Plasma levels of luteinizing hormone and prolactin in relation to double brooding in Great Tit (Parus major)

1.   Introduction

Among animals, multiple brooding is a common reproductive strategy in a variety of taxa (Verhulst et al., 1997 and references therein). However, multiple breeding strategies may exist within a population with some individuals breeding once while others are able to breed more than once and are often termed facultative multiple breeders (Nagy and Holmes, 2005; Monroe et al., 2008; Husby et al., 2009; Carro et al., 2015; Hoffmann et al., 2015). Producing one or more clutches per year involves complex changes in reproductive state and behavior, which are mainly controlled by the hypothalamus-pituitary-gonad (HPG) axis (Zhang et al., 2014, 2019; Gao et al., 2018). In birds, the hypothalamus secretes gonadotrophin-releasing hormone-I (GnRH-I) and vasoactive intestinal peptide (VIP) (Pawson and McNeilly, 2005; Christensen and Vleck, 2008). These stimulate the pituitary to synthesize and release hormones, such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL). It is widely believed that LH and FSH can initiate reproductive behaviors, and PRL can play a crucial role in the onset and maintenance of broodiness (Sharp et al., 1998; Silverin et al., 1999; Sockman et al., 2000; Dawson and Sharp, 2010; Zhang et al., 2017a; Smiley, 2019).

For most birds distributed at mid- and high latitudes, the photoinduced increase in LH secretion in spring is accompanied by an increase in PRL secretion (Dittami, 1981; Hissa et al., 1983; Dawson and Goldsmith, 1984, 1985; Stokkan et al., 1988; Silverin, 1991). In general, plasma LH reaches a peak in the stage of sexual behavior before egg-laying and promotes initial egg-laying behavior, while plasma PRL rises significantly from the beginning of egg-laying and reaches a high level during the brooding period (Sharp et al., 1998; Angelier and Chastel, 2009). However, the concentrations of plasma LH will drop to the basic low level after the young have fledged (Wingfield and Farner, 1993) and increase again if parent birds initiate the next breeding attempts within the same breeding season (Sharp et al., 1988). Although the concentrations of PRL are the highest during the brooding period in most cases (Buntin et al., 1996; Chastel et al., 2005), several studies also found that plasma PRL drops shortly after hatching and is maintained at a moderate level during the brooding period (Goldsmith, 1982; El Halawani et al., 1988). Additionally, some studies have found that temperature, food availability, breeding experience and breeding parameters would also affect hormone levels of birds (Silver et al., 1980; Cate et al., 1993; Sockman et al., 2000; Angelier et al., 2006; Silverin et al., 2008; Ouyang et al., 2011; Riechert et al., 2014; Ryan et al., 2014; Valle et al., 2015; Smiley and Adkins-Regan, 2016a, Smiley and Adkins-Regan, 2016b). Therefore, the secretion patterns of LH and PRL may be different among breeding stages in different species over time.

Previous studies on some passerine bird species reported that the secretion patterns of PRL and LH were similar in female and male individuals (Hiatt et al., 1987; Silverin, 1991, 1994). While females and males often play different roles during the reproductive period (Stodola et al., 2009), the secretion patterns of LH and PRL in different sexes may be different during the breeding season (Sharp et al., 1986, 1998). A few studies have found that the PRL levels of females were higher than those of males in some bird species that are only provided with parental care by females, whereas the results might be reversed if only males provide parental care (Oring et al., 1988; Sharp et al., 1998; Vleck, 1998). Goldsmith (1982) still found that PRL levels of females remained high during the brooding period but males only showed slight increase in PRL in canaries (Serinus canarius) which raised their offspring together. These sex differences of hormonal were common in birds (Goldsmith, 1982; Lormée et al., 2000; Greives et al., 2016; Wojczulanis-Jakubas et al., 2018; Tolla and Stevenson, 2020).

Great Tits (Parus major), a secondary cavity nester, are an abundant species in our study area, Zuojia Nature Reserve, Jilin Province, Northeastern China. They are facultative multiple breeders, as some individuals can reproduce a second brood after the first one has succeeded, and the mean clutch size of the first brood is commonly bigger than that of the second brood (see details in section Materials and Methods). As a model species, Great Tits are widely used in studies worldwide, such as ecology, endocrinology, and so on (e.g. Silverin et al., 1999; Verboven et al., 2001; Ahola et al., 2007; Schaper et al., 2011; Ouyang et al., 2013; Vatka et al., 2021). However, only a few studies have focused on the temporal trend of hormone levels between reproductive stages (Silverin, 1991; Silverin et al., 1999; Ouyang et al., 2013). The present study aims to test whether there are differences in the LH and PRL levels of both females and males at different stages (pre-breeding period, first and second brooding periods) within the same reproductive season. Based on the previous studies about the functions of LH and PRL in the reproduction process (see above), we predicted (1) that Great Tits had significantly higher LH and lower PRL concentrations in the pre-breeding period than brooding period. Considering that breeding parameters were different between the two brooding periods, we predicted (2) that individuals in the first brooding period have higher PRL concentrations and lower LH concentrations than those in the second brooding period. For tits, females are responsible for hatching and provisioning young; while males are responsible for nest defense and other responsibilities in addition to providing food for chicks (Matysioková and Remeš, 2010; Yu et al., 2017). Due to females and males often playing different roles during the reproductive period (Sharp et al., 1986, 1998), we predicted (3) that Great Tits would show sex differences in LH and PRL concentrations.

2.   Materials and methods

2.1   Study area and study species

This study was carried out in March–July of 2018 and 2019 in Zuojia Nature Reserve (44°1′–45°0′ N, 126°0′–126°8′ E). We monitored a population of Great Tits nesting in nest boxes during the breeding seasons. In our population, Great Tits began to occupy the territory in early March, added nesting material to the nest box in early April, and began to lay eggs in mid-to-late April. The mean brood size of the first and second broods was 10.57 ​± ​1.45 and 7.94 ​± ​1.34, respectively. In both broods, the incubation period was 10–13 days, and the brooding period was 15–18 days. In addition, the chicks were raised by both males and females. Between the first and second breeding attempts, tits usually do not switch mates (Fan et al., 2021). The nest boxes were fixed on a tree 3 ​m above the ground, facing random directions. In our research area, the number of nest boxes distributed annually was maintained at approximately 450. We visited the nest boxes at least once a week to record breeding attempts and reproductive data (e.g., laying date, clutch size, hatching date, brood size, fledged young) (Yu et al., 2020).

2.2   Blood sampling

In 2018 and 2019, blood samples (Table 1) were collected from adult male and female Great Tits at three stages: (1) the pre-breeding period (from mid-March to early April), (2) the stage of feeding hatched 6–7 days chicks of the first clutch, that is, the first brooding period, and (3) the stage of feeding hatched 6–7 days chicks of the second clutch, that is, the second brooding period. Among them, the number of recaptured individuals each year is shown in Table 1. In the pre-breeding period, adults were trapped with a cage and conspecific song playbacks. The cage was divided into two floors. There was a speaker (Royqueen M300, Shenzhen, China) at the lower level that played the conspecific songs continuously to lure other Great Tits. On the upper level, there was a food container with mealworms and a small wooden stick. When the tits outside were attracted to the food and stepped on the stick, the mechanism was triggered, and they were locked in the cage. Tits were trapped less than 10 ​min and most of them were feeding (the worms were their most favorite food) or drinking in the cage. All tits were ringed with numbered copper rings for individual identification and then released alive after blood sampling (≤100 ​μL, 1% body weight). To minimize stress, the whole process from adults capture to release was finished within 2–3 ​min. In the brooding periods of the first and second clutches, the parents were captured in the nest box using a spring trap when they entered to feed nestlings (6–7 days old). Most parents were also caught in less than 10 ​min, then they were collected for blood samples (≤100 ​μL) and ringed within 2–3 ​min. The estimated handling time was independent of the determination of LH and PRL in birds (Ryan et al., 2014). Then, all the blood samples were kept on ice for up to 8 ​h until centrifuged at 3000 r/min for 10 ​min. The resultant blood plasma was then used to measure the plasma LH and PRL concentrations. The plasma samples were injected into a 100-μL centrifuge tube containing an anticoagulant. The resultant blood cells were used for extraction of DNA, which was injected into a 1.5 ​mL centrifuge tube containing 95% alcohol for storage, and then the sex of the individual was determined using P2P8 primers (Griffiths et al., 1998). Table 1 gives details on the sampling of individuals during the different stages. All of the blood samples were immediately stored at −80 ​°C until assayed. Hormone analysis was performed every year after blood samples were collected.

Table  1.  Blood sample sizes at different breeding stages in Great Tits.

	Year	Breeding stage	N	No. of recaptured individuals
Female	2018	Pre-breeding	16	0
		First brooding	51	0
		Second brooding	18	6 (FB)
	2019	Pre-breeding	20	0
		First brooding	67	8 (LYFB)
		Second brooding	16	4 (FB)
Male	2018	Pre-breeding	81	0
		First brooding	42	1 (PB)
		Second brooding	17	3 (FB)
	2019	Pre-breeding	32	2 (LYFB)
		First brooding	61	5 (LYFB)
		Second brooding	15	4 (FB)
PB, the pre-breeding period; FB, the first brooding period; LYFB, the first brooding period in the previous year.

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2.3   Hormone analysis

The Great Tit plasma LH and PRL levels were measured using chicken enzyme immunoassay kits (Rsbio Company, CK-EN95591 and CK-EN95943, respectively) according to the instructions. This method has been previously demonstrated for Tree Sparrows (Passer montanus), Asian Short-toed Lark (Calandrella cheleensis), and Zebra Finch (Taeniopygia guttata) (Zhang et al., 2014, 2017; Smiley and Adkins-Regan, 2016a). To confirm that the kits can be used for Great Tits, we diluted the plasma pool by 1, 1/2, 1/4, 1/8, 1/16, and 1/32 (Chastel et al., 2005). Since the LH and PRL dilution curves of Great Tit plasma were parallel to the standard ELISA kit curves (Appendix Fig. S1), we confirmed that these kits can be used to reliably assess relative levels of LH and PRL in the plasma of Great Tits. Assay sensitivity of LH and PRL were 1.00 ​ng/mL. Because the plasma volume of some samples was insufficient to measure LH and PRL concentrations, for accuracy, all plasma samples were diluted five times with PBS buffer and measured three times. The measurement results were multiplied by five to obtain the final results. The intra-assay variations of LH and PRL were 2.4% and 2.3%, respectively. The inter-assay ranges of LH and PRL were 3.7%–7.8% and 4.8%–9.7%, respectively.

2.4   Data analysis

The LH and PRL concentrations of female Great Tits were significantly higher than those of males during the breeding stages (Mann-Whitney U test, all P ​< ​0.001; Table 2). Then, we analyzed the hormones of males and females separately. The Kolmogorov-Smirnov test was used to check the normality of LH and PRL concentrations. Both hormones showed a normal distribution, so we ran linear mixed models (LMMs) to study the effect of the breeding stage on the LH and PRL concentrations of male and female Great Tits, respectively. For models, we analyzed the LH/PRL concentrations as dependent variables, with the breeding stage as the fixed effect. Even though blood samples were taken in 2018 and 2019, the annual change trend of hormone concentrations was similar when we separately analyzed LH and PRL of females and males each year (Appendix Table S1). So, we chose the year and individual identity as random effects. To probe into significant effects further, we performed post hoc pairwise comparisons between reproductive stages (i.e., the pre-breeding period vs. the first brooding period; the first brooding period vs. the second brooding period; the pre-breeding period vs. the second brooding period). In the comparison between the first and the second brooding period, we also treated the first egg-laying date (Julian date, in days after April 1st that year) and the brood size as fixed effects, and the partner-ID within a pair as the random effect. Occasionally, we only captured one of a breeding pair (n ​= ​67), and we randomly assigned a new ID to the pair-partner that was not captured. We used likelihood ratio tests (LRT) to obtain the significance of the random effects (Yu et al., 2017). The Bonferroni correction was used to correct the P-values (Yu et al., 2017). Data analyses were performed in R version 4.0.3 using the “lme4” and “lmtest” packages and IBM SPSS software version 21.0. All statistical tests were two-tailed, and probability levels < 0.05 were considered significant.

Table  2.  Results of Mann-Whitney U test between sexes in the breeding stages of Great Tits.

Breeding stage	Hormone	Female (ng/mL)	Male (ng/mL)	Z	P
Pre-breeding	LH	7.88 ± 0.37	3.48 ± 0.13	−7.574	< 0.001∗∗
	PRL	28.01 ± 1.05	20.59 ± 0.64	−5.410	< 0.001∗∗
First brooding	LH	6.97 ± 0.18	3.22 ± 0.10	−11.407	< 0.001∗∗
	PRL	45.89 ± 0.82	20.95 ± 0.59	−12.525	< 0.001∗∗
Second brooding	LH	6.15 ± 0.22	4.08 ± 0.19	−3.503	< 0.001∗∗
	PRL	43.43 ± 1.52	23.58 ± 1.27	−6.364	< 0.001∗∗
LH, the luteinizing hormone; PRL, the prolactin; The values are means ± SE; *P < 0.05; ∗∗P < 0.01.

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3.   Results

The concentrations of plasma LH and PRL were significantly different during the breeding stages between male and female Great Tits (P ​< ​0.019 for all multiple comparisons). Females in the brooding period of the first and second clutches had lower LH and higher PRL concentrations than those in the pre-breeding period (Table 3; Fig. 1). In males, plasma LH concentrations in the pre-breeding period were significantly lower than those in the second brooding period (P ​= ​0.045) but had no difference from that in the first brooding period (Table 3; Fig. 1). Plasma PRL concentrations did not differ between the pre-breeding period and the first/second brooding period (Table 3; Fig. 1). In the comparison between the first and the second brooding period, there was no significant difference in LH and PRL concentrations in females between the first and the second brooding periods, but plasma LH and PRL concentrations in males in the first brooding period were significantly lower than those in the second brooding period (Table 4). For the first egg-laying date and the brood size, no significant effects were found in females and males. The test of the random effects indicated significant effects of years (LRT: P ​< ​0.001 for all tests) and non-significant effect of individual identity and partner-ID (LRT: P ​> ​0.05 for all tests).

Table  3.  Results of post hoc comparisons of LH and PRL concentrations between the pre-breeding period, the first and second brooding periods of Great Tits.

Sex	Response variable	Explanatory variable	F	P	Post hoc P
						PB	FB
Female	LH	Breeding stage	10.003	< 0.001∗∗	FB	0.002∗∗
					SB	0.009∗∗	0.131
	PRL		92.171	< 0.001∗∗	FB	< 0.001∗∗
					SB	< 0.001∗∗	0.901
Male	LH	Breeding stage	5.4215	0.005∗∗	FB	1.000
					SB	0.045	< 0.001∗∗
	PRL		4.0251	0.019∗	FB	0.367
					SB	0.351	0.009∗∗
Results are from linear mixed models. LH, the luteinizing hormone; PRL, the prolactin; Post hoc P values were adjusted by Bonferroni correction; PB, the prebreeding period; FB, the first brooding period; SB, the second brooding period. ∗P < 0.05; ∗∗P < 0.01.

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Figure  1.  Concentrations of luteinizinghormone (A) and prolactin (B) at different stages of Great Tits. LH, the luteinizing hormone; PRL, the prolactin; PB, FB, SB refer to the pre-breeding period, first brooding period, and second brooding period, respectively. The upper-case letters and the lower-case letters represent the significant difference of each breeding stage in males and females, respectively. Data points that share the same letter are not significantly different. Data are means ​± ​SEM.

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Table  4.  Results of the effects of the breeding stage, clutch size, Julian date, on LH and PRL concentrations in Great Tit during the first and the second brooding period.

Sex	Response variable	Explanatory variable	F	P
Female	LH	Breeding stage	0.448	0.505
		Clutch size	0.166	0.684
		Julian date	0.023	0.879
	PRL	Breeding stage	0.145	0.704
		Clutch size	0.367	0.545
		Julian date	0.464	0.497
Male	LH	Breeding stage	11.304	< 0.001∗∗
		Clutch size	0.929	0.337
		Julian date	3.671	0.058
	PRL	Breeding stage	4.145	0.044
		Clutch size	0.106	0.746
		Julian date	0.022	0.883
Results are from linear mixed models. LH, the luteinizing hormone; PRL, the prolactin; ∗P < 0.05; ∗∗P < 0.01.

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4.   Discussion

Consistent with the findings of Silverin (1991), we observed that female Great Tits exhibited significantly higher plasma LH concentrations and lower PRL concentrations in the pre-breeding period than those in the two brooding periods. The main physiological function of LH is to stimulate the gonads to initiate breeding activities, so the concentrations should be increased in the pre-breeding period (Sharp et al., 1998; Greives et al., 2016). The function of PRL has long been considered to be the maintenance of incubation and parental care behavior in birds (Goldsmith et al., 1984), and the high concentrations in the brooding period is most likely to maintain post-hatch care (Goldsmith, 1982; Hiatt et al., 1987; Sockman et al., 2010; Krause et al., 2015; Smiley and Adkins-Regan, 2016a, Smiley and Adkins-Regan, 2016b). For male Great Tits, there was no difference in the concentrations of LH and PRL between the pre-breeding period and the first brooding period. In male Mallards (Anas platyrhynchos) and Canaries (Serinus canarius), who do not participate in incubation but do feed the young, PRL concentrations remain low from the pre-breeding period to the brooding period (Goldsmith and Williams, 1980; Goldsmith, 1982; Basso and Richner, 2015). In addition, PRL levels of male Gambel's White-crowned Sparrows (Zonotrichia leucophrys gambelii) were rising immediately at the time of territory establishment and remained high throughout the breeding season (Krause et al., 2015). Thus, we thought that hormone concentrations of male tits were elevated from the pre-breeding period and remained high to the first brooding period. Thus, the results we found were partly consistent with predictions (1).

In females, there were no differences in the concentrations of LH and PRL between the two brooding periods, which is similar to previous studies on Song Sparrow (Melospiza melodia) and White-crowned Sparrow (Zonotrichia leucophrys) (Hiatt et al., 1987). It is possible that females need to make great efforts to maintain chick-rearing behaviors in multiple breeding. However, the LH and PRL levels of the second chick-rearing period were higher than those of the first chick-rearing period in male Great Tits. Higher PRL concentrations are generally associated with increased reproductive success and tend to come with reproductive experience (Smiley and Adkins-Regan, 2016a, Smiley and Adkins-Regan, 2016b). Some studies have shown that reproductively experienced birds have higher PRL levels than inexperienced birds (Angelier et al., 2006, 2007; Riechert et al., 2012). Individuals who produce a second clutch within a year could be thought to have more reproductive experience than those who produce a single clutch (Smiley and Adkins-Regan, 2016a, Smiley and Adkins-Regan, 2016b). However, few studies have reported the effect of LH on double or multiple broods of male birds. Similar to our results, Wingfield and Farner (1993) also found the LH concentrations of male tits remained high during the whole reproductive process. Here, we suggest that male tits with higher levels of LH and PRL were more likely to maintain the second brood. The results provided no support to our prior prediction (2). In addition, the first egg-laying date and the brood size were not found to have effects on LH and PRL concentrations between the two brooding periods. Photoperiod is the principal cue used to time each stage, allowing birds to adapt their physiology in advance of predictable environmental changes (Dawson, 2008). While, non-photoperiodic signals, such as temperature and rainfall, can directly affect the exact timing of egg-laying and the latter stages of ovulation (Caro et al., 2006; Ball and Ketterson, 2008; Dawson, 2008; Schaper et al., 2012; Zhang et al., 2017b). Here, we thought that the internal program for the absolute levels of hormones might be regulated independently of environmental input once the decision to breed has been made. However, we did not examine the influences of weather on hormones, although that could be explored in future research.

As predicted (3), we found that females had higher PRL and LH concentrations than males during the reproductive phase; moreover, the change trends of LH and PRL between females and males were different from the pre-breeding period to the first and second brooding periods (Table 2; Fig. 1). Studies have shown that sex differences of some birds in hormone levels are due to the sex-biased investment in reproduction (Lormée et al., 2000; Li et al., 2015). In female-biased incubation and biparental care species, females' parental investment is always greater than males during breeding (Li et al., 2016). For example, females may have higher PRL levels than males when they put more effort into reproduction (Oring et al., 1988; Gratto-Trevor et al., 1990). Therefore, we suggest that the sexual dimorphism of hormones during the breeding cycle in Great Tits most likely correlates with differences in the responsibilities that males and females assume throughout reproduction.

5.   Conclusion

In general, the current study provides new data for changes in LH and PRL between brooding attempts per year in passerine birds. According to our results, we conclude that there are sex-based differences between LH and PRL at different stages of reproduction. The changes in LH and PRL in both males and females should be related to their physiological functions. Especially for males, individuals with higher levels of LH and PRL are more likely to maintain second clutches. The differences in reproductive hormones between single and multi-brooded individuals may be an important goal for future research.

Authors’ contributions

HW and JY conceived and designed the study. XL and WX conducted the experiments in the field. XL, JY, and ZW analyzed the data and wrote the paper. JY, HW, and XL proofread and made comments to the manuscript. All authors read and approved the final manuscript.

Ethics statement

The experiments comply with the current laws of China. Fieldwork was carried out under the permission of the Zuojia Nature Reserve. Experimental procedures were permitted by the National Animal Research Authority at Northeast Normal University (approval number: NENU-20080416). In addition, standard procedures were followed, such as the ARRIVE guidelines for animal research reports.

Declaration of competing interest

The authors declare that they have no competing interests.

Appendix A.   Supplementary data

The following is the Supplementary data to this article:

Acknowledgements

We are grateful to Na Zhao, Li Shen, Jing Yue, Junlong Yin, Jian Fang, and Liufang Wang for their assistance in fieldwork. We also thank the Zuojia Nature Reserve for the support and permission to carry out this study. This work was supported by the National Natural Science Foundation of China (No. 31770419 and 31971402 to HW, 32001094 and 31870368 to JY).