DOI: 10.5061/dryad.9ghx3ffs3
title: Steroid hormone profiles in cowbirds and redwings

description: Avian brood parasites display enhanced annual fecundity compared to other
 passerine birds. Adult female Brown-headed Cowbirds (Molothrus ater), a
 brood parasite in the Icterid family, are estimated to lay 40- 50
 eggs/year. Some studies suggest annual fecundity in various parasitic
 species can exceed even this upper estimate. However, boosting
 reproductive output to this extent may come at the cost of reduced or lost
 parental care. Steroids, such as testosterone, may act as a pivot point
 that balances the fitness consequences between reproduction and parental
 behavior. Moreover, steroids are targets of selection that shape life
 history strategies and can therefore potentially contribute to the
 evolution of novel behavioral phenotypes. Here, we examine how
 reproductive steroids may mediate a possible tradeoff between increased
 annual fecundity and parental care in female cowbirds. We compare seasonal
 fluctuations in steroid profiles and follicular development in cowbirds
 and Red-winged Blackbirds (Agelaius phoeniceus), a related non-parasitic
 Icterid species. We aim to identify differences in seasonal steroid
 patterns that correspond to the striking divergence in life history
 strategies between these species.  We also use GnRH
 administration in these two species to determine whether species variation
 in the response to GnRH maps onto the differences in behavioral
 phenotypes. We identified several mechanisms that would enhance annual
 fecundity in the cowbird and one mechanism that would do this at the cost
 of parental care: elevated testosterone. We show that female cowbirds are
 reproductively ready before female Red-winged Blackbirds, a preferred and
 abundant host of the cowbird at our study site. We find no significant
 association between estrogen and follicular size in cowbirds, whereas this
 association is apparent in Red-winged Blackbirds. However, follicular
 development is associated with testosterone, and female cowbirds exhibit a
 pattern of elevated testosterone throughout the breeding season compared
 to the Red-winged Blackbird. These steroid profiles indicate divergence in
 the physiological mechanisms that regulate oocyte development in these
 related species. Moreover, cowbirds produce testosterone significantly
 quicker and more robustly in response to GnRH administration compared to
 female Red-winged Blackbirds. Divergence in the steroid seasonal profile
 and responsivity to GnRH in cowbirds, particularly with respect to
 testosterone, indicates the HPG axis exhibits consequential modifications
 in cowbirds that can enhance reproduction while simultaneously inhibiting
 caregiving behaviors.
description: Seasonal profiles of reproductive steroids
 Female Brown-headed Cowbirds (n = 18) and Red-winged Blackbirds
 (n = 14) were captured using walk-in traps and mist nets in Ransom and
 Cass counties of North Dakota during the non-breeding and the peak
 breeding season in 2021 (May-June). Traps and mist-nets were continuously
 monitored to ensure the birds were removed within five minutes of capture.
 Birds were immediately euthanized via rapid decapitation for a separate
 study. This allowed us to measure the size of the largest follicle using
 calipers and collect blood to be stored on ice until centrifugation.
 Collections started May 1 (pre-breeding in North Dakota) and concluded
 June 25 (peak breeding). We categorized female cowbirds and Red-winged
 Blackbirds into three categories: pre-breeding, breeding onset, or peak
 breeding. These categories were defined by observing the breeding activity
 (or lack thereof) of female Red-winged Blackbirds, the most abundant
 preferred host available to cowbirds at this location.  The pre-breeding
 condition was defined as no nest building or egg laying activity present,
 but males are present, singing, and establishing territories (May 1-14).
 We captured no female Red-winged Blackbird in this stage because they are
 either less abundant or significantly more sedentary than males at this
 time (Wright and Wright, 1944; Holm et al., 1973). The breeding onset
 condition was marked by the first nest-building activity of a female
 Red-winged Blackbird. In this category, female Red-winged Blackbirds began
 building nests, but no eggs were observed in nests yet (May 15-22). The
 final category is the peak breeding season, which was marked by eggs
 observed in nests and provisioning behaviors present in female Red-winged
 Blackbirds (May 23 to June 25). These are subjective categories based on
 the observations of our field crew; however, these categories do coincide
 with the timing that female Red-winged Blackbirds transition from breeding
 to parental behaviors as described by other studies conducted at similar
 latitudes in North America (Wright and Wright, 1944; Holm et al., 1973).
 The sample sizes of each species in these categories include cowbirds:
 pre-breeding (n = 7); onset of breeding (n = 6), and peak breeding (n =
 5); red-wings: onset of breeding (n = 6) and breeding (n = 8).  We
 measured 17-β estradiol (E2), testosterone (T), and follicular sizes
 across these breeding categories in both species. GnRH
 administration Breeding female Brown-headed Cowbirds (n
 = 10) and Red-winged Blackbirds (n = 8) were captured using walk-in and
 drop-down traps during the breeding season of 2022 at the Marine Nature
 Study Area and Lido Beach Nature Area of NY (red-wings) and the Balcones
 wildlife refuge in Marble Falls, Texas (cowbirds). Blood samples were
 obtained from each female via the branchial vein within five minutes of
 capture. This initial blood sample was used to obtain baseline hormone
 measurements. Immediately following blood collection, females were
 injected with 1.5 µg of gonadotropin-releasing hormone (GnRH; Bachem
 catalog # 4030773) intramuscularly in a 50 μl volume dissolved in
 phosphate-buffered saline (Jawor et al., 2006; Burns et al., 2014; Rosvall
 et al., 2013; 2014). Treated birds were placed in a cage approximately
 18"L x 14"W x 24"H so that additional blood samples could
 be collected at 30 min (timepoint 2), 1 hour (timepoint 3), and 2 hours
 (timepoint 4) after GnRH treatment. Red-wing females were released with a
 color band in case of repeated capture, whereas female cowbirds were
 euthanized. Blood samples were frozen until centrifugation. Plasma was
 used to measure testosterone so that we could identify the immediate
 steroid response from the gonads, which initially produces testosterone
 before it is aromatized into estrogen. We then measured E2 with subjects
 that had remaining plasma: Brown-headed Cowbirds (n = 7); Red-winged
 Blackbirds (n =7). All procedures described here were
 conducted in accordance with Hofstra IACUC procedures as well as federal
 (MB96705A-0), NY state (1181), Texas state (SPR-0521-069), and North
 Dakota state (GNF05428921) scientific collecting permits.
  Steroid hormone assays Circulating T and E2
 concentrations were quantified as described in Lynch et al., 2018;
 Pellicano, 2019; Lynch and Wilczynski, 2005; 2006; 2008; Lynch et al.,
 2006. Briefly, steroids were extracted from plasma using 3 ml of diethyl
 ether. Extracted steroids were resuspended in assay buffer for T and E2
 assays using ELISA kits from Arbor Assays (17-β estradiol: cat # KB30-H1;
 Ann Arbor, Michigan) and Cayman Chemical (testosterone; cat # 582701; Ann
 Arbor, Michigan). These kits have been validated for use in birds in our
 previous studies (Lynch et al., 2018; Pellicano, 2019). Both steroids were
 measured using a single standard curve, thereby precluding an inter-assay
 variation measurement. Estradiol EIA kits have 100% cross reactivity with
 17β-Estradiol, and all other reported cross reactivities were less than
 1%. Testosterone kits have 27.4% cross reactivity with
 5a-dihydrotestosterone, 18.9% cross reactivity with
 5b-dihydrotestosterone, 4.7% cross reactivity with methyl testosterone,
 3.7% cross reactivity with androstenedione, and 2.2% cross reactivity with
 11-keto testosterone. All other cross-reactivities were less than 1%. The
 detection limit is 3.9 pg/ml for testosterone and 3.75 pg/ml for
 estradiol. We used a two-way ANOVA for independent
 samples to determine whether there was an interaction between the two
 species across breeding stages. This analysis was used for steroid hormone
 data as well as follicular size. However, because we collected cowbirds in
 three breeding stages, whereas Red-winged Blackbirds could only be
 collected in two of these stages, we did an additional analysis to
 determine if pre-breeding hormone concentration or follicular size in
 cowbirds was different from the early breeding state in cowbirds and
 Red-winged Blackbirds. We used a t-test for independent samples and
 selected tests for unequal variances where data exhibited
 heteroscedasticity.  Steroid hormone concentrations were logarithmically
 transformed to achieve a normal distribution. Alpha values were set at
 0.05. We also used regression to examine the
 relationship between follicular size and steroid concentration in both
 species. These correlational analyses will allow us to identify species
 differences in the relationship between follicular growth and
 steroid<sub> </sub>concentration. These variables should be
 correlated if a typical associative breeding pattern is present.
 Finally, we examined species comparisons in T and
 E<sub>2</sub> across four timepoints in the GnRH study using a
 2x4 two-way ANOVA with one factor as a repeated measure. Hormone
 concentrations were logarithmically transformed to achieve a normal
 distribution. Raw data is represented in all graphs for
 simplicity.
description: # Steroid hormone profiles in cowbirds and redwings
 [https://doi.org/10.5061/dryad.9ghx3ffs3](https://doi.org/10.5061/dryad.9ghx3ffs3) ## Description of the data and file structure ### Files and variables #### File: archived\_data\_sheet\_organized.xls #### **Description:**  BHCO = Brown-headed Cowbird RWBL = Red-winged Blackbird T = testosterone (pg/ml = picogram per milliliter) E2 = 17 beta estradiol (pg/ml = picogram per milliliter) T-p1-4 and E2-p1-4 = time point 1-4 for blood collection (p1=baseline or prior to GnRH injection and t2-4 = 30, 60 and 120 minutes after GnRH injection) lrgst follicle = largest ovarian follicle as measured in millimeters (mm) GnRH = gonadotropin releasing hormone (pg/ml = picogram per milliliter) species (GnRH trt) = species with the GnRH treatment ## Code/software NA ## Access information NA