Mating

Mating takes place throughout the season of activity but is most common in spring--soon after emergence from hibernation--and in autumn. Turtles frequently copulated in the laboratory in spring and autumn. Copulation was observed under natural conditions on several occasions but only once at the Damm Farm.

Norris and Zwiefel (1950:4) saw two captive individuals of _T. o.

luteola_ copulating on 12 August; copulation lasted two hours.

Brumwell (1940:391-2) gave the following description of mating in _T.

o. ornata_. A male pursued a female for nearly half an hour, first nudging the margins of her sh.e.l.l and later approaching her rapidly from the rear and hurling himself on her back in an attempt to mount, at the same time emitting a stream of liquid from each nostril. The liquid was presumably water; both s.e.xes had imbibed water in a pond just before courtship began. Brumwell suggested that pressure on the plastron of the male had forced the water out his nostrils. The pair remained in the coital position for 30 minutes after the male had achieved intromission. In another instance, Brumwell (_loc. cit._) saw four males pursuing a single female, the males exhibiting the same behavior (nudging and lunging) outlined above. Males that attempted to mount other males were repelled by defensive snapping of the approached male. The female also snapped at some of the males that tried to mount her. One male was finally successful in mounting and was henceforth unmolested by the other males. Brumwell suggested that sh.e.l.l biting and tapping may be methods of s.e.x-recognition.

In the several instances of mating that I observed, the male, after mounting the sh.e.l.l of the female (Pl. 28), gripped her, with the first claws of his hind feet, just beneath her legs or on the skin of the gluteal region and, with the remaining three claws, gripped the posterior edges of her plastron. In most instances the female secured the male's legs by hooking her own legs around them. The coital position of _T. ornata_ seems to differ from that of _T. carolina_, at least in regard to the position of the male's legs. The coital positions of _T. carolina_ ill.u.s.trated by Cahn (1937:94, Fig. 13) are physically impossible for _T. ornata_.

In _T. ornata_ the pressure exerted on the male's legs by the female probably impairs circulation and probably is painful to the male, especially after coitus, when the male falls backward but is still held by the female. The heavily developed musculature of the legs of males may be an adaptation to strengthen the legs for this temporary period of stress. Evans (1953:191) and Cahn and Conder (1932:87-88) observed the hind legs of males of _T. carolina_ to be noticeably weakened after copulation, causing the males to remain inactive for several hours.

Evans (_op. cit._) observed 72 matings of _T. carolina_ and divided the process into three phases as follows: 1) circling, pushing and biting by the male; 2) mounting (female with sh.e.l.l closed); and, 3) coition (female with sh.e.l.l open). Penn and Pottharst (1940:26) reported that captive _T. carolina_ in New Orleans mated chiefly under conditions of optimum temperature (21 to 27 C.) and high humidity; some matings took place in a pool of water. Males pushed females about after mating, often rolling them over several times.

Because ornate box turtles observed by me were able easily to right themselves from an inverted position on substrata of all kinds, males left lying on their backs after copulation are probably in no danger of perishing in this position, as was suggested by Allard (1939) for _T. carolina_.

Insemination

Oviducts of several females were flushed by means of a pipette to determine whether they contained sperm. Approximately half of the females captured in May, 1956, had sperm in their oviducts, but females captured in June and July did not. Sperm flushed from the oviducts were in clumps of several hundred and showed no sign of motility a few minutes after the female was anesthetized with chloroform. No sperm were found in the oviducts of immature females but one female of nearly adult size was observed in copulation with a mature male.

Thorough examination of microscopic sections of oviduct (taken at various times in the season of activity) usually revealed a few sperm lodged in the folds (Pl. 19, Fig. 8) of the cephalic as well as the caudal portion of the tube, but no specialized seminal receptacles such as occur in snakes (Fox, 1956) were present. Fertilization without reinsemination probably occurs in _T. ornata_. Ewing (1943) and Finneran (1948:126) reported that females of _T. carolina_ produced fertile eggs for periods of four and two years, respectively, after being removed from all contact with males.

s.e.xual Cycle of Males

Testes were preserved in each month from April to October. The following description of spermatogenesis is based chiefly on material collected in 1955, although testes were preserved also in 1954.

Comparison of material obtained in 1954 and 1955 revealed that spermatogenesis began earlier and was more advanced on any given date in 1955 than in 1954.

Testes of mature individuals are pale yellow and slightly oblong. The epididymis is ordinarily dark brown or black and contrasts sharply with the color of the testes. Size of testes was expressed as the average length (greatest diameter) of both testes. Testes are smallest in April, immediately after emergence from hibernation, and largest in early September (Pl. 20, Figs. 3-4). They are nearly spherical when of maximum size; increase in bulk, therefore, is relatively greater than the increase in size shown in Figure 3. They increase in size from April until early June, recede during most of June, and again increase in size in July and August. They remain large from early September until hibernation is begun, becoming only slightly smaller in late September and October.

Increase in size following emergence from hibernation may be due in part to proliferation of the sustentacular cytoplasm. Decrease in size in early June is correlated with the end of the period of most active mating; maximal size is coincident with the peak of the spermatogenic cycle in early September.

[Ill.u.s.tration: FIG. 3. Seasonal fluctuations in size (average greatest diameter) of testes in _T. o. ornata_ as determined by examination of 40 specimens from eastern Kansas.]

Spermatogenesis (refer to Pl. 19, Figs. 1-5) begins in early May when a few spermatogonia appear in the seminiferous tubules. The histological appearance of testes preserved in April and May is much the same. Nuclei of Sertoli cells, which outnumber the spermatogonia, are evident at the periphery of the tubules and the clear cytoplasm of the cells extends into and nearly fills the lumina. The few darkly stained spermatids that are present in April are cells that probably were produced in the previous summer. Sperm are present in small groups within the sustentacular cytoplasm, but ordinarily are absent in the lumina.

Primary spermatocytes appear in the tubules from mid-May to early June. By mid-May there are practically no sperm at any place in the tubules. The sustentacular cytoplasm has a less compact arrangement in late May than in April.

Spermatogenesis is well under way by mid-June; at this time, two or three distinct layers of primary and secondary spermatocytes are present and these cells outnumber the Sertoli cells. The lumina are filled with cellular detritus and are no longer bordered by a clear ring of sustentacular cytoplasm. No sperm are present.

Spermatids appear in late June and a few of them undergo metamorphosis in early July; by mid-July, spermatids and secondary spermatocytes are the dominant cells in the seminiferous tubules, although spermatogonia are still active.

By late August, cl.u.s.ters of sperm and metamorphosing spermatids surround the Sertoli cells; large numbers of sperm as well as sloughed cells representing various spermatogenic stages are present in the lumina. Secondary spermatocytes are still evident near the periphery of the tubules but they are much less numerous than spermatids. The germinal epithelium is still semiactive and small groups of primary spermatocytes are present in nearly all of the tubules.

The spermatogenic cycle is completed in the latter half of October when most of the spermatozoa pa.s.s into the epididymides. A few spermatozoa and spermatids remain in the seminiferous tubules during hibernation. Although no testicular material was obtained from hibernating turtles, comparisons of sections made in October and April show that the germinal epithelium remains inactive from autumn until spring. Possibly some spermiogenesis takes place in the early phases of hibernation or in the period in late autumn when turtles are intermittently active. It is uncertain whether the reorganization of the sustentacular cytoplasm occurs in autumn, in spring, or in the course of hibernation.

The seminiferous tubules of immature males are small, lack lumina, and contain a few large but inactive spermatogonia (Pl. 19, Fig. 6). The testes of specimens that were nearly mature contained primary and secondary spermatocytes but lacked lumina; it was thought that such individuals would have matured in the following summer and bred in the following autumn.

Mature sperm were found in epididymides at all times of the year but were most numerous in spring and autumn, the period between spermatogenic cycles (Pl. 19, Fig. 7). Sperm expelled from the epididymides in autumn matings are seemingly replaced by others from the seminiferous tubules; the epididymides become much smaller when their supply of sperm is nearly exhausted after spring mating.

Risley (1938:304) found the testes of the common musk turtle, _Sternotherus odoratus_, to be largest in August and smallest in early May. Recession of testes in spring was coincident with the period of active breeding; increase in size, later in the season, corresponded to increasing spermatogenic activity and enlargement of seminiferous tubules. Altland (1951:600-603) found the spermatogenic cycle of _Terrapene carolina_ to be nearly like that of _Sternotherus odoratus_. Fox (1952) found that testes of garter snakes (_Thamnophis sirtalis_ and _T. elegans_) in California reached a peak of spermatogenic activity in midsummer, regressed in the latter half of the summer, and were inactive in winter.

The spermatogenic cycle of _T. ornata_ as here reported, differs in no important respect from those of _Thamnophis_, _Sternotherus odoratus_, or _Terrapene carolina_, except that in _T. ornata_ the cycle begins and ends somewhat later in the season of activity. In most of the lizards that have been studied (Fox, 1952:492-3), spermatogenesis reaches a peak in spring (more or less coincident with the mating period and with ovulation) and the germinal epithelium remains active in winter. _Sternotherus_, _Terrapene_, and _Thamnophis_ are alike in completing spermatogenesis late in the season and storing spermatozoa, in the seminiferous tubules or in the epididymides, during hibernation.

It is noteworthy that, in the turtles and snakes mentioned above, sperm produced in autumn are used to fertilize eggs laid in the following year, and mating [with the exception of _Thamnophis elegans_, (Fox, 1956)] occurs in both spring and autumn. It is not definitely known in any of these instances, whether sperm resulting from autumn or spring inseminations (or both) fertilize the eggs.

Risley (1933:693) found motile sperm in the oviducts of female _Sternotherus odoratus_ that had recently emerged from hibernation; he believed that spring mating, although it commonly occurred, was not necessary to fertilize eggs. Disadvantages, if any, of completing spermatogenesis well in advance of ovulation seem to be at least partly counteracted by two annual mating periods or by mating throughout the season of activity.

s.e.xual Cycle of Females

The following account of oogenesis is based on examination of preserved ovaries from 68 mature specimens. The ages of most specimens were known, inasmuch as the specimens were used in studies of growth as well as gametogenesis. Other data were obtained from adult females that were dissected but not preserved, and from immature females.

[Ill.u.s.tration: FIG. 4. Seasonal fluctuations in ovarian weight in _T. o. ornata_, as determined by examination of 60 specimens from eastern Kansas.]

Size of ovarian follicles was determined by means of a clear plastic gauge containing notches 5, 10, 15, 20, and 25 millimeters wide. The number of follicles within a given size range could be quickly determined by finding the smallest notch into which the follicles fit.

It was necessary to weigh all ovaries after preservation since some of them had not been weighed when fresh. Since all ovarian samples were preserved in the same manner, weights remained relatively the same.

Preserved material was lighter than fresh by an average of 13 per cent. Follicles less than one millimeter in diameter were not counted.

Corpora lutea and corpora albicantia were studied under a binocular dissecting microscope. No histological studies were made of the female reproductive system.

Ovarian follicles and oviducal eggs were recorded separately for the right and left sides. Each ovary was always kept a.s.sociated with the oviduct of the same side, but in some instances it was not recorded whether the organs were left or right.

Ovaries ordinarily weighed most in October, March, and April, when most females contained enlarged follicles, and least in August and September when the supply of enlarged follicles was usually exhausted (Figs. 4 and 5).

[Ill.u.s.tration: FIG. 5. The seasonal occurrence of enlarged ovarian follicles in females of _T. o. ornata_, expressed, For each month, as the percentage of total females that contained two or more follicles having diameters greater than 15 mm. Total number of females in each of the samples is shown in parentheses at the top of each bar.]

The ovarian cycle begins in July or August, after ovulation has occurred. At that time many minute follicles form on the germinal ridges of the ovaries. On the basis of the material that I examined, it seems that ovarian follicles either grow to nearly mature size in the season preceding ovulation and remain quiescent over winter or grow rapidly in the period of approximately six weeks between spring emergence and ovulation. Altland (1951:603-5) reported that the former condition was the usual one in _T. carolina_; he suggested that possibly some of the enlarged follicles were absorbed during hibernation.

Examination of yolks of oviducal eggs revealed that follicles mature when they reach a diameter of 16 to 20 millimeters and a weight of two to two and one-half grams (Pl. 20, Fig. 1).

The enlarged follicles remaining on the ovaries after ovulation (excluding those smaller than six mm.) can be grouped according to diameter as: large (greater than 15 mm.), medium (11 to 15 mm.), and small (six to 10 mm.). Ten females collected in the period from June 2 to 8, after they had ovulated, all had follicles falling in at least one of these size groups, and eight had follicles falling in two or more of the groups. In females having enlarged follicles of more than one of the size groups, there were several follicles in each of two groups and no follicles, or only one follicle, in the remaining group.

Enlarged follicles represent future clutches but whether the enlarged follicles will be ovulated in the same season or in a later season is questionable.

Evidence found in the present study suggested that at least a few females lay more than one clutch of eggs per year. Among 34 specimens obtained in June and July, eight (24 per cent) had corpora lutea (or easily discernible corpora albicantia) and at least two follicles more than 15 millimeters in diameter; in three specimens (9 per cent) the ovaries bore fresh corpora lutea (representing recent ovulations) and a set of older corpora lutea (representing ovulations that had occurred several weeks previously). It was thought that each of these eleven females (33 per cent of sample) had produced or would have produced two clutches of eggs in the season of its capture. The number of large follicles present after the first set of ovulations (mean, 3.5) was fewer in most instances than the average clutch-size (see below), indicating that second clutches are smaller than first clutches. Smaller second clutches were found also in _T. carolina_ (Legler, 1958).

Further evidence for multiple clutches was the absence of enlarged ovarian follicles in some females obtained in September. Atretic follicles, ordinarily orange, brown, or purplish, were observed on the ovaries of many of the females examined; in most instances, not more than two follicles of the small or medium size groups were atretic.

Atresia was in no instance great enough to account for the complete loss of enlarged follicles.

Further study probably will show that many of the females laying in May and early June lay again before the end of July, and that eggs in the oviducts of females captured in the latter month frequently represent second clutches. Under favorable conditions, eggs laid by the end of July would have a good chance of hatching before the advent of cold weather in autumn; turtles hatching too late to escape from the nest could burrow into its sides and probably escape freezing temperatures.

Cagle's findings concerning _Pseudemys scripta_ (1950:38) and _Chrysemys picta_ (1954:228-9) suggest that these species lay more than one clutch per season, at least in the southern parts of their ranges. Carr (1952) indicated that multiple layings were known in most species of marine turtles (families Dermochelydae and Chelonidae) and strongly suspected in other species. Other turtles recorded to have produced multiple clutches in a single season (based chiefly on captive specimens or cultured populations) include: the starred tortoise, _Geochelone elegans_ (Deraniyagala, 1939:287); the Asiatic trionychid, _Lissemys punctata_ (_op. cit._:304); the diamond-backed terrapin, _Malaclemys terrapin_ (Hildebrand and Prytherch, 1947:2); and the j.a.panese soft-sh.e.l.led turtle, _Trionyx j.a.ponicus_ (Mitsukuri, 1895, cited by Cagle, 1950:38).

There is a marked alternation of ovarian activity in _T. ornata_, one ovary being more active than its partner in a given season. The less active ovary is more active than its partner in the following season.

For example, a specimen killed in July had four corpora lutea on the right ovary and two on the left and there were five enlarged follicles (of the medium size group), representing the next set of eggs to be ovulated, four on the left ovary and one on the right. Similar alternation of ovarian activity was observed, to a greater or lesser extent, in nearly all of the females examined. Many subadult females that were approaching their first breeding season (as evidenced by the presence of large ovarian follicles but no indication of former ovulation) had but one active ovary. This may account in part for the tendency of small, young females to lay clutches smaller than average.

One ovary may become senile in old females before its partner does; this may explain the occasional absence or atrophy of one ovary in large females that I have examined.

In all the specimens examined, it was evident that ovulation had occurred or would occur in two successive seasons. Senile or young females might, however, be expected to skip a laying season if only one ovary was functioning.