Part 5

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.

Sexual 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.

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, clusters 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 pass 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.

Sexual Cycle of Females

The following account of oAÂgenesis 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.

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 associated 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).

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).