porcine [43], canine[68] and bovine[85] species. extracted samples, could be due to the elimination of The performance of a reduction/alkylation procedure interfering substances but also due to the loss of the would be recommended at the initial stage of the analyti- oxytocin of the sample during the procedure [66]. cal validation of any assay to determine which forms of •  The loss of correlation between analytical values ​​ oxytocin can recognize. and biological effects that can be produced after the process of extraction [92]. Sample concentration •  Analytical extraction is expensive and time Due to the low oxytocin levels in human saliva, it has consuming. been described that for some methods, a sample concen- However, there are some available commercial kits that tration of at least 4-fold is necessary [3]. With this pro- recommend the extraction of saliva samples for its mea- cedure, the oxytocin concentration in saliva would be in surement, like Arbor assay kit [93] or Cayman kit [69]. a reliable part of the standard curve of some commercial Despite to this, it is demonstrated the validity of measur- kits [72]. The most common procedure to do the sample ing oxytocin using the Arbor Assays and the Cayman kit concentration is by lyophilization. As an example, for without extraction in plasma of mice (r = 0.95 between doing a concentration of 4-fold by this method, a proce- extracted and non-extracted samples with Arbor Assays) dure that can be followed is to dry 1 ml of supernatant [94] and in saliva of dogs [67] (r = 0.80 in case of Arbor from each sample at 4 °C in a lyophilizer. Then the lyoph- Assays and r = 0.61 in case of Cayman between extracted ilized material is reconstituted in 250 µL, resulting in a and non-extracted samples). However, these results sample with a salivary concentration 4 times higher than are not so clear with other assays, such as in the case the original [72]. Other authors chose to evaporate saliva of Enzo kit, which shows less correlation than in case samples and suspended with the assay buffer for oxytocin of Arbor Assays between extracted and non-extracted measurements [71]. plasma sample of mice (r = 0.676), so there are differences In veterinary medicine, a previous concentration for between kits and it would be important to evaluate the the measurement of oxytocin in plasma has been rec- need of sample extraction in each assay, as a part of its ommended for some methods and species. For example, validation. before its use [94]. 10-fold in sheep, goat and pigs, 15-fold in horses and 20-fold in the cow has been recommended for the Enzo Reduction/alkylation Life Sciences kit [81]. However, with other methods like Oxytocin in blood can bind to larger proteins, but Arbor Assays kit, Cayman kit or AlphaLISA technology also there is a fraction which is free. Brandtzaeg et al. it is not necessary to concentrate the dog saliva [77], pig [83] developed a reduction/alkylation procedure that saliva [43] and plasma samples of dog and cat [81]. broke the bonds between oxytocin and plasma proteins enabling the detection of total oxytocin, not only the Storage oxytocin that is “free”. When they applied reduction/ Although no reports in saliva have been found, it is alkylation to plasma samples, obtained large increases described that the storage of samples in a range of tem- in detectable oxytocin of human plasma compared with peratures from 0 to 37ºC until 18 h or several freeze/ non-treated plasma. In addition, reduction/alkylation thaw cycles have minimal effects on oxytocin stability plasma samples yielded excellent linearity and paral- in plasma samples [82]. In urine samples, stability tests lelism when measured with commercial EIA kits. They revealed that oxytocin concentrations degrade over time indicated that total oxytocin may in many cases be better when stored at 4 °C but are little affected by repeated suited as a biomarker than the free fraction of oxytocin. thawing [95]. This reduction and alkylation process could also affect to the molecular conformation of oxytocin by breaking the disulfide bridge and producing a linear form [39, 83]. López-Arjona et al. BMC Veterinary Research (2023) 19:96 Page 7 of 11 Assays for oxytocin measurement EIA The oxytocin sequence structure is highly conserved EIA assays have some advantages over available RIA among mammals [96], so the different methods used for kits, such as a longer shelf life of the reagents, the lack oxytocin measurement could recognized the oxytocin in of radioactive materials and a wider detection range. all the species of mammals, however, it should be taken Moreover, EIA requires sample volumes much smaller in consideration that different species have diverse oxyto- than those necessary for the RIA, although it depends cin concentrations in biological fluids. primarily on whether an extraction is performed prior to The main types of assays used for oxytocin measure- assaying. The first EIA suitable for measuring oxytocin ments in saliva are: in plasma was developed by Prakash et al. [101], show- ing very close agreement with RIA method in the same RIA samples after extraction. Although it was the method that was firstly used for the In a study by MacLean et al. (2018), oxytocin con- measurement of oxytocin [97, 98], it has some limitations centrations in dog saliva using mass spectrometry such as: (HPLC-MS) and EIA techniques were determined, dem- •  Low sensitivity in some cases that limits the onstrating a high correlation (Arbor assays: r = 0.80, Cay- detection of low oxytocin concentrations, existing man kit: r = 0.61) of oxytocin levels in the same samples various reports in which RIAs were not sensitive analyzed with and without extraction. However, the oxy- enough for detecting oxytocin in human plasma tocin concentrations detected by EIA were much higher [47, 55, 89, 99], although some RIAs have very low than those for mass spectrometry. This could be probably detection limits such as 0.5 pg/sample for human because the HPLC-MS detects the specific compound saliva [71]. based on a known mass-to-charge ratio. By contrast, EIA •  Use of radioactive material and need of special may recognize not only the primary form of the target conditions in the laboratory for its performance. analyte, but also structurally related molecules, including In some cases, the low sensitivity can be observed even precursor forms and biologically related metabolites [38]. after extraction and 10-fold concentration of human Table 3 provides information about EIA kits that have plasma samples, with 90% of human plasma samples been used for oxytocin measurements in saliva and other showing values below the limit of detection of the RIA kind of samples. As it can be observed there is a variabil- [82]. This has been also described in other species such as ity in the detection limit of the different kits; with the EIA rat where Vecsernyés et al. [100] reported basal oxytocin kit developed by Assay Designs, Inc. reporting a mini- for male rats to be 9.6 pg/ml, but the minimum detection mum detection limit of 4.68 pg/mL [92], whereas the kit limit for that assay was near 10 pg/ml. of Cayman has a detection limit of 20 pg/mL. In general, it is a lack of knowledge about the form of oxytocin and Table 3 Commercial EIA kits used for oxytocin measurements in saliva (EIA: enzyme immunoassay) Commercial kit Method Sensitivity Species Saliva processing Other matrix samples used Arbor assay EIA 16.38 pg/ml Dog [67] Concentration Urine Western lowland gorilla [93] ALPCO EIA 15 pg/ml Rats [102] Concentration Plasma Human [62, 103] Cerebro- spinal fluid Urine Assay Designs EIA 4.68 pg/ml Human [3, 35, 36, 92] Concentration Plasma Cayman Chemical EIA 20 pg/ml Dog [77, 78] Extracted and Plasma Pig, cattle, goat [69] non-extracted Enzo Life Sciences EIA 15 pg/ml Dog, cat, cow, horse, pig, goat, Lyophilized and 4-fold Plasma sheep [81] concentrated Urine Dog [104] Cerebro- Mice [105] spinal fluid Pig [106] Wild chimpanzees [107] Human [74, 108] López-Arjona et al. BMC Veterinary Research (2023) 19:96 Page 8 of 11 possible oxytocin metabolites that the different assays extraction, although last reports do not support this pro- detect. cedure. (c) There is a wide range of different assays for oxytocin measurements and in any case, it is important HPLC-MS to use assays fully validated showing an adequate accu- HPLC is a technique that separates molecules based on racy and precision to measure oxytocin in saliva in the their physical properties. It has been used for oxytocin species planned to be studied. In addition, it would be of quantification in saliva of dogs [67]. They found that 9 interest to know which form of oxytocin (free, linked to of 20 total samples were below the limit of detection of proteins and/or oxytocin metabolites) the assay is mea- the assay (~ 2 pg/mL) and the remaining samples had a suring. Overall, there is a major need of more studies, median concentration of 18 pg/ml (range = 8–49 pg/mL). knowledge, and assay standardization efforts in order to This assay showed a positive correlation with extracted progress in the adequate and proper use of saliva for the and non-extracted samples measured by EIA. measurement of oxytocin and also to determine all its Recently, various researchers have used liquid chroma- possible applications. tography coupled to tandem mass spectrometry to assess Acknowledgements oxytocin levels in rat, human or mice [109, 110]. Also, Not applicable. others reported plasma oxytocin levels, following extrac- tion, within the range of 1–10 pg/mL [83, 111]. How- Authors’ contributions Conceived and designed the review: JJC and SMS. Performed the review: MLA, ever, Wang et al. [84] used a LC-MS method for oxytocin MB, SMS. Wrote and revised the paper: MLA, MB, JJC, SMS. All authors read and measurement in dog saliva with higher values ranged approved the final manuscript. between 0 and 5000 pg/ml. Funding This study has been supported by the Spanish “Agencia Estatal de AlphaLISA investigación” (Grant Reference PDC2021-121291-I00 / AEI / https://doi. AlphaLISA technology (PerkinElmer) is an amplified org/10.13039/501100011033) and the European Union – NextGenerationEU. ML-A has a post-doctoral fellowship “Juan de la Cierva Formación” supported luminescent proximity homogenous assay that uses two by the Spanish Ministry of Science and Innovation (FJC2021-047105-I), Spain. bead types, donor and acceptor. The AlphaLISA assay is based on the principle that, when a laser beam is shone Data Availability The data generated and analyzed during the current study are available from on a donor bead, a single oxygen molecule is generated, the corresponding authors on reasonable request. producing excitation at an adjacent acceptor bead and amplification of a fluorescent signal, which is detected Declarations and quantified. Some advantages of this type of immu- noassay in comparison with conventional EIA are the Competing interests The authors declare that they have no competing interests. small sample volume and requires fewer steps than EIA, with no need for washings. There are few studies that Ethical approval and Consent to Participate have measured oxytocin with AlphaLISA technology Not applicable. in saliva or other samples. In saliva of pig, canine and Consent for publication bovine species, AlphaLISA methods have shown a good Not applicable. linearity, precision and recovery [43, 68, 85, 86]. In addi- tion, they can measure different oxytocin forms, such as Received: 10 February 2023 / Accepted: 14 July 2023 free, bound, or other metabolites of oxytocin, depending on the antibody against oxytocin used in the immunoas- say. Moreover, the AlphaLISA was sensitive for detecting changes in oxytocin measurements in different situations References of stress or welfare in these species. 1. Beard R, Stucki A, Schmitt M, Py G, Grundschober C, Gee AD, et al. Building bridges for highly selective, potent and stable oxytocin and vasopressin Conclusions analogs. Bioorg Med Chem. 2018;26:3039–45. 2. Kocyigit ÜM. 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