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Effects of hemicastration and castration on foam production and its relationship with fertility in male Japanese quail
Theriogenology 58 (2002) 29±39
Effects of hemicastration and castration on foam production and its relationship with fertility in male Japanese quail Jag Mohan*, Ram Phal Moudgal, Kochiganti Venkata Hanumat Sastry, Jagbir Tyagi, Rajvir Singh Division of Physiology and Reproduction, Central Avian Research Institute, Izatnagar, UP-243 122, India Received 15 September 2000; accepted 30 April 2001
Abstract Healthy heavy body weight strain of adult male Japanese quail (Coturnix coturnix Japonica) of the same age were used in this study to observe the effect of hemicastration and castration on the frequency of foam discharges from cloacal gland and other related parameters. The quails were housed in individual cages and divided into four groups: control (intact birds), sham-operated control (intact birds with incision), hemicastrated, and castrated groups of birds. Hemicastration and castration were carried out surgically at 10 and 13 weeks of age, respectively. Subsequently, 3 weeks after castration birds were examined for different parameters. Hemicastration caused a signi®cant (P < 0:05) drop in the foam discharge frequency, weight of the foam, and the level of testosterone in the plasma, whereas in castrated group these variables were observed nil or negligible as compared to both of the controls. A suppressive effect of hemicastration was also noticed on the body weight, area of the cloacal gland, as well as percent fertility. Castration induced the drastic regression of the cloacal gland and a signi®cant reduction (P < 0:05) of the body weight was also noted as compared to other groups. Frequencies of foam discharges were twice the number in daytime (06:00±18:00 h) than night, irrespective of the groups. In another study, the effect of characteristics of foam or foam glands on fertility was examined in 77 male birds paired with females. Several characteristics of foam and foam glands were examined for infertility, such as light yellow foam, dark yellow foam, smaller area of foam gland (below 225 mm2), hardness of foam gland and certain unknown factors that contributed 0.23, 0.68, 5.23, 2.27 and 3.64% infertility, respectively. An overall 12.1% birds were found to be infertile. This data indicated that smaller sizes of foam glands might re¯ect the poorest fertilizing ability of the male birds. From this study, it may be concluded that the cloacal gland may be considered as an external indicator of testicular function of the birds. Characteristics of the cloacal gland or foam or both may be used as a
* Corresponding author. Tel.: 91-581-446-420; fax: 91-581-447-321. E-mail address: [email protected] (J. Mohan).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 8 6 3 - 4
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simple and effective tool to predict the fertilizing ability of an individual male in view of the very small semen ejaculate that is very dif®cult to collect and evaluate for fertility. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Japanese quail; Cloacal gland; Foam; Castration; Testes
1. Introduction The development of secondary sexual characteristics in domestic fowl, such as comb and wattles, have been used as external indicators for determining the sex of birds and the stage of sexual maturation. Androgens are required to induce the growth of the comb and wattles in domestic fowl [1]. Similarly, an androgen dependent cloacal gland in male Japanese quail (Coturnix coturnix Japonica) was found as a reliable indicator for testicular function [2,3]. The cloacal gland has not been found in other genus of birds, therefore, they are speci®c only to the member of the genus Coturnix [4]. There is a high correlation between the size of the cloacal gland and the weight of the testes. Both increase with the age of the birds [5,6]. Paired males have larger cloacal glands and testicular size and higher plasma levels of testosterone and LH when compared with the isolated males [7]. The cloacal gland of sexually active male Japanese quail under the in¯uence of testosterone produce the foamy material constantly, irrespective of the time of the day [2]. Administration of testosterone to the castrated male Japanese quail stimulates the growth of the cloacal gland and synthesis of the foamy material within the gland [8]. The physiological signi®cance of foamy material in Japanese quail reproduction has been the subject of con¯icting reports for over three decades [9±11]. Hemicastration causes hypertrophy of the testes in some mammalian species, such as rabbits, mice, rats, hamsters, and rams [12±16], and results in changes in the plasma gonadotrophin level, testicular weight and spermatogenesis. Also after hemicastration in avian species a compensatory hypertrophy of the remaining testis in domestic fowl was reported [17]. To our knowledge, no information is available on the effects of castration and hemicastration on the foam discharge pattern and various other variables, such as size of foam gland, foam weight, testosterone level in blood and fertilizing ability of quail spermatozoa. However, a few reports are available on the effects of castration (not hemicastration) and hormone treatment on the histology and development of the cloacal gland [2,8]. In addition, to date no conclusive evidence is present in literature regarding the function of foam in Japanese quail reproduction. In view of the complete lack of information on the above areas, this work was carried out to investigate the effects of hemicastration and castration on the foam production and certain other variables as described previously. Further, the direct evaluation of the fertilizing ability of male Japanese quail by semen examination is dif®cult because it is very dif®cult to obtain the neat semen without any contamination with foam. As the cloacal gland is androgen dependent, it is hypothesized whether the size of cloacal gland or frequency of foam discharges from this gland could be used as an indirect tool to predict the fertilizing ability of particular males.
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2. Materials and methods Heavy body strain of sexually mature males (>8 weeks of age) of Japanese quail (C. coturnix Japonica) was used in this investigation. They were kept in individual cages and were maintained under the uniform husbandry conditions with the temperature between 25 and 30 8C. They were given normal quail breeder ration and water ad libitum with a constant light 14 h per day during the experiment. During the course of the experiment the same technical personnel were allowed to feed, water and collect the data from birds. At the age of 10 weeks the birds were arbitrarily divided into four groups of 18 birds in each. The ®rst group served as control (intact birds), whereas second group was the sham-operated control group with an incision in the last intercostal space of the birds under the local anesthetic (xylocaine 2%). In the third and fourth group, the left testis was removed surgically through an incision made between the last two intercostal ribs under the local anesthesia [17]. All the birds were hemicastrated within 2±3 days. In this similar way after 3 weeks of hemicastration, the remaining testis (right) was also removed only in half of the hemicastrated birds. Now, birds missing one or both of the testes have been termed as hemicastrated (third group) and castrated (fourth group), respectively. Subsequently, 3 weeks after castration the number of birds were ®xed at six in each group. They were examined for foam discharge frequency and certain other variables, like size of the cloacal gland, foam production, testosterone level in plasma, and fertilizing ability of birds. To examine the foam discharge pattern during 24 h at different times of intervals morning, afternoon and night, the foam masses discharged from the cloacal glands of male birds were counted in fecal trays kept under each bird. After each count of the foam discharge, trays were cleaned properly and replaced immediately in the cages for the next count of foam discharge from the birds. To determine the foam produced by each bird, foam was collected [18] in airtight glass bottles to prevent evaporation. Quantitative measurement of foam was done immediately using electronic balance up to fourth digit. The size of the cloacal gland was measured by employing Vernier callipers as per the method described by Siopes and Wilson [5]. For the hormone assay, the blood samples were collected from the wing vein of the male using heparinized syringes. All the samples were immediately centrifuged and plasma was taken out for hormone assay. Subsequently, testosterone was assayed in duplicate in all the samples using a Spactromax-190 micro plate reader by using the testosterone kit (ELA, DSL-10-4000) received from Diagnostic System Laboratories Inc., Webster, TX). To examine their fertilizing ability, male birds were paired in suitable cages with equal number of healthy females (>10 weeks of age) so that each adult male (control or hemicastrated) was allowed to mate with one female (1:1). After 2 weeks of pairing, eggs were collected daily for 2 weeks from females of each group and averaged for two replications (one replication per week). Experimental eggs were stored under standard conditions. Subsequently, all the eggs were incubated for 10 days in hatchery. Thereafter, eggs were broken to con®rm the fertilization macroscopically by the presence of developing embryo. The egg fertility was determined as the ratio of the number of fertile eggs to the number of total eggs set in the incubator. In a second experiment designed to study the relationship between the characteristics of foam or foam glands with the fertilizing ability of male Japanese quail, foam was
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collected from 77 heavy body weight strain, sexually active male Japanese quail and classi®ed on the basis of color: normal white, light yellow, and dark yellow foam. A foam gland was identi®ed as a small size if the area of the gland was below 225 mm2. Determination of hardness of the foam gland was evaluated on the basis of the pressure applied by ®ngers around the cloacal gland at the time of foam collection. Under most of the cases foam could be obtained by applying gentle pressure around the cloacal gland. In this regard the cloacal gland behaved just like a tube of toothpaste. We could not categorize some cases of infertility in birds for which both males and females may be responsible and termed these cases as unknown factors of infertility. Signi®cant differences between means were compared by employing Student's t-test. The correlation coef®cient between sizes of cloacal gland and male reproductive indicators (testicular weight, testosterone level, and foam production) were also determined as per the standard method [19].
Table 1 Normal values (mean S:E:M:, 11 replicates) of foam discharge frequency of each bird in various groups of Japanese quail at different daily intervals Intervals
Bird number
Control group
Sham-operated control group
Hemicastrated group
Morning (06:00±12:00)
1 2 3 4 5 6
5.44 9.33 7.88 6.55 11.32 8.10
6.77 8.39 7.11 7.57 8.37 6.90
5.22 5.62 6.77 4.00 6.66 5.57
Average value range Afternoon (12:00±18:00)
1 2 3 4 5 6 Average value range
Night (18:00±06:00)
1 2 3 4 5 6 Average value range
0.77 0.80 0.65 0.60 0.74 0.67
8.10 0.84 5.44±11.32 5.11 8.84 6.00 7.22 10.77 7.59
0.33 0.41 0.60 0.53 0.85 0.23
7.59 0.82 5.11±10.77 6.11 9.33 7.22 9.44 10.66 8.55
0.43 0.35 0.32 0.67 0.47 0.73
8.55 0.67 6.11±10.66
0.65 0.44 0.33 0.68 0.47 0.66
0.44 0.33 0.62 0.33 0.60 0.29
7.51 0.29 6.77±8.39
5.64 0.41* 4.00±6.77
8.15 8.10 7.75 7.15 8.55 6.47
4.11 3.88 5.55 2.88 7.00 4.68
0.44 0.35 0.64 0.32 0.58 0.29
0.33 0.33 0.25 0.47 0.68 0.33
7.69 0.31 6.47±8.55
4.68 0.58* 2.88±7.00
7.44 4.77 8.65 7.19 8.75 7.05
7.44 4.77 4.71 3.11 6.11 5.23
0.44 0.23 0.42 0.49 0.65 0.35
7.69 0.35 6.70±8.75
0.44 0.33 0.25 0.38 0.73 0.62
5.22 0.59* 3.11±7.44
* Average mean values of hemicastrated group only differed signi®cantly (P < 0:05) from other groups (Student's t-test). Castrated group produced no foam.
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3. Results A summary of the normal values on the frequency of foam discharge during day and night hours in control, sham-operated control and hemicastrated group of heavy body weight strain of male Japanese quail is presented in Tables 1 and 2. Ejection of foam from the cloacal gland was examined at ®xed timings: 12:00, 18:00 and 06:00 h for the interval of morning (06:00±12:00 h), afternoon (12:00±18:00 h) and night (18:00±06:00 h), respectively. Frequency of foam discharge from the cloacal gland was evaluated for 11 days within 30 min of these ®xed timings in all the groups. The foam discharge pattern for castrated group was not shown in Table 1, as there was no foam production in this group. The frequency of foam discharge was signi®cantly (P < 0:05) reduced in hemicastrated group in comparison to other control groups at all the intervals examined in this study. It was observed in all the groups that foam discharge from cloacal gland was nearly double in daytime (06:00±1800 h) than night (18:00±06:00 h). On an average, the normal control birds discharge the foam after each hour (Table 2). No signi®cant differences were observed in body weight and sizes of cloacal glands between hemicastrated and control groups, whereas in the castrated group the body weight of birds was reduced signi®cantly (P < 0:05) and the cloacal glands regressed drastically (Table 3). A signi®cant (P < 0:05) reduction in average foam weight, as well as testosterone level in plasma, was noticed in hemicastrated group when compared with the controls. Similarly, a Table 2 Foam discharge frequency in different groups of Japanese quail during day and night (mean S:E:M:, 11 replicates) Intervals
Control group
Sham-operated control group
Hemicastrated group
Night (18:00±06:00) Day (06:00±18:00) Night day (24 h)
8.55 0.67 15.69 1.66 24.24 2.33
7.69 0.35 15.20 0.60 22.89 0.95
5.22 0.59** 10.32 0.99** 15.54 1.58**
** Mean values of hemicastrated group by row differed signi®cantly (P < 0:01) from control and shamoperated group (Student's t-test). Castrated group did not discharge foam.
Table 3 Effects of hemicastration and castration on the different variables of Japanese quail (mean S:E:M:, n 6, except fertility) Group
Body weight (g)
Size of cloacal gland (mm2)
Foam weight (ng)
Testosterone (ng/ml)
Fertility (%)
Control group Sham-operated control group Hemicastrated group Castrated group
238 17.50 233 14.31
355.20 19.77 366.83 15.11
21.51 0.35 20.27 0.43
1.693 0.150 1.625 0.170
91 87
17.55 0.79* 0.00
0.921 0.210* 0.010 0.003
79 Not examined
a
218 11.33a 317.53 17.21 181 13.89a,* Regressed
Body weight data was given after deducting the corresponding testicular weight. Mean values of hemicastrated and castrated group differed signi®cantly (P < 0:05) from other groups (Student's t-test). *
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Table 4 Effects of the characteristics of foam and foam gland on fertility of Japanese quail Characteristics of foam and foam gland
Total bird no.
Total eggs
Total infertile eggs
Total fertile eggs
Fertility (%)
Infertility (%)
Normal white foam Light yellow foam Dark yellow foam Smaller area of foam gland (below 225 mm2) Hardness of foam gland Unknown factors
47 1 1 13
262 9 16 65
0 1 3 23
262 8 13 42
100.00 88.88 81.25 64.61
0 11.12 18.75 35.39
7 8
40 48
10 16
30 32
75.00 66.66
25.00 33.34
Grand total
77
440
53
387
87.95
12.05
drop in percent fertility was also found in the same group (Table 3). An overall positive correlation was observed for the size of cloacal gland with testicular weight (R2 0:36, P < 0:01), testosterone level (R2 0:40, P < 0:005) and foam production (R2 0:16, P < 0:10) of the male Japanese quail. In the second experiment, it was observed that 47 birds were producing normal white foam expressing 100% fertility. Deviation from normal white foam such as light yellow or dark yellow foam caused the reduction of 11.1 and 18.7% infertility in those birds
Fig. 1. A total of 12.05% infertility attributed by characteristics of foam glands in male Japanese quail.
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producing such kind of foam. Two birds produced yellow foam out of total 77 birds (Table 4). Birds containing smaller sizes of foam glands (<225 mm2) attributed more (35.4%) towards infertility. In the present study during foam collection, it was realized that seven male birds required comparatively more ®nger pressure around the cloacal gland to get the foam released than others (hardness of foam gland) and a 25% decline in fertility was noted in these birds. On the basis of the color of foam or characteristics of foam gland we could not categorize eight birds, because of which 33.3% infertility in this study was related to certain unknown (mixed) factors. An overall 12.1% infertility was revealed in the group of 77 male Japanese quail (Table 4). The overall relationship of different variables of characteristics of foam or foam glands towards total infertility (12.1%) were as follows: light yellow foam (0.2%), dark yellow foam (0.7%), smaller size of foam gland (5.2%), hardness of foam gland (2.3%) and other unknown factors (3.6%). Among these characteristics, the smaller sizes of foam glands played a prominent role in re¯ecting the infertility in male Japanese quail (Fig. 1). 4. Discussion In the present study, effects of hemicastration and castration were investigated on foam discharge pattern (in 24 h) in association with size of cloacal gland, foam weight, testosterone level and fertility. The experimental results indicated that in the hemicastrated group there was signi®cant (P < 0:05) reduction in foam discharge, foam weight and level of testosterone in plasma. Hemicastration also caused a decline in the sizes of the cloacal gland and fertility when compared with controls (Tables 1 and 3). The cloacal gland of Japanese quail is androgen dependent [2,3]. The reduction in the area of cloacal gland and percent fertility in the hemicastrated group appears to be mediated by reduction in circulating testosterone (Table 3). In fact, in the mechanism of hemicastration a relaxation of the pituitary gonadal and sertoli-inhibin axes occurs which causes the reduction in the amount of inhibin and testosterone plasma concentration [16,20,21]. Possibly, this may be the cause of marked reduction of testosterone levels in the plasma of male Japanese quail (Table 3). Plasma testosterone concentration correlated positively with the body weight [22,23]. Therefore, a drastic reduction in the testosterone concentration in this study may be responsible for the decreased body weight in hemicastrated and castrated groups of birds (Table 3). In contrast to our results, Wilson and de Reviers [24] and Onuora [17] reported that after hemicastration in immature birds there is an increases in plasma gonadotrophin concentration and testicular hypertrophy in cockerels with consequent enhancement of spermatogenesis [17]. This may be due to the species difference as the reproductive system of male Japanese quail is unique among the avian species due to the presence of the cloacal gland. Secondly, in the present study we employed sexually mature birds instead of immature. It is advocated that the extent of testicular hypertrophy due to hemicastration depends on the age at castration. Little or no effect is observed when the sexually active mature males are hemicastrated [12,25], which suggests that at the stage of maturity, the normal components of the spermatogenesis are established. In the absence of the compensatory mechanisms in mature birds, it would appear, therefore, that the number of sertoli cells and seminiferous
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tubules were reduced nearly 50% in mature hemicastrated birds compared with intact ones, thereby reducing the daily production of spermatozoa. The number of sertoli cells and seminiferous tubules is closely associated with the process of spermatogenesis [26]. Keeping in view the above facts, this may be the probable reason of reduction in fertility in hemicastrated birds in the present study (Table 3). Since the mating behavior of bird is associated with testosterone levels [27], a reduction in the level of testosterone in hemicastrated birds may also be partly responsible for reduced mating rates causing poor fertility in the hemicastrated group. A reduction in the foam discharge frequency (Table 1) was associated with a signi®cant drop in the area of the cloacal gland (P < 0:05), as well as weight of foam in hemicastrated group compared with control groups (Table 3). There was also a signi®cant (P < 0:01) reduction in the frequency of foam that discharges during the night when compared with daytime, irrespective of the type of groups (Table 2). This may be associated with the reduced activity of birds during night. In this regard, we do not have data in literature to compare with ours. Under the natural condition of copulation the foamy material along with semen is transferred by the male into the cloaca of female [2,28]. Perez and Sandoval [29] suggested that mixing of foam with semen prevents loss of spermatozoa when the female takes ¯ight. Wetherbee [30] advocated that foam might act as an analogue to the secretion of mammalian Cowper's gland aiding in sperm transfer. In addition to the reproductive process, most of the time (70±80%) the foam discharges were found associated with the fecal matter (data not shown). Ikeda and Taji [28] revealed that the rates and extents of foam discharges by cloacal gland appear to be random. Removal of the both testes in the castrated groups of birds cause the profound regression of cloacal glands and the complete cessation of foam discharge frequency or foam production from the cloacal gland along with negligible testosterone levels in plasma (Tables 1 and 3). This is in agreement with the observations of Siopes and Wilson [5] and Follett and Maung [6]. These researchers found a high degree of correlation between the weight of the testes and the sizes of cloacal gland. Further, cloacal gland growth and development is androgen dependent [2,3] and may not survive in the absence of testosterone as reported in the present study (Table 3). Our results clearly indicate that foam production by quail is not possible without testes. Earlier, Wilson et al. [31] and Sachs [2] have shown a positive relationship between the presence of foam in cloacal gland and weight of the testes. The mean testicular weight associated with the ®rst appearance of foam in cloacal gland was 377 mg and this occurred at the mean age of 29 days [5]. A close association of the hypertrophy of the cloacal gland with the enlarged area of the testes along with a high level of egg fertility was reported [3,32,33]. Another experiment was conducted to establish the relationship between the characteristics of foam or foam glands and infertility. The highest infertility [13] was found in the group of birds that has the smallest area of foam gland, whereas the lowest infertility [2] was recorded in the group of quails producing yellow foam (Table 4, Fig. 1). The relationship of testicular size and color of foam with fertility is in agreement with our earlier ®ndings observed in other strains of Japanese quails [34]. Japanese quails normally produce white foam and alteration from this to yellow foam may be the cause of infertility in the present study due to some abnormality in biochemical composition of yellow foam (Table 4, Fig. 1). The association of poor fertility and hatchability with abnormal-colored
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semen in turkeys has been well established [35,36]. Thurston [36] reported 14% of male turkeys in a research breeder ¯ock produced semen with yellow seminal plasma and abnormal germ cells. Abnormal spermatozoa and spermatids were among the ®rst irregularities to be associated with yellow semen production in turkeys [35,37]. An increase in the number of seminal macrophages [36] and the possible association of a herpes virus [38] were later added as characteristics of yellow semen. An elevated concentration of seminal plasma protein, cholesterol, acid phosphatase and aspartate amino transaminase were also highly correlated with the presence of a yellow color of semen and abnormal spermatids in turkeys [39]. To our knowledge, there are no reports available in the literature regarding the detailed chemical analysis of foam. Infertility contributed due to the hardness of the foam gland may be associated with certain problems linked with the connective tissue of the gland that is composed of collagen, secretory cells and elastic ®bers. The causes linked with the infertility could not be identi®ed (Table 4, Fig. 1). Probably, for those factors both the males and the females may be responsible. The physiological signi®cance of foam in quail reproduction is still controversial. Some workers reported that foam has detrimental or neutral effect on the spermatozoa [9,11], whereas others suggested that it is bene®cial [10,40]. Based on the overall results of this investigation it was noted that the secretion of thick foam from cloacal gland somehow might have a positive effect on the fertilizing ability of Japanese quail producing the foam. From the foregoing discussion it may be summarized that level of testosterone, in plasma, testicular size, area of cloacal gland and foam production all are positively correlated with the fertilizing ability of male Japanese quail (Table 3). Therefore, when males have been investigated for reproductive functions, the area of cloacal gland or foam production may be considered as a criterion that permits repeated examinations without bleeding, surgery or killing of the birds unlike, hormone assay and testicular measurement, respectively. It may, therefore, further be supplemented from this research that observations on cloacal gland and foam production may serve as a simple and effective tool for screening the infertile males from the breeding ¯ocks of Japanese quail. References [1] Etches RJ. Growth and sexual maturation. Reproduction in poultry. Wallingford (UK): CAB International, 1996. p. 74±105. [2] Sachs BD. Photoperiodic control of the cloacal gland of the Japanese quail. Science 1967;157:201±3. [3] Sachs BD. Photoperiodic control of reproductive behavior and physiology of the Japanese quail. Horm Behav 1969;1:7±24. [4] Klemm RD, Knight CE, Stein S. Gross and microscopic morphology of the glandula proctodealis (foam gland) of coturnix. J Morph 1975;141:171±84. [5] Siopes TD, Wilson WO. The cloacal gland an external indicator of testicular development in coturnix. Poult Sci 1975;54:1225±9. [6] Follett BK, Maung SL. Rate of testicular maturation in relation to gonadotrophin and testosterone levels in quail exposed to various arti®cial photoperiods and natural day lengths. J Endocrinol 1978;78: 267±80. [7] Delville Y, Sulon J, Hendrick JC, Balthazart J. Effect of the presence of females on the pituitary testicular activity in male Japanese quail (Coturnix coturnix Japonica). Gen Comp Endocrinol 1984;55: 295±305.
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[36] Thurston RJ. Physio-pathological studies of semen production in the domestic turkeys. Ph.D. dissertation. University of Missouri±Columbia, 1976. [37] Saeki K, Brown KI. Effect of abnormal spermatozoa on fertility and hatchability in the turkey. Poult Sci 1962;41:1096±100. [38] Adidinger HK, Thurston RJ, Solorzano RF, Biellier HV. Herpes virusÐa possible cause of infertility in male turkey. Arch Gesamate Virus Forsch 1974;46:370±6. [39] Hess RA, Thurston RJ. Protein cholestrol, acid phosphatase and aspartate aminotransaminase in the seminal plasma of turkeys producing normal white or abnormal yellow semen. Biol Reprod 1984;31: 239±43. [40] Fujihara N, Koga O. Physiological function of the dorsal proctodeal gland foam of the male quail. In: Proceedings of the World Quail Conference. Tallinn, USSR, 1991. p. 78±83.
Effects of hemicastration and castration on foam production and its relationship with fertility in male Japanese quail Jag Mohan*, Ram Phal Moudgal, Kochiganti Venkata Hanumat Sastry, Jagbir Tyagi, Rajvir Singh Division of Physiology and Reproduction, Central Avian Research Institute, Izatnagar, UP-243 122, India Received 15 September 2000; accepted 30 April 2001
Abstract Healthy heavy body weight strain of adult male Japanese quail (Coturnix coturnix Japonica) of the same age were used in this study to observe the effect of hemicastration and castration on the frequency of foam discharges from cloacal gland and other related parameters. The quails were housed in individual cages and divided into four groups: control (intact birds), sham-operated control (intact birds with incision), hemicastrated, and castrated groups of birds. Hemicastration and castration were carried out surgically at 10 and 13 weeks of age, respectively. Subsequently, 3 weeks after castration birds were examined for different parameters. Hemicastration caused a signi®cant (P < 0:05) drop in the foam discharge frequency, weight of the foam, and the level of testosterone in the plasma, whereas in castrated group these variables were observed nil or negligible as compared to both of the controls. A suppressive effect of hemicastration was also noticed on the body weight, area of the cloacal gland, as well as percent fertility. Castration induced the drastic regression of the cloacal gland and a signi®cant reduction (P < 0:05) of the body weight was also noted as compared to other groups. Frequencies of foam discharges were twice the number in daytime (06:00±18:00 h) than night, irrespective of the groups. In another study, the effect of characteristics of foam or foam glands on fertility was examined in 77 male birds paired with females. Several characteristics of foam and foam glands were examined for infertility, such as light yellow foam, dark yellow foam, smaller area of foam gland (below 225 mm2), hardness of foam gland and certain unknown factors that contributed 0.23, 0.68, 5.23, 2.27 and 3.64% infertility, respectively. An overall 12.1% birds were found to be infertile. This data indicated that smaller sizes of foam glands might re¯ect the poorest fertilizing ability of the male birds. From this study, it may be concluded that the cloacal gland may be considered as an external indicator of testicular function of the birds. Characteristics of the cloacal gland or foam or both may be used as a
* Corresponding author. Tel.: 91-581-446-420; fax: 91-581-447-321. E-mail address: [email protected] (J. Mohan).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 8 6 3 - 4
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simple and effective tool to predict the fertilizing ability of an individual male in view of the very small semen ejaculate that is very dif®cult to collect and evaluate for fertility. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Japanese quail; Cloacal gland; Foam; Castration; Testes
1. Introduction The development of secondary sexual characteristics in domestic fowl, such as comb and wattles, have been used as external indicators for determining the sex of birds and the stage of sexual maturation. Androgens are required to induce the growth of the comb and wattles in domestic fowl [1]. Similarly, an androgen dependent cloacal gland in male Japanese quail (Coturnix coturnix Japonica) was found as a reliable indicator for testicular function [2,3]. The cloacal gland has not been found in other genus of birds, therefore, they are speci®c only to the member of the genus Coturnix [4]. There is a high correlation between the size of the cloacal gland and the weight of the testes. Both increase with the age of the birds [5,6]. Paired males have larger cloacal glands and testicular size and higher plasma levels of testosterone and LH when compared with the isolated males [7]. The cloacal gland of sexually active male Japanese quail under the in¯uence of testosterone produce the foamy material constantly, irrespective of the time of the day [2]. Administration of testosterone to the castrated male Japanese quail stimulates the growth of the cloacal gland and synthesis of the foamy material within the gland [8]. The physiological signi®cance of foamy material in Japanese quail reproduction has been the subject of con¯icting reports for over three decades [9±11]. Hemicastration causes hypertrophy of the testes in some mammalian species, such as rabbits, mice, rats, hamsters, and rams [12±16], and results in changes in the plasma gonadotrophin level, testicular weight and spermatogenesis. Also after hemicastration in avian species a compensatory hypertrophy of the remaining testis in domestic fowl was reported [17]. To our knowledge, no information is available on the effects of castration and hemicastration on the foam discharge pattern and various other variables, such as size of foam gland, foam weight, testosterone level in blood and fertilizing ability of quail spermatozoa. However, a few reports are available on the effects of castration (not hemicastration) and hormone treatment on the histology and development of the cloacal gland [2,8]. In addition, to date no conclusive evidence is present in literature regarding the function of foam in Japanese quail reproduction. In view of the complete lack of information on the above areas, this work was carried out to investigate the effects of hemicastration and castration on the foam production and certain other variables as described previously. Further, the direct evaluation of the fertilizing ability of male Japanese quail by semen examination is dif®cult because it is very dif®cult to obtain the neat semen without any contamination with foam. As the cloacal gland is androgen dependent, it is hypothesized whether the size of cloacal gland or frequency of foam discharges from this gland could be used as an indirect tool to predict the fertilizing ability of particular males.
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2. Materials and methods Heavy body strain of sexually mature males (>8 weeks of age) of Japanese quail (C. coturnix Japonica) was used in this investigation. They were kept in individual cages and were maintained under the uniform husbandry conditions with the temperature between 25 and 30 8C. They were given normal quail breeder ration and water ad libitum with a constant light 14 h per day during the experiment. During the course of the experiment the same technical personnel were allowed to feed, water and collect the data from birds. At the age of 10 weeks the birds were arbitrarily divided into four groups of 18 birds in each. The ®rst group served as control (intact birds), whereas second group was the sham-operated control group with an incision in the last intercostal space of the birds under the local anesthetic (xylocaine 2%). In the third and fourth group, the left testis was removed surgically through an incision made between the last two intercostal ribs under the local anesthesia [17]. All the birds were hemicastrated within 2±3 days. In this similar way after 3 weeks of hemicastration, the remaining testis (right) was also removed only in half of the hemicastrated birds. Now, birds missing one or both of the testes have been termed as hemicastrated (third group) and castrated (fourth group), respectively. Subsequently, 3 weeks after castration the number of birds were ®xed at six in each group. They were examined for foam discharge frequency and certain other variables, like size of the cloacal gland, foam production, testosterone level in plasma, and fertilizing ability of birds. To examine the foam discharge pattern during 24 h at different times of intervals morning, afternoon and night, the foam masses discharged from the cloacal glands of male birds were counted in fecal trays kept under each bird. After each count of the foam discharge, trays were cleaned properly and replaced immediately in the cages for the next count of foam discharge from the birds. To determine the foam produced by each bird, foam was collected [18] in airtight glass bottles to prevent evaporation. Quantitative measurement of foam was done immediately using electronic balance up to fourth digit. The size of the cloacal gland was measured by employing Vernier callipers as per the method described by Siopes and Wilson [5]. For the hormone assay, the blood samples were collected from the wing vein of the male using heparinized syringes. All the samples were immediately centrifuged and plasma was taken out for hormone assay. Subsequently, testosterone was assayed in duplicate in all the samples using a Spactromax-190 micro plate reader by using the testosterone kit (ELA, DSL-10-4000) received from Diagnostic System Laboratories Inc., Webster, TX). To examine their fertilizing ability, male birds were paired in suitable cages with equal number of healthy females (>10 weeks of age) so that each adult male (control or hemicastrated) was allowed to mate with one female (1:1). After 2 weeks of pairing, eggs were collected daily for 2 weeks from females of each group and averaged for two replications (one replication per week). Experimental eggs were stored under standard conditions. Subsequently, all the eggs were incubated for 10 days in hatchery. Thereafter, eggs were broken to con®rm the fertilization macroscopically by the presence of developing embryo. The egg fertility was determined as the ratio of the number of fertile eggs to the number of total eggs set in the incubator. In a second experiment designed to study the relationship between the characteristics of foam or foam glands with the fertilizing ability of male Japanese quail, foam was
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collected from 77 heavy body weight strain, sexually active male Japanese quail and classi®ed on the basis of color: normal white, light yellow, and dark yellow foam. A foam gland was identi®ed as a small size if the area of the gland was below 225 mm2. Determination of hardness of the foam gland was evaluated on the basis of the pressure applied by ®ngers around the cloacal gland at the time of foam collection. Under most of the cases foam could be obtained by applying gentle pressure around the cloacal gland. In this regard the cloacal gland behaved just like a tube of toothpaste. We could not categorize some cases of infertility in birds for which both males and females may be responsible and termed these cases as unknown factors of infertility. Signi®cant differences between means were compared by employing Student's t-test. The correlation coef®cient between sizes of cloacal gland and male reproductive indicators (testicular weight, testosterone level, and foam production) were also determined as per the standard method [19].
Table 1 Normal values (mean S:E:M:, 11 replicates) of foam discharge frequency of each bird in various groups of Japanese quail at different daily intervals Intervals
Bird number
Control group
Sham-operated control group
Hemicastrated group
Morning (06:00±12:00)
1 2 3 4 5 6
5.44 9.33 7.88 6.55 11.32 8.10
6.77 8.39 7.11 7.57 8.37 6.90
5.22 5.62 6.77 4.00 6.66 5.57
Average value range Afternoon (12:00±18:00)
1 2 3 4 5 6 Average value range
Night (18:00±06:00)
1 2 3 4 5 6 Average value range
0.77 0.80 0.65 0.60 0.74 0.67
8.10 0.84 5.44±11.32 5.11 8.84 6.00 7.22 10.77 7.59
0.33 0.41 0.60 0.53 0.85 0.23
7.59 0.82 5.11±10.77 6.11 9.33 7.22 9.44 10.66 8.55
0.43 0.35 0.32 0.67 0.47 0.73
8.55 0.67 6.11±10.66
0.65 0.44 0.33 0.68 0.47 0.66
0.44 0.33 0.62 0.33 0.60 0.29
7.51 0.29 6.77±8.39
5.64 0.41* 4.00±6.77
8.15 8.10 7.75 7.15 8.55 6.47
4.11 3.88 5.55 2.88 7.00 4.68
0.44 0.35 0.64 0.32 0.58 0.29
0.33 0.33 0.25 0.47 0.68 0.33
7.69 0.31 6.47±8.55
4.68 0.58* 2.88±7.00
7.44 4.77 8.65 7.19 8.75 7.05
7.44 4.77 4.71 3.11 6.11 5.23
0.44 0.23 0.42 0.49 0.65 0.35
7.69 0.35 6.70±8.75
0.44 0.33 0.25 0.38 0.73 0.62
5.22 0.59* 3.11±7.44
* Average mean values of hemicastrated group only differed signi®cantly (P < 0:05) from other groups (Student's t-test). Castrated group produced no foam.
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3. Results A summary of the normal values on the frequency of foam discharge during day and night hours in control, sham-operated control and hemicastrated group of heavy body weight strain of male Japanese quail is presented in Tables 1 and 2. Ejection of foam from the cloacal gland was examined at ®xed timings: 12:00, 18:00 and 06:00 h for the interval of morning (06:00±12:00 h), afternoon (12:00±18:00 h) and night (18:00±06:00 h), respectively. Frequency of foam discharge from the cloacal gland was evaluated for 11 days within 30 min of these ®xed timings in all the groups. The foam discharge pattern for castrated group was not shown in Table 1, as there was no foam production in this group. The frequency of foam discharge was signi®cantly (P < 0:05) reduced in hemicastrated group in comparison to other control groups at all the intervals examined in this study. It was observed in all the groups that foam discharge from cloacal gland was nearly double in daytime (06:00±1800 h) than night (18:00±06:00 h). On an average, the normal control birds discharge the foam after each hour (Table 2). No signi®cant differences were observed in body weight and sizes of cloacal glands between hemicastrated and control groups, whereas in the castrated group the body weight of birds was reduced signi®cantly (P < 0:05) and the cloacal glands regressed drastically (Table 3). A signi®cant (P < 0:05) reduction in average foam weight, as well as testosterone level in plasma, was noticed in hemicastrated group when compared with the controls. Similarly, a Table 2 Foam discharge frequency in different groups of Japanese quail during day and night (mean S:E:M:, 11 replicates) Intervals
Control group
Sham-operated control group
Hemicastrated group
Night (18:00±06:00) Day (06:00±18:00) Night day (24 h)
8.55 0.67 15.69 1.66 24.24 2.33
7.69 0.35 15.20 0.60 22.89 0.95
5.22 0.59** 10.32 0.99** 15.54 1.58**
** Mean values of hemicastrated group by row differed signi®cantly (P < 0:01) from control and shamoperated group (Student's t-test). Castrated group did not discharge foam.
Table 3 Effects of hemicastration and castration on the different variables of Japanese quail (mean S:E:M:, n 6, except fertility) Group
Body weight (g)
Size of cloacal gland (mm2)
Foam weight (ng)
Testosterone (ng/ml)
Fertility (%)
Control group Sham-operated control group Hemicastrated group Castrated group
238 17.50 233 14.31
355.20 19.77 366.83 15.11
21.51 0.35 20.27 0.43
1.693 0.150 1.625 0.170
91 87
17.55 0.79* 0.00
0.921 0.210* 0.010 0.003
79 Not examined
a
218 11.33a 317.53 17.21 181 13.89a,* Regressed
Body weight data was given after deducting the corresponding testicular weight. Mean values of hemicastrated and castrated group differed signi®cantly (P < 0:05) from other groups (Student's t-test). *
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Table 4 Effects of the characteristics of foam and foam gland on fertility of Japanese quail Characteristics of foam and foam gland
Total bird no.
Total eggs
Total infertile eggs
Total fertile eggs
Fertility (%)
Infertility (%)
Normal white foam Light yellow foam Dark yellow foam Smaller area of foam gland (below 225 mm2) Hardness of foam gland Unknown factors
47 1 1 13
262 9 16 65
0 1 3 23
262 8 13 42
100.00 88.88 81.25 64.61
0 11.12 18.75 35.39
7 8
40 48
10 16
30 32
75.00 66.66
25.00 33.34
Grand total
77
440
53
387
87.95
12.05
drop in percent fertility was also found in the same group (Table 3). An overall positive correlation was observed for the size of cloacal gland with testicular weight (R2 0:36, P < 0:01), testosterone level (R2 0:40, P < 0:005) and foam production (R2 0:16, P < 0:10) of the male Japanese quail. In the second experiment, it was observed that 47 birds were producing normal white foam expressing 100% fertility. Deviation from normal white foam such as light yellow or dark yellow foam caused the reduction of 11.1 and 18.7% infertility in those birds
Fig. 1. A total of 12.05% infertility attributed by characteristics of foam glands in male Japanese quail.
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producing such kind of foam. Two birds produced yellow foam out of total 77 birds (Table 4). Birds containing smaller sizes of foam glands (<225 mm2) attributed more (35.4%) towards infertility. In the present study during foam collection, it was realized that seven male birds required comparatively more ®nger pressure around the cloacal gland to get the foam released than others (hardness of foam gland) and a 25% decline in fertility was noted in these birds. On the basis of the color of foam or characteristics of foam gland we could not categorize eight birds, because of which 33.3% infertility in this study was related to certain unknown (mixed) factors. An overall 12.1% infertility was revealed in the group of 77 male Japanese quail (Table 4). The overall relationship of different variables of characteristics of foam or foam glands towards total infertility (12.1%) were as follows: light yellow foam (0.2%), dark yellow foam (0.7%), smaller size of foam gland (5.2%), hardness of foam gland (2.3%) and other unknown factors (3.6%). Among these characteristics, the smaller sizes of foam glands played a prominent role in re¯ecting the infertility in male Japanese quail (Fig. 1). 4. Discussion In the present study, effects of hemicastration and castration were investigated on foam discharge pattern (in 24 h) in association with size of cloacal gland, foam weight, testosterone level and fertility. The experimental results indicated that in the hemicastrated group there was signi®cant (P < 0:05) reduction in foam discharge, foam weight and level of testosterone in plasma. Hemicastration also caused a decline in the sizes of the cloacal gland and fertility when compared with controls (Tables 1 and 3). The cloacal gland of Japanese quail is androgen dependent [2,3]. The reduction in the area of cloacal gland and percent fertility in the hemicastrated group appears to be mediated by reduction in circulating testosterone (Table 3). In fact, in the mechanism of hemicastration a relaxation of the pituitary gonadal and sertoli-inhibin axes occurs which causes the reduction in the amount of inhibin and testosterone plasma concentration [16,20,21]. Possibly, this may be the cause of marked reduction of testosterone levels in the plasma of male Japanese quail (Table 3). Plasma testosterone concentration correlated positively with the body weight [22,23]. Therefore, a drastic reduction in the testosterone concentration in this study may be responsible for the decreased body weight in hemicastrated and castrated groups of birds (Table 3). In contrast to our results, Wilson and de Reviers [24] and Onuora [17] reported that after hemicastration in immature birds there is an increases in plasma gonadotrophin concentration and testicular hypertrophy in cockerels with consequent enhancement of spermatogenesis [17]. This may be due to the species difference as the reproductive system of male Japanese quail is unique among the avian species due to the presence of the cloacal gland. Secondly, in the present study we employed sexually mature birds instead of immature. It is advocated that the extent of testicular hypertrophy due to hemicastration depends on the age at castration. Little or no effect is observed when the sexually active mature males are hemicastrated [12,25], which suggests that at the stage of maturity, the normal components of the spermatogenesis are established. In the absence of the compensatory mechanisms in mature birds, it would appear, therefore, that the number of sertoli cells and seminiferous
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J. Mohan et al. / Theriogenology 58 (2002) 29±39
tubules were reduced nearly 50% in mature hemicastrated birds compared with intact ones, thereby reducing the daily production of spermatozoa. The number of sertoli cells and seminiferous tubules is closely associated with the process of spermatogenesis [26]. Keeping in view the above facts, this may be the probable reason of reduction in fertility in hemicastrated birds in the present study (Table 3). Since the mating behavior of bird is associated with testosterone levels [27], a reduction in the level of testosterone in hemicastrated birds may also be partly responsible for reduced mating rates causing poor fertility in the hemicastrated group. A reduction in the foam discharge frequency (Table 1) was associated with a signi®cant drop in the area of the cloacal gland (P < 0:05), as well as weight of foam in hemicastrated group compared with control groups (Table 3). There was also a signi®cant (P < 0:01) reduction in the frequency of foam that discharges during the night when compared with daytime, irrespective of the type of groups (Table 2). This may be associated with the reduced activity of birds during night. In this regard, we do not have data in literature to compare with ours. Under the natural condition of copulation the foamy material along with semen is transferred by the male into the cloaca of female [2,28]. Perez and Sandoval [29] suggested that mixing of foam with semen prevents loss of spermatozoa when the female takes ¯ight. Wetherbee [30] advocated that foam might act as an analogue to the secretion of mammalian Cowper's gland aiding in sperm transfer. In addition to the reproductive process, most of the time (70±80%) the foam discharges were found associated with the fecal matter (data not shown). Ikeda and Taji [28] revealed that the rates and extents of foam discharges by cloacal gland appear to be random. Removal of the both testes in the castrated groups of birds cause the profound regression of cloacal glands and the complete cessation of foam discharge frequency or foam production from the cloacal gland along with negligible testosterone levels in plasma (Tables 1 and 3). This is in agreement with the observations of Siopes and Wilson [5] and Follett and Maung [6]. These researchers found a high degree of correlation between the weight of the testes and the sizes of cloacal gland. Further, cloacal gland growth and development is androgen dependent [2,3] and may not survive in the absence of testosterone as reported in the present study (Table 3). Our results clearly indicate that foam production by quail is not possible without testes. Earlier, Wilson et al. [31] and Sachs [2] have shown a positive relationship between the presence of foam in cloacal gland and weight of the testes. The mean testicular weight associated with the ®rst appearance of foam in cloacal gland was 377 mg and this occurred at the mean age of 29 days [5]. A close association of the hypertrophy of the cloacal gland with the enlarged area of the testes along with a high level of egg fertility was reported [3,32,33]. Another experiment was conducted to establish the relationship between the characteristics of foam or foam glands and infertility. The highest infertility [13] was found in the group of birds that has the smallest area of foam gland, whereas the lowest infertility [2] was recorded in the group of quails producing yellow foam (Table 4, Fig. 1). The relationship of testicular size and color of foam with fertility is in agreement with our earlier ®ndings observed in other strains of Japanese quails [34]. Japanese quails normally produce white foam and alteration from this to yellow foam may be the cause of infertility in the present study due to some abnormality in biochemical composition of yellow foam (Table 4, Fig. 1). The association of poor fertility and hatchability with abnormal-colored
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semen in turkeys has been well established [35,36]. Thurston [36] reported 14% of male turkeys in a research breeder ¯ock produced semen with yellow seminal plasma and abnormal germ cells. Abnormal spermatozoa and spermatids were among the ®rst irregularities to be associated with yellow semen production in turkeys [35,37]. An increase in the number of seminal macrophages [36] and the possible association of a herpes virus [38] were later added as characteristics of yellow semen. An elevated concentration of seminal plasma protein, cholesterol, acid phosphatase and aspartate amino transaminase were also highly correlated with the presence of a yellow color of semen and abnormal spermatids in turkeys [39]. To our knowledge, there are no reports available in the literature regarding the detailed chemical analysis of foam. Infertility contributed due to the hardness of the foam gland may be associated with certain problems linked with the connective tissue of the gland that is composed of collagen, secretory cells and elastic ®bers. The causes linked with the infertility could not be identi®ed (Table 4, Fig. 1). Probably, for those factors both the males and the females may be responsible. The physiological signi®cance of foam in quail reproduction is still controversial. Some workers reported that foam has detrimental or neutral effect on the spermatozoa [9,11], whereas others suggested that it is bene®cial [10,40]. Based on the overall results of this investigation it was noted that the secretion of thick foam from cloacal gland somehow might have a positive effect on the fertilizing ability of Japanese quail producing the foam. From the foregoing discussion it may be summarized that level of testosterone, in plasma, testicular size, area of cloacal gland and foam production all are positively correlated with the fertilizing ability of male Japanese quail (Table 3). Therefore, when males have been investigated for reproductive functions, the area of cloacal gland or foam production may be considered as a criterion that permits repeated examinations without bleeding, surgery or killing of the birds unlike, hormone assay and testicular measurement, respectively. It may, therefore, further be supplemented from this research that observations on cloacal gland and foam production may serve as a simple and effective tool for screening the infertile males from the breeding ¯ocks of Japanese quail. References [1] Etches RJ. Growth and sexual maturation. Reproduction in poultry. Wallingford (UK): CAB International, 1996. p. 74±105. [2] Sachs BD. Photoperiodic control of the cloacal gland of the Japanese quail. Science 1967;157:201±3. [3] Sachs BD. Photoperiodic control of reproductive behavior and physiology of the Japanese quail. Horm Behav 1969;1:7±24. [4] Klemm RD, Knight CE, Stein S. Gross and microscopic morphology of the glandula proctodealis (foam gland) of coturnix. J Morph 1975;141:171±84. [5] Siopes TD, Wilson WO. The cloacal gland an external indicator of testicular development in coturnix. Poult Sci 1975;54:1225±9. [6] Follett BK, Maung SL. Rate of testicular maturation in relation to gonadotrophin and testosterone levels in quail exposed to various arti®cial photoperiods and natural day lengths. J Endocrinol 1978;78: 267±80. [7] Delville Y, Sulon J, Hendrick JC, Balthazart J. Effect of the presence of females on the pituitary testicular activity in male Japanese quail (Coturnix coturnix Japonica). Gen Comp Endocrinol 1984;55: 295±305.
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