An investigation of syrinx morphometry and sound frequency association during the chirping period in lovebirds ( Agapornis fischeri)

Background: In the issue of biodiversity, the domestication of birds as pets and trade animals requires special attention as a conservation effort. Lovebirds ( Agapornis fischeri) are popular birds worldwide, due to their varied ornamentation and melodic chirping sound. Syrinx structure is suspected to be the main source of sound production during the chirping period. This study aimed to investigate syrinx morphometry and its correlation with sound frequency produced in lovebirds. Methods: A total of 24 lovebirds of different ages and gender were investigated. Polymerase chain reaction method was performed to determine lovebird gender, meanwhile bird age was identified based on post-hatch recordings at the breeding farm. Thus, we enrolled male (n=12) and female (n=12) lovebirds aged 2 (n=4), 3 (n=4), and 4 (n=4) months in the investigation group, respectively. Fast Fourier Transform (FFT) was performed to evaluate sound frequency during chirping period. Then, syrinx morphometry was identified using a topographic approach and methylene blue staining. Each variable was evaluated with Image J software and vernier caliper. Results: Based on a topographical approach, we reported the general cartilage structure of the tracheosyringeal, bronchosyringeal, paired protrusions, tracheolateral muscles, sternotracheal muscles, and syringeal muscles in lovebird syrinx. In particular, the tympaniform membranes lateral lead a crucial role in modulating the frequency of male lovebirds more significantly (p=0,009) compared to female. On the other hand, the tympaniform membranes lateral dexter (p=0,02) and sinister (p=0,05) in females showed wider compared to male. We also reported a negative correlation between sound frequency compared to tympaniform membranes lateral dexter (y = -913,56x + 6770,8) and sinister (y = -706,16x + 5736). Conclusions: It can be concluded that the tympaniform membranes lateral produced the lovebirds’ primary sound. The sound frequency of male lovebirds was higher compared to female, however negatively correlated with the area of tympaniform membranes lateral.


Introduction
In the last decade, various birds have been domesticated as pets.In Indonesia, lovebirds (Agapornis fischeri) are popularly reared in captivity and are traded pets.Lovebird is an attractive ornamental bird because it has a characteristic feather color pattern and distinctive behavior.In addition, the sound of birds chirping and variations in sound can attract the attention of breeders to rear lovebirds. 1Lovebird is a small parrot that often participates in chirping contests, thereby increasing its economic value as a traded pet.In Indonesia, the lovebird contests are organized by enthusiastic bird communities to evaluate the criteria for plumage color and chirp production.However, the risk of bird population sustainability must be a concern 2 In some groups of avians, the syrinx is a crucial organ for producing sound and melody during chirping. 3The chirping variation is a manifestation of the articulation formed by the syrinx cartilage.In previous studies, syrinx organs were morphometrically revealed in pigeons, 4 parakeets and canaries, 5 chickens and rock dove, 6 sparrows 7 and penguins. 8The syrinx will stimulate the vibrations reflected from each cartilage to produce sound frequency. 9Lovebirds voice articulation develops in five phases from 0 to 91 days old, thereafter lovebirds can produce clear sound with different configurations.The duration of the sound and chirping frequency produced by the syrinx is significantly increased due to the reflection of the tympaniform membrane lateral. 10pair of tympaniform membrane lateral are found in the lateral wall of the syrinx at the tracheobronchial junction.This structure extends from the tympanum to the first bronchial ring in chickens and pigeons or is composed of short membranes between the bronchosyringeal rings. 11The tympaniform membrane medial connects the pessulus to the medial portion of the primary bronchus.This structure connects the ring portion of the bronchosyringeal cartilage dorsoventrally. 12The labia are a pair of elastic cords found in the lateral and medial tympaniform membranes that physiologically produce the primary sound. 13e difference between male and female lovebirds requires further information, in particular, to determine the eligible age for the Lovebirds contest.Age may influence syrinx morphometry and normal function of the tympaniform membrane lateral to produce sound.In addition, it is very important to investigate the association of syrinx morphometry and sound frequency.This study was expected to provide an evidence of syrinx topography and morphometry in lovebirds and their association on the sound frequency produced with differences in gender and age.

Ethics statement
This study was approved by the Ethical committee of animal care and use, Universitas Airlangga (No.445/HRECC.FODM/X/2020).This study was also reported according to the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines 2.0: author checklist (see Reporting guidelines 45 ).All efforts were made to ameliorate any suffering of animals.
Based on legislation from the Ministry of Forestry and Biodiversity, Republic of Indonesia with reference number (No.20/2018) and Government Regulation with reference number (No.8/1999), Fischer's Lovebird is not included in the red list in Indonesia.In addition, the owners of the rearing farm held special conservation permission and is reported periodically with an increase in the Fischer's Lovebird population at the rearing farm.

REVISED Amendments from Version 1
In the abstract section, we have added study locations, brief descriptions of statistical methods, interpretation of results, and modification of conclusions.In the introduction, we explained the urgency of the lovebird contest and the information gaps during the study.In the methods section, we have clarified the PCR method, reference to Federer's formula, the sequence of randomization and blinding of samples, the reaction of LD50 during the euthanasia stage, and statistical methods to evaluate the data.We also briefly describe the discussion of context-related syrinx adaptation prior to the physiology of vocal production in birds.We have modified the conclusions according to the biological implications and study limitations.We have corrected the reference section, minor typos, and grammar errors and we would like to thank the reviewers for their valuable comments which improved the quality of the manuscript.
Any further responses from the reviewers can be found at the end of the article

Experimental design
The sample size was determined using Federer's formula: t(n-1) ≥ 15 with a combination of age and gender for six groups.This formula design was considered due to two aspects of the same thing i.e. randomization and replication are necessary to obtain a valid estimate of the error variance of a contrast. 14There were four replications in each group, meaning a total of 24 lovebirds of different ages and gender (12 males and 12 females) were investigated.Inclusion criteria were that the birds were healthy (no animals were excluded).These lovebirds were reared in a fenced enclosure, fed millet seeds, and fresh water ad libitum.Our study was conducted on 2, 3, and 4-month-old (mo) birds based on posthatch recordings at the breeding farm.

Sex determination in lovebirds
Meanwhile, sexing was performed on feather samples using the polymerase chain reaction (PCR) method.Deoxyribonucleic acid (DNA) isolation was performed using the Gsync DNA Extraction Kit protocol (Geneaid Biotech Ltd, Taiwan).DNA extraction was carried out overnight to produce clear DNA bands.DNA fragment amplification was carried out targeting the CHD gene using primers and reference primary markers (Table 1).The reaction mixture of bird DNA was mixed as much as 25 μl consisting of 12,5 μl MyTaq™ DNA Polymerase (Labgene Scientific SA, Switzerland), 1 μl forward primer, 1 μl reverse primer, and 9,5 μl isolated DNA.Thereafter, the mixture was incubated in a PCR machine followed by pre-denaturation at 94°C for 2 mins, denaturation at 94°C for 20 sec, annealing at 46°C for 30 sec, extension at 72°C for 40 sec, and final extension at 72°C for 10 mins.All stages were repeated for 40 cycles.The results of the electrophoresis were then determined by comparing all samples with a 100 bp HyperLadder™ marker (Bioline, UK).The results of the electrophoresis are available in Underlying data. 48Visualization was revealed PCR products of 400 bp and 350 bp for females, meanwhile only 400 bp for males.Of the 33 collected samples, only male (n=12) and female (n=12) lovebirds aged 2 (n=4), 3 (n=4), and 4 (n=4) mo were enrolled in the investigation group, respectively. 48Randomization and blinding of the collected samples were performed in replicated gender and age groups using the following steps: (1) Lovebird populations aged 2, 3, and 4 months in the respective fenced enclosures were assigned as the investigated group, (2) lovebirds were caught one by one randomly and then their gender was determined using the PCR method, (3) only 4 male and 4 female lovebirds in the respective age were enrolled and labeled as samples, (4) this method was repeated for the 3 and 4 month age groups.

Fast Fourier Transform (FFT) method
We evaluated each bird separately.The Fast Fourier Transform (FFT) 17 method was performed to record the lovebirds' sound frequency in an insulation room to avoid noise.The analog recording process was performed in an interval of 30 mins to record the number of bird sounds.Analog data on the recording device was transferred to a computer using a soundcard and then converted into WAV (Microsoft Waveform Audio Files) format files using Microsoft Sound Recording software (Windows Voice Recorder for Windows 10, Microsoft Corporation) (see Underlying data 49 ).The visualization of voice phrases described the characteristics of the sound in a time interval in the form of a spectrogram (see Underlying data 50 ).In addition, the visualization of sound phrases was observed as a continuous wave to reduce errors due to noise and phase differences in the FFT method.
Methylene blue staining 18 Euthanasia was performed by injection of a lethal dose (LD50) of 99.5 mg/kg BW of ketamine intramuscularly then observed for 8-15 mins. 19The dissection procedure was performed on the dorsal recumbency with a focus on the thoracic cavity and then the topography of the dorsal, ventral, and lateral site of the syrinx was observed.The lower respiratory organs were dissected and then stored for 2 Â 24 h using 10% formalin solution, then as a stopping point, moved the organs into 70% alcohol solution.Syrinx organs were immersed in 1% methylene blue for 15 mins and then rinsed using 50% and 70% alcohol.

Code
Nucleotide Primer NP-F 15 5'-GAGAAACTGTGCAAAACAG-3' Forward P2-R 15 5'-TCTGCATCGCTAAATCCTTT-3' Reverse MP-R 16 5'-AGTCACTATCAGATCCGGAA-3' Reverse tracheolateral muscle (TLm), sternotracheal muscle (STm), profundal syringeal muscle (SPm), and superficial syringeal muscle (SSm).Meanwhile, syrinx morphometric were evaluated for the following variables: tympaniform membrane lateral dexter (TMLd), tympaniform membrane lateral sinister (TMLs), upper trachea (upT), middle trachea (mT), lower trachea (loT), paired protrusions dorsal (PPd), and paired protrusions ventral (PPv).Each variable was observed using a stereo microscope and then evaluated using Image J version 1.8.0 software. 20e area of tympaniform membrane lateral dexter and sinister were determined according to the curved line around the lateral side of the tympaniform membrane lateral.The cranial and caudal margins of the tympaniform membrane lateral are the paired protrusions and bronchosyringeal cartilages (Figure 1A). 4 The length of the paired protrusions dorsal and paired protrusions ventral were determined according to the cranial to caudal line.This line is emphasized from the end profundal syringeal muscle to the bronchosyringeal-1 cartilage (Figure 1B). 5 For trachea diameter, evaluation was performed on the upper trachea, middle trachea, and lower trachea sides using a vernier caliper (Figure 1C).

Statistical analysis
The normality of the data was analyzed using the Shapiro-Wilk test with a normality value of p > 0,05.All the data were expressed as mean AE standard error (SE) then analyzed using multivariate analysis of variance (MANOVA) followed by Duncan's comparison test.Values were considered significantly different at p < 0,05.The sound frequency of male and female birds were analyzed using independent T-test with a significance level (p < 0,05).Meanwhile, to investigate the association between the area of tympaniform membrane lateral and the lovebirds sound frequency produced, an association test was performed.Statistical analysis was performed using SPSS v.25 software (IBM, USA).

Topographic anatomy and morphometry of the syrinx
The raw, uncropped images associated with syrinx morphometry and topography are available in Underlying data. 46The topographical observations of lovebird syrinx showed that there was no anatomical topographic variation across the various age and gender groups.Syrinx location in the thoracic cavity tends to the left side compared to the esophagus, on the dorsocranial of the heart and caudal of the crop.Tracheal organs were found in the ventral esophagus.The trachea in the cervical region was found on the left side of the esophagus, before the thoracic cavity of the trachea is on the dorsal side of the esophagus.The crop continues to the caudal side of the transformed esophagus parallel to the syrinx.The esophagus continues to the caudal side by crossing to the dorsal bifurcation of the trachea then being on the ventral of the lung and transformed into the proventriculus (Figure 2).
Using methylene blue staining, clearly visible tympaniform membrane lateral was observed from both primary bronchi.The cranial margin of the tympaniform membrane lateral is paired protrusions covered by profundal syringeal muscle and the caudal margin of the tympaniform membrane lateral is the bronchosyringeal-1 cartilage.An evidence revealed that pessulus and labia were not found in the lovebird syrinx.It was found tympanum modification in the form of caudal protrusion called processus tympanicus on the dorsal and ventral sides.Processus tympanicus structure was found between a pair of paired protrusions which are tympanic plates and are connected to the cranial side of the tympaniform membrane lateral.Paired protrusions and tympanum structures are part of the tracheosyringeal cartilage.Paired protrusions is tracheosyringeal-1 cartilage, meanwhile tympanum is composed of three cartilages i.e. tracheosyringeal 2-4 (Figure 3).Bronchosyringeal cartilage was found on the cranial side of the trachea bifurcation and the caudal side of the tympaniform membrane lateral.All samples from each gender and age group had three pairs of bronchosyringeal cartilage on the right and left sides (Figure 4).
Observation of the muscles revealed that in all gender and age groups the lovebird syrinx was identified as tracheolateral muscle, sternotracheal muscle, profundal syringeal muscle and superficial syringeal muscle.Tracheolateral muscle was found on the lateral side of the trachea.Meanwhile, sternotracheal muscle was found on the lateral side of the lower trachea and then split into two in the dexter and sinister syrinx (Figure 5).The muscular structure gets thinner on the mediastinal side and eventually forms the sternotracheal muscle tendon, which then attaches to the lungs and extrapulmonary bronchi.Sternotracheal muscle on both sides looks asymmetrical, however the sinister is more inclined to the dorsal compared to the dexter is inclined ventrally.Profundal syringeal muscle was found from tympanum to paired protrusions with the caudal end of the muscle connected to the cranial portion of the tympaniform membrane lateral.Superficial syringeal muscle was identified from the lateral side of tympanum to the primary bronchus.The difference was found in the location of the connection of the caudal end of the profundal syringeal muscle and the bronchial cartilage.In groups aged 2 and 3 mo, both male and female, it was reported that there was a connection of the caudal end of the profundal syringeal muscle with the bronchus-2 cartilage, meanwhile, in the aged 4 mo group, both male and female, it was found in the bronchus-3 cartilage (Figure 6).
In this syrinx morphometric study, it was reported that the area of the tympaniform membrane lateral dexter and sinister increased with the increasing age of lovebirds.Tympaniform membrane lateral dexter and sinister in female lovebirds were observed more widely compared to male lovebirds.In the paired protrusions variables, upper, middle, and lower tracheal diameters, there was no significant difference reported in gender and age groups (Table 2).The full data associated with the Syrinx morphometry and sound frequency evaluation is available in Underlying data. 45

Sound frequency evaluation
In this present study, the evaluation of sound frequency was performed only on lovebirds aged 4 mo because at that age they are considered a mature lovebird during contest.The sound frequency produced by male lovebirds (3500,00 AE 204,12) was significantly higher compared to female lovebirds (2687,50 AE 62,50) (Table 3).This study also revealed that there was a negative association between sound frequencies compared with the area of tympaniform membranes lateral dexter (y = -913,56x + 6770,8) and sinister (y = -706,16x + 5736) (Figure 7).This finding indicated that the smaller area of lateral tympaniform membranes followed by an increase in the sounds frequency produced by the syrinx cartilage articulation.

Discussion
Modification of the syrinx in male birds is related to dimorphism of muscle mass and nerve fiber components in the laryngeal canal.Sexually dimorphic vocal behavior arises from each vibration reflected by the pesulus and often represents a more significant evolution in bird vocal complexity.This adaptation illustration allows the potential to generate an enhanced frequency range. 21The syrinx is a vocal organ in the avian family with the physiological principle of flowing air from the lungs across the narrow bronchotracheal tract and vibrating the tympaniform membranes lateral and medial. 22In respective bird species there is a variety of sounds due to differences in the syrinx anatomical structure. 23n this present study, we observed a modified syrinx cartilage in the form of processus tympanicus and paired protrusions which are only owned by the Psittacidae bird family. 24Paired protrusions are classified into tracheosyringeal cartilages with a composition similar to the tympanum compared to the structure of the bronchosyringeal cartilages. 25 a previous study involving male Budgerigars (Melopsittacus undulatus), the tympanum was composed of six elements, 26 meanwhile in this study the tympanum was only composed of three elements.Lovebird syrinx was found on the caudal side of the lower trachea and not yet connected to the trachea bifurcation.The syrinx type in the Psittacidae family has a single set of membranes so that it produces only a single sound, unlike other bird species with the tracheobronchial syrinx type and can produce multiple sounds independently. 27The sternotracheal muscle was identified from the lower trachea and then transformed into a tendon that connects to the lung area near the end of the extrapulmonary bronchus called the septum obliquum. 28 another study, the sternotracheal muscle appeared asymmetrical because the syrinx crossed with the esophagus, thus the muscle junction was not parallel between the dexter and sinister sides. 29Another characteristic of the Psittacidae family, it has the Syringeal profundus and superficial muscle which is found in all gender and age groups with insertions starting from the cranial tympanum.Lovebirds only have two intrinsic muscles, i.e. the syringeal profundus and superficial muscles compared to other species of chirping birds due to the muscle structure is more complex, i.e. the tracheobronchial dorsal, ventral and brevis muscles and the syringeal dorsal and ventral muscles.In this syrinx morphometric study, there were significant differences in the area of tympaniform membranes lateral dexter and sinister in lovebirds aged 2, 3, and 4 mo.The area of the tympaniform membranes lateral increases with age in both male and female lovebirds.Tympaniform membranes lateral are functional membranes that produce melodic sounds in the Psittacidae family. 31The development of the tympaniform membranes lateral is triggered by the development of the chirping sound.The rapid development that occurs in pubertal birds will then decrease gradually at aged 9 mo. 32 another study, tympaniform membranes lateral were reported to be larger in male pigeons than in female pigeons. 33his is supported by studies of the duration and number of chirping periods being greater in females than males.Studies on artificial vocal cords reported that the larger area of the vibrating membrane produces lower sound frequencies. 34The area of the tympaniform membranes can be one of the factors that trigger the difference in the sound of chirping in male and female lovebirds.In addition, the body weight of female lovebirds is significantly heavier than males.Generally, bird species with larger bodies have lower sound frequencies. 35Hormonal factors also affect the character of the sound produced. 36Repeated administration of testosterone in perdix birds resulted in a longer duration of chirping with a lower frequency. 37The same study stated that birds treated with testosterone showed a significant widening of the lumen of the trachea and bronchi and a thicker syrinx membrane. 38mpaniform membranes lateral in chickens are composed of connective tissue and an extracellular matrix consisting of carbohydrates, hyaluronic acid and collagen. 39The vocal folds are sensitive to the hormone estrogen.The decrease in estrogen has an impact on changes in the expression of molecules in the extracellular matrix, whereas testosterone does not affect the extracellular matrix. 40Studies on sparrows after hatching at 3, 10 and 17 days old showed that androgen receptors can be found in the muscles around the syrinx and estrogen receptors are found in many syrinx chondrocytes. 41strogens and androgens can affect the development of the syrinx of birds after hatching in the skeletal and muscular parts, but their effect on the differentiation process is unidentified.Hormones have an influence on the nervous system which controls sound production.Testosterone has two effects, i.e. anatomically controlling the growth of vocal control stations and physiologically controlling the number of enzymes required during neural transmission. 42cal control stations on the central nervous system have androgen and estrogen receptors that affect sound sensitivity. 43tudies on adult canaries require estrogen to produce a chirp with a high rate of repeating syllables. 44The duration of the  chirp, the length of the chirping element and the frequency range can be influenced by the hormone testosterone but only in certain species so that there are differences in the expression of hormone receptors in the central nervous system which differ in each species and even gender.Various factors that influence the anatomical and morphometric differences of the lovebird syrinx and its relation to the characteristics of the chirping sound produced need to be studied further.

Conclusions
In conclusion, we identified tympanum, processus tympanicus, tracheosyringeal cartilage, bronchosyringeal, bronchi, trachea bifurcation, paired protrusions, tracheolateral muscle, sternotracheal muscle, profundal and superficial syringeal muscle in lovebird syrinx.Our study emphasized that tympaniform membranes lateral initiates the lovebirds' primary sound, specifically in male birds.This finding also highlighted that the narrower tympaniform membranes lateral reflects the enhanced frequency of chirping in a single vocal period.
• FL1.wav (Sound recording for female lovebird sample No.1).I have read the paper and in my opinion this paper is well written and has scientific merits.In Indonesia lovebirds are popular ornamental birds kept for their beautiful colours and melodious chirps.In fact these birds are used in competition for their chirping ability.This study investigated the chirping frequency in relation to the morphology of the syrinx, which is where the voice originate.The description of methodology was clear and appropriate for this kind of study.
The results of this study were well presented and showed significant relationships between sound frequency and syrinx.
Overall, an interesting paper.

Conclusions:
Try and beef up this section especially as it relates to biological implication and future study.

References
Please revisit for accuracy and correct formatting.
○ NB: There is a need for language editing of the entire text.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Animal Breeding and Genetics
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Figure 2 .
Figure 2. Location of the syrinx to (A) the heart and (B) the esophagus in the thoracic cavity.
Values are expressed in mean AE SE (n = 4 samples for each different gender and age groups).Multivariate analysis of variance was carried out followed by Duncan's comparison test

Table 3 .
Sound frequency of male and female lovebirds aged 4 months old.

the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests:
No competing interests were disclosed.Experimental design: Try and separate experimental design from PCR part of the study.Provide a reference for the Federer's formula: t(n-1) ≥ 15, and why is it adopted in this study considering the sample size of only 24 lovebirds?Methylene blue staining: How soon does lethal dose (LD50) have effect on the bird?Did you test for the homogeneity of variance and normality of your data?You are mixing up Correlation and Regression (Association).Although the two has to do with 'Relationship', they provide different values.Sound frequency evaluation: You should have tested sound frequency for other ages as it would have provided additional information for comparison.Briefly discuss syrinx morphometrics in the context of adaptation before relating with sound frequency.
○ Methods: ○ ○ What gel and dye were used for the PCR? ○ 'Of the 33 samples'.There is a need to elaborate on this.○ Briefly describe the term 'blinding'.○ ○ Statistical analysis: ○ ○ Results: ○ Discussion: