Evaluation of Steroid Sex Hormone Levels in Cockatiels During Their Reproductive Cycle

Sexual steroid hormone concentrations differ with respect to the different stages of animal life. For example, hormone concentrations in the reproductive stage show a periodic variation. Studies on variations in sexual hormones in parrots using non-invasive methods as well as the effect of nest-boxes in parrots are less. This study aimed to detect the levels of fecal steroid hormones during the cockatiel reproductive cycle and to investigate the effect of nest-box placement on reproduction. There were 24 mating pairs of cockatiels, and the study was conducted over 13 months. Based on their reproductive status, individual birds were divided into pre-reproduction (PR), incubation (IN), and parent period (PA) in the pre-test; then, the nest box was either temporarily removed for 20 days (RN) or continuously provided (pre-reproduction, PR). The estrogen concentrations measured in the last stage of the PR period and the initial stage of the IN period were significantly higher than those measured in the PA stage, and there were no significant differences in testosterone and progesterone concentrations throughout the reproductive cycle. The nest-box treatments (NB) indicated that eggs were laid 6-13 days after the new box was hung; however, in the pre-test, there was an interval of 30-40 days before the next mating pairs started the next reproductive stage or no reproduction. Estrogen concentrations in PR and testosterone concentrations in RN were significantly higher than those in the other periods during the nest-box treatments. In conclusion, estrogen showed the most significant change among the fecal steroid hormones measured. Nest boxes serve as a positive stimulus for parrot reproduction management and can help non-seasonal reproductive parrots to rapidly enter the next breeding cycle.


Introduction
Cockatiels (Nymphicus hollandicus) are granivorous, sexually dimorphic parrots, native to the arid grassland and savannah regions of central Australia (Shields, Yamamoto, & Millam, 1989;Martin & Millam, 1995).They are monogamous birds, in which both parents contribute to incubation and the feeding of nestlings, of which there are often three to six per clutch (Forshaw & Cooper, 1981).In southern Australia, the wild cockatiel populations breed seasonally, but are more or less dependent on rainfall further north.In Taiwan, which lies in a temperate zone area, cockatiels are non-seasonal breeders, breeding both in and out of season.
Monitoring the concentrations of hormones can provide insight into the mechanistic aspects of bird mating behavior.Hormone levels are associated with sexual, reproductive, courtship, parental, aggressive, and feeding behaviors, and many studies have revealed seasonal variation in the steroid sex hormones of avian species (Silver, Reboulleau, Lehrman, & Feder, 1974;Schumacher & Balthazart, 1983;Williams, 1992;Kotrschal, Dittami, Hirschenhauser, Moestl, & Peczely, 2000).Avian steroid sex hormones can be analyzed using a number of different source materials, including plasma, feces, urine, and feathers.Collecting and analyzing animal feces has become an effective non-invasive approach for the study of animal endocrine status and stress physiology (Whitten, Brockman, & Stavisky, 1998), and an increasing number of studies are using fecal steroid measurements to assess reproduction, stress, and aggression in populations of animals (Khan, Altmann, Isani, & Yu, 2002a).In the present study, we used feces to assay hormone levels in relation to cockatiel mating and reproductive behavior, as described previously (Whitten et al., 1998;Khan, Altmann, Isani, & Yu, 2002b;Goymann, 2005).
Research on the relationship between nest box use and avian endocrine responses has, to date, tended to be relatively limited.For breeders of artificial breeds, the re-set of a nest-box can be used to regulate the reproduction status of parrots that are unable to reproduce for a long time period, providing more economic benefits.Furthermore, parrots play an important role in agricultural ecology and contribute to ecological balance.Our research is beneficial for the reproduction of parrots.Most captive avian species can rapidly enter the next reproductive cycle and produce more offspring, leading to the perpetuation of some avian species.Success in artificial propagation in avians can reduce the pressure of hunting for the wild animal.In this study, by altering the availability of nest boxes, we were able to observe the ability of cockatiels to regulate physiological steroid hormone levels.We speculated that the replacement of nest boxes would stimulate cockatiels to rapidly enter the next reproductive cycle.The objectives of this study were to (1) observe the cockatiel reproductive cycle, (2) assay the levels of steroid sex hormones in fecal samples from mating pairs of cockatiels, and (3) study the effect of nest box replacement on cockatiel reproduction.

Animal Maintenance
Twenty-four pairs of cockatiels (Nymphicus hollandicus) were obtained from bird shops in Chiayi, Taiwan.All the birds used in this study were born between April and June 2011, and fecal samples were collected when the birds were 11 months old, an age at which these birds are sufficiently mature to breed.Pairs of cockatiels were housed in cages that were approximately 132,000 cm 3 (40 cm × 55 cm × 60 cm) in size, within light-controlled rooms with a photoperiod of 10 h light: 14 h dark, at the National Chiayi University Animal Research Farm.Each cage contained wooden perches.A commercial complex cereal grain diet (Extra, Taiwan) and water were continuously provided.The average weight of the cockatiels was 102.4 ±13.5 g.

Reproductive Cycles
Reproductive cycles were divided into three periods: pre-reproduction (PR), incubation (IN), and parental (PA).The PR period was defined as the mean time before cockatiels began to lay eggs, the IN period was when the cockatiels laid their first egg and incubated their eggs until nestlings hatched, and the PA period was when parents began to feed their nestlings.The duration of the three periods was 40, 19, and 20 days, respectively.The reproductive cycles could either be complete (including PR, IN, and PA periods) or incomplete (including only PR and IN periods).
Nest behavior is a necessary element of cockatiel reproduction.Birds that are captive-bred typically lay and incubate their eggs in nest boxes.We determined that the cockatiels that laid eggs and successfully hatched nestlings continued to do so when provided withe nest boxes.The cockatiel pairs (24 pairs) were divided into two groups according to their reproductive status.In one group, we re-set the nest box (NB group), whereby the box, which was external and could be easily dismantled, was removed and a new nest box was rehung after an interval of 20 days, and the dung tray of which was regularly cleaned before feeding.In the other group, the original nest box was maintained (CON group) and regularly cleaned.The NB group was further divided into the period of nest box removal (RN period, 20 days), pre-reproduction period (PR, hanging of the new nest box, 6-13 days), incubation period (IN, 19 days), and parental period (PA, 20 days).The samples of droppings produced during each period were pooled (RN1-5, PR1-7, IN1-5, and PA1-4) for steroid hormone analysis.Each period was subdivided into different stages to determine the fluctuation in steroid hormones.

Dropping Collection, Storage, and Hormone Extraction
For validation of assays, each morning, we collected all the fresh droppings produced by the captive cockatiels in each cage and placed these into clear tubes.Prior to feeding, we renewed the dung trays in each cage, and collected the droppings deposited within 1-1.5 hours after feeding to ensure freshness.Approximately one to two tubes of droppings were collected from the cages, each of the latter of which was considered as an experimental unit.The tubes were stored frozen at -80 °C until the samples were used for steroid hormone extraction.We used a fecal steroid hormone extraction method that has previously been described by (Staley, Blanco, Dufty, Wildt, & Monfort, 2007) with minor modifications.For extraction, feces (0.05-0.08 g) were suspended in 1.5-2 mL of 80% ethanol, vortexed, incubated in a water bath at 55 °C for 20 min, and then centrifuged at 500-2,000 × g for 15 min (the g-value being dependent on the amount of solid droppings).The supernatant was then decanted into clean tubes.This step was repeated three times using the original sample to maximize the amounts of fecal steroid sex hormones that could be extracted.The final extract was re-suspended in 1 mL methanol, vortexed and sonicated, and then diluted (1:50-1:500 for fecal estrogen metabolites (fEM); 1:40-1:250 for fecal progesterone metabolites (fPM); 1:8-1:64 for fecal androgen metabolites (fAM)) in assay buffer (for fA and fE metabolite extracts: steroid diluent, for fPM extracts: phosphate buffer).All the tubes were stored at -80 °C until used for assay of the steroid sex hormones in fecal samples.The hormone recovery procedure conducted in the study has previously been described by Wasser, Monfort, Southers, and Wildt (1994), and Khan et al. (2002).Estrogen, testosterone, and progesterone (Sigma-Aldrich Co., LLC., USA) were prepared by dissolving in 80% methanol to give 3000 pg/mL solutions.To two tubes, we added 1 mL of one of the aforementioned hormone solutions (estrogen, testosterone, and progesterone), giving six tubes in total, and a further two tubes for each hormone were left empty as controls.All the tubes were stored frozen at -80 °C.From randomly selected dropping samples, we took 10-mg amounts and transferred these to each of the 12 tubes, followed by the addition of 500 µL 80% ethanol.The droppings and ethanol in the tubes were mixed for 20 min (at 50 C).Thereafter, all the tubes were centrifuged at 500-2,000 × g for 15 min (the g-value being dependent on the amount of solid droppings).The supernatant was then decanted into clean tubes.This step was repeated three times on the original sample to maximize the amount of steroid sex hormone that could be extracted.

Fecal Steroid Sex Hormone ELISA Assays
The concentrations of steroid sex hormones (estrogen, testosterone, and progesterone) in fecal samples were determined from duplicate tubes with enzyme-linked immunoassay (ELISA), using a 17β-Estradiol high sensitivity ELISA kit, a Testosterone high sensitivity ELISA kit, and a Progesterone ELISA kit (Enzo Life Sciences Inc., Farmingdale, NY, USA), respectively, following the manufacturer's instructions.

Statistical Analysis
We independently analyzed the assay data for steroid sex hormones in the fecal samples collected from 24 pairs of cockatiels.The statistical analysis used in this study was based on generalized linear model (GLMs; SAS 9.1) that has previously been described by Blas, Lopez, Tanferna, Sergio, and Hiraldo (2010) with differences considered significant at P < 0.05.

Fecal Steroid Hormone Levels Change After Mating in Cockatiels
The reproductive cycle after cockatiel mating and the curve of steroid hormone levels in cockatiel feces are shown in Figure 1.At the end of the pre-reproduction period and the initial stage of the incubation period (PR4, PR5, and IN1), the estrogen concentrations were significantly higher than those in the other PR stages (P < 0.05).
The highest concentration of estrogen was 165% higher than those in the other stages.However, the testosterone concentration in the early stage of the reproduction period (PR1) was higher than that in the other stages, whereas the progesterone concentration in the early stage of incubation was higher than that in later stages, although the differences were not significant.
The higher level of estrogen at the end stage of the pre-reproduction (PR) period and the early stage of the incubation period (IN) indicates that there was a spike in estrogen production before ovulation in the reproduction cycle.This could be because estrogen can enhance ovum oogenesis (Ma & Yang, 1999;Peh, Wu, & jas.ccsenet.Paired cockatiels generally require a transition period of 30-40 days after completing one reproductive cycle before entering the next reproductive cycle.However, when nest boxes were removed for a time before being replaced, we found that some cockatiels required a transition period of only 6-13 days prior to entering the next cycle, although we did not observe this pattern in all mating pairs.Previous research suggests that reproductively experienced cockatiels typically begin laying eggs within 21 days after nest box presentation (Shields et al., 1989); however, the responses of our cockatiels may have been influenced by individual differences in reproductive and environmental conditions.

Table 1 .
The extract recovery rates of fecal steroid sex hormones in cockatiels