In Texas, the Balcones Escarpment (BE) is a southwest-to-northcentral oriented inactive fault line that creates a strong west to east ecological boundary [15Cadenasso ML, Pickett STA, Weathers KC, Jones CG. A framework for a theory of ecological boundaries. Bioscience 2003; 53(8): 750-8.
[http://dx.doi.org/10.1641/0006-3568(2003)053[0750:AFFATO]2.0.CO;2] , 16Abbott P, Woodruff CM Jr. The balcones escarpment: Geology, hydrology, ecology, and social development in Central Texas 1986.]. West of the BE, the Edwards Plateau ranges in elevation from 180 m to nearly 900 m with a semi-arid climate dominated by juniper-oak vegetation, receiving an average of 380-860 mm of rainfall per year, with the majority occurring in May or June [17Griffith G, Bryce S, Omernick J, Rogers A. Cartographers. Ecoregions of Texas 2007.]. East of the BE, lower elevation (0-180 m) and a more temperate climate characterize the more mesic habitats of the Blackland Prairie and Post-Oak forests nearer the BE, and the Piney Woods of the interior coastal plains in eastern-most Texas [17Griffith G, Bryce S, Omernick J, Rogers A. Cartographers. Ecoregions of Texas 2007., 18Webb WL. Biogeographic regions of Texas and Oklahoma. Ecology 1950; 31(3): 426-33.
[http://dx.doi.org/10.2307/1931496] ]. Rainfall in these ecoregions is fairly evenly distributed annually with an average of 700-1000 mm in the Post-Oak and Blackland Prairie regions and 900-1300 mm in the Piney Woods [17Griffith G, Bryce S, Omernick J, Rogers A. Cartographers. Ecoregions of Texas 2007., 18Webb WL. Biogeographic regions of Texas and Oklahoma. Ecology 1950; 31(3): 426-33.
[http://dx.doi.org/10.2307/1931496] ].

With this change in elevation, vegetation and rainfall regimes on and east of the BE coincide with a longitudinal distributional barrier for numerous vertebrate taxa, including birds [19Goetze JR. Distribution, natural history, and biogeographic relationships of mammals on the Edwards Plateau of Texas 1995.-21Owen JG, Dixon JR. An ecogeographic analysis of the herpetofauna of Texas. Southwest Nat 1989; 34(2): 165-80.
[http://dx.doi.org/10.2307/3671726] ]. In addition to distributional range limits occurring at the escarpment, some species-pairs are thought to hybridize at this ecological interface [22Smith HM, Buechner HK. The influence of the Balcones Escarpment on the distribution of amphibians and reptiles in Texas. Bullentin of the Chicago Academy of Sciences 1947; 8(1): 1-16., 23Dixon KL. Contact zones of avian congeners on the southern Great Plains. Condor 1989; 91(1): 15-22.
[http://dx.doi.org/10.2307/1368143] ]. Avian species-pair recently confirmed to be hybridizing at this interface are the Black-crested Titmouse (Baeolophus atricristatus) and Tufted Titmouse (B. bicolor) [23Dixon KL. Contact zones of avian congeners on the southern Great Plains. Condor 1989; 91(1): 15-22.
[http://dx.doi.org/10.2307/1368143] ]. Black-crested Titmice are primarily found in the more arid-scrubby habitat to the west and south (to include northern Mexico) of the BE ecotone but are occasionally found east of the BE [24Patten MA, Smith-Patten BD. Black-crested Titmouse (Baeolophus atricristatus). [Internet]. Cornell Lab of Ornithology 2008. Available from: http://bna.birds.cornell.edu.lib-ezproxy.tamu.edu:2048/bna/ species/717/articles/introduction, 25Ritchison G, Grubb TC Jr, Pravosudov VV. Tufted Titmouse (Baeolophus bicolor). [Internet]. Cornell Lab of Ornithology 2015. Available from: http://bna.birds.cornell.edu/bna/species/086/articles /introduction]. Tufted Titmice are found in the more mesic, deciduous forests east of the BE and throughout the eastern United States and Canada [25Ritchison G, Grubb TC Jr, Pravosudov VV. Tufted Titmouse (Baeolophus bicolor). [Internet]. Cornell Lab of Ornithology 2015. Available from: http://bna.birds.cornell.edu/bna/species/086/articles /introduction, 26Dixon KL. An ecological analysis of the inter-breeding of crested titmice in Texas. Univ Calif Publ Zool 1955; 54(3): 125-206.].

While these non-migrating sister species share similar diet, breeding/nesting behavior, and general patterns of intraspecific communication, they differ in plumage, song, morphology and habitat preference [24Patten MA, Smith-Patten BD. Black-crested Titmouse (Baeolophus atricristatus). [Internet]. Cornell Lab of Ornithology 2008. Available from: http://bna.birds.cornell.edu.lib-ezproxy.tamu.edu:2048/bna/ species/717/articles/introduction, 25Ritchison G, Grubb TC Jr, Pravosudov VV. Tufted Titmouse (Baeolophus bicolor). [Internet]. Cornell Lab of Ornithology 2015. Available from: http://bna.birds.cornell.edu/bna/species/086/articles /introduction]. Currently ranked as separate species, their taxonomic status has fluctuated between species and subspecies because ornithologists have postulated hybridization [24Patten MA, Smith-Patten BD. Black-crested Titmouse (Baeolophus atricristatus). [Internet]. Cornell Lab of Ornithology 2008. Available from: http://bna.birds.cornell.edu.lib-ezproxy.tamu.edu:2048/bna/ species/717/articles/introduction, 25Ritchison G, Grubb TC Jr, Pravosudov VV. Tufted Titmouse (Baeolophus bicolor). [Internet]. Cornell Lab of Ornithology 2015. Available from: http://bna.birds.cornell.edu/bna/species/086/articles /introduction]. Using morphological measurements and crest plumage, Dixon identified several hybrid zones, with the primary one located in central Texas [26Dixon KL. An ecological analysis of the inter-breeding of crested titmice in Texas. Univ Calif Publ Zool 1955; 54(3): 125-206.]. He estimated this zone to be approximately 50 km wide and 175 km long (Fig. 1) [23Dixon KL. Contact zones of avian congeners on the southern Great Plains. Condor 1989; 91(1): 15-22.
[http://dx.doi.org/10.2307/1368143] , 26Dixon KL. An ecological analysis of the inter-breeding of crested titmice in Texas. Univ Calif Publ Zool 1955; 54(3): 125-206.]. Curry and Patten recently investigated plumage and morphological variation within this central hybrid zone and their findings support Dixon’s assessments of hybridization [27Curry CM, Patten MA. Current and historical extent of phenotypic variation in the Tufted and Black-crested titmouse (Paridae) hybrid zone in the southern Great Plains. Am Midl Nat 2014; 171(2): 271-300.
[http://dx.doi.org/10.1674/0003-0031-171.2.271] , 28Curry CM, Patten MA. Shadow of a doubt: premating and postmating isolating barriers in a temporally complex songbird (Passeriformes: Paridae) hybrid zone. Behav Ecol Sociobiol 2016; 70(8): 1171-86.
[http://dx.doi.org/10.1007/s00265-016-2126-y] ]. In a related study, Vaughn and Voelker (in progress) provide genetic evidence of hybridization in birds captured near the BE.

Since this hybrid zone occurs at a strong ecotone, we aim to investigate which blood gases and electrolytes differ between Tufted and Black-crested Titmice and assess if the differences are linked to climate and/or habitat. In a previous study of a suite of passerines, including titmice, we observed differences in venous blood analytes between individuals sampled east of the BE, relative to those sampled west of the BE [29Heatley JJ, Cary J, Kingsley L, Beaufrere H, Russell KE, Voelker G. Midazolam sedates Passeriformes for field sampling but affects multiple venous blood analytes. Vet Med (Auckl) 2015; 6: 61-9.
[http://dx.doi.org/10.2147/VMRR.S71402] [PMID: 30155435] , 30Heatley JJ, Cary J, Russell KE, Voelker G. Clinicopathological analysis of Passeriform venous blood reflects tranitions in elevation and habitat. Veterinary Medicine: Research and Reports 2013; 4: 21-9.]. We hypothesize that, between species, differences in blood analyte concentrations will be strongly correlated with habitat or climatic differences associated with the different ecoregions east and west of the BE. We also aim to report the role that other non-habitat variables (age, sex, and methodology) have on blood gas and electrolytes within two closely related species. The aim is to expand the knowledge of the variability of blood gas and electrolytes in wild passerines as well as provide a better understanding of ecological impacts on physiological differences between closely related, hybridizing species.

From March-August in 2010-2012, we captured 54 titmice (Baeolophus spp.) on private properties in central Texas (Fig. 1). Most birds were collected via mist nests from 0630-1200 CST; two were caught between 1700-2000 CST. We collected 26 titmice west of the Balcones Escarpment; two of these were captured west of the contact zone (Table 1) [26Dixon KL. An ecological analysis of the inter-breeding of crested titmice in Texas. Univ Calif Publ Zool 1955; 54(3): 125-206., 27Curry CM, Patten MA. Current and historical extent of phenotypic variation in the Tufted and Black-crested titmouse (Paridae) hybrid zone in the southern Great Plains. Am Midl Nat 2014; 171(2): 271-300.
[http://dx.doi.org/10.1674/0003-0031-171.2.271] ]. East of the BE, we collected 28 titmice. In a related project, we determined that plumage is not a reliable indicator of hybridization. Therefore, for this study, we used mitochondrial DNA for species assignment for all individuals in this study.

After removal from the mist net, titmice were held in cloth bags for 5-15 min before blood collection. As part of a larger study, Titmice captured in 2012 (n = 26) were administered 0.01-0.03 mL midazolam (3.9 +/-1.8 mg kg^-1; 5mg ml^-1 Injection USP Hospira, Inc.) intra-nasally prior to blood collection for sedation [29Heatley JJ, Cary J, Kingsley L, Beaufrere H, Russell KE, Voelker G. Midazolam sedates Passeriformes for field sampling but affects multiple venous blood analytes. Vet Med (Auckl) 2015; 6: 61-9.
[http://dx.doi.org/10.2147/VMRR.S71402] [PMID: 30155435] ]. Midazolam is a safe and effective sedative of wild birds [31Mans C, Guzman DSM, Lahner LL, Paul-Murphy J, Sladky KK. Sedation and physiologic response to manual restraint after intranasal administration of midazolam in Hispaniolan Amazon parrots (Amazona ventralis). J Avian Med Surg 2012; 26(3): 130-9.
[http://dx.doi.org/10.1647/2011-037R.1] [PMID: 23156974] -33Schaffer DPH, de Araújo NLLC, Raposo ACS, Filho EFM, Vieira JVR, Oriá AP. Sedative effects of intranasal midazolam administration in wild caught Blue-fronted Amazon (Amazona aestiva) and Orange-winged Amazon (Amazona amazonica) Parrots. J Avian Med Surg 2017; 31(3): 213-8.
[http://dx.doi.org/10.1647/2016-201] [PMID: 28891701] ]. Clinical effects of sedation were observed and recorded on a sedation scale [29Heatley JJ, Cary J, Kingsley L, Beaufrere H, Russell KE, Voelker G. Midazolam sedates Passeriformes for field sampling but affects multiple venous blood analytes. Vet Med (Auckl) 2015; 6: 61-9.
[http://dx.doi.org/10.2147/VMRR.S71402] [PMID: 30155435] ]. Following rest or sedation plus rest, we obtained blood samples (200-500 µL) via jugular venipuncture and immediately transferred to lithium heparin Microtainer tubes (Sarstedt Inc., USA) for anticoagulation prior to analysis.

Fig. (1) Map of locations for sampling (square) of Tufted and Black-crested Titmice. Counties west of the Balcones Escarpment (dashed line) include Kerr (1), Comal (2), Hays (3), and Travis (4). Counties east of the Escarpment are Williamson (5), Bastrop (6), Caldwell (7), Grimes (8), and Tyler (9). The extent of Titmouse species’ distribution range in Texas (dotted line) and central contact zone [26Dixon KL. An ecological analysis of the inter-breeding of crested titmice in Texas. Univ Calif Publ Zool 1955; 54(3): 125-206.] (shaded oval) are also shown. Map modified from EnchantedLearning.com (2018).

Table 1
Titmice (Baeolophus spp.) based on species (mtDNA clade), age, sex, and capture location. Balcones Escarpment consists of “West” and “Contact zone.”

Samples were analyzed in the field using an i-STAT 1 system (Abbott Laboratories, USA), following standard field protocols [30Heatley JJ, Cary J, Russell KE, Voelker G. Clinicopathological analysis of Passeriform venous blood reflects tranitions in elevation and habitat. Veterinary Medicine: Research and Reports 2013; 4: 21-9.]. Cartridges that evaluated blood gases were used first to minimize the change in analytes based on time-lapse from sampling [29Heatley JJ, Cary J, Kingsley L, Beaufrere H, Russell KE, Voelker G. Midazolam sedates Passeriformes for field sampling but affects multiple venous blood analytes. Vet Med (Auckl) 2015; 6: 61-9.
[http://dx.doi.org/10.2147/VMRR.S71402] [PMID: 30155435] ]. Remaining cartridges were analyzed within 5 min of blood collection. Venous blood analytes measured by the i-STAT 1 system were pH, carbon dioxide partial pressure (pCO₂), oxygen partial pressure (pO₂), lactate, ionized calcium (iCa⁺), glucose, sodium (Na⁺), potassium (K⁺), chloride (Cl^-), blood urea nitrogen (BUN), and hematocrit (Hct). The i-STAT 1 system also determined, via calculations, bicarbonate (HCO₃), total carbon dioxide (tCO₂), base excess (BEecf), dissolved venous oxygen (sO₂), and hemoglobin (Hb) [34Abbott Point of Care i-STAT System Manual: Hematocrit/HCT and calculated hemogloblin/HB 2016., 35Abbott Point of Care i-STAT System Manual: PCO2 and calculated values for HCO3, base excess and anion gap 2017.]. Packed red blood cell volume (PCV) was also determined via standard centrifugal methods [30Heatley JJ, Cary J, Russell KE, Voelker G. Clinicopathological analysis of Passeriform venous blood reflects tranitions in elevation and habitat. Veterinary Medicine: Research and Reports 2013; 4: 21-9.].

Following sampling, we humanely euthanized and deposited birds as museum specimens at the Biodiversity Research and Teaching Collections (BRTC) at Texas A&M University (College Station, TX). Morphological and genetic information from these birds will be used in future studies. This research was conducted under required Texas Parks and Wildlife (permit# SPR-0209-016) and United States Fish and Wildlife Permits (permit #188 MB205752) and with approval of the Texas A&M University Institutional Animal Care and Use Committee (AUP# 2012-6).

All venous blood analytes (n = 16) were assessed for normality using a Shapiro-Wilk Goodness of Fit Test (α = 0.05). We transformed analytes, which were not normally distributed and tested for, and removed, any outliers. Thus, for all subsequent analyses, we used the log of lactate and tCO₂, the reciprocal of Hct, and the reciprocal of the exponent of pH. As obtaining the reciprocal square root failed to normalize BUN data, we categorized this analyte as less than 3 mg/dL or greater or equal to 3 mg/dL or greater. We performed non- parametric tests for further statistical analysis of this analyte.

We further explored possible confounding factors. We tested all analytes against age (juvenile, adult), sex (male, female), and administration of midazolam (yes, no) as well as differences between populations based upon capture locality (west, contact zone, east) and location relative to BE (east, west) (Fig. 1, Table 1). For each analyte and factor, heteroscedasticity was tested using a 2-sided, 2 factor F Test and the 3 factor Bartlett test. If variances were unequal, as in Hct, we utilized Welch’s test to assess significant differences between means, and we weighted models to account for heteroscedasticity. For variables with equal variance, we used a one-way analysis of variance (ANOVA) tests and paired T-tests to compare population differences and the effect of sample size (α = 0.05).

We further tested related or similar variables using correlation analysis and Bland Altman plots to assess interactions between multiple variables for those analytes showing significance. The i-STAT 1 system calculates sO₂ from measurements of pO₂, pH, and HCO₃. The matched-paired analysis confirmed a correlation of sO₂ and pO₂, as well as sO₂ with pH and HCO₃ [35Abbott Point of Care i-STAT System Manual: PCO2 and calculated values for HCO3, base excess and anion gap 2017.]. Therefore, we opted not to analyze sO₂ in future analyses and focus on the measured value of pO₂. The i-STAT 1 system calculates hemoglobin by using the measured hematocrit and multiplying by a factor of 0.34, creating a manufactured value [34Abbott Point of Care i-STAT System Manual: Hematocrit/HCT and calculated hemogloblin/HB 2016.]. We confirmed the correlation between Hct and Hgb (0.925, p <0.001) and then focused on Hct differences across variables. Hematocrit (Hct) from the i-STAT 1 system and PCV determined via centrifuge were compared via the Tukey Mean Difference plot. We incorporated rainfall data and geographic location in two-way ANOVAs or linear regressions to determine if there was a biological reason for those analytes showing statistical significance. All values in the results section are means with standard error, unless reported otherwise. All significance tests set alpha at 0.05.

We analyzed venous blood samples from 54 titmice (Baeolophus spp.) (22 juveniles, 33 adults) (Table 1). In the field, using plumage, 27 were identified as Black-crested Titmice (Baeolophus atricristatus), 23 as Tufted Titmice (B. bicolor), and four as hybrids. Mitochondrial DNA, from a related genetic study, identified the four phenotypic hybrids as one Black-crested and three Tufted Titmice (Vaughn and Voelker, in progress). Two phenotypic Black-crested Titmice captured east of the BE (Grimes and Williamson counties) actually classified as Tufted Titmice from mtDNA [24Patten MA, Smith-Patten BD. Black-crested Titmouse (Baeolophus atricristatus). [Internet]. Cornell Lab of Ornithology 2008. Available from: http://bna.birds.cornell.edu.lib-ezproxy.tamu.edu:2048/bna/ species/717/articles/introduction].

Sex and age of titmice affected relatively few analytes. Females had increased potassium concentrations compared to males (ANOVA, F₂ = 4.77, p = 0.03, Table 2). Hematocrit, as measured with an i-STAT 1 system (Hct), failed to agree with the traditional centrifuge method (PCV) (Tukey Mean Difference -12.6, SE 0.9, p <0.001) (Table 2). We confirmed the correlation of Hct and Hb (0.93, p <0.001) and thus focused on Hct differences based on age and sex. Hematocrit (Hct) was higher in adult titmice compared to juveniles (Transformed 1/Hct, Welch’s Test, t₁ = 2.03, p = 0.05) and concentrations of hemoglobin (Hb) showed a similar trend (Welch’s Test, t₁ = 1.97, p = 0.05) (means, SE in Table 2).

Table 2
Venous blood analyte values from collected Titmice based on age, sex, and midazolam administration.

Midazolam administration affected all blood analytes except pH, Hct, PCV, and Hb (Table 3). Midazolam administration was associated with increased values of pCO₂, bicarbonate, tCO₂, base excess, glucose, and sodium but lower values of pO₂, sO₂, lactate, potassium, chloride, and blood urea nitrogen. Midazolam administration was associated with relatively decreased pO₂ in juveniles compared with adults receiving midazolam, while pO₂ was comparably lower in adults than juveniles, regardless of the administration of midazolam (ANOVA, F₃ = 6.47, p <0.001), (Table 4, Fig. 2).

Fig. (2) Venous partial pressure of oxygen (pO2) from different ages of Titmice (Baeolophus spp.) both east (open squares) and west (filled squares) of the Balcones Escarpment and with and without administration of Midazolam. Juvenile titmice had lower pO2 values after administration of midazolam (p = 0.01), based on an interaction of midazolam and age (p = 0.01, adj R2= 0.23).

Table 3
Venous blood analyte values from collected Titmice based on capture location and location relative to Balcones Escarpment.

Table 4
Effect of multiple variables on blood venous analytes collected from Tufted and Black-crested Titmice.

Blood glucose concentrations of titmice differed significantly based on population (west, contact zone, east) (Table 3). Titmice captured within the contact zone had the highest glucose concentrations, followed by western and then eastern populations (Table 3). Administration of the sedative midazolam also increased glucose concentrations, but the location of capture was a stronger influence of glucose than sedative (east vs. west of BE) (2-way ANOVA, Table 4).

Blood glucose concentrations were related to relative rainfall (higher or lower) and “days since rainfall” (Table 4). For titmice captured west of the BE, blood glucose concentrations were increased in months of high rainfall. However, for birds captured east of the BE, no difference of blood glucose was noted in association with levels of monthly rainfall (Fig. 3). In titmice captured west of the BE, length of time since rainfall was positively correlated with higher blood glucose concentrations occurring after 12-25 days after rainfall (Linear Regression R² = 0.40, ANOVA, F₃ = 9.83, p <0.001) (Table 4, Fig. 4).

Other venous blood analytes that varied significantly based on population, primarily between the contact zone and eastern populations included pO₂, sO₂, pH and HCO₃. Both pO₂ and sO₂ values were significantly lower in individuals within the contact zone compared to the eastern population (Table 3). Although the western population had the highest values for pO₂ and sO₂, these did not differ significantly from contact zone or eastern population values (Table 3). Midazolam administration resulted in decreased pO₂ values, with a more pronounced effect in Titmice west of the BE (ANOVA, F₁ = 5.10, p =0.03, Table 3). No interaction of capture location and midazolam administration was apparent (ANOVA, t₂ = -0.99, p = 0.33, Table 4).

Of 16 blood venous blood analytes assessed in two parapatric species of titmice, only blood glucose concentrations were statistically significantly different east and west of the BE, with relatively increased values west of the BE. A small sample size from the western population and inherent analyte variability likely prevented statistical distinctions between the western and hybrid zone populations (Table 3). The difference in glucose east and west of the BE was also observed in our related studies of multiple Texas passerine species [29Heatley JJ, Cary J, Kingsley L, Beaufrere H, Russell KE, Voelker G. Midazolam sedates Passeriformes for field sampling but affects multiple venous blood analytes. Vet Med (Auckl) 2015; 6: 61-9.
[http://dx.doi.org/10.2147/VMRR.S71402] [PMID: 30155435] , 30Heatley JJ, Cary J, Russell KE, Voelker G. Clinicopathological analysis of Passeriform venous blood reflects tranitions in elevation and habitat. Veterinary Medicine: Research and Reports 2013; 4: 21-9.]. Although pO₂ and sO₂ also differed between titmice east and west of the BE, differences resulted from age, sex, and/or midazolam administration.

In general, within bird species, glucose can vary by species, age, time of year, or can be elevated by physiological or psychological stress [36Lill A. Sources of variation in blood glucose concentrations of free-living birds. Avian Biol Res 2011; 4(2): 78-86.
[http://dx.doi.org/10.3184/175815511X13073729328092] -39Kaliński A, Bańbura M, Glądalski M, et al. Long-term variation in blood glucose concentration in nestling Great Tits (Parus major). Avian Biol Res 2015; 8(3): 129-37.
[http://dx.doi.org/10.3184/175815515X14294426911072] ]. In birds, blood glucose concentrations are modulated by Glucocorticoid (GC) production pathways via two different receptors. Baseline GC production is mediated by the mineralocorticoid receptor from which increased production can occur following an increase in energetic demands, thermoregulatory changes, or even a period of low food abundance [37Tomasek O, Bobek L, Kralova T, Adamkova M, Albrecht T. Fuel for the pace of life: Baseline blood glucose concentration co-evolves with life-history traits in songbirds. Funct Ecol 2019; 33(2): 239-49.
[http://dx.doi.org/10.1111/1365-2435.13238] -40Hau M, Casagrande S, Ouyang JQ, Baugh AT. Chapter two - glucocorticoid-mediated phenotypes in vertebrates: Multilevel variation and evolution advances in the study of behavior 48 2016; 41-115.]. Stress-induced GC production, however, is mediated through glucocorticoid receptors that upregulate gluconeogenesis based on 2-10 fold higher concentration of glucocorticoid than baseline.

Fig. (3) Venous blood glucose concentrations (mg dL-1) of Titmice captured west and east of the Balcones Escarpment (BE) based on relative monthly rainfall data. Black-crested Titmice are represented by solid triangles and Tufted Titmice represented with open triangles. Titmice captured west of the BE had relatively increased concentrations of glucose than those captured east (R2=0.33, p<0.001). Concentrations were further elevated west of the escarpment during months of relatively high rainfall (BE p<0.001, relative rainfall p = 0.34, BE*relative rainfall p = 0.03; whole model R2 = 0.38, p<0.001; Table 4).

Fig. (4) Venous blood glucose concentrations (mg dL-1) of Titmice captured east (open square, dashed fit line) and west (filled square, solid fit line) of the Balcones Escarpment and the number of days since the last rain event. Titmice captured west of the Balcones Escarpment had relatively increased blood glucose concentrations during periods of prolonged dryness (Adj. R2 east = 0.00, Adj. R2 west = 0.24; whole model Adj. R2 = 0.30, p <0.001).

Titmice captured west of the Balcones Escarpment have relatively increased glucose values compared with that east of the BE (Tufted Titmice), irrespective of sedative administration. Black-crested and Tufted Titmice share many life history similarities but differ in their habitat preference [24Patten MA, Smith-Patten BD. Black-crested Titmouse (Baeolophus atricristatus). [Internet]. Cornell Lab of Ornithology 2008. Available from: http://bna.birds.cornell.edu.lib-ezproxy.tamu.edu:2048/bna/ species/717/articles/introduction, 25Ritchison G, Grubb TC Jr, Pravosudov VV. Tufted Titmouse (Baeolophus bicolor). [Internet]. Cornell Lab of Ornithology 2015. Available from: http://bna.birds.cornell.edu/bna/species/086/articles /introduction]. Black-crested resides in the semi-arid scrub habitat of west Texas where precipitation is more sporadic, a factor that can lead to unpredictable food resources and induce physiological stress [17Griffith G, Bryce S, Omernick J, Rogers A. Cartographers. Ecoregions of Texas 2007., 41Nielsen-Gammon JW. Changing climate of texas. Impact of global warming on texas 2^nd ed. 2011; 39-68.-43Yang LH, Bastow JL, Spence KO, Wright AN. What can we learn from resource pulses? Ecology 2008; 89(3): 621-34.
[http://dx.doi.org/10.1890/07-0175.1] [PMID: 18459327] ]. Low food abundance can increase baseline glucose concentrations. Survival and adaptation to a continually altered physiological state, due to constant stress, is known as the Chronic Stress Hypothesis [44Cherel Y, Robin J-P, Maho YL. Physiology and biochemistry of long-term fasting in birds. Can J Zool 1988; 66(1): 159-66.
[http://dx.doi.org/10.1139/z88-022] -46Fokidis HB, des Roziers MB, Sparr R, Rogowski C, Sweazea K, Deviche P. Unpredictable food availability induces metabolic and hormonal changes independent of food intake in a sedentary songbird. J Exp Biol 2012; 215(Pt 16): 2920-30.
[http://dx.doi.org/10.1242/jeb.071043] [PMID: 22837467] ]. In an arid habitat, chronic stress may be due to scarce or unpredictable food or water sources and, over the long term, such stresses can induce adaptations to increase survivability [44Cherel Y, Robin J-P, Maho YL. Physiology and biochemistry of long-term fasting in birds. Can J Zool 1988; 66(1): 159-66.
[http://dx.doi.org/10.1139/z88-022] , 46Fokidis HB, des Roziers MB, Sparr R, Rogowski C, Sweazea K, Deviche P. Unpredictable food availability induces metabolic and hormonal changes independent of food intake in a sedentary songbird. J Exp Biol 2012; 215(Pt 16): 2920-30.
[http://dx.doi.org/10.1242/jeb.071043] [PMID: 22837467] , 47Williams J, Tieleman BI. Physiological ecology and behavior of desert birds.Current Ornithology Current Ornithology 16 2001; 299-353.
[http://dx.doi.org/10.1007/978-1-4615-1211-0_6] ].

Evidence for a an increased natural glucose concentration based on intermittent food/water resources is shown by the further increase in Black-crested glucose concentration following rain events, a pattern not observed in Tufted Titmice. In arid and semi-arid regions, rainfall amplifies primary productivity and triggers an increase in insect and fruit abundance, stimulating foraging by secondary and tertiary consumers [43Yang LH, Bastow JL, Spence KO, Wright AN. What can we learn from resource pulses? Ecology 2008; 89(3): 621-34.
[http://dx.doi.org/10.1890/07-0175.1] [PMID: 18459327] , 48Ostfeld RS, Keesing F. Pulsed resources and community dynamics of consumers in terrestrial ecosystems. Trends Ecol Evol (Amst) 2000; 15(6): 232-7.
[http://dx.doi.org/10.1016/S0169-5347(00)01862-0] [PMID: 10802548] , 49Wenninger EJ, Inouye RS. Insect community response to plant diversity and productivity in a sagebrush–steppe ecosystem. J Arid Environ 2008; 72(1): 24-33.
[http://dx.doi.org/10.1016/j.jaridenv.2007.04.005] ]. We suspect that sporadic rain showers temporarily increase food resources, causing hyperphagia or an increase in metabolic activity (a known correlate of glucose concentrations). A rise in blood glucose has been observed in organisms exposed to unpredictable or pulse food resources as a result of a change in diet (e.g., sugar-filled berries) or a product of hyperphagia [46Fokidis HB, des Roziers MB, Sparr R, Rogowski C, Sweazea K, Deviche P. Unpredictable food availability induces metabolic and hormonal changes independent of food intake in a sedentary songbird. J Exp Biol 2012; 215(Pt 16): 2920-30.
[http://dx.doi.org/10.1242/jeb.071043] [PMID: 22837467] ]. We observed blood glucose concentrations were highest in birds captured 12-25 days following rain, likely a result of the time lag between rainfall and pulse food resources from increased primary productivity [38Jimeno B, Hau M, Verhulst S. Corticosterone levels reflect variation in metabolic rate, independent of ‘stress’. Sci Rep 2018; 8(1): 13020.
[http://dx.doi.org/10.1038/s41598-018-31258-z] [PMID: 30158537] , 47Williams J, Tieleman BI. Physiological ecology and behavior of desert birds.Current Ornithology Current Ornithology 16 2001; 299-353.
[http://dx.doi.org/10.1007/978-1-4615-1211-0_6] , 48Ostfeld RS, Keesing F. Pulsed resources and community dynamics of consumers in terrestrial ecosystems. Trends Ecol Evol (Amst) 2000; 15(6): 232-7.
[http://dx.doi.org/10.1016/S0169-5347(00)01862-0] [PMID: 10802548] ]. Glucose concentrations in Tufted Titmice, on the other hand, did not increase following rain events, lending support to the idea that these species, living in mesic habitats, have a continual food source year round and, thus, are not prone to infrequent bouts of low food sources. It is interesting to note that the two phenotypically (plumage) Black-crested Titmice captured east of the BE did not express similar glucose concentrations to those captured west. The adult male Black-crested Titmouse (Williamson county) had a glucose concentrations slightly less than the average of birds west of the BE (354 mg/dL, x = 367 mg/dL), but the juvenile male Black-crested (Grimes county) had a glucose value less than the average of birds east of the BE (288 mg/dL, x = 294 mg/dL). Both birds received a sedative and were captured in late spring; however, the adult male was captured just east of the BE (closer to the hybrid zone). Therefore, though these birds look Black-crested and have Tufted Titmouse DNA, their glucose values appear to match with longitude/habitat.

If habitat drives glucose, as our data suggests, it may play an important role in species distribution and reduce genetic introgression across the hybrid zone. Black-crested Titmice have been observed east of the BE, but Tufted Titmice are rarely observed west of the BE [24Patten MA, Smith-Patten BD. Black-crested Titmouse (Baeolophus atricristatus). [Internet]. Cornell Lab of Ornithology 2008. Available from: http://bna.birds.cornell.edu.lib-ezproxy.tamu.edu:2048/bna/ species/717/articles/introduction, 25Ritchison G, Grubb TC Jr, Pravosudov VV. Tufted Titmouse (Baeolophus bicolor). [Internet]. Cornell Lab of Ornithology 2015. Available from: http://bna.birds.cornell.edu/bna/species/086/articles /introduction]. Tufted Titmice may have low survival west of the BE due to physiological intolerance to unpredictable food and water resources. Therefore, the physiology of glucose metabolism could act as a limiting factor to the colonization west of the BE by the Tufted Titmouse.

Other venous blood analytes were influenced by age, sex, season, and sedative administration. Although saturated oxygen and base excess were higher in males, especially those administered midazolam, we suspect that the sex difference is an artificial finding based on sample size and influence of midazolam.

Both sex and midazolam administration influenced venous blood potassium concentrations. Adult females showed relatively increased venous blood potassium concentrations compared to other groups. The most common cause of hyperkalemia in human and veterinary medicine is red blood cell lysis [50Asirvatham JR, Moses V, Bjornson L. Errors in potassium measurement: A laboratory perspective for the clinician. N Am J Med Sci 2013; 5(4): 255-9.
[http://dx.doi.org/10.4103/1947-2714.110426] [PMID: 23724399] ]. However, in avian species, the effect of hemolysis on potassium concentrations has not been evaluated to our knowledge. In this study, however, no adult female received the sedative midazolam. Thus, adult females may have undergone more stress and struggle during sampling resulting in a sample with relatively increased cellular lysis prior to analysis. During stress or strenuous exercise, increased blood pressure and muscular contractions trigger the kidneys to release potassium to ensure adequate sodium-potassium pump activity [51Siegel HS. Physiological stress in birds. Bioscience 1980; 30(8): 529-34.
[http://dx.doi.org/10.2307/1307973] , 52Stranahan AM, Lee K, Mattson MP. Central mechanisms of HPA axis regulation by voluntary exercise. Neuromolecular Med 2008; 10(2): 118-27.
[http://dx.doi.org/10.1007/s12017-008-8027-0] [PMID: 18273712] ]. Furthermore, as all females were captured during nesting or breeding season, females could have experienced greater energetic demands, and handling and blood sampling may have been more problematic without sedation. The capture of both sexes outside the breeding season could confirm if the increased potassium concentrations in female Titmice are consistently present, or they result from increased energetic demands related to breeding. Recording a hemolysis score for the blood samples could have facilitated the determination of the effect of hemolysis, if any, upon potassium concentrations in our samples [53Yoo G, Kim J, Uh Y, Yoon KR, Park SD, Yoon KJ. Scoring system for detecting spurious hemolysis in anticoagulated blood specimens. Ann Lab Med 2015; 35(3): 341-7.
[http://dx.doi.org/10.3343/alm.2015.35.3.341] [PMID: 25932443] ].

As found in our study, juvenile passerine birds of many species often have a relatively decreased hematocrit compared to adults [54Potti J. Variation in the hematocrit of a passerine bird across life stages is mainly of environmental origin. J Avian Biol 2007; 38(6): 726-30.
[http://dx.doi.org/10.1111/j.2007.0908-8857.04073.x] ]. Hematocrit’s difference between ages is likely a product of body size [55Senar JC, Pascual J. Keel and tarsus length may provide a good predictor of avian body size. Ardea 1997; 85(2): 269-74.]. The relationship was strongest with males, as expected in Titmice, since males are larger than females [25Ritchison G, Grubb TC Jr, Pravosudov VV. Tufted Titmouse (Baeolophus bicolor). [Internet]. Cornell Lab of Ornithology 2015. Available from: http://bna.birds.cornell.edu/bna/species/086/articles /introduction, 27Curry CM, Patten MA. Current and historical extent of phenotypic variation in the Tufted and Black-crested titmouse (Paridae) hybrid zone in the southern Great Plains. Am Midl Nat 2014; 171(2): 271-300.
[http://dx.doi.org/10.1674/0003-0031-171.2.271] ]. Since hematocrit varies greatly between individuals due to health, hydration, life history, and more, the unexplained high variance is unsurprising. The observed difference of Hct, between sexes, might have been stronger with a larger sample size of adult females and less disparity of the ratio of juveniles and adults.

Similar to other studies, we also found a discrepancy between PCV and measured Hct, with PCV values consistently greater than Hct [30Heatley JJ, Cary J, Russell KE, Voelker G. Clinicopathological analysis of Passeriform venous blood reflects tranitions in elevation and habitat. Veterinary Medicine: Research and Reports 2013; 4: 21-9., 34Abbott Point of Care i-STAT System Manual: Hematocrit/HCT and calculated hemogloblin/HB 2016.]. The discrepancy of methods for determination avian hematocrit is likely explained by the relative difference in the avian red blood cell shape, volume and retained nucleus, compared to the human red blood cell [29Heatley JJ, Cary J, Kingsley L, Beaufrere H, Russell KE, Voelker G. Midazolam sedates Passeriformes for field sampling but affects multiple venous blood analytes. Vet Med (Auckl) 2015; 6: 61-9.
[http://dx.doi.org/10.2147/VMRR.S71402] [PMID: 30155435] , 56Yaw TJ, Gentry J, Ratliff C, et al. Venous blood analytes and osmolality of rehabilitated juvenile Black-bellied Whistling Ducks (Dendrocygna autumnalis). J Avian Med Surg 2019; 33(2): 123-32.
[http://dx.doi.org/10.1647/2016-194] [PMID: 31251499] , 57Pistone J, Heatley JJ, Campbell TA, Voelker G. Assessing Passeriformes health in South Texas via select venous analytes. Comp Biochem Physiol B Biochem Mol Biol 2017; 210: 64-71.
[http://dx.doi.org/10.1016/j.cbpb.2017.06.002] [PMID: 28630012] ]. Packed cell volume (as determined by centrifugation), rather than Hct (as determined by electrical resistance via the i-STAT 1), provides consistently lower results and should remain the gold standard for use in passerine species. Packed cell volume (PCV or Hct), the ratio of red blood cells to total blood volume expressed as a percent, can indicate dehydration or fitness based on an individual’s ability to transport oxygen [34Abbott Point of Care i-STAT System Manual: Hematocrit/HCT and calculated hemogloblin/HB 2016.]. Larger organisms usually have increased metabolic demands based on increased body and organ size and greater demand for oxygen transport [58Glazier DS. Effects of metabolic level on the body size scaling of metabolic rate in birds and mammals. Proc Biol Sci 2008; 275(1641): 1405-10.
[http://dx.doi.org/10.1098/rspb.2008.0118] [PMID: 18348961] , 59Minias P. The use of haemoglobin concentrations to assess physiological condition in birds: A review. Conserv Physiol 2015; 3(1)cov007
[http://dx.doi.org/10.1093/conphys/cov007] [PMID: 27293692] ]. In addition, adult birds and those with androgens, rather than estrogens, also tend toward relatively increased red blood cell mass with resulting increased PCV [60Johnstone CP, Lill A, Reina RD. Use of erythrocyte indicators of health and condition in vertebrate ecophysiology: A review and appraisal. Biol Rev Camb Philos Soc 2017; 92(1): 150-68.
[http://dx.doi.org/10.1111/brv.12219] [PMID: 28075072] , 61Scanes CG. Allometric and phylogenic comparisons of hematological parameters between and within birds and mammals. Int J Veterin Health Sci Res 2016; 4(5): 123-9.]. However, Hct should be further evaluated in these species with a larger adult sample size and with individuals from across the full distribution of the species to ensure other factors, such as latitude or elevation, are not influencing Hct or PCV.

Administration of midazolam affected many blood gas, and electrolyte values often used to determine health in wild birds. Interpretation of avian health in wild birds is inherently complicated as capture, handling, and blood collection are stressful events for free-living birds [62Le Maho Y, Karmann H, Briot D, et al. Stress in birds due to routine handling and a technique to avoid it. Am J Physiol 1992; 263(4 Pt 2): R775-81.
[PMID: 1415787] -64Newman SH, Carter HR, Whitworth DL, Zinkl JG. Health assessments and stress response of Xantus’s murrelets to capture, handling, and radio-marking. Mar Ornithol 2005; 33: 147-54.]. Physicians and dentists have used midazolam for decades to reduce procedural stress in children [65Kupietzky A, Houpt MI. Midazolam: A review of its use for conscious sedation of children. Pediatr Dent 1993; 15(4): 237-41.
[PMID: 8247896] , 66Lee-Kim SJ, Fadavi S, Punwani I, Koerber A. Nasal versus oral midazolam sedation for pediatric dental patients. J Dent Child (Chic) 2004; 71(2): 126-30.
[PMID: 15587094] ]. Midazolam is a benzodiazepine that boosts inhibitory neurotransmitters, decreases neuron excitability and results in sedation, anxiolysis, and/or hypnosis depending upon the dose administered [65Kupietzky A, Houpt MI. Midazolam: A review of its use for conscious sedation of children. Pediatr Dent 1993; 15(4): 237-41.
[PMID: 8247896] ]. However, hypoxia, reduced respiration, reduced memory, and stimulation of a stress endocrine response may also occur [29Heatley JJ, Cary J, Kingsley L, Beaufrere H, Russell KE, Voelker G. Midazolam sedates Passeriformes for field sampling but affects multiple venous blood analytes. Vet Med (Auckl) 2015; 6: 61-9.
[http://dx.doi.org/10.2147/VMRR.S71402] [PMID: 30155435] , 65Kupietzky A, Houpt MI. Midazolam: A review of its use for conscious sedation of children. Pediatr Dent 1993; 15(4): 237-41.
[PMID: 8247896] ]. In multiple avian species, the intranasal administration of midazolam has been shown to reduce the stress of handling and venipuncture [67Vesal N, Eskandari MH. Sedative effects of midazolam and xylazine with or without ketamine and detomidine alone following intranasal administration in Ring-necked Parakeets. J Am Vet Med Assoc 2006; 228(3): 383-8.
[http://dx.doi.org/10.2460/javma.228.3.383] [PMID: 16448361] ].

In our study, midazolam administration influenced all analytes we measured in Titmice except pH, Hct, and Hb. For both species, irrespective of geographic location, Titmice given midazolam had, on average, higher pCO₂, HCO₃, tCO₂, base excess, glucose, and sodium values. Midazolam lowered the venous partial pressure of oxygen (pO₂), and oxygen saturation (sO₂), as well as concentrations of lactate, potassium, chloride, and BUN. Except for glucose, changes in all analytes paralleled those seen following the administration of midazolam to humans [68Tucker MR, Ochs MW, White RP Jr. Arterial blood gas levels after midazolam or diazepam administered with or without fentanyl as an intravenous sedative for outpatient surgical procedures. J Oral Maxillofac Surg 1986; 44(9): 688-92.
[http://dx.doi.org/10.1016/0278-2391(86)90036-4] [PMID: 2943882] ].

While the location of capture was a stronger predictor of blood glucose concentrations than sedative administration, glucose concentrations were unexpectedly higher in birds receiving midazolam. Gluconeogenesis occurs near the end of the stress response pathway to meet the increased energetic demands of the stressor [51Siegel HS. Physiological stress in birds. Bioscience 1980; 30(8): 529-34.
[http://dx.doi.org/10.2307/1307973] , 69Blas J. Stress in Birds.Sturkie’s Avian Physiology 6^th ed. 2015; 769-810.
[http://dx.doi.org/10.1016/B978-0-12-407160-5.00033-6] ]. Production of glucose is a vital component of the negative feedback mechanism regulating the sympathetic (aka “fight or flight”) nervous system. Even after a stress stimulus is removed, glucose concentrations may continue to rise based on the time lag between neuroendocrine communication and the hepatic metabolic response [51Siegel HS. Physiological stress in birds. Bioscience 1980; 30(8): 529-34.
[http://dx.doi.org/10.2307/1307973] , 70Braun EJ, Sweazea KL. Glucose regulation in birds. Comp Biochem Physiol B Biochem Mol Biol 2008; 151(1): 1-9.
[http://dx.doi.org/10.1016/j.cbpb.2008.05.007] [PMID: 18571448] ]. Mist-net capture, handling, and blood sampling each induce stress on wild birds, so increased glucose concentrations were expected in birds [64Newman SH, Carter HR, Whitworth DL, Zinkl JG. Health assessments and stress response of Xantus’s murrelets to capture, handling, and radio-marking. Mar Ornithol 2005; 33: 147-54., 71Harms CA, Harms RV. Venous blood gas and lactate values of mourning doves (Zenaida macroura), boat-tailed grackles (Quiscalus major), and house sparrows (Passer domesticus) after capture by mist net, banding, and venipuncture. J Zoo Wildl Med 2012; 43(1): 77-84.
[http://dx.doi.org/10.1638/2011-0114.1] [PMID: 22448512] , 72Harms CA, Jinks MR, Harms RV. Blood gas, lactate, and hematology effects of venipuncture timing and location after mist-net capture of Mourning Doves (Zenaida macroura), Boat-tailed Grackles (Quiscalus major), and House Sparrows (Passer domesticus). J Wildl Dis 2016; 52(2)(Suppl.): S54-64.
[http://dx.doi.org/10.7589/52.2S.S54] [PMID: 26845300] ]. The apparent ineffectiveness of midazolam to reduce glucose concentrations is likely a product of the lag time between capture until midazolam administration. Although we obtained blood 0-5 min following sedation, a stressful event (mist-net capture) has already occurred prior to anxiolytic administration. Further stress, from handling, during sedative administration, could also cause blood glucose concentrations to continue increasing during blood collection despite sedative administration. To better assess the efficacy of midazolam for stress reduction in captured birds, future studies could measure blood glucose concentrations and or corticosterone at various time intervals (e.g., 15-60 min) following capture handling and midazolam administration. Although midazolam changed multiple venous blood analytes similarly to humans experiencing anxiolysis, this sedative’s impact on blood gases and electrolytes in passerine birds needs further investigation.

We administrated midazolam to titmice in 2012 to reduce stress and facilitate blood collection while assessing health. Providing a rest period after capture and before blood collection may also reduce stress, as indicated by blood gases and other variables [71Harms CA, Harms RV. Venous blood gas and lactate values of mourning doves (Zenaida macroura), boat-tailed grackles (Quiscalus major), and house sparrows (Passer domesticus) after capture by mist net, banding, and venipuncture. J Zoo Wildl Med 2012; 43(1): 77-84.
[http://dx.doi.org/10.1638/2011-0114.1] [PMID: 22448512] , 72Harms CA, Jinks MR, Harms RV. Blood gas, lactate, and hematology effects of venipuncture timing and location after mist-net capture of Mourning Doves (Zenaida macroura), Boat-tailed Grackles (Quiscalus major), and House Sparrows (Passer domesticus). J Wildl Dis 2016; 52(2)(Suppl.): S54-64.
[http://dx.doi.org/10.7589/52.2S.S54] [PMID: 26845300] ]. Blood samples from Mourning Doves (Zenaida macroura), Boat-tailed Grackles (Quiscalus major), and House Sparrows (Passer domesticus), immediately after capture or after a 45-60 min delay, lacked differences in pH for any species, while blood gas values (pCO₂, pO₂, and HCO₃^-) and PCV varied based on species. However, lactate significantly decreased based on a rest period in all species. We found similar results in Titmice when comparing birds receiving a sedative to those without the sedative. However, this comparison should be reviewed with caution, as the authors studied two Passeriformes species and a Columbiformes species that were larger than the Titmice of this study [72Harms CA, Jinks MR, Harms RV. Blood gas, lactate, and hematology effects of venipuncture timing and location after mist-net capture of Mourning Doves (Zenaida macroura), Boat-tailed Grackles (Quiscalus major), and House Sparrows (Passer domesticus). J Wildl Dis 2016; 52(2)(Suppl.): S54-64.
[http://dx.doi.org/10.7589/52.2S.S54] [PMID: 26845300] ].