Conservation genetics of a resident population of Greylag Goose (Anser anser) at the southernmost limit of the species' distribution in Europe

1.   Introduction

Sperm competition is a phenomenon that occurs in the female oviduct where the sperm of multiple males compete to fertilize a limited number of eggs and is a representative example of postcopulatory sexual selection (Parker, 1970, 1984; Møller and Ninni, 1998). Sperm competition has been observed in various animal taxa, ranging from insects to vertebrates. In birds, the strength of sperm competition is associated with the type of mating system and the strength and duration of pair bond (Birkhead, 1987; Birkhead and Montgomerie, 2020). For example, sperm competition is likely to occur more intensely in polyandrous species, where a single female mates with multiple males than that in monogamous species (Birkhead, 1995). Even in monogamous species, however, competition can be strong when females have a chance to mate with two or more males, for example through extra-pair copulation (Parker, 1984; Briskie et al., 1997; Wink and Dyrcz, 1999). Sperm competition is known to affect sperm characteristics, such as longevity (Immler et al., 2007; Helfenstein et al., 2008), size (Briskie and Montgomerie, 1992; Briskie et al., 1997; Carballo et al., 2019), motility (Froman and Feltmann, 1998; Birkhead et al., 2005; Kleven et al., 2009) and morphology (Lüpold et al., 2009; Bennison et al., 2016; Mccarthy et al., 2021). As a result, sperm competition is a driver for the evolution of sperm diversity. Furthermore, strong sperm competition often results in the evolution of other adaptive behaviors, such as mate guarding by males and sexual conflicts in mating numbers (Briskie, 1992; Stockley, 1997; McKinney and Evarts, 1998).

The foremost step in the study of avian sperm biology and sperm competition is to confidently collect sperm from males. Although birds reproduce by internal fertilization, their genital anatomy and copulation process are strikingly different from those of mammals. Most male birds (approximately 97%) have a cloaca that is different from the external phallus, typical of most mammals, and couples mate by rubbing their cloacae (the so-called "cloacal kiss") instead of intromitting (Montgomerie, 2010). The size and shape of cloaca vary among birds (Quay, 1984; Oliveira et al., 2004; Brennan et al., 2007). In passerines, for example, some species such as the Vesper Sparrow (Pooecetes gramineus) and Clay-colored Sparrow (Spizella pallida) have a spherical cloacal protuberance form of cloaca while others such as the Horned Lark (Eremophila alpestris) show cylindrical form (Salt, 1954). Furthermore, the diameters and heights of the cloacal protuberance also differ among birds (Briskie, 1993). Together, these factors may make it difficult to collect avian sperm using a single universal technique. The most primitive way to collect sperm from birds is to kill the male bird and open its abdomen to squeeze out the semen. However, although collection via dissection would be a reliable method for collecting semen, there are important arguments regarding the ethics of this practice, as well as the impossibility of repeated collection. In the mid-1930, the induction of ejaculation by stimulating the cloacal protuberance or the abdomen around the cloaca (i.e., cloacal massage) began to be used (Ivanov, 1913; Craft et al., 1926; Burrows and Quinn, 1935). This method does not require any special tools, and has the advantages of repeatedly obtaining semen from the same individual (Lake, 1957; Gee et al., 2004; Shanmugam et al., 2012; Girndt et al., 2017). Therefore, the cloacal massage method has been widely used in various species, including passerines and non-passerines (Owen, 1941; Wolfson, 1952; Briskie and Montgomerie, 1992; Hemberger et al., 2001), although its effectiveness can vary among species with success rates ranging from 5 to 90% depending on the species (Sontakke et al., 2004; Della et al., 2011; Humann-Guilleminot et al., 2018). Alternatively, other attempts have been made to collect sperm, such as feces collection, wherein sperm may be obtained from feces during the breeding season (Immler and Birkhead, 2005; Calhim et al., 2007), and electro-stimulation, which induces ejaculation using weak electrical stimulation (Watanabe, 1957; Setioko and Hetzel, 1984; Lierz et al., 2013; Frediani et al., 2019).

In this study, we proposed a new method for collecting semen from brood parasitic Cuculus cuckoos. Brood parasitism is a breeding strategy in which biological parents shift their parental roles, such as egg incubation and provisioning, to other species (i.e., hosts) by secretly laying eggs in host nests (Payne, 1977). Breeding systems without parental care are common in invertebrates and lower vertebrates such as fish, amphibian, and reptiles. However, it is rare in higher vertebrates, including birds and mammals, wherein at least one parent provides care to raise offspring (Balshine, 2012). This unique breeding strategy may have a profound effect on sexual relationship and breeding behavior, such as the type of mating system, strength of pair bond, and spacing behavior during the breeding season. In the Common Cuckoo (Cuculus canorus), for example, various types of mating systems, from monogamy to promiscuity, and social systems, from territoriality to dominance hierarchy, have been reported (Nakamura and Miyazawa, 1997; Marchetti et al., 1998; Bolopo et al., 2017). In a radio-tracking study, Yun et al. (2019) showed that the Lesser Cuckoo (Cuculus poliocephalus) exhibits a scramble competition mating system without territoriality and proposed that the absence of parental care was the most likely reason for this unusual breeding system in higher vertebrates. Furthermore, Lee et al. (2019) suggested that prolonged mating periods due to lack of parental care may cause sexual conflicts over mating frequency in the common cuckoo, which may lead to sexual harassment by males and the counter-evolution of female-specific color polymorphism to escape it. Despite these unusual breeding systems and complex sexual relationships, few studies have documented postcopulatory sexual selection, such as sperm competition, in brood parasitic cuckoos.

This lack of research is mainly due to the difficulty in capturing enough cuckoos in the field, but most importantly by the technical difficulties associated with obtaining sperm from captured cuckoos. The cloacal shape of cuckoos is a typical non-passerine form, making it difficult to visually confirm whether they are sexually mature, which can be seen in the protruding cloaca of passerines (Salt, 1954; Birkhead et al., 1993; Tuttle et al., 1996). In addition, Bae et al. (2020) used the cloacal massage and feces collection methods to collect semen samples from wild cuckoos, but their success rates were very low (approximately 30% and 10%, respectively), indicating that traditional methods are likely to be ineffective in Cuculus cuckoos. Here, we introduced a new method, called "urodeum stimulation method (UroS method)" to effectively collect sperms from wild cuckoos. Specifically, we first explain how to apply the UroS method to wild cuckoos and then describe the properties of secretions obtained from 76 Common Cuckoos using the UroS method. Finally, we showed its success rate and discussed how to improve its efficiency. We believe that this method will facilitate sperm research in brood parasitic cuckoos, as well as in other non-passerine birds.

2.   Methods

2.1   Fieldwork

Fieldwork to capture cuckoos was conducted across South Korea during the breeding season (May–July) in 2022. We captured cuckoos using mist nets with dummy cuckoos and playback of female calls, and the sex of cuckoos was classified by call (e.g. "cu-coo" for male and "bubbling call" for female) and behavior (e.g. copulation attempt for male) during capture. Upon capture, we applied the UroS method to obtain semen samples, and a capillary tube (length: 75 ​mm, internal diameter: 1.2 ​mm, capacity: 70 ​μL) with the sample was placed next to a ruler and photographed using a smartphone (SM-G986N; Samsung, Suwon, Rep. Korea), for which we later calculated the sample volume using the cylindrical volume formula (V=πr²h). The sample was then placed and preserved in a 1.5 ​mL Eppendorf tube (EP tube) with 500 ​μL of 5% formaldehyde diluted in phosphate-buffered saline (Bae et al., 2020). Before adding the sample to the EP tube, we mixed the sample and a small aliquot (5–10 ​μL) of 5% formaldehyde on the lid of the EP tube using a pipette to appropriately separate the sperm.

2.2   Sample preparation and observation

Stored samples were checked for sperm presence using a light microscope and phase contrast microscope (DM1000; Leica, Wetzlar, Germany). Before microscopic observation, 20 ​μL of 0.4% trypan blue solution (Sigma-Aldrich; St. Louis, USA) and 20 ​μL of the sample were mixed in a new EP tube and left overnight (Louis and Siegel, 2011). Aliquots of the stained samples were observed at 200 ​× ​to 1000 ​× ​magnification, and images were captured using an attached digital camera (Gryphax Arktur; Jenoptik, Jena, Germany).

2.3   Statistical analysis

The difference in sample volumes according to the presence or absence of sperm was tested using the Wilcoxon rank sum test. The relationship between sampling time and sperm presence was tested using a generalized linear model with binomial error structure and logit link function, in which the response variable was the presence or absence of sperm in the sample, and the explanatory variable was the time at which the sample was collected. Both analyzes were performed using R version 4.2.0 (R Core Team, 2022).

3.   Results

3.1   UroS method

This method is easy to apply in the field and can be performed by one or two people. First, the bird's neck was held between the index and middle fingers with its back resting against the palm of the hand, while the rest of the fingers naturally wrap around the bird holding it tightly. Second, the cloacal area was placed upward to improve visibility, and a capillary tube was inserted approximately 5 ​mm into the cloaca. Third, the urodeum was stimulated by rotating the capillary tip for approximately 20–40 ​s, which usually rotates approximately 20–30 times in 10 ​s. This led the cloaca to repeatedly contract and relax and the fluid to exit (Fig. 1). When two people were involved in the collection, one person held the bird with its cloaca facing the other person who stimulated the cloaca and collected the fluid using a capillary tube (Appendix Video S1).

Figure  1.  Images showing the process of the urodeum stimulation method. (A) Positioning the abdomen upward for a better view of the cloacal area; (B) Inserting a capillary tube into the cloaca, which was then rotated; (C) Collection of semen.

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Supplementary video related to this article can be found at doi:10.1016/j.avrs.2023.100085

3.2   The efficiency of UroS method

The UroS method was applied to 82 male Common Cuckoos that were captured in the field, and fluid samples were collected from 76 cuckoos (acquisition rate ​= ​92.7%). The volume and color of the ejected fluid varied. The average volume was 17.4 ​± ​12.3 ​μL (n ​= ​74; two samples were excluded due to missing photos), ranging from 1.3 to 58.4 ​μL (Fig. 2), and colors ranged mostly from a bright white to a yellowish brown, but dark brown was also observed. In addition, small dark particles visible to the naked eye (possibly fecal debris) were occasionally observed in the fluid (Fig. 3).

Figure  2.  Distribution of sample volumes collected via the urodeum stimulation method (n ​= ​74). The dashed line is the mean value and the rug plots under the histogram represent the actual volume of samples.

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Figure  3.  Fluid samples in the capillary tubes. The characteristics of samples vary in volumes (A) and colors (B). Four capillaries (partial pictures) from four different male common cuckoos are presented together in each panel, in which fluid are between the vertical black bars. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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Among the 76 fluid samples, sperm was found in 43 samples (observation rate ​= ​56.5%; overall 52%, n ​= ​82; Fig. 4A), and most of the remaining 33 samples without sperm were found to contain a mixture of uric acid and fecal matter (Fig. 4C). However, sperm was observed along with fecal debris (i.e. the color of the fluid is a yellowish or dark brown) in 13 samples (30% of all samples, Fig. 4B), suggesting that the color and volume of the samples were not necessarily related to the presence or absence of sperm (sample volume ​− ​sperm presence: 15.3 ​± ​9.3 ​μL, n ​= ​41; sperm absence: 19.9 ​± ​14.8 ​μL, n ​= ​33; Wilcoxon rank sum test: W ​= ​576, p ​= ​0.28). In addition, the probability of sperm presence was not related to the time the sample was taken (GLM: estimate ​= ​0.05, 95% confidence interval: −0.04 to 0.13, p ​= ​0.3, Fig. 5).

Figure  4.  Samples observed under a light microscope. (A) Sauropsid-type sperm of the common cuckoo (1000 ​× ​); (B) Sperm (red circle) in fecal matters (1000 ​× ​); (C) fecal matters without sperm (200 ​× ​). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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Figure  5.  The probability of sperm presence in the fluid obtained by the UroS method as a function of the time the sample is taken. Each dot represents different individuals and the response variable is binary: sperm presence (1) or absence (0). A fitted line (blue) is shown with its 95% confidence intervals (gray). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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4.   Discussion

In this study, we introduced the UroS method to collect semen from the Common Cuckoo and verified its effectiveness. The UroS method is similar to the widely used cloacal massage method in that it stimulates the cloaca but from the inside rather than around. The major advantage of this method is that live sperm can be collected by one person in a short time (less than 1 ​min), while minimizing sample loss by stimulating the cloaca directly with a capillary tube that absorbs the ejected semen. In addition, the success rate (52%) was significantly higher than that of the cloacal massage method (28.5%, Bae et al., 2020) in the Common Cuckoo, and can also be effectively applied to other Cuculus cuckoos, such as C. optatus and C. micropterus (Lee et al., unpublished data).

The cloaca consists of three sections: coprodeum, urodeum, and proctodeum (King, 1981). The reproductive tract where semen passes is connected to the urodeum (Pollock and Orosz, 2002); therefore, stimulation of the urodeum may induce semen ejaculation. However, the cloaca is also the terminus of the digestive and urinary tracks, in the coprodeum and urodeum, respectively (Klasing, 1999; Gee et al., 2004). Therefore, stimulation around the cloaca may lead to ejaculation of feces, urine, and semen, which is one of the side effects of the conventional method (Lake, 1957; Blanco et al., 2002; Cheng et al., 2002; Frediani et al., 2019). In this study, 43% of fluid samples (n ​= ​76) were found to be a mixture of urine and feces without semen, indicating the limitations of our method. However, the presence of urine and fecal matter did not necessarily indicate the absence of sperm because both sperm and fecal matter were found in 13 samples (17%, n ​= ​76). In particular, when fecal debris and sperm were observed together, the color of the fluid sample was different from that of semen (i.e., white to ivory), hindering the ability to determine the presence of sperm based on the color of the sample. Since the observation of sperm is difficult in the presence of impurities, these substances can be minimized by washing the inside of the cloaca with clean water (like the cloacal lavage method, Quay, 1984) before performing the UroS method. In addition, if the insertion depth and rotation angle of the capillary tube are properly applied, unnecessary stimulation that induces the expulsion of feces and urine can be minimized. However, further studies are necessary to elucidate the correlation between capillary-handling skills and semen ejaculation.

Sperm depletion due to frequent mating may also explain this result. In this study, we used a cuckoo dummy with a playback of female calls to lure wild cuckoos to capture sites, and three males attempted to mate with the dummy before being caught. We obtained fluid samples from two of these males using the UroS method, but those did not contain any sperm. This was probably because they had pre-ejaculated when mating with the dummy. In fact, other sperm collection methods use a female dummy in species, such as in the Houbara Bustard (Chlamydotis undulata), Mallard (Anas platyrhynchos) and Sand Martin (Riparia riparia) (Wishart and Wilson, 1999; Samour, 2004; Helfenstein et al., 2008). Catching cuckoos without a dummy may be an easy alternative to increase the chance of obtaining sperm using the UroS method, but it may also decrease the chances of catching cuckoos. Therefore, the use of a dummy is recommended, but cuckoos should be prevented from coming into direct contact with the dummy by using a wire mesh, for example.

In conclusion, we introduced a new method called the UroS method to collect semen from Cuculus cuckoos, evaluated its efficiency in 76 cuckoos, and proposed ideas to improve its applicability. Our method is easy to apply in a short time and can effectively collect live sperm from the cloaca, allowing the study of sperm properties such as sperm motility and sperm concentration as well as morphology. In addition, we believe that this method can also be applied to other medium-sized non-passerines, where capillary tubes can be inserted into the cloaca. We anticipate that our method will facilitate the study of sperm biology and competition in a variety of birds, including Cuculus cuckoos, and contribute to species restoration through artificial insemination for endangered bird species.

Authors' contributions

JWL, HNK, and JSG conceived the idea; HL, HNK, JSG, MCC, SY, SJJ and JWL performed fieldwork; HL conducted observation; HL and JWL analyzed data; HL, JWL, and JCY wrote the manuscript. All authors read and approved the final manuscript.

Ethics statement

Cuckoos were captured with the permission of the local governments, and bird care was approved by the Kyung Hee University Animal Ethics Committee.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank Jisoo Han for helping us in the field and Geun-Won Bae for a useful comment. We are also grateful to Hye-Kyoung Moon for her valuable help in using a microscope. This research was financially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by The Ministry of Education (NRF-2020R1I1A2063567), for which we are most grateful.