We recorded the egg-laying patterns of Magang geese, oviposition time distribution during the day, and oviposition intervals or cycle lengths. In addition, we measured the expression of reproductive hormones throughout the cycle, and the expression of genes involved in hormonal regulation in the context of ovarian follice development in order to understand the regulatory mechanisms of egg-laying in Magang geese. The results of this study constitute the first thorough investigation of oviposition characteristics and regulation in geese.
Most oviposition occurs during the day and scarcely at night in Magang geese. This is somewhat different from European graylag geese (Anser anser) reported by Çelebi and Güven , which lay eggs mostly during the early part of the day. Oviposition is thought to be regulated by an interaction of endogenous rhythms with daily photoperiod , which determines neuroendocrine regulation of ovulation. It appears that Magang geese differ from European geese in the photo-entrainment of oviposition. In this regard, Magang geese are similar to the chicken which also lays eggs during the day, but unlike the duck, a waterfowl that lays eggs at night or before photophase.
The oviposition interval of Magang geese, being 46.8 h on average, is consistent with previously reported observations [2, 12]. This means egg-laying occurs every other day, and will make the daily laying rate close to 50%. This laying rate is rarely observed under practical production conditions, as the actual laying rate seldom reaches 30% , possibly due to variability between individual birds and the presence of non-laying birds in the flock, as observed by Yang et al. . In this study, some geese laid only very few eggs (4 to 5 eggs), while some laid up to 12 eggs before establishing incubation behavior. These latter good layers also had oviposition intervals stably close to 48 h, compared with the rather irregular intervals that could last up to 80 to 90 h in some poor laying geese. These phenomena are quite different from the short intervals, as short as 36 h, in some prolific geese that had peak laying rate exceeding 60% . Therefore, in selection program for Magang geese, the target should be set for selecting high number of eggs and also stable oviposition intervals. In addition, the oviposition interval became increasingly longer towards the end of the clutch. Lengthening of the oviposition interval at the end of the clutch might result from the lowering of LH secretion caused by rising secretion of PRL , which would slow follicle maturation.
Follice developmental speed was recorded by marking follicle diameter with Sudan Black dye. This not only unraveled the time course required for SYFs to develop to ovulation, 18 days, but also helped to construct a mathematical model of follicle growth that was hitherto unknown. The 18 days for SYFs to develop to ovulation could be established by laying of the previous 8 to 10 eggs in a clutch, but it is an accurate reflection of natural conditions. It took between 20 to 25 days from the end of incubation to laying a new clutch of eggs in Magang geese . In other words, the time required for LWFs to develop to ovulatory maturity is 20 or so days on average. In addition, during practical production, the laying-incubation cycle in Magang geese is approximately 50 days . This consists of the 7 to 10 days for terminating incubation behavior, the 20 to 25 days required for initiation of laying, and the 18 to 20 days required to lay the whole clutch of eggs.
During the stages of egg-laying, ovarian follicles of different sizes are exposed to the same endocrine milieu, which affects each individual follicle through the expression of receptors on the cell surface. The factors involved in ovarian follicular development and ovulation include pituitary gonadotropins  and the autocrine/paracrine factors secreted by the follicles themselves [8, 13]. We characterized the expression profiles of these factors in this study. The dramatic upregulation of LHR and inhibin alpha subunit, in the largest F1 follicle, and also changing patterns of other genes in granulosa and theca layers, were all similar in Magang goose to those reported for chicken [14–17]. These results indicate that the progression of follicular development and the molecular mechanisms involved, i.e., the endocrine regulation by gonadotropin and autocrine/paracrine regulation by inhibin/activinR, are much similar for geese and chickens even though the former have lower laying rates than the latter .
Apart from the aforementioned differences in chickens, geese have much longer oviposition cycles. This could be due to the maturation process and hormonal profile, which affects the oviposition cycle. In this study, the concentrations of 6 hormones or metabolite were measured within a single ovulation cycle. We found that the changing hormonal patterns were consistent with those previously reported for chickens [6, 8, 10] and Graylag goose . Among the hormones, PGF2α plays a pivotal role in oviposition cycle regulation in avian species, such that the enhanced synthesis and release of uterotonic PGF2α stimulates uterine muscle contraction, which culminates in expulsion of the egg [12, 18]. The blood concentrations of PGFM, the metabolic molecular form of PGF2α, are normally measured to reflect changes in PGF2α secretions [12, 18]. In this study, the single peak in plasma PGFM was detected during the narrow oviposition time window, similar to previous results in chickens and geese [12, 18]. PGF2α is synthesized in uterine and follicle tissues and its concentration peaks can be detected in the blood of chickens independent of oviposition . Therefore, analyzing the expression and structure of the genes associated with PGF2α synthesis and their association with oviposition cycle length and egg-laying rate in geese are of particular interest.
For progesterone, another hormone instrumental to ovulation, the plasma concentrations during the oviposition cycle were comparable to those of Magang geese (Anser cygnoids) under laying state , but much lower than those reported for Graylag geese (Anser anser) . These differences may result from interspecies differences in progesterone secretion or from differences in assay methods. This issue needs to be clarified in future studies. Nevertheless, the interoviposition progesterone variation pattern in Magang geese strikingly resembles that reported for Graylag geese , with the preovulatory P4 increasing at 14 h and peaking 3 h before oviposition. Considering the interval between the preovulatory P4 peak and ovulation is about 3.5 hours in both geese and chickens [6, 10, 11], even though the goose ovulation cycle is about 22 h longer than in chickens, goose F1 follicles clearly take longer to mature. In other words, follicular development and maturation immediately after ovulation occurs at a slower pace in geese. This may be a factor in the prolonged oviposition cycle of geese and may reduce egg-laying frequency and rate.
Immediate to ovulation, both plasma concentrations of activin and inhibin decreased dramatically. This indicated the mature F1 follicle also secrets large amount of activin in Magang geese. This phenomen is contrary to situation in the chicken that activin was considered to be mainly secreted by less developed F4 to F3 follicles . Also, the residual inhibin concentration nearing 50 pg/ml was about half the preovulatory peak of 110 pg/ml, indicating the F2 follicle also secreted copious amount of inhibin, rather than the small amount in the chicken model . Ready secretion of inhibin by F2 was also seen by the rapid rebound of plasma concentration in less than 5 hr after the ovulation, also followed by follistatin concentration rebounds. Compared with inhibin secretion rebound at 10 hr post-ovulation in chicken hens whose ovipositin interval was only 24 hr or so and new SYF recruitment occurs daily , post-ovulatory inhibin secretion in Magang geese was recovered highly rapid. Moreover, these infer that, during laying a sequence or clutch of eggs, copious amount of these hormones are always present in blood circulation. Since inhibin and follistatin counteract the activin’s role of promoting small follicle development , the continued presence of inhibin and follistatin in blood may inhibit new SYF recruitment into preovultory LYF development after Magang geese entering into lay , causing clutch egg size limited to no more than the number of LYFs present before laying of the first egg in a sequence. This also explains taking place of the near 20-day interruption between two clutches of eggs as discussed above, and also in some non-incubating geese .
Within 10 h to 15 h after ovulation, the hormonal profile is characterized by the static secretion of progesterone, transient secretions of activin and inhibin, and robust secretions of estradiol and follistatin. Activin A enhances granulosa cell proliferation and mediates expression of FSHR and LHR [8, 13]. The increase in follistatin secretion during the first 12 after ovulation should counteract the effects of low transient activin A secretions to delay follicular maturation during this stage and to inhibit progesterone secretion. Nevertheless, increasing gonadotropin secretions following ovulation of the F1 follicle and subsequent withdrawal of negative feedback [6, 19] may stimulate the remaining F2 follicle and enhance estradiol secretion.
From mid-cycle onwards, i.e., 15 h after ovulation, the plasma activin A concentrations continued to increase with inhibin concentrations (though at a lower magnitude), whereas follistatin concentrations remained stable. These changes in hormonal balance or increase in “activin tone” may regulate the second stage of follicular development or maturation, i.e., functional upgrading of the F2 follicle to F1.
As the F1 follicle continues to mature, plasma activin A concentrations continue to increase, peaking at 34 h after ovulation, or 14 h before the next ovulation. This activin A peak occurred concomitantly with the fall of follistatin concentrations, but with preovulatory increases in P4 and estradiol. Considering the plasma inhibin concentrations remain stable during this stage, the “activin tone” is undoubtedly strengthened, facilitating final maturation of the F1 follicle that ultimately triggers the preovulatory LH surge and ovulation. The earlier decrease in plasma E2 concentrations compared with P4 before ovulation is also consistent with the phenomenon observed in chickens, wherein the more mature F1 follicle secretes less E2 . This indicates that as the preovulatory F1 follicle matures, gonadal steroidogenesis shifts from E2 to P4.