Bulletin J I I ILLIN-OIS SLt\xirsLl IIistO]:*3r The Reproductive Cycle of the Raccoon in Illinois C. Sanderson Nalbandov NATURAL HISTORY SURVEY NOV 141973 LIBRARY 3F ILLINOIS ITMENT OF REGISTRATION AND EDUCATION IRAL HISTORY SURVEY DIVISION NA, ILLINOIS THE LIBRARY 01; THE my 7- 1973 VOLUME 31, ARTICLE JULY, 1973 ILLINOIS tii.ra.1 History Survey SXILLETIN The Reproductive Cycle of the Raccoon in Illinois Sanderson bandov LINOIS lENT OF REGISTRATION AND EDUCATION iL HISTORY SURVEY DIVISION I ILLINOIS VOLUME 31, ARTICLE 2 JULY, 1973 STATE OF ILLINOIS DEPARTMENT OF REGISTRATION AND EDUCATION BOARD OF NATURAL RESOURCES AND CONSERVATION Dean BarbinoeRj Ph.D., Chairman; Thomas Park, Ph.D., Biology: L. L. Sloss, Ph.D., Geology: (Vacant), Chemistry: Robert H. Anderson, B.S.C.E.. Engineering; Charles E. Olmsted, Ph.D., Forestry; W. L. EVERITT, E.E., Ph.D., Representing the President of the University of Illinois; Roger E. Beyler, Ph.D., Representing the President of Southern Illinois Universitii. NATURAL HISTORY SURVEY DIVISION. Urbana. SCIENTIFIC AND TECHNICAL STAFF George Spbugel, Jr.. Ph.D., Chief Alice K. Adams, Secretary to the Chief inois Section of Economic Entomology William H. Luckmann, Ph.D.. Entomologist and Head Willis N. Bruce, Ph.D., Entomologist Wayne L. Howe, Ph.D., Entomologist Stevenson Moore, III, Ph.D., Entomologist, Exten- HowABD B. Petty, Ph.D., Entomologist, Extension James E. Appleby, Ph.D., Associate Entomologist Edward J. Armbrust, Ph.D., Associate Entomologist Marcos Kogan, Ph.D., Associate Entomologist Joseph V. Maddox. Ph.D., Associate Entomologist Ronald H. Meyer, Ph.D., Associate Entomologist Robert D. Pausch. Ph.D.. Associate Entomologist Ralph E. Sechriest, Ph.D., Associate Entomologist John K. Bouseman, M.S., Assistant Entomologist George L. Godfrey. Ph.D.. Assistant Entomologist William G. Ruesink, Ph.D., Assistant Entomologist James R. Sanborn, Ph.D.. Assistant Entomologist Douglas K. Sell. B.S., Assistant Entomologist Clarence E. White. B.S.. Assistant Entomologist Keun S, Park. M.S.. Assistant Chemist Sue E. Watkins. Supervisory Assistant Donald E. Kuhlman. Ph.D., Assistant Professor, Extension RoscOE Randell. Ph.D.. Assistant Professor, Exten- Tim Cooley, M.A.. Assistant Specialist. Extension John F. Walt. M.S.. Assistant Specialist, Extension Jean G. Wilson, B.A.. Supervisory Assistant Natalie Ekl B.A.. Research Assistant Stephen K. Evrard. B.S., Research Assistant Raymond A. Kotek. M.Mus., Research Assistant Martha P. Miller M.S.. Research Assistant Barbara E. Peterson. B.S., Research Assistant Annemarie Redbobg. B.S., Research Assistant Keturah Reinbold. M.S.. Research Assistant Nancy Tsung. M.S., Resenrch Assistant Stephen Roberts, B.S., Junior Professional Scientist John T. Shaw, B.S., Junior Profe. Denise a. Cope. B.S., Techn Lowell Davis. Technical Assistant Lu-PlNG Kan. M.S., Technical Assistant Mary Kathryn McClendon. B.S.. Tcchnii Ching-chieh Yu. Ph.D.. Technical Assistant al Scientist t Linda Klippert. B.S., Technical Assistant Mary Frances Martin, Technical Assistant Kenneth R. Walker. Technical Assistant C. Russell Rose. Field Assistant Section of Founistic Surveys and Insect Indentificotion Philip W. Smith. Ph.D.. Taxonomi3t and Head Wallace E. LaBerge. Ph.D.. Taxonomist Milton W. Sanderson. Ph.D.. Taxonomist Lewis J. Stannard, Jr.. Ph.D.. Taxonomist Larry M. Page, Ph.D.. Assistant Taxonomist John D. Unzicker. Ph.D.. Assistant Taxonomist Donald W. Webb, M.S.. Assistant Taxonomist Bernice p. Sweeney. Junior Professional Scientist Section of Wildlife Research Glen C. Sanderson. Ph.D., Wildlife Specialist and Head Frank C. Bellro'se, B.S., Wildlife Specialist Richard R. Graber, Ph.D., Wildlife Specialist Harold C. Hanson, Ph.D., Wildlife Specialist Ronald F. Labisky, Ph.D., Wildlife Specialist William L. Anderson. M.A., Associate Wildlife Specialist W. W. Cochran. Jr.. B.S., Associate Wildlife Specialist William R. Edwards. M.S.. Associate Wildlife Specialist Jack A. Ellis. M.S.. Associate Wildlife Specialist Charles M. Nixon. M.S., Associate Wildlife Specialist Kenneth E. Smith, Ph.D., Associate Chemist ROREBT E. Greenberg, M.S., Assistant Wildlife Specialist G. Blair Joselyn, M.S., Assistant Wildlife Specialist David R. Vance M.S., Assistant Wildlife Specialist Ronald L. Westemeier, M.S., Assistant Wildlife Specialist Ronald E. Duzan, Junior Professional Scientist Helen C. Schultz. M.A., Technical Assistant Eleanore Wilson. Technical Assistant Robert D. Crompton, Field Assistant James W. Seets, Laboratory Assistant Section of Botany and Plant Pothology J. Cedbic Carter. Ph.D.. Plant Pathologist and Head Robert A. Evers, Ph.D.. Botanist Junius L. Forsberg. Ph.D.. Plant Pathologist Eugene B. Himelick, Ph.D.. Plant Pathologist R. Dan Neely, Ph.D., Plant Pathologist D. F. Schoeneweiss. Ph.D.. Plant Pathologist J. Leland Crane. Ph.D., Associate Mveologist Walter Habtstirn. Ph.D.. Assistant Plant Pathol- ogist Betty S. Nelson. Junior Professional Scientist Gene E. Reid, Technical Assistant Section of Administrative Services Robert O. Watson. B.S.. Administrator and Head Supporting Services Vernon F. Billman. Maintenance Supervisor WiLMA G. Dillman. Property Control and Trust Robert O. Ellis. Assistant for Operations Lloyd E. Huffman. Stockroom Manager J. William Lusk. Mailing and Distribution Service Melvin E. Schwartz. Financial Records James E^ Sergent. Greenhouse Superintendent Pifblications and Public Relations Owen F. Glissendorf, M.S.. Technical Editor Robert M. Zewadski. M.S.. Associate Technical Editor Shirley McClellan. Assistant Technical Editor Lloyd LeMere, Technical Illustrator Wilmeb D. Zehr. Technical Photographer Section of Aquatic Biology George W. Bennett. Ph.D.. Aquatic Biologist and Head D. Homer Buck, Ph.D.. Aquatic Biologist R. Weldon Larimore. Ph.D., Aquatic Biologist Robert C. Hiltibban. Ph.D.. Biochemist William F. Childers. Ph.D., Associate Aquatic Biologist Donald F. Hansen, Ph.D., Associate Aquatic Bi- ologist Richard E. Sparks. Ph.D.. Assistant Aquatic Bi- ologist Arnold Gnilka. Ph.D.. Junior Professional Scientist Richard J. Baue. M.S.. Research Assistant Dennis L. Dooley, Technical Assistant CONSULTANTS AND RESEARCH AFFILIATES: Systematic Entomology. Rodebick R. Ibwin. Chi- cago, Illinois; Wildlife Reseabch. Willabd D. Klimstra, Ph.D., Professor of Zoology and Director of Co- operative Wildlife Research. Southern Illinois University; Parasitology, Norman D. Levine, Ph.D., Profes- sor of Veterinary Parasitology, Veterinary Research, and Zoology and Director of the Center for Ecology, University of Illinois; Entomology, Robebt L. Metcalf. Ph.D., Professor of Zoology and of En- tomology and Head of the Department of Zoology, University of Illinois; and GILBERT P. WaLDBAUER. Ph.D., Professor of Entomology, University of Illinois; Statistics, HORACE W. NORTON, Ph.D., Professor of Sta- tistical Design and Analysis, University of Illinois. Technical library Doris F. Dodds. M.S.L.S., Technical Librarian Doris L. Sublette. M.S.L.S.. Assistant Technical Libr CONTENTS acknowiedgments 29 Methods 30 Seasonal Cycle of the Gonads 30 Captive Raccoons 30 Males 31 Females 31 Mean Birth Date of Raccoon Litters 32 Secondary Sex Ratios 32 Estrous Cycle and Ovulation 32 Estrous Cycle 32 Ovulation 32 Interstitial Tissue 33 Histology 33 Placental Scars 33 Morphology of the Reproductive Tracts 33 Males 33 Females 34 EflEects of Castration 34 Males 34 Females 34 Effects of Exogenous Hormones 34 Males 34 Females 34 Uterine Milk 35 Results and Discussion 35 Seasonal Cycle of the Gonads 35 Males 35 Females 43 Mean Birth Date of Litters 45 Estimating Birth Dates of Raccoons 46 Secondary Sex Ratios 47 Estrous Cycle, Ovulation, and Pseudopregnancy 48 Estrous Cycle 48 Ovulation 53 Pseudopregnancy 55 Percentage of Yearling Females That Were Sexually Mature 56 Secretion of Progesterone by Corpora Lutea 57 Pigmentation of Mammae 57 Interstitial Tissue 57 Placental Scars 61 Morphology of the Reproductive Tracts 65 Males 65 Females 66 Effects of Castration 68 Males 68 Females 68 Effects of Exogenous Hormones 70 Males 70 Females 71 Uterine Milk 78 Summary 79 Literature Cited 82 Index 84 This report is printed by authority of the State of Illinois, IRS Ch. 127, Par. 58.12. It is a contribution from the Section of Wildlife Research of the Illinois Natural History Survey. Glen C. Sanderson is Wildlife Specialist and Head, Section of Widlife Research, Illinois Natural History Survey. A. V. Nalbandov is Professor of Animal Science, Physiology, and Zoology, University of Illinois. (50105—5M—7-73) The Reproductive Cycle of the Raccoon in Illinois ALTHOUGH THE RACCOON (Procyon lotor) is a commonly recog- nized, widely distributed, and abundant North American mammal, little has been known about its reproductive cycle except the season of birth, the number of young per litter, and the duration of the gesta- tion period. Basic information on the length of the estrous cycle, whether ovu- lation is spontaneous or induced, the period of sexual activity in the male, the occurrence of pseudopregnancy, the roles of the various hormones in reproduction, and the anatomy of the reproductive tracts has been either lacking or frag- mentary. The objectives of this study were to gather data on the reproductive cycle and the basic anatomy of the reproduc- tive system of the raccoon and to in- vestigate those aspects of the raccoon's reproductive physiology that gave prom- ise of increasing our knowledge in the general field of mammalian reproductive physiology. This study was part of an effort to obtain a refined understanding of the population dynamics of the species. Other aspects of the study will be pub- lished elsewhere. ACKNOWLEDGMENTS Prior to 1961 this work was supported by Illinois Federal Aid Project VV-56-R, the Illinois Department of Conservation, the U.S. Bureau of Sport Fisheries and Wildlife, and the Illinois Natural History Survey, cooperating. During 1961, 1962, and 1963 partial support for these studies was contributed by the National In- stitutes of Health under Research Grant Glen C. Sanderson A. V. Nalbandov 7849. The remainder of the support for this study was provided by the Illinois Natural History Survey. We thank Dr. T. G. Scott, former Head of the Section of Wildlife Research at the Survey, for his encouragement and advice throughout the study, and Dr. H. W. Norton, Professor of Stu:.istical De- sign and Analysis in the Department of Animal Science at the University of Illi- nois College of Agriculture, for his help with the statistical analyses. We also thank Dr. Jean W. Graber, former Re- search Assistant Professor of Animal Science, University of Illinois, who pre- pared most of the histological sections and provided other valuable assistance, and the senior author's wife, Beverley C. Sanderson, who drew the sketches of the male and female reproductive tracts and helped in many other ways. W. D. Zehr, Illinois Natural History Survey Tech- nical Photographer, and G. G. Mont- gomery, former Survey staff member, assisted with the photomicrographs. R. J. Ellis was employed as Research As- sociate on Project W-56-R from Feb- ruary 1, 1961 through June 30, 1962 and contributed specimens and other assist- ance to this study. G. G. Montgomery was employed as Research Associate on Research Grant 7849 and made many contributions to the study. C. L. Foley, Illinois Department of Conservation, Paris, 111., supplied live raccoons for this study and was helpful to the project in other respects. Present and past em- ployees of the Natural History Survey who contributed specimens and informa- tion to the study include Dr. B. J. Verts, Dr. G. L. Storm, Dr. R. D. Andrews, Dr. Frontispiece.—Cages used to hold raccoons in Urbono, 111. Each double cage held either one pair, one female and her young, or 1-3 adult raccoons in each half. The outside dimensions of the cages were 3 feel (width) X 4 feet (height) X 6 feet (length). Each cage was divided crosswise through the middle with wire and hod a nest box on each end. The wire was 1.5-inch mesh, 14-gauge hexagonol netting. The nest boxes had wire bottoms, and wooden bottoms were in- serted on lop of the wire in winter. A hinged wire top on eoch nest box filled under the removable lid. 29 30 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 R. R. Graber, and others. Helen C. Schultz of the Survey staff and Robert M. Zewadski, Associate Technical Editor of the Survey, edited the manuscript. Dr. H. W. Norton and Dr. A. Sydney Johnson, Associate Director, Institute of Natural Resources at the University of Georgia, Athens, reviewed the man- uscript and made many valuable sugges- tions. We are especially grateful to Clifford, Albert, and Robert Perardi (Perardi Brothers Fur and Wool Company, Farm- ington, 111.) for their active and en- thusiastic cooperation with our study. METHODS SEASONAL CYCLE OF THE GONADS Each year from 1955 through 1961 the senior author examined dead raccoons at a number of fur houses in central Illinois. The majority of the raccoons were exam- ined at Farmington in Fulton County and Colchester in McDonough County. Most or all of these animals came from within the range of Procyon lotor hirtus (Gold- man 1950:24). During the hunting and trapping season, which usually occurred during November through January (but occasionally included late October), large numbers of recently killed raccoons were sold to fur-buying establishments and pelted. Often a majority of the ac- ceptable carcasses were dressed and frozen prior to being sold for human food. Thus, from the large number of raccoons examined, numerous data were recorded and many organs suitable for gross examination were collected as the animals were being skinned. The present report deals principally with the reproductive organs of the rac- coon. Before the animals were skinned, one testis and epididymis were removed from each male, and the condition of the nipples of each female was recorded. All pertinent information was recorded sep- arately for each animal. After the rac- coons were pelted, the complete repro- ductive tracts were removed from fe- males and were placed separately in 1- pint plastic bags to prevent the tissues from drying. Each plastic bag was placed in a small paper bag on which the data were recorded. The specimens were usually examined in the laboratory the day after collection but sometimes were examined on the day they were collected. The testes were weighed to the nearest 0. 1 gram. A drop of fluid collected from the tail of the epididymis was diluted with a drop of normal saline solution and examined un- der the microscope for the presence of sperm. Both ovaries were examined vi- sually and weighed to the nearest 0.1 mg. Raccoons found dead or collected by trapping and shooting specifically for au- topsy were processed in the same general manner as those examined in fur houses. A small number of raccoons, obtained from sources other than fur buyers, came from the southern and eastern sections of Illinois within the range of P. I. lotor (Goldman 1950:24). Gonads from both sexes were collected from adult and juvenile raccoons each month. Several gonads were removed immediately after the deaths of the animals and were preserved and pre- pared for histological study. The aver- age monthly weights of the gonads from all of the raccoons studied, both those freshly killed and those dead for several j hours, were used in constructing graphs! showing the seasonal gonadal weights for * juveniles and adults of both sexes. His- tological examinations of the testes, ep- ididymides, ovaries, and uteri contrib- uted information regarding the seasonal sexual cycle. CAPTIVE RACCOONS For many phases of the study captive ij raccoons were kept in outdoor cages in I Urbana, 111. Most of these animals were I trapped in the wild, both as adults and! juveniles, and some as small young, most- ly in Champaign, Piatt, Edgar, and Car- roll counties. 111. We estimated the ages^ of wild raccoons at the times of their cap-l July, 1973 Sanderson & Nalbandov : Reproductive Cycle of the Raccoon 31 ture (Sanderson 1961a). Some animals used for the study were born in captivity —some were conceived in captivity and others were born in captivity to females that were pregnant when captured. Captive raccoons were usually paired and held as one male and one female per cage. Pregnant females were isolated prior to parturition; the males were not returned while the young were with the females. Some females were isolated to determine whether ovulation in the rac- coon is induced or spontaneous. In some cases three or more animals—juveniles of both sexes and surplus males—were held in a single cage. The captives were given fresh food and water daily. The main diet was Dog Checkers or Laboratory Checkers, manufactured by the Ralston Purina Company. Occasionally the diet was supplemented by chickens, fish, eggs, and other available fresh foods. Captive raccoons that died or were killed were processed as described above, except that all of the gonads, after being weighed, were preserved for histological study. Usually a section of the uterus and occasionally accessory organs of the reproductive tract were also preserved for histological examination. Males The annual reproductive cycle in several captive male raccoons was deter- mined by restraining each male in a wire cone at irregular intervals through- out the year and collecting a drop of fluid from the tail of the epididymis. The tail of the epididymis was forced against the skin of the scrotum; then a pointed scalpel was used to prick through the skin, and a drop of fluid was collected on a glass slide. The drop was diluted with normal saline solution and ex- amined under the microscope for the presence of sperm. After the collection of the epididymal fluid, the animal was returned to its cage with no further treat- ment. No infection or other troubles resulted from this treatment. Occasionally, a captive male, or a wild male that had been livetrapped and was to be released at the point of capture for another phase of the study, was uni- laterally castrated to obtain a testis and epididymis for study. Captive males that fathered young were assumed to have had sperm in their epididymides at the time that they impregnated the females. Females The reproductive cycle of captive fe- male raccoons was studied by examining the ovaries and uteri during laparotomies of anesthetized animals. The anesthetic used was pentobarbital sodium admin- istered at the rate of 1 cc per 4 pounds of body weight. Given intraperitoneally, it usually produced surgical anesthesia in 10-30 minutes; however, individual re- s]5onses to the anesthetic varied, and animals that required more anesthetic were given larger doses the second time laparotomies were performed. The raccoon is resistant to infection and withstands surgical incursions well. Instruments were washed in 70-percent alcohol but were not sterilized. As many as 12 laparotomies were performed on one female over a period of several months, sometimes on subsequent days, sometimes two or three times in 1 week, but usually from 2 weeks to several months apart. Animals were usually given penicillin after each operation al- though no infections developed when it was not used. Surgical silk or cat gut was used to close the peritoneal linings and muscle; these sutures were not re- moved until a subsequent laparotomy was performed. Wound clips, used to close the skin, were removed approx- imately 10 days after the operation. To examine ovaries for evidence of ovulation, it was usually necessary to slit the ovarian capsules. Because the cut edges of the capsules did not always grow together, this procedure was omitted when examining females that were being held to produce young. The uterus was gently withdrawn from the body cavity for examination and gross measurement. 32 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 In several cases one or both ovaries were removed for study. Uterine sec- tions were taken from living females for histological study of the development of the endometrium. MEAN BIRTH DATE OF RACCOON LIHERS The mean date of birth was deter- mined for 20 litters conceived in the wild in the northern half of Illinois. Of these 20 litters, 7 were born in captivity. The potential birth dates of the others, most of which were examined in female raccoons found dead along roadways, were estimated by measuring the uterine swellings in the manner described by Llewellyn (1953:321). Data obtained during the present investigation were also used in estimating the probable birth dates. Because Llewellyn (1953:321) recorded measurements of only three embryos in one litter at three different stages and at birth, several embryos were measured in captive females during this study. Although the dates of conception were not known, the maximum measure- ments of the uterine swellings were plot- ted in relation to the number of days prior to the known birth dates. Many wild females were examined throughout the year for pregnancy, lactation, and the presence of fresh placental scars and corpora lutea. This information helped to determine the limits of the breeding season in wild raccoons. SECONDARY SEX RATIOS Secondary sex ratios were obtained by examining 83 embryos and young at birth in 26 litters and by determining the sex of 54 wild raccoons less than 2 months old from 23 litters. Chi-square tests were used to test whether the sex ratio of the wild young less than 2 months of age was different from equal- ity and from the ratio of the embryos and young at birth. ESTROUS CYCLE AND OVULATION Estrous Cycle Estrous cycles were determined for individual captive female raccoons by examining the ovaries at or near ovula- tion and then reexamining the ovaries at intervals until the animals ovulated again. The raccoon's main breeding season was interrupted throughout much of Illi- nois by colder - than - normal temper- atures and deep snows in 1960. Obser- vations of livetrapped raccoons and the body weights of young, wild raccoons weighed during the fall and winter of 1960 indicated that some raccoons were bom later than normal during that year. Lenses collected from several young rac- coons during the hunting and trapping season of 1960-1961 were used to esti- mate the months of birth for these juveniles ( Sanderson 1 96 1 6 : 482-485 ) . The time intervals between the peaks of estimated birth dates were assumed to represent the average interval between ovulations for wild raccoons in central Illinois. Cotton swabs were used to take daily vaginal smears from several captives in an attempt to delineate the estrous cycle. Observations of vulval swelling, size and pigmentation of the nipples, and general disposition of the animals were made each time the animals were handled. Vaginal tissues were removed from sev- eral females for histological study. Ovulation Each of two females was placed alone in a small cage in the fall of 1960 to obtain information on the mechanism of ovulation and on pseudopregnancy. These females could see other raccoons but could not come into physical contact with them. Also, one pet female, re- ported by the owner to have had no con- tact with other raccoons, was observed. Individual corpora lutea were studied in these females during a series of laparot- omies. Some of the corpora lutea in the ova- ries of three females were marked with India ink—and the locations of all corpora lutea were mapped. By follow- ing the fate of the marked and mapped corpora until they disappeared, we found that mapping the corpora lutea was as July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 33 reliable a method of determining their life-spans as was marking them with ink. Mapping was used in subsequent studies. At each initial obser\ation the ovary was forced through the slit ovarian capsule, the corpora were examined for color and measured grossly, and their locations in the ovary were mapped. INTERSTITIAL TISSUE Ovarian interstitial tissue was studied in wild raccoons on which observations as to pregnancy and lactation had been made, in several captive females treated with various hormones prior to the re- moval of the ovaries, and in untreated captives whose breeding histories were known. A uterine section was usually obtained when ovaries were collected, and the condition of the endometrium was studied in relation to the degree of development of the interstitial tissue. Representative sections selected from each ovary and uterus were photo- graphed by mounting the slide in the carrier of a photographic enlarger and projecting the image directly onto 4- X 5-inch contrast process ortho sheet film. Prints 8 X 10 inches were made on F5 Kodabromide paper. By examining the photographs, we determined the abun- dance and distribution of cells of each type in the interstitial tissue in relation to the development of the endometrial glands, the time of year, the age of the animal, and the stage of the reproductive cycle. HISTOLOGY Tissues were preserved in Bouin's solution or in 10-percent formalin neutralized with either MgCO, or CaCOa. The organs preserved in Bou- in's solution were left for an indefinite period, but those preserved in 10-percent formalin were transferred to 70-percent alcohol after 48-72 hours. With a few exceptions, all tissues prepared for his- tologcial examination were stained with hematoxylin and eosin. The ovaries of a few females that had died some time prior to the preservation of the organs were sectioned at 15-20 microns; the number of corpora lutea was our main interest in these ovaries. In all other cases the sections were cut 6 microns thick. The preserved organs were em- bedded in paraffin and sectioned and mounted by routine methods. PLACENTAL SCARS In dead female raccoons placental scars were counted, using transillumina- tion. The uterus was then slit and the inside surfaces were examined for scars. In captixe pregnant females the uter- ine swellings were measured and the locations of the embryos were mapped during laparotomies. After parturition the presence and persistence of placental scars at the sites of known placental attachment were studied during a series of laparotomies. The scars were ex- amined in living animals by gently pull- ing the uterus far enough out of the body cavity to allow it to be transilluminated. Uterine sections containing scars at \arious stages were removed from living females at intervals for histological study. MORPHOLOGY OF THE REPRODUCTIVE TRACTS Males A few complete male reproductive tracts were removed and preserved for histological study. The entire tract from one male, and individual accessory organs from a few additional males, were sectioned. India ink was injected into one vas deferens of a fresh specimen until the ink ran out the urethral opening of the penis. The tract was then preserved and sectioned for histological study to trace the duct system, containing parti- cles of India ink, through the prostate gland. A schematic diagram of the male re- productive system was sketched from a fresh specimen that had been partially dissected but was sufficiently undisturbed to show its relationships to adjacent structures, A complete reproductive tract that had been dissected and pre- served was used for reference. 34 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 Females A schematic diagram of the reproduc- tive tract (frcm one female) was prepar- ed from a fresh tract that had been suffi- ciently dissected to reveal its conforma- tion but that maintained its position relative to adjacent structures. One en- tire tract that had been removed and preserved was used for reference. EFFECTS OF CASTRATION Males Four captive male raccoons were castrated at ages ranging from 72 days to approximately 9 months to study the effects of castration on the development of the penis bone, the opening of the preputial orifice, and the age at which the epiphyses close in the radius and ulna. These studies were not completed because the four animals died of various causes at dififerent ages; the one that lived the longest attained an age of approximately 22 months. Females One female raccoon, born in captivity, was 3 months of age when castrated; the second, born in the wild, was estimated to be 4 months old when castrated. Several adult females were also castrated to study the effects of castration on vaginal smears, the vaginal epithelium, the uterus, and the closure of the epiphyses in the radius and ulna. Vaginal tissues and uterine sections were taken from castrated females at intervals. These tissues were prepared for histological study and used for com- parison with similar tissues from females believed to be anestrus. The females castrated as adults were also used to study the effects of various exogenous hormones on vaginal smears, the vaginal epithelium, and the development of the endometrium. Two pregnant females were castrated as the first phase of a study of the effect of castration on pregnancy. The first fe- male, with four embryos, was castrated 38 days (estimated time) after concep- tion. The second female, with five em- bryos, was castrated approximately 1 1 days after conception. These females were observed daily after castration for signs of abortion. A second laparotomy was performed on the first female 21 days after castration and on the second female 19 days after removal of the ovaries. EFFECTS OF EXOGENOUS HORMONES Males Two captive adult male raccoons were used for preliminary studies of the effects of androgen on spermatogenesis. Begin- ning in August, near the midpoint of sexual inactivity, injections of testos- terone cyclopentylpropionate (Res. No. 8961-1, Upjohn) were administered to both of these males. The first male re- ceived seven subcutaneous injections of 30 mg each at 3-day intervals. Immediately before the first injection of the hormone the left testis and ep- ididymis were removed from each an- imal. The testis was weighed and a smear from the tail of the epididymis was examined for the presence of sperm. Each testis and epididymis was prepared for histological study. The first male was killed 21 days after receiving the first androgen injection, and the right testis, right epididymis, and the prostate were removed. The testis was weighed and a smear from the tail of the epididymis was examined for sperm. The second male was similarly treated but received four injections of 12 mg each and was killed 15 days after the first injection was administered. Females Several attempts were made to cause the growth and development of Graafian follicles and to cause ovulation by in- jecting various hormones into female raccoons. The hormones used were pregnant mare's serum (PMS, Upjohn), the pituitary gonadotropins (FSH and LH, Armour) , estradiol cyclopentylpro- pionate (ECP, Upjohn), estradiol valer- ate (estradiol, Squibb), hydroxyproges- terone caproate (progesterone, Squibb), chorionic gonadotropin (CGH, Up- July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the RACCoo^ 35 John), and human menopausal gonado- tropin (HMG-J5, Statens Seruminstitut, Copenhagen). Because these hormones were administered by many different routes and at many different dosage levels and time intervals, the methods used are discussed in connection with the particular animals involved or are given in the tables where the results from the individual animals are sum- marized. UTERINE MILK Studies were made to determine the hormone or hormones responsible for the secretion of uterine milk by the endo- metrial glands and to learn the nature of this secretory material. Ovaries and uteri were sectioned and stained from 18 raccoons—all were collected during the breeding season and some of them were pregnant—in which corpora lutea were present and from 89 raccoons—collected throughout the year—whose ovaries con- tained no corpora lutea. None of these 107 raccoons had been injected with hormones. In all cases the endometrial glands were examined for the presence of secretory materials. Various hormones were administered to castrate females, uterine sections were removed at varying time intervals, and the endometrial glands were examined by histological methods for the presence of secretory material. The hormones used on individual castrate and intact females to study hormonal control of the secretion of uterine milk were progester- one and ECP, ECP alone, and progester- one alone; however, progesterone alone was not given to any castrate animal for a sufficient time to determine whether it would cause the uterine glands to se- crete. Also studied were the direct and secondary effects of PMS, FSH, and LH, used primarily in attempts to cause the growth of Graafian follicles and to cause ovulation, and the production of secre- tory material by endometrial glands in intact females. Methods described by Pearse (1960: 265-271) and Lillie (1954: 274-299) were used to demonstrate the nature of the material observed in the lumina of the endometrial glands. Uterine sections from three female raccoons that had material present in the endometrial glands were used. The uterine section from one was fixed in 10-percent for- malin neutralized with CaCO,. The uterine section from another was fixed in Bouin's solution, and the section from a third was fixed in 10-percent formalin neutralized with MgCOj. All of these tissues were imbedded in paraffin for sectioning, and control slides were used in each case. RESULTS AND DISCUSSION SEASONAL CYCLE OF THE GONADS Males The age at which male raccoons reach sexual maturity may vary from one re- gion to another. In Michigan, on the basis of meager circumstantial evidence, Stuewer (1943i: 72) concluded that males "are probably not sexually mature by the first breeding season after their birth." In Illinois Pope (1944: 91) had two captive males—of parent stock sup- posedly "from northern Illinois or some adjacent region"—that mated success- fully before they were 1 year of age. Stuewer (1943^:63) reported that the testes of juveniles and yearlings were in an abdominal position; those of adults were usually descended during the breed- ing season and, though variable in posi- tion, during the remainder of the year were most often in the coelom. Stuewer's evidence suggested that testis size might reflect the capacity to breed. He measur- ed the lengths of testes in the scrotum with an accuracy of approximately 5 mm. Stuewer (19436: 64) concluded that if "testis size is significant, males are prob- ably capable of breeding at all times of year after reaching maturity." Asdell (1946: 136), on the basis of Stuewer's work but omitting his qualifications, stat- ed that the male raccoon was capable of matin? at any time. Nalbandov (1958: 162) cited the male raccoon as 36 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 a species in which spermatogenesis is continuous although the breeding season of females is restricted to late winter and early, spring. The data in Table 1 and Fig. 1 show that raccoon testes grew at a rather uni- form rate from birth until about 10 months of age (through the February after birth ) , when the average weight of one testis was 5.6 grams. The testes of juvenile males showed the most rapid gains in weight between December and February. The average weight of a testis from a juvenile male in November was only 30 percent of the average weight in February. The sample sizes for February, March, and April were small, but there was an indication that the weights of testes in juveniles declined after February. After April testicular weights of juveniles were included with those of adults because a majority of the juvenile males were sexually active by April. In our experience raccoon testes were nearly always found in the scrotum, even at birth, Stuewer's (19436: 63) statements to the contrary notwithstand- ing. They were more prominent in adults than in juveniles, and most prom- inent in adults during the breeding sea- son. Even in immature animals the testes were rarely withdrawn into the body cavity. In Illinois a majority of the male rac- coons reached sexual maturity as year- lings. Although the presence of sperm in the epididymis does not necessarily indicate sexual potency, it does indicate that an animal is in or approaching the period of sexual activity. No juvenile male had sperm in its epididymis prior to October (Table 1). In October the epididymides of about 9 percent of the juveniles contained sperm; by February this figure had increased to 87 percent. An extrusible penis was another indica- tion of a juvenile's stage of sexual de- - velopment (Sanderson 1961a: 14). Oc- casionally, a male was found with a non- extrusible penis but with sperm in its epididymides. Among juvenile males, 5 percent had extrusible penes in Septem- ber. This figure had increased to about 67 percent by February and March but declined slightly in April. These data in- dicated that, in Illinois, from one-half to two-thirds of the juvenile male rac- coons are sexually mature by the time they are 1 year old (Table 1 ) . By sex- ually mature we mean that the male has an extrusible penis and a relatively high concentration of sperm in the epi- didymides. Comparison of data from juvenile and adult male raccoons shows that juveniles became sexually mature 3-4 months later in the year than did adults. Several ? 4 »- X a 3 UJ » 2 1- MAY JUNE JULY AUG. SEPT. OCT. NOV. OEa JAN. FEB. MAR. APR. Fig. 1 .—Seasonal variations in the average weight of one testis in adult and juvenile raccoons. With each mean are the number of observations and a vertical line representing the mean plus or minus one standard error. All animals were token in Illinois from November 1955 through April 1961. The data ore given in Table 1. [uly, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 37 E-5 g. 20 c - .S'S jco^^ ^jTi-c^yp ooo oooo oooo I CO cTi o in en (r> ( -H ^ CO o to en ^ I oococNc^j "^cMtO'—' intDomooooo ci^^'-'CM cnincoco t; e I- 0! r; S 3 U — -";;?g;~ons— ^U CJ3SS «m-|^| < ^o o (£> r^ CM 00 CM *-H r-* CM tJ- to in [ 1-* CO CM to to ^ . o o ^^ r* lo CO o I CO to CM ^ to CM (O CM c^ r^ o,^ :^ —^Tf .^ ^ «*• CM in CM CO o 00 to to Tf in CO CO o in i^ '-* CTi ^--^ CM CM ^ '^ 'inm^"^ cooir^co^o 'cMcoOj*-; ^'-jr^o^cqco i^r^tor^ r^r-^totor-^'O i-H CO T^* O tJ* CO in in tj- to in to o to T}- o x^ r^ -H in to to CM ^ Oi CM Th CM to CM CM O^ 00 ci —' CO -^ o^ r^ ^ CM CM CM ^ ^ CO CMCM CMrhcOX^COCl ^ ' CM CM CM CM -— ^H CO * *oin r^cMo^inoo 1-^ * ',-ito '-it^CMOjOt^ O ^-^ CMCMCOCO—icO oor^'^ino o^inmcMOito CMOO —•'-iCO '-OCOtOCMTf inr^co^^ Oiinr^-HOco ocotooin mcM^HTj-cMto ^^^^CM '-'CMCMCMCM^ toocMCM '*'r-»cMr^cMin .toincoco co'^totocoCT) 'cM-^tOTj* inr^CTJi-Htor^ '^-hcmcoo o^tocioqcMT^ '5 do E c t; S O oji- t „^^ 2*' 5 'c S c/3 rt c/3 1—icMco-^in tor^cooiO'-' inmininm mminintoto "1oJhc^4co-^ 5»n^r-i.coa)0 Smmintnin .S^^^^mmmtD OClCTiO^ClO^ — 0^0^0100^<7) S ^ O July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 43 cant differences in the average weights of adult testes occurred from November to January. This finding was not unex- pected, because virtually all adult males were capable of breeding by November but only 8 percent of the juveniles had sperm in the epididymides during No- vember (Table 1). There were some statistically significant annual differences in the weights of testes, but the meanings of these differences were not clear. Females The ovaries of raccoons showed a nearly steady rate of growth from birth in April through the following November (Table 5 and Fig. 5). In contrast, the testes of juveniles showed their most rapid increases in weight between De- cember and February (Fig. 1). The ovaries of juveniles reached their maximum average weight in November, approximately 3 months prior to the peak of the breeding season. The heaviest normal ovaries encountered were found during November in juveniles; the average weights are shown in Table 5 and Fig. 5 and 6. In October, Novem- ber, and December the ovaries of juve- niles weighed more than the ovaries of parous raccoons. The average weights of ovaries for the two groups of females in January were practically identical (Fig. 5). _ The ovaries of juvenile (nulliparous) females showed a significant decline in average weight from November through January, and perhaps through March, but the sample sizes, for February, March, and April were too small to be definitive. The small sample of juveniles for these latter 3 months resulted partly from classi- fying raccoons as nulliparous (juveniles) or as parous or pregnant. During those 3 months many females approximately 1 year of age were either pregnant or par- ous, and hence their ovaries were placed Table 5.—Average weights of ovaries by month in the raccoon.' 44 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 300 July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 45 O lO r^ C£> T^ ^CM ^ ^ ^ lO r^ O — ' lO O (£1 ^— lo -H CO O t£) CO Ol O Oi ^ ^ a^ o • CO en ( Ol CTl Oi ^ a*o*o.SP t- fc C CTJ weight was reached in November. The ovaries of parous raccoons declined sig- nificantly (f<0.01) in average weight from November to December but again increased in weight during January. By April the ovaries of parous raccoons had reached their peak average weight for the year, slightly heavier than in Novem- ber. The average weight of adults' ovaries in April was a little more than 1.6 times their average weight in July, in contrast to the approximately 2.8-fold increase in average weight reported for the testis in the adult between the low average of July and the high of Decem- ber. From the study of ovaries collected during all months and seasons, we gained the impression that differences in weight existed from month to month and year to year. For example, the total weight of both ovaries of parous females averaged nearly 350 mg in November 1958 but only 219 mg in November 1959 (Table 6). Ovaries from nulliparous females killed in November 1958 also weighed consid- erably more on the average than did ovaries from nulliparous females killed in November 1959. Less striking varia- tions were noted for other months and years. There were also annual difTerences in the average weights of ovaries from parous females but no significant difTer- ences in those from nulliparous females. MEAN BIRTH DATE OF LIHERS Wood (1955:409-410) concluded that 7 of the 16 females he examined in Texas had mated by the end of February, but the earliest pregnancy he recorded was March 18. George & Stitt (1951: 218) found three litters that were born during March 1950 in Michigan after an unseasonably warm January. Berard (1952:248) observed a lactating female in West Virginia that he estimated had given birth no earlier than August 15, whereas normal births in that area usu- ally occur before May 15. Dorney (1953: 123) weighed young raccoons taken in Wisconsin from November 25 through December 22, 1950 and concluded that 46 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 Table 7.—Months of birlh of raccoons in the northern half of Illinois as determined by actual births or as estimated from examination of embryos.* Month Number of Litters Conceived in Number of Adult Wild Captivity That Females Examined Were Born in Month for Pregnancy* Designated Number of Litters Conceived in the Wild That Had Actual or Potential Birth Date in Month Designated" January February March April May June July August 202 6 15 18 9 6 11 4 All embryos were examined between April 2, 1957 and Ju ' Many nonpregnant, adult females examined from April t ' Potential birth dates were estimated (Fig. 8). .e 24, 1961. August we: ; lactating. "a sizable percentage" of the young had been born later than usual in that year. He suggested that the cold spring weather in 1950 had decreased raccoon mobility and thus had decreased the normal num- ber of early conceptions. A similar situ- ation, discussed later, apparently occurred in Illinois during the breeding season in 1960. The reports cited emphasize the varia- tion in birth dates that is normal in the raccoon. Most raccoons in the northern half of Illinois are born during April (Table 7). The mean date of birth for 20 litters conceived in the wild, 7 of which were born in captivity, was April 18; the earliest date of birth was March 9, and the latest, June 24. The potential birth dates of embryos measured in dead Table 8. —- Estimated number of days prior to birth based on the measurement of uterine swell- ings in captive raccoons. Largest Measurement July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 47 DAYS PRIOR TO BIRTH Fig. 7.—Sizes of uterine swellings in raccoons at various numbers of days preportum. Ttie line was fitted by least squares, not including Llewellyn's data. Tfie dash line, on extension of the line to conception 63 days preportum, is not based on data. The size used for the uterine swelling at conception was 5 mm, the approximate overage diameter of the uterus during estrus. The data ore given in Table 8. the embryo. In the later stages the swell- ings were elongate and the measurement approximated that of the crown-rump measurement. The dates of mating were not known, but it was possible to graph the size of the uterine swellings in relation to the number of days prior to parturition (Fig. 7 ) . The line was fitted by least squares and gave a good fit for uterine swellings between 20 and 60 mm in size. When we used this line to estimate the dates of birth for eight litters, the maximum error \\as 4 days when the uterine swellings were between 20 and 60 mm. In one litter uterine swellings larger than 60 mm were measured and in another litter uterine swellings smaller than 20 mm were measured. Measure- ments of the swellings in these two litters appear to indicate slower-than-average growth from conception to the 20-mm size and faster-than-average growth from the 60-mm size to birth. Measurements of uterine swellings made during this study were similar to those reported by Llewellyn (1953:321). Our data and Llewellyn's make it possible to estimate the date of birth (Fig. 7). If we assume a gestation period of 63 days, which many authors agree is average for the raccoon, it is possible also to estimate the date of conception. SECONDARY SEX RATIOS Incidental information collected during the present study indicated that the sex 48 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 Table 9.—Secondary sex ratios in raccoons." July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 49 Tab'e 10.—Approximate number of days bet* ovulations in five capti' Estimated Date of First Ovulation Estimated Date of Second Ovulation Days Between Ovulations Remarks 2-10 Before 3-14 Before 3-10 3-2 (±2) 1-29 5-10 5-26 5-11 6-23 ( : 6-16 89 During first pregnancy, carried embryos half way or more to term but resorbed them. 70* Pseudopregnant 62* Pseudopregnant 84 Pseudopregnant 141'' Carried embryos to term each time. Young of first pregnancy all dead 4 days postpartum. ^ The interval between the births of two litters in one On the basis of our observations of five captive raccoons for which the approxi- mate dates of the first and second ovula- tions were known (Table 10), we found that the inter\-al between ovulations in captive raccoons in Urbana, 111., varied approximately from 80 to 140 days—and not invariably 4 months, as reported by Whitney & Underwood (1952:83). The shorter intervals that we observed agree with Millard's (1939:28-29) data. He obtained two litters in one breeding sea- son from 6 of 10 captive raccoons in Wis- consin whose young were removed on the day of birth and whose mates were returned 3 days later. Seven of the fe- males were observed to mate 10-16 days after the young were bom. If we assume a gestation period of 63 days and that the female raccoon ovulates on the day of mating, ovulations in Millard's animals occurred 73-79 days apart. Under normal circumstances wild, adult female raccoons in Illinois rarely skip a breeding season. Special circum- stances may interfere with the regular breeding cycle, causing a higher-than- normal percentage of the litters to be born late (Dorney 1953:123). Such interfer- ence occurred in some sections during the 1960 breeding season in Illinois. Temper- atures at the Urbana and Peoria weather stations (U.S. Weather Bureau 1960) were average for January 1960, but mean temperatures in February were 15.7° F [9.0° C] below normal. Snowfall at Urbana and Peoria for February' and March 1960 ranged from 8 to 16 inches [20.3-40.6 cm] per month higher than the average for the preceding 10 years. On the Allerton Park Study Area (Piatt County, east-central Illinois) young rac- coons were caught in live traps beginning in early Jiuie of each year from 1957 through 1961, with the exception of 1960. In 1960 the first young were livetrapped after September 1 even though trapping was conducted during the entire summer. Eyes were collected from 257 juvenile raccoons killed by hunters and trappers o\'er a wide area centered aroimd Farm- ington in west-central Illinois during the 1960-1961 hunting season. The lens tech- nique (Sanderson 19616:482-485) was used to estimate birth dates. The lenses indicated that the peak of births in 1960 occurred in mid-April, the usual time, but that a second, smaller peak occurred at the first of July, about 1 1 weeks later. These two peaks were separated by about the length of one estrous cycle, as it was estimated from our observations of captive raccoons. According to the lens data, approximately 16 percent of the young were born during August, Septem- ber, and October in 1960—later than the latest date of birth reported in Table 7 — indicating that under some circumstances a substantial number of wild raccoons have had more than one estrous cycle in a year. In view of Millard's (1939:28-29) success in getting two litters in one season from captive raccoons and because the present study demonstrated that some of our captive pseudopregnant and pregnant females had second heat periods in cap- tivity, it was, at first, surprising that so 50 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 few second litters were conceived in cap- tivity during our study. Millard's (1939) objective was to rear a large number of young raccoons for restocking purposes, and no doubt he disturbed his animals as little as possible. Our study, on the other hand, required frequent handling of the animals and their subjection to laparotomies. Only two pregnancies are known to have resulted from second ovu- lations during our study. The first female became pseudopregnant after her first ovulation, and the single embryo from her second ovluation was resorbed; the second female give birth to her second litter in August, 141 days after the first litter was born. She had killed the last surviving young of her first litter 4 days postpartum. Female raccoons will not ovulate and come into estrus so long as they are nurs- ing young. Young were removed at birth from four female raccoons and 5 days after birth from one female. All of these females were returned to their mates when the young were removed, but no second matings were observed and no second pregnancies resulted. Young were removed from six females at periods vary- ing from 17 days to 6 weeks after birth, and the males were returned to the fe- males. One female was given a drug that caused her to abort or resorb her young. Her mate remained with her at all times. However, no second pregnancies resulted in any of these animals. In addition to these females several others underwent periods of pseudopregnancy during the normal breeding season while remaining with their mates through the summer. No late pregnancies resulted. Possibly some of the males were no longer capable of fertilization (Fig. 3 and 4) by the time their mates experienced their second es- trous cycles. Our data make it clear that in Illinois it is possible for raccoons to ovulate two times during one season and even to give birth to two litters. However, to give birth to the second litter, the female must lose her first litter on or shortly after the day of birth. We have no evidence that ovulations occur after lactation ceases in the raccoon. In any case, it appears that laccoons must nurse for 2-3 months in the wild and that probably they usually nurse for 3-5 months (Stuewer 1943a: 213; Montgomery 1969:155-158). The vaginal smear is frequently used to determine the stage of the reproductive cycle in the laboratory mouse, rat, and guinea pig. Stockard (1932:1612-1627) gives a general review. Although this technique can theoretically be applied to other species, many problems occur with species that have relatively long periods of proestrus and estrus. Nalbandov (1958:103-104) pointed out that all mammalian females show changes in their vaginal histology during the estrous cycle. He further reports: "The vaginal-smear technique is most useful, however, with animals having short estrous cycles . . .; in animals with longer cycles . . . vagi- nal changes lag from one to several days behind ovarian changes, and vaginal smears are therefore less re- liable indicators of ovarian events." Stuewer (1943i):64) observed that from 1 to 2 weeks elapsed from the onset of vaginal swelling in the raccoon until the female would receive the male. After a receptive period of about 3 days, 3 or 4 weeks elapsed before the vulva returned to normal appearance. Whitney & Un- derwood (1952:83) reported that the onset of the mating cycle could be recog- nized by a thickening or swelling of the vagina and vulva and traces of bloody fluid (absent in some females), and that the female would accept the male at the onset of 4he mating cycle and was recep- tive for a period of 3-6 days. We obtained estrous-type vaginal smears for a period of several weeks in our raccoons; examples of these smears are shown in Fig. 8. An estrous-type . smear was obtained from a castrated fe- J male 36 days after the end of treatment 1 with estradiol and progesterone (Fig. 8B) . One captive female must have mat- ed during the 7 days between the taking of two vaginal smears; she gave birth July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 51 '-^ > ^. S-»' 4 ^^-jt,,,.^' 'k-V%*^ ' # A -J^^^̂'^ B^.- -^^ "^X^i^ 'f^TC't ^;:;,v;- Fig. 8.—Voginal smears from captive raccoons representing various stages of the estrous cycle. A, female 1292; ovaries removed May 14, 1958; smear taken July 23, 1959. B, castrated female 1297; second ovary removed August 13, 1957; smear taken May 16, 1958, 36 days after treatment with estradiol and progesterone ended. C, nulliparous female 2525; smear taken December 4, 1958. D, female 2959; smear taken January 11, 1958. E, female 2959; smear token March 7, 1958. F, female 2959; smear taken March 14, 1958; female 2959 must hove mated between these two dotes, because she gove birth to young on May 13, 1958. G, adult female 1786; smear token April 12, 1957. H, female 2114; smear taken February 21, 1958; uterine swellings were 8 mm in diameter. The smears were stained with Wright's blood stain and ore shown 62 times actual siie. 52 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 60 days after the second smear was taken (Fig. 8£ and F). On the basis of the results obtained from this study, we conclude that the days when a female raccoon will receive a male can not be identified by examination of vaginal smears. The vaginal smear appeared to be no more specific than gross vulval swelling—which can be observed much more readily. Leucocytes (Fig. 8C and G) were seen in vaginal smears from rac- coons only infrequently. The paucity of July, 1973 Sanderson & Nalbandov : Reproductive Cycle of the Raccoon 53 vaginal smears containing leucocytes sug- gested that the raccoon may pass through metestrus in a relatively short time. Many of the difficulties inherent in using vaginal smears may be avoided by taking vaginal tissue for biopsies— a simple procedure in the raccoon. Sam- ples of vaginal tissues were removed from both anesthetized and unanesthetiz- ed animals (Fig. 9). However, vaginal tissues from castrated females (Fig. 9 A and B) showed that the histology of the vaginal epithelium is not a reliable indi- cator of estrus in the raccoon. Ovulation Whitney & Underwood (1952:84), without citing evidence, reported that in the raccoon "sufficient stimulation is produced during copulation to insure ovulation." Llewellyn & Enders (1954(3: 440) removed one ovary from each of four sexually mature raccoons that had been isolated from males before and during the normal breeding season. They found "well developed follicles" in each ovary but no corpora lutea and, on the basis of this evidence, suggested that ovulation in the raccoon is not spontane- ous but is induced by copulation. In our study one female was approxi- mately 5 months old when captured about 5 months before the breeding season. She was isolated for 3 months prior to the breeding season. Her nipples were moderately stimulated but unpigmented when a laparotomy was first performed on her 3 months after she was isolated. The ovaries were each 8X5 mm—small for ovaries with corpora lutea—yet each ovary had two corpora lutea, each 5 mm in diameter. Thirty-five days later the corpora lutea were essentially unchanged in size and appearance. Sixty days after the first examination the ovaries were 10 X 5 mm, and the corpora lutea were slightly paler and were between 3 and 4 mm in diameter. Ninety-three days after the initial observation no traces of the corpora lutea were visible. A second female that was isolated was approximately 40 days old when cap- tured. She was isolated 2 months prior to the breeding season, and the first lap- arotomy was performed on her 2 months after the isolation began. Her nipples were only slightly stimulated, and her ovaries, measuring 8X4 mm, contained no corpora lutea. The left ovary had one clear follicle and the right ovary had two, each follicle measuring 2 mm in diameter. Nine days later the right ovary had four freshly ovulated follicles, each 3 mm in diameter. The left ovary had a single follicle of the same size with a tiny hole in its highest point. It was believed that this female had ovulated no more than 2 days earlier. Twenty-six days after the freshly ovu- lated follicles were observed, the right ovary was 11X5 mm and had three corpora lutea, each 4 mm in diameter. Either the fourth follicle in the ovary did not form a corpus luteum, or it was obscured by one of the other corpora. The left ovary was 10 X 5 mm and had one corpus luteum of the same size as those in the right ovary. Eighty-one days after the freshly ovu- Fig. 9 (Page 52).—Photomicrographs of vaginal biopsies from captive raccoons representing various stages of the estrous cycle. A, female 1292; ovaries removed May 14, 1958; biopsy performed July 23, 1959. B, castrated female 1297; second ovary removed August 13, 1957; biopsy performed May 16, 1958, 36 days after treatment with estradiol and progesterone ended. C, female 2114; biopsy performed February 21, 1958; uterine swellings 8 mm in diameter. D, nulliparous female 2525; biopsy performed December 4, 1958. E, female 1297; biopsy performed July 23, 1959; no treatment with estradiol ond progesterone after April 3, 1958. F, female 1298; biopsy performed October 5, 1959; ovulated about September 24, 1959 as a result of injections of pregnant mare's serum. G, female 1782; biopsy performed October 29, 1957; treated with estradiol beginning October 17, 1957; ovaries removed June 10, 1957. H, female 2959; biopsy performed Jonuory 27, 1960; ovulated after December 18, 1959 as a result of treotment with follicle-stimulating hormone and luteinizing hormone; corpora lutea present. I, female 2805; biopsy performed February 24, 1960; fresh corpora lutea present. The sections were stained with hematoxylin and eosin and are shown 122 times actual size. 54 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 lated follicles were observed, the ovaries were each 8X3 mm; the four corpora lutea, each now 3 mm in diameter, were still present. By 102 days after the cor- pora were first seen, four whitish corpora albicantia (not examined histologically), each measuring 1 mm in diameter, had formed at the sites of the preceding cor- pora lutea. At this time there were also two follicles, each 2 mm in diameter, in the right ovary and one of similar size in the left ovary. The female was judged ready to ovulate a second time. Thirty-one days later one corpus lute- um was found in the left ovary and five or more were found in the right ovary; each corpus luteum was approximately 5 mm in diameter. The left ovary was removed (133 mg) and sectioned, but no ovum was found in the corpus luteum. Thus, this animal had probably ovulated. Also, the secretory material in the endo- metrial glands indicated that progesterone had been secreted. Seventy-three days after the follicles were examined, the corpora lutea were still present but mea- sured only 2 mm in diameter. A female found when approximately 3 weeks old was kept as a house pet until the middle of April, when she was about 1 year of age. At that time she suddenly became vicious, severely biting both own- ers. She remained the most vicious rac- coon we have seen among the many dozens of wild, captive, and pet raccoons that we have handled. According to her owners, she had never come into contact with other raccoons. At the time her behavior changed, her nipples were mod- erately stimulated and moderately pig- mented, indicating that she was either pregnant or pseudopregnant. When first examined, her ovaries were 9X6 mm and 7X5 mm, respectively, and con- tained a total of five corpora lutea, each 3 mm in diameter. The size of the cor- pora indicated that they were regressing when examined, because newly formed corpora lutea in the ovaries of raccoons are approximately 5 mm in diameter. Thirty-five days after the initial examina- tion all five corpora lutea were plainly visible but were regressing and were slightly smaller than when first examined. P'orty-eight days later (83 days after the first examination) no traces of the corpora lutea could be seen by gross examination. The data on these three isolated fe- males, one of which ovulated twice in one season, show that the raccoon is a spontaneous ovulator, and refute Llewel- lyn & Enders' (1954a: 440) interpretation of their observations. The statement of Whitney & Underwood (1952:84) that ovulation in the raccoon is dependent upon copulation is not true for captive raccoons in Illinois. In many captive raccoons, especially those reared as pets, the onset of estrus and pseudopregnancy was apparent from changes in behavior. A docile house pet sometimes suddenly became vicious and unmanageable. In all such cases that we examined, corpora lutea were present in the ovaries. The formation of corpora lutea was invariably accompanied by changes in the uteri and nipples whether the animal was pregnant or only pseudo- pregnant. The nipples always enlarged, and some became heavily pigmented, some became only slightly pigmented, and still others remained unpigmented. With the onset of pseudopregnancy the uteri became turgid and opaque and were considerably enlarged from their size dur- ing anestrus; however, they were not flu- id filled and somewhat rubbery, as they were during estrus. A female raccoon born in April was reared as a pet until the following Janu- ary, when she became too unruly for the owners to handle and was donated to our project. She is described here as representative of the females, housed with other raccoons, that ovulated but did not become pregnant. According to her own- ers, she had not come into contact with other raccoons before she was donated to our project. Two days after we re- ceived her, she was placed in a cage' with four yearling males. Forty-nine days later her nipples were tiny and white, but after 21 more days (March 28) they were elongated and black, indicating that she was either pregnant or pseudopreg- July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 55 nant. Five days later her left ovary was removed, and histological examination showed four freshly formed corpora lutea. Histological examinations of the ova- ries from this nonisolated female, from one isolated female, from three nonpreg- nant wild females, and from two addi- tional nonisolated, nonpregnant, captive females revealed no ova in the corpora lutea. No substantial difTerence was not- ed between the corpora lutea of the iso- lated nonpregnant and of the nonisolated nonpregnant females. Ovaries from sev- eral nonpregnant females housed with other females, or with males, were ex- amined during and after the breeding season. In several cases these ovaries had corpora lutea, which were grossly identi- cal to those seen in females isolated prior to the breeding season and to corpora lu- tea in pregnant females. Thus, we con- cluded that corpora lutea in both isolated pseudoprcgnant and nonisolated pseudo- pregnant females formed from ovulated follicles and not from luteinization of fol- licles. Normal-appearing corpora lutea were also formed in the ovaries of a fe- male in which ovulation was induced by exogenous hormones. Data gathered from examination of 13 captive raccoons indicated that corpora lutea persist in pregnant females until parturition. Observations on four of these captives indicated that corpora lutea dis- appeared 14-16 days after parturition if the young were taken from the mother within 5 days after birth. In one of these four the corpora lutea were not present 16 days after parturition; in an- other they were present 14 days after parturition. One female ovulated, apparently for the second time in the season, about May 1 1 . She mated, and one embr^'o was implanted; approximately 20 days after ovulation the embryo was dead. Traces of one corpus luteum were still present in each ovary approximately 52 days after ovulation, and, on the basis of size and appearance, we concluded that they undoubtedly persisted for a maximum of 60 days. In five nursing females the corpora lutea disappeared before the ovaries were examined from 11 to 35 days postpartum. A sixth female examined 11 days after parturition had four regressing corpora lutea, each 3 mm in diameter, in her left ovary and none in the right ovary. The corpora were those observed when she was first examined 34 days before the birth of her young. She was examined again 20 days after giving birth, when only four corpora albicantia were pres- ent in her left ovary. Thus, in this nurs- ing female, the corpora lutea disappeared between 1 1 and 20 days after parturition. Corpora lutea were not found in histo- logical preparations of ovaries from two wild, lactating females, nor by gross ex- amination of the ovaries from six other wild, lactating females. Pseudopregnancy Our data indicate that corpora lutea persisted for about the same length of time in captive pseudoprcgnant raccoons as they did in those that give birth to young. Corpora were present 61 days but not 82 days after the estimated date of ovulation in one pseudoprcgnant captive. Three other pseudoprcgnant females showed similar periods of pseudopreg- nancy, although the data for these fe- males were less precise than were the data for the first. The persistence of corpora in females that went at least half- way to term did not appear to differ sig- nificantly whether the young were abort- ed, were resorbed, or were born and were removed at birth or nursed until weaned. In one female, discussed in the preceding section, the young were re- sorbed at an early stage and the corpora lutea disappeared no more than 60 days after ovulation. In some species pseudopregnancy may equal normal pregnancy in duration, but in most animals it lasts about half as lon^ (Nalbandov 1958:218). Our obser- vations indicated that all captive rac- coons that ovulated, but did not become pregnant, undeiwent a period of pseu- dopregnancy much as does the dog. In the raccoon pseudopregnancy lasted ap- 56 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 proximately the same length of time as does normal pregnancy and followed ovulation. Our observations of wild female rac- coons during the breeding season indi- cated the relative incidence of pregnan- cies and pseudopregnancies, and supplied substantiating evidence that corpora lutea disappear in wild, lactating females, as in captives, shortly after they have given birth. Histological sections were made of ovaries collected from March through June (1957 through 1961) from 15 wild females 2 years of age or older. Six were pregnant, four were pseudo- pregnant, and five had recently given birth. Corpora albicantia were present in the ovaries of four of the five parous females, and corpora lutea were present in all of the pregnant and pseudopreg- nant animals. The fifth parous female, collected March 1, had recently given birth or aborted, as indicated by the fresh placental scars in her enlarged uter- us and the four corpora lutea in her ovaries; however, she was not lactating. Corpora lutea were not found in histo- logical sections of the ovaries from 39 young-of-the-year, 14 yearling (12-20 months old), and 10 adult wild raccoons collected from July through January. Of 15 wild, parous female raccoons collected from Februray through Sep- tember, only 1 had freshly ovulated folli- cles in February, and another had cor- pora lutea in March. None of the re- maining 13 females, including 6 that were lactating, had corpora lutea. As mentioned earlier, histological examina- tions of ovaries from lactating, captive females indicated that corpora lutea dis- appear between 1 1 and 20 days after parturition, regardless of whether the fe- males nurse their young. From February through June (1957 through 1961) we made 30 observations on 24 captive female raccoons 2 years of age or older. Five were caught only a few days prior to examination. Of the 30 observations, 18 were of pregnant ani- mals, 8 were of pseudopregnant females, and 4 were of animals neither pregnant nor pseudopregnant when examined. The one animal that accounted for two of the four latter observations had an abnor- mally large uterus but inactive ovaries in 1959. Her uterus was enlarged but her ovaries were small when she was exam- ined in May of 1957. Thus, she did not represent the norm. The other two ob- servations of females that were neither pregnant nor pseudopregant were of adult females that had given birth to litters in previous years; each was examined once during subsequent mating seasons. Be- cause each was examined only once dur- ing the breeding season of the year in which corpora lutea were not found, it is conceivable that they had undergone pseudopregnancy but that the corpora had regressed before they were examined. Thus, evidence from both captive and wild females indicated that a majority of the females 2 years of age or older were either pregnant or pseudopregnant each year. Every year during the hunting and trapping season a small percentage of females, judged to have ovulated on the basis of the stimulated or pigmented nip- ples, or both, were without uterine pla- cental scars. During the fur seasons in Illinois from 1956-1957 through 1960- 1961, uteri were examined from 284 fe- males that appeared, on this basis, to have ovulated, and 7 (2.5 percent) had no placental scars. The evidence indicated that these animals had been only pseudo- pregnant. Some annual variation occurs in this characteristic. During the 1960- 1961 fur season, all 77 females judged, upon examination of their nipples, to have ovulated had placental scars in their uteri. PERCENTAGE OF YEARLING FEMALES THAT WERE SEXUALLY MATURE Of 21 captive female raccoons approxi- mately 1 year of age examined from Feb- ruary through June, 1 1 were either preg- nant or pseudopregnant, but 10 were sex- ually immature. Histological sections of the ovaries from nine wild yearlings col- lected from February through August showed no corpora lutea in the five non- July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 57 pregnant females nor in the two lactating females, but corpora were present in the ovaries of the two pregnant yearlings. Gross examination of the ovaries from five wild, nulliparous yearlings collected from March through August showed that the ovaries of four contained no corpora lutea, but that three corpora lutea were present in one female collected in May. Thus, 10 of 21 captive yearlings and 9 of 14 wild yearlings were sexually im- mature. During two fur seasons in Illinois (1959-1960 and 1960-1961) nulliparous adults with tiny unpigmented nipples ac- counted for 15 of 164 (9.2 percent) adult female raccoons examined. These nulli- parous adults, with tiny unpigmented mammae, probably did not ovulate dur- ing the first breeding season after their birth. SECRETION OF PROGESTERONE BY CORPORA LUTEA The period of the production of pro- gesterone by corpora lutea in the raccoon is unknown, but circumstantial evidence indicates that corpora lutea probably secrete progesterone as long as they are present (discussed later in connection with the production of uterine milk) . One female had five corpora lutea, each 5 mm in diameter, when first examined on April 10. At that time we traumatized her left uterine horn by inserting a nee- dle into the uterine lumen two times, each time scratching the entire length of the inside of the uterine horn with the point of the needle as it was withdrawn. Eight days later the ovaries and corpora were unchanged in gross size and appear- ance. The left uterine horn showed no evidence of trauma, but there is no direct evidence that the uterus of the raccoon will respond to traumatization with a decidual reaction in the presence of pro- gesterone. PIGMENTATIO:^ OF MAMMAE Several female raccoons were studied to establish a possible physiological cause for the pigmentation or npigmentation of nipples. Some pseudopregnant yearling females developed heavily pigmented nip- ples, whereas others did not. The pres- ence or absence of pigment was not cor- related with nursing, abortion, resorp- tion of embryos, age at first estrus, or any other factors we could*recognize. Unpig- mented nipples remained so throughout life, but lightly pigmented nipples some- times became darker with age. The pig- ment was not sloughed after nursing as Snyder & Christian (1960:650) found in the woodchuck (Marmota monax). INTERSTITIAL TISSUE Many studies were conducted before 1920 on the interstitial tissue in mam- malian ovaries. Interstitial tissue is present in greater or lesser amounts in the ovaries of some species and is ap- parently absent in others. Little is knovvTi about its function. His (1865) was ap- parently the first to describe interstitial tissue cells in mammalian ovaries and to discuss their importance. Allen (1904: 120, 141) concluded that interstitial cells were formed from connective tissue dur- ing a process of degeneration in both the testis and ovary, and noted many points of similarity between the cells of .the interstitial tissue and the lutein cells of corpora lutea. Kingsbury (1914:86) dis- cussed the interstitial cells in the do- mestic cat (Felis catus) and recognized the lipoid nature of the granules in these cells, but he found no evidence that the cells constitute morphologically an intra- ovarian gland. He also reported their presence in immature, newly bom, and fetal kittens. Rasmussen (1918:395) believed that in the woodchuck th" interstitial cells proliferated from t :ainal epithelium during adult life, l found a marked seasonal variation in the number of in- terstitial cells and in the amount of lipoid present in them in the woodchuck. These cells gradually increased in number dur- ing hibernation and hypertrophied rapid- ly immediately after hibernation (Ras- mussen 1918:371-372). Maximum num- bers were seen in females that did not 58 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 become pregnant until late in the breed- ing season. Retrogression began with pregnancy and the growth of corpora lutea and continued until July. The ovar- ian interstitial cells were minimal in size in late summer and early autumn but then began to enlarge. After an exten- sive review of the literature, Rasmussen concluded, in accord with the vast ma- jority of the investigators, that the inter- stitial cells come either directly from the connective tissue (stroma) of the ovary, or indirectly from the theca interna of atretic follicles. According to Corner (1932: 1597), the stroma of the rabbit ovary consists so largely of epithelioid cells heavily laden with lipoid granules that the entire organ is a solid mass of interstitial cells in which the follicles and corpora lutea are em- bedded. This finding led to the concept that the ovarian stroma in this and simi- lar species was a gland of internal secre- tion, the so-called interstitial gland. Em- bryological study showed that interstitial cells were largely derived from the theca interna of atretic follicles and that inter- stitial cells were found in many species at a very early stage of embryonic differ- entiation, in which case they seemed to be produced by the modification of the cells of the stroma and of the various epithelial proliferations. The pig ovary (Corner 1932:1597) contains epithelioid cells only in follicles and corpora lutea, the stroma cells being simply fibroblasts. Corner (1932: 1597) reported that the cat ovary was between the extremes rep- resented by the rabbit and pig ovaries. In the adult human ovary there appeared to be epithelioid cells only in follicles and corpora lutea. It is conceivable that interstitial cells, whether found in great numbers in the stroma of the rabbit or in thin layers in atretic follicles in humans, are function- ally the same, but proof is lacking (Cor- ner 1932:1597). Comer (1932:1598) further reported that in all of the species he studied the interstitial cells contained granules of neutral fat or, at least, of lipoids, which reduce osmic acid and stain with Sudan III. Some workers are ready to assume that the lipoids found in the interstitial cells represent a true internal secretion. Much of the older work, mentioned by Stafford & Mossman (1945:97), showed that in some mammals the de- velopment of ovarian interstitial tissue is at its maximum during proestrus and estrus and that all of the animals in- cluded in this group, most of which breed annually or semiannually, have long re- productive cycles. The literature reports no evidence of ovarian interstitial tissue in laboratory rodents, which have short estrous cycles, and there is no easily dis- cernible cycle in the amount or state of interstitial tissue that could be correlated with pregnancy in the guinea pig. There is a trend toward a maximum amount of interstitial tissue in the cortex near estrus and into early pregnancy and a minimum in midpregnancy. The high and low in the medulla seemed to occur a week or two later than in the cortex, suggesting that in the guinea pig medullary inter- stitial tissue originates from that of the cortex. Patzelt (1955) studied the interstitial tissue in several carnivores and empha- sized that age, time of year, and stage of the reproductive cycle greatly affected the interstitial cells. He also pointed out that other investigators considered thecal cells, which are traced back to the par- ticularly active atresia of follicles during . pregnancy, to be closely associated with I the cells of the corpora lutea. Thus, Alt- I mann (1927) thought it conceivable that only a topographical contrast existed be- tween thecal granulosa and lutein cells. Patzelt (1955) regarded the intersti- tial tissue cells as producers of hormones and as a storage place for the substance necessary for the formation of new folli- cles and for propagation in general. The basis for his ideas was the fact that the lipoid-containing cells are variously de- rived within the rudimentary ovary from germ layers, thecal cells, and cells of the surrounding stroma, and that it is not possible to demarcate the source of the interstitial cells. He usually found that ovarian interstitial cells were filled with i July, 1973 Sanderson &Nalbandov: Reproductive Cycle of the Raccoon 59 stored lipoids after a heat period and during pregnancy. After parturition a decrease in stored lipoids occurred that led to a functional dimorphism simultan- eously with the formation and maturation of new follicles. Hansson (1947) concluded that the abundance of interstitial tissue in the mink [Mustela vison) indicated that the tissue performed a special task. Because anestrus in the mink lasts from May to January, when no follicular growth be- yond the vesicular stage takes place, the interstitial tissue may serve as a regu- lator during this time, governing sexual differentiation. A preliminary study of the abundance of interstitial tissue in histological sec- tions of the ovaries of 119 raccoons taken in all months indicated that interstitial tissue cells were abundant at some stamps of the reproductive cycle, often occupying as much as 50-90 percent of the space in the ovary. However, interstitial tissue cells were seldom abundant when corpora lutea were present. Of 21 pairs of ovaries with corpora lutea, only 3 had significant amounts of interstitial tissue. One of these is shown in Fig. lOG. Females less than about 2 months of age did not have large amounts of inter- stitial tissue in their ovaries. With this exception the ovaries of females less than 12 months old contained more, both relatively and absolutely, of this tissue, on the average, than did the ovaries of older females. Seasonal trends in the abundance of interstitial tissue were apparent in ovaries with no corpora lutea. The ovaries re- moved from 16 adults from January through June contained little interstitial tissue. Ovaries removed from 26 adults killed from July through December con- tained more interstitial tissue than did those collected earlier in the year. No trend was apparent in the amount of interstitial tissue within the July-Decem- ber period. During this interval ovaries from adults did not contain as much interstitial tissue as did ovaries from fe- males less than 12 months old. Ovaries from raccoons less than 12 months of age showed less seasonal vari- ation in the abundance of interstitial tissue than did the ovaries from older animals. Small amounts of interstitial tissue were present in ovaries removed from seven juveniles in May and June, when most young were less than 2 months old. The ovaries excised from 24 juveniles in July, August, and September contained more interstitial tissue than did the ovaries examined in May and June, but the differences among the amounts of interstitial tissue found in July, August, and September were slight. The greatest abundance of interstitial tissue was dis- covered in 16 pairs of ovaries taken from juveniles during October and November. The maximum ovary weights recorded during this study were those of juvenile females in November (Table 5). Nine pairs of ovaries were examined from fe- males not yet 1 year old killed during the period December through April. The abundance of interstitial tissue in these ovaries did not appear to differ from that in the ovaries of juveniles examined from July through September. In spite of marked seasonal and age differences in the abundance of intersti- tial tissue, there was no apparent correla- tion between its abundance and the size or amount of coiling of the uterine glands. The development of the uterine glands and the presence of secretory ma- terial in these glands were largely de- pendent upon the presence of corpora lutea. Three sources of interstitial tissue have been suggested, germ layers, thecal cells, and cells of the surrounding stroma, and all three may be present in the raccoon. Small amounts of interstitial tissue were present in some raccoon ovaries at birth. Judging from appearance alone, we be- lieve it probable that some interstitial tissue in the raccoon is formed from degenerating follicles. Several cases simi- lar to the one shown in Fig. 10..4 were seen during this study. In other ovaries there appeared to be a streaming of the cells as the interstitial tissue formed, presumably from the germinal epitheli- 60 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 One striking feature of interstitial cells although the luteal cells were generally was their resemblance to luteal cells, larger (Fig. 10) . Under the microscope A « '^.:^'-^'%£!mr^^^>W.y^:^i 9f July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 61 these two kinds of cells appeared more alike than the photographs in Fig. 10 indicate. PLACENTAL SCARS Deanesly (1935:464) first reported that she could recognize parous uteri in the stoat (Mustela erminea) by the pres- ence of pigment granules that later work- ers called placental scars. Deno (1937: 433, 445) found that placental scars were produced in the mouse by accumu- lations of hemosiderin in the cells of the reticulo-endothelial system and that the placental scars were associated with the involuting metrial gland. Deno (1941) later reported that placental scars were visible m both the rat and mouse for a year or longer. Conaway (1955:516-517) stated : "The placental scars of the rat ap- pear as yellow to black pigmented areas along the utero-mesometrial border. Their origin seems identi- cal with that of the scars in the mouse .... In both the rat and mouse, the metrial gland is a promi- nent structure at the base of the placenta .... Presumably it is formed by an extension of the decidual re- sponse into the connective tissue of the myometrium. The pigment-lad- en cells are concentrated in this area between the longitudinal and circu- lar muscle layers although some are found in the deeper stroma of the endometrium. As the age of the scar increases the pigmented area may decrease in size and appear darker in color." Sooter (1946:69-70) counted placen- tal scars to determine the numbers of young produced by muskrats (Ondatra zibethicus) although no critical work has been done to determine whether the num- ber of placental scars corresponds to the number of young born. Elder (1952) reported the failure of placental scars to reveal breeding history in captive mink. Brambell & Mills (1948:241), working with the European rabbit (Oryctolagus cuniculus), again pointed out "that although there is little likeli- hood of failure to detect implanta- tion sites containing living embryos the possibility remains of the disap- pearance before full term of sites in which the embryos had died and were reabsorbed soon after implan- tation or, more probably, that such sites might be overlooked, through becoming less conspicuous, and hence omitted from the counts." In laboratory rats and wild brown rats placental scars were only a crude indica- tion of the number of young produced (Davis & Emlen 1948: 166), with errors as high as 100 percent in either direction. Conaway (1955:531) found that pla- cental scars in the laboratory rat were always formed if all embryos were re- sorbed after the 11th day of pregnancy, whereas total resorption prior to this time never caused the formation of scars. If some of the embryos were resorbed, death on the seventh day or later resulted in scar formation at all resorption and term sites. If some embryos were resorbed be- tween the 8th and 11th days and the remainer after that, scars were formed at all sites. The size and appearance of resorption scars were similar to those of term scars. Momberg & Conaway ( 1956: 379) found that 32 of 312 placental scars from previous pregnancies were overlapped by scars of second pregnan- Fig. 10 (Poge 60).—Photomicrographs of luteal and interstitial cells of raccoons, showing similari- ties in the two. A, female 2137; luteal cells (X 94); ovary removed March 19, 1958; pregnant. B, fe- male 1292; luteal cells (X 94); ovary removed May 14, 1958; ovulation caused by injections of preg- nant mare's serum. C, female 2805; luteal cells (X 94) ; ovary removed February 24, 1960; fresh corpora lulea resulted from natural ovulations; pseudopregnant. D, female 2232; interstitial cells (X 94); ovary removed August 7, 1958; wild animal approximately 3 months old. E, female 2403; interstitial cells (X 94); ovary removed November 6, 1958; wild animal 7 months old. F, female 2234; inter- stiiiol cells (X 94); ovary removed August 8, 1958; wild animal 3 months old. G, female 1292; luteal cells (X 375); ovary removed May 14, 1958; ovulation caused by injections of pregnant mare's serum. H, female 2242; interstitial cells (X 375); ovary removed August 27, 1958; wild animal 4 months old. The sections were stained with hematoxylin and eosin. 62 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 cies in the white rat. They could not always recognize the superposed scars by gross examination, but microscopic recognition was possible. The placenta of the raccoon was first described by Watson (1881:280-296). The zonary placenta of the raccoon is similar to that of other carnivorous mam- mals. Watson (1881:279) noted: "The placenta formed a complete ring, but at the centre of its widest part, i.e., opposite the back of the foetus, there was a spot similar to that figured by Daubenton in the placenta of Martes domestica, and described by Bischoff in that of Lutra vulgaris, Mustcla foina and Mustela martes, where the substance of the placenta was deficient. This defi- ciency involved the entire thickness of the placenta, so that a probe could be passed from the uterine to the chorionic surface of the organ without injury to its substance." The placenta of Procyon is truly decidu- ous in character, as it is in the dog, cat, fox, and seal. According to the classifi- cation of Mossman (1937:224), the rac- coon placenta is endotheliochorial. Pla- cental scars in the raccoon were apparent- ly first noted by Stuewer (19436:68), who autopsied a female raccoon in May and found four placental scars in the uterus; he believed they indicated that four young had been born. Sanderson (1950:399) examined uteri from six cap- tive females and concluded that "pla- cental scars may be an accurate measure of litter size in raccoons." If placental scars are to be useful in estimating the reproductive performance of a species, several facts about them must first be known. Pertinent questions are: (1) Is one placental scar formed for each implantation site regardless of the fate of the developing embryo? (2) If the answer to the first question is no, then what stages of embryonic develop- ment result in the formation of placental scars? (3) Is it possible to differentiate placental scars formed from embryos that go to term from those formed from em- bryos that are aborted or resorbed? (4) How long do the placental scars persist, and is the length of time they persist affected by the female's subsequent breed- ing hijtory? (5) Are the placental scars recognizable at all seasons of the year? (6) If the placental scars persist beyond a subsequent pregnancy, is it possible to recognize scars representing litters from different years? Some preliminary infor- mation on all of these questions has been obtained. In only 2 of 27 litters with a total of 98 embryos in 2 of 20 captive female raccoons that we examined did we find discrepancies between the number of em- bryos observed and the number of pla- cental scars identified later. One female (No. 2960) had four embryos, estimated to be 30 days of age when examined on May 26, but five grossly identical pla- cental scars when the uterus was re- moved 6 months later. The additional scar may have represented a litter of one from a previous year. If so, the scar was overlooked when this same uterus was examined during the fall before the four embryos were observed. The extra scar may have also represented an additional embryo that was aborted or resorbed prior to the time the four embryos were examined. The second female (No. 4022) had two live and one dead em- bryo when first examined on February 19. She gave birth to two live young 29 days later, but when her uterus was examined 11 days after parturition, there were two grossly identical scars in each horn. There were four corpora lutea in her left ovary and none in the right. Thus, the additional scar observed at the second laparotomy was probably from an embryo that was aborted or re- sorbed prior to the first examination. A captive female raccoon (No. 2960) had four embryos, estimated to be 30 days of age, when examined. She was j given a drug, Malucidin, that caused 1 either abortion or resorption. The em- bryos were gone 13 days later, and the sites of attachment were indicated by large bumps. When the uterus was re- moved 6 months later, five placental scars were identified by slight bumps. We July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 63 split the uterine horns and identified the five placental scars as typical for captive females. (Possible differences in placental scars of captive and wild animals are dis- cussed below.) Thus, in this female, one placental scar was formed for each of the four embryos even though all four embryos were either aborted or resorbed at midterm. As has been discussed, the fifth scar either persisted from the previ- ous year or resulted from an embryo resorbed prior to the first examination when the four embryos were about 30 days of age. Another captive female raccoon (No. 3333) had four live embryos and one that was being resorbed in her uterus on March 21. It was estimated that the embryo being resorbed had died 30 days after conception. The young were born 33 days after the initial examination. Sixteen days after the birth of the litter the placental scar representing the re- sorbed embryo was smaller than the others, but 47 days after parturition no gross difference could be detected among the five scars. One pregnant captive female raccoon (No. 2824) was castrated approximately 50 days prepartum, but her embryos con- tinued to grow for about 20 days before they were aborted and resorbed. A second captive pregnant female (No. 2151) was castrated appro.ximately 30 days prepartum, and her young were aborted about 1 week prepartum. Two months after abortion or resorption the placental scars in these females could not be differentiated grossly from those formed by normal embryos born at term. Female No. 2824 was killed 4.5 months after she was castrated. When she was killed, only one placental scar was found, both before and after the uterus was split, even though the exact locations of the embryos were known. The one scar was dark and broad, and appeared to be typical of those formed from young bom during the current breeding season. The scar was formed at the site of one of three embryos present 19 days after cas- tration. All three of the embryos were aborted prior to 26 days after castration. The data from the two castrated fe- males (No. 2824 and 2151) that lost their young and from six intact captive females that resorbed or aborted some or all of their embryos indicated that one placental scar was formed for each embryo that existed for appro.ximately 30 days, whether or not any embryo went to term. We have made some observations on the persistence of placental scars in the raccoon (Table 11). Placental scars were present, although indistinct, in one female (No. 2959) when her uterus was removed nearly 19 months after her young were born. Scars were visible 12 and 17 months after parturition in another female (No. 1786), but could not be seen in her enlarged uterus stimu- lated by hormones 14 and 2 1 months after parturition. Her ovaries were re- moved approximately 1 year after the birth of her young, and, after castration, she was treated with estradiol and pro- gesterone at various intervals. These treatments may have affected the rate of disappearance of her scars. A third fe- male (No. 2779) had placental scars for 14 months, but not 23 months, after Table 11.— Persistence of plocentol scars in coptive raccoons. 64 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 parturition, and a fourth female (No. 2125) retained placental scars nearly 17 months after parturition. None of these females gave birth in the second year. There was no macroscopic evidence of placental scars from a 1958 litter (Fe- male 2124) 18.5 months postpartum, but histological examination revealed a few scattered pigment granules, and scars from a 1959 litter were prominent. Thus, the 1958 scars disappeared, for practical purposes, prior to 18.5 months after parturition, when she had a litter the following year. All female raccoons had placental scars when examined from 2 to 10 months after the birth of their young. The evidence indicated that if a female failed to give birth to a litter in the next year, placental scars persisted for approximately 19 months in captives, but not as long as 24 months. If a cap- tive gave birth to a litter the next year, scars from the first litter persisted for 10 or more months but not as long as 19 months. One captive raccoon became pregnant at the second ovulation during one sea- son. The single embryo, in the process of being resorbed when it was first ob- served, was estimated to be 20 days old. Twenty-one days later the site of placen- tal attachment was readily identified as a blimp 8X7 mm in size; 61 days after the initial observation no trace of the scar could be seen. Thus, this scar dis- appeared between 21 and 61 days after the resorbing, 20-day embryo was ob- served. The absence of living embryos in this captive may have been an im- portant factor in the rapid disappear- ance of the scar. The variability in the length of time that placental scars were visible in the raccoon after parturition is shown in Table 11. In two females scars were not grossly visible in their stimulated uteri during or near estrous cycles of the ensuing years, because their enlarged uteri caused a dififusion of the pigment granules of the scars, making them in- visible. Scars in these females were again visible macroscopically when the uteri regressed. Scars from a litter born in May 1958 (discussed above) could not be seen (No. 2124, Table 11) 18.5 months later (De- cember 1959) even though their exact locations were known and the uterus was removed and split. After we sectioned the site of one scar, we were able to identify a few scattered pigment granules in the endometrium. Two scars from a litter born in April 1959 were easily identified macroscopically in this same uterus 8 months (December 1959) after the birth. In a second female (No. 2959, Table 1 1 ) scars from young born in May were not visible with translucent light after the uterus, which was stimulated, was removed 18.5 months later. All four scars from this litter were located and were identified by the slight bumps vis- ible at the placental sites. After the uterus was opened, all four scars were visible as pale, brownish areas, but they might have been overlooked had not their exact locations been known. When one of these scars was examined his- tologically, moderate numbers of pigment granules were seen in clumps and scat- tered in the endometrium and in the ad- jacent myometrium. The distribution of pigment granules in the uteri of two wild females was stud- ied in histological sections. Each of these females had four scars at autopsy. Pigment granules in the uterus of one fe- male were somewhat scattered but seem- ed to concentrate in a ring deep in the endometrium near the myometrium. Many pigment granules were scattered throughout the endometrium of the uterus of the other female. Pale placental scars were often diffi- cult to see in situ in a live animal, and early in the study some scars may have been overlooked. We believe that, after we became experienced in looking for scars, no visible placental scar was over- looked, but they could not be seen in pregnant females and females at or near estrus. When the scars had practically . disappeared, they could be observed only j by splitting the uterus. Thus, these pale scars would be overlooked when examin- ing live females by laparotomy. If the July, 1973 Sanderson & Nalbandov: Reproductive Cvcle of the Raccoon 65 uterus of a live female was stimulated, many of the placental sites could best be identified by slight, opaque bumps rather than by the pigmentation. Identification of the location of scars by the presence of bumps was possible for several weeks after parturition, when the uterus was still stimulated, as well as in the stimu- lated uterus at or near estrus. After the uterus regressed, scars were usually read- ily visible as bumps or could be identi- fied by using translucent light to observe the pigmented areas. The pigmented areas could also be located when the uterus was opened or by histological ex- amination. Placental scars seem to persist longer in wild raccoons than they do in captives. The placental scars of captives that we examined from October through January after the births of their litters were gen- erally pale brown, small, and slightly opaque. A majority of the wild females examined during these same months had larger, more opaque scars, often black. Many (55.2 percent in 1959 and 41.0 percent in 1960) uteri of wild, parous females had more than one group of scars, which differed in size and density (Fig. 11 ). Presumably these scars were from difTerent years; however, some might have been from difTerent litters born in 1 year. There was no evidence that as many as 40 or 50 percent of the wild females gave birth to second litters during a single season. Thus, placental scars probably persist for 20 months or longer in many — perhaps in all — wild females. In the few wild females with three groups of scars, the first group may have persisted for as long as 32 months. The placental scars of raccoons are useful for estimating litter size and rate of productivity. However, these scars must be used with caution, and care must be taken to separate properly the groups of scars. We do not know for certain the significance of multiple groups of scars. We can say with reasonable con- fidence that each embryo that reaches 1 month of age is represented by one scar for 10 or more months. Scars in wild females with only one group of scars probably reflect implantation rates for the preceding breeding season. Most single groups of placental scars occur in females that have mated successfully only once. MORPHOLOGY OF THE REPRODUCTIVE TRACTS Males The duct system and accessory glands in the reproductive system of the male raccoon (Fig. 12) are similar to those found in the dog, as described and shown by Nalbandov (1958: 42-44). Seminal vesicles are lacking, as they are in the dog, fo.x (Vulpes fulva) , and wolf (Canis lupus). The Cowper's glands (bulbo- urethral glands) are also absent. The walls of the vasa deferentia thicken prior to entering the prostate and form the ampullae. The ampullae and the urethra Fig. 11.—Raccoon uterus (X 0.75) split to show two groups of placental scars. This female was killed on January 23. Two light scars were only barely visible in the photogroph but were readily visible in the fresh specimen. Their locations and densities relotive to the three dark scars ore indicated by the light stippling (arrows). 66 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 Ampulla Prostot Vas Deferens -Schematic drawing (side view) of the reproductive system (X 0.85) of an adult molelFig. 12.. raccoon. unite inside the prostate to form a com- mon duct. The many compartments of the prostate gland open into this duct system. The OS penis or os baculum (bone of the penis) is well developed in the rac- coon. Its stage of development has been used to separate males into two age groups (Sanderson 1950: 395-396; 1961a: 11-14) . The os baculum was once used by tailors as a ripping tool for taking out basting threads (Jaeger 1947: 297). We found several raccoon bacula that had been broken and then healed. Sand- erson (1950: Plate 11) showed a photo- graph of some of these bones. Our data from wild males shed some light on pos- sible causes for these broken bones. Dur- ing four hunting and trapping seasons in Illinois (1957-1958 through 1960- 1961), 7,233 bacula from juvenile rac- coons were examined. Forty-three (0.6 percent) of these had been broken but were healed or healing, and 238 (3.3 per- cent) were freshly broken. At the same time, 4,152 bacula from adults were ex- amined. Eighty-six (2.1 percent) of these had been broken but were healed, whereas 41 (1.0 percent) were freshly broken. These data indicate that most of the breaks in the os baculum of the raccoon occur in juveniles. The bacula of juven- iles are much softer and more easily broken than are those of adults. Hunters often shake a raccoon out of a tree and let their dogs fight it. Fighting with" dogs could account for the freshly broken bones found in both adults and juveniles, and the more durable bones of adults would explain the smaller percentage of freshly broken bacula found in older raccoons. Females The raccoon uterus (Fig. 13) is some- what intermediate between the bicornuate uterus found in the pig and insectivores, and the bipartite uterus found in the cat and dog. There is a single cervix and the horns are distinct, but after the horns join externally to form the single, small uterine body, the uterine lumina remain separate — even though this separation is not apparent from the outside — to a point near the cervix. Llewellyn & Enders (19546: 439) re- moved one ovary, ovarian capsule, ovi- duct, and proximal end of the uterine horn in each of two raccoons. After closing the cut ends of the uteri with sutures, they released the females. When retrapped the next year, each female was carrying three embryos, two each in the normal horns and one each in the ovar- iectomized horns. Thus, even though the internal separation of the uterus ex- tends nearly to the cervix, ova can pass from one uterine horn to the other. In our study some indirect evidence of transuterine migration of ova was noted. In a few cases more embryos were found July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 67 Fimbria Burso ovarii »« 'M,,irU- Co I on "_ . * V liii»^..i» I ' Vagina Cervix Uterine Body Placental scor- ^^ Urethra Uterine tiorn Oviduct Ovary Fig. 13.—Schematic drawing (ventral view) of the reproductive tract (X 0.5) of a parous female raccoon. in a uterine horn than there were corpora lutea in the corresponding ovary, but the total number of corpora lutea present in both ovaries was usually the same as the number of embryos or placental scars present in both uterine horns. The ovary in the raccoon, ovoid in shape, is completely surrounded by the bursa ovarii (Fig. 13). This sac is in- tact except for a small slit on one side, not large enough to permit passage of the ovary as in the mink [Mustela vison) , dog, and fox. One of our captive fe- males had a congenital deficiency of the bursa that was large enough to permit passage of the right ovary. This opening was slightly dorsal to the normal slit in the bursa but was not connected with it. The left ovarian bursa was normal. This captive was the only such animal among several hundred examined. Watson (1881: 273-274) observed one raccoon and reported that the ovary was destitute of any peritoneal pouch or pavilion such as formed an almost complete sac in many animals. The fimbria is extensive, and in the estrous female the edge of the fimbria is bright red and protrudes through the slit in the capsule. This bit of fimbria grossly resembles the gills of a fish. The fimbria joins with the end of the oviduct. The oviduct is highly convoluted and makes an almost complete circle around the ovary before entering the uterus (Fig. 13). Two, three, and sometimes four ova were observed in a single follicle. When an ovary contained one follicle with multiple ova, several other follicles with multiple ova were usually present. Approximately 25 female raccoons from Iowa and approximately 25 from Illinois were examined for the presence of the OS clitoridis. A bone — 11 mm in length — was found in only one clitoris. Rinker (1944: 91) found four ossa cli- toridae in four female raccoons examined in Kansas, but found no bones in the clitoria of four other females from a "distant locality," apparently in Kansas. Burt (1960: 8) used Rinker's observa- tion as the basis for stating that the os clitoridis is present in the raccoon. Sanderson (1950: 398) found only one OS clitoridis among 100 female raccoons in Missouri. Because only a small per- centage of females examined from Mis- 68 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 soiiri, Iowa, and Illinois had ossa clitor- idac, there may be geographic variation in the j)resence of this bone. Its presence is not of general occurrence in raccoons in all localities. EFFECTS OF CASTRATION Males Some effects of castration on the de- velopment of the OS baculum in the rac- coon have been discussed by Sanderson (1961a: 13-14). The information in that report, with additional observations, is presented here. The lack of sex hor- mones in males was reflected by the much shorter and thinner bacula in castrated animals in comparison with bacula from intact animals of similar ages (Sanderson 1961a: Fig. 4). The lack of sex hormones became apparent at 8- 1 1 months of age in castrated males. Sanderson (1950: 396) showed that in intact males the penis normally became extrusible at about 10 months of age, but a castrate male (Sanderson 1961o: Fig. 6, No. 59) had a nonextrusible penis and a small baculum at 22 months of age. This baculum was only slightly longer and heavier than one from a castrate raccoon only 10 months of age (Sander- son 1961a: Fig. 5, No. 209), but both were much shorter and thinner than were the bacula from intact males 18-23 months of age (Sanderson 1961a: Fig, 6) . The baculum from the castrated rac- coon 22 months of age was dense like an adult bone and not spongy at the base as were bacula of similar size from rac- coons 12 months of age and younger. Thus, we concluded that the level of sex hormones affected the enlargement of the preputial orifice and maturation of the penis bone but had little or no effect on the development of the baculum prior to 7 months of age. Castration in males also apparently caused a slight delay in the closure of the epiphyseal cartilage in the radius and ulna, but because most of the castrat- ed males in this study died of disease at early ages, not enough information was available to demonstrate this relation- shin conclusively. Epiphyseal plates were classified as closed (without cartilage), thin (inter- mediate condition), or broad (with a thick plate of cartilage) (Sanderson 1961a: 7). One castrated male had broad epiphyses at 1 7 months of age and thin epiphyses at 20 months of age. His epiphyses were still thin when he died at 22 months of age. When examined, 35 intact males with broad epiphyses were 15 months of age or less, whereas 14 of 17 males (82 percent) with thin epi- , physes were 13-19 months of age. Epi- physeal plates in 11 of 13 intact males closed between 16 and 21 months of age (Sanderson 1961a: 16). The effects of castration on epiphyseal closure merit further study. Females The time of closure of epiphyses in fe- males was much like that in males, but the greater variation in the upper ages of females with thin epiphyses indicated that epiphyseal closure was delayed in some females or occurred later in some than in others. One factor that perhaps influences age at epiphyseal closure is the level of cir- culating hormones. Two females were castrated to study the effects of the ab- sence of ovarian hormones on epiphy- seal closure. One female, born in the wild, was castrated at an estimated age of 4 months, and one, born in captivity, was 3 months old when castrated. The first had broad epiphyses at 14 months of age, thin epiphyses at 20 months of age, and thin epiphyses when she died at 23 months of age. The second had broad epiphyses at 22 months of age, thin epi- phvscs at 25 months, and nearly closed epiphyses at 27 months of age. Epiphy- seal development and closure in these two castrated females were delayed in comparison with the rate of development-' and closure found in the average intact female. Four additional females were castrated at estimated ages ranging from 13 to 24 months. Our observations sug- gested that the removal of the ovaries, even after raccoons had reached sexual maturity but before the epiphyses closed, delayed the rate of epiphyseal closure. July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 69 Major factors that may have contrib- uted to the variations we observed in age at epiphyseal closure in female rac- coons were ( 1 ) age at first mating, ( 2 ) hormone secretion level, and (3) quality and quantity of nutrition. Factors influ- encing age at epiphyseal closure should be studied further because the available data are somewhat contradictory. Thus, our data and those of Sanderson (1961a: 10-11) suggest that epiphyseal closure in the castrated female raccoon is delayed in comparison with that found in the average intact female but falls within the limits of variability for intact females. In mammals castration after implanta- tion and during the first third or first half of pregnancy usually leads to abor- tion or resorption of the fetuses (Nalban- dov 1958:221). In some mammals the ovaries are required throughout gestation, but other mammals do not lose their young after castration, once the crucial period is past. Two pregnant females were castrated to learn vvhether the raccoon is a species in which pregnancy is maintained after castration. To establish limits after which castration is tolerated, these two animals were castrated approximately 1 1 and 38 days after conception, respectively. (It had been established earlier that perform- ing laparotomies on pregnant raccoons did not interfere with pregnancy.) The first of these two pregnant female raccoons that we castrated was born in 1959 and reared as a pet. She gave birth to a litter in 1960. On February 5, 1961 she forcibly repelled the approaches of her mate. Twenty-five days later she had three embryos in her left uterine horn and two in the right but only four cor- pora lutea. Each uterine swelling was 10 mm in diameter. We estimated the embryos to be 11 days old, suggesting that mating had occurred about February 17. Both ovaries were removed on March 2, and 19 days later two embryos in the left uterine horn were being resorbed. These two swellings were almost as large as the other three, but the surfaces were collapsed and flaccid, not turgid like those of a normal swelling. The embryos were still present at both sites. The remaining three embryos, 45 X 20 mm, looked almost normal, except that the swellings appeared less round and turgid than normal swellings are. We could not discern whether the embryos were alive or dead. We estimated that, if they were alive, they would be born in 25 days ( Fig. 7 ) . Thus, these embryos had a normal rate of growth for 19 days after the castration of the female. No embryo was found 26 days after castration. From gross appearances we concluded that the last three embr\'os were aborted and the first two were resorbed. Twenty-eight days after this female raccoon was castrated, her mate was re- turned to her cage. The next day, only 3-10 days after her young were aborted, this pair was observed in copulation. This activity suggests the possibility that postpartum heats, which occur in several species such as the sow and mare, may not be dependent upon the presence of the ovaries. This female escaped 3 months after she was castrated and was taken in a steel trap 52 days later. After she was killed, it was discovered that she was lactating profusely. When the mammary gland was sliced with a scalpel, the entire cut area immediately filled with milk. She was lactating more than 4.5 months after her ovaries had been removed and 4 months after her young had been resorbed and aborted. However, the second pregnant female that was castrated showed no indication of lactation 5 months after re- moval of her ovaries. No traces of ovarian tissue were found during the autopsy performed on the first of these females. The uterus, measur- ing 7X4 mm, was turgid and appeared similar to uteri of animals at estrus, but sectioning showed the endometrium to be devoid of even traces of glands. Other female raccoons, months after being cas- trated, had thick epithelia lining their vaginas (Fig. 9A) , suggesting the possi- bility of an extraovarian source of estro- gen in the castrated female. The second of the pregnant raccoons that was castrated was placed in captivity in 1958 when she was about 2 months 70 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 old. She had two embryos in each uterine horn on February 28, 1961, when both ovaries were removed (approximately 38 days after conception and 25 days before ])arturition) , but the left ovary had three corpora lutea and the right ovary only one corpus luteum. Nineteen days after castration and 6 days prior to expected parturition, one dead embryo weighing 43 grams was found in her nest box. The average birth weight of eight newly born raccoons that we weighed was 61.8 grams. The embryo was well developed but did not have much hair, and its hair was shorter than in most young at birth. Twenty-one days after castration the uterus contained enlarged areas where the young had been attached. One of these sites was opened and examined. Detritus was present, but there was no other evi- dence of resorption, which was occasion- ally seen in both wild and captive fe- males. Thus, all four embryos were prob- ably aborted about 1 week prepartum. Although the two females were cas- trated at different stages of pregnancy, the embryos apparently persisted for about the same length of time in each — 19 days after castration—33 days short of term for the first female and approxi- mately 7 days short of term for the second. — EFFECTS OF EXOGENOUS HORMONES Males Two male raccoons were studied to learn whether injections of androgen would initiate or prolong spermatogenesis during the male's period of summer steril- ity. The first animal chosen was an adult male at least 20 months old when he was captured. In August (during the period of sexual inactivity) the left testis was removed, weighed (1.6 grams), and preserved for histological study. The average weight of one testis from an adult in August was 2.6 grams (Table 1). Sperm could not be found in the epididy- mis, but spermatogenesis was occurring in a few seminiferous tubules. This male was given six testosterone doses of 30 mg each subcutaneously over a period of 18 days. He was killed 21 days after the removal of his left testis and the first injection of testosterone. The right testis weighed 1.6 grams, and no sperm were present in either the seminiferous tubules or epididymis. Histological comparison of the two testes and the epididymides showed slight changes that we attributed to the testosterone injections. Both before and after the hormone treatment most sper- matogenic cells were approximately 8 mi- crons, and the nuclei 3 microns, in di- ameter. However, after testosterone in- jections a few of the cells were as large as 1 1 microns in diameter. The lumina of the seminiferous tubules remained about the same size after the treatment as they were before, but after the injections the cells of the seminiferous tubules were more scattered than they were before treatment. Sperm were present in the seminiferous tubules prior to treatment, but not afterwards. Sperm could not be found in either epididymis, one of which was removed and examined before and the other after the homone treatment. The epithelial lining of the tubules was 46 microns tall prior to treatment and 30 microns after treatment (each height is an average of five measurements) , indi- cating degenerative changes, perhaps caused by the hormone. The average outside diameter of the tubules was 140 microns prior to treatment and 65 mi- crons afterwards. A second male raccoon, captured when approximately 3 months of age, was reared as a pet. He was approximately 16 months old when his left testis, weigh- ing 3.3 grams, was removed in August. The epididymis contained many motile sperm. He was treated with four doses of 12 mg each of testosterone over a 13-day period. He was killed 15 days after the removal of the left testis and the first injection of the hormone. At that time his right testis weighed 2.5 grams, and many motile sperm were in the epididymis. His- tological examination revealed few chang- es in the cells of the seminiferous tubules. July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 71 A few sperm were present in the semini- ferous tubules both before and after treat- ment. After treatment sperm were not found in a section of the epididymis, but a few were observed in a drop of fluid collected from the tail of the epididymis. Females Ovulation can be induced during anes- trus in several species of domestic and laboratory animals by the injection of gonadotrophic hormones. Hammond (1952:218) used pregnant mare's serum (PMS) as a follicle-stimulating agent and chorionic gonadotropin to cause ovula- tion in ranch mink. We made several attempts, using 19 individuals, to cause the growth and de- velopment of follicles and to cause ovu- lation in the raccoon by injecting hor- mones. Only four individuals ovulated, and three of these cases involved the use of PMS (Table 12). Only the four cases in which o\ulation occurred are discussed. In one scries of experiments various dosages of follicle-stimulating hormone (FSH) given subcutaneously were fol- lowed by luteinizing hormone (LH) given intravenously. Later FSH and LH were mixed and given subcutaneously, followed by intravenous injection of LH. With one exception all attempts using FSH and LH were unsuccessful in caus- ing ovulation. In some cases normal-ap- pearing follicles were numerous in the ovaries after injections of FSH and mix- tures of FSH and LH, but attempts with LH and with a mixture of LH and FSH to cause the follicles to ovulate were un- successful. The ovaries generally were overstimulated ; that is, they were larger than normal and contained more follicles than normal. The successful ovulation that did not involve injections of PMS occurred in a female raccoon (the first female in Table 12) approximately 44 months old, weigh- ing 6.7 kg. Each ovary was 11X6 mm, with no follicles or corpora lutea ap- proximately 2 months prior to the breed- ing season. A section of her uterus was removed when the ovaries were measured (approximately 2 months prior to the breeding season) so that we could study the pigment granules. The next day sub- cutaneous injections of a mixture of 10 Armour units (AU) each of FSH and LH were begun. These injections were given for 10 days, and on the 12th day a mixture of 80 units each of FSH and LH was injected intravenously. At that time each ovary was 12X8 mm and con- tained 10-20 clear follicles, each about 1 mm in diameter. On the 13th day 100 units each of FSH and LH were injected intraperitoneally as a mixture. On the 16th day the left ovary was 18 X 9 mm and contained approximately 20 follicles, each about 2 mm in diameter, but ovu- lation had not occurred. The left ovary weighed 760 mg, compared with an aver- age weight of about 137 mg for one ovary of parous or pregnant females uuring the mating season (Table 5) . When the rac- coon was killed 45 days after the first injection, her right ovary weighed 290 mg and contained 1 1 corpora lutea. In 1 second series of experiments PMS was injected into eight females in at- tempts to cause the development of folli- cles and to cause ovulation. Three of these attempts were successful. The first female was approximately 2 years of age and had been in captivity for more than a year when hormone treatments vvere begun. We injected 100 international units (lU) of PMS subcutaneously each day for 12 days and 500 lU each on the 13th and 16th days. Thirteen days later, 28 days after the treatment was begun, the uterus and both ovaries were removed. The contents of the oviducts and uterine horns were flushed out, but no ova or blastocysts were found. Each ovary con- tained approximately 30 corpora lutea. Even though no ovum was recovered, the abnormally large number of corpora lutea containing no ova indicates that this female probably ovulated. The secretory material found in the lumina of the uter- ine glands indicated that progesterone had probably been secreted. The second female was captured when she was at least 18 months of age; how- ever, she was not injected with hormones until she was about 53 months old. Sub- 72 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 Q S Si > o 3 . J3 JS T-H hJ " s i ti- a § g 3 s fc. '^ tl > u <=> ,^ *"• M '^ fc-B- ai- 4 4 .. c g S .. or- •" oi ,m >» to .5 lo I S S 2 WW M Pn Oi (4 b > Ehhhh — "; o o 3 (M CJ IT) S S "c " a CT> «, C 01 ip S B di Oh Oh CLi tJS t3C> —« lo -H m July, 1973 Sanderson & Nalbandov : Reproductive Cycle of the Raccoon 73 i s- 2 '^ c -^ B E2 6 8 o, p — — — Ol C3 I > C^^ O — J *^ o . 1^ x^SJ^r b's S a > 5 oMO ^ — * S E?8 •no is bn in^ o ;s c« 6 1 • • 7 • E c -g ^ " "5 >>" « f' ^ >o o t > V'^.'^. o CTi O (^ ^ CO C - S |j2 cy2 U3 W t/3 SIS 0U| CL, en S EC 22 o o 22 o oo o— m 2P C HH H-l NH m o o o -; lO o o -• P„R, I J- C^J Cl I I i(2 s 74 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 Q S 2 6 o •" >ri " in 'o CO Q. n .„ I o 2 6 4- •* 60 " 42 o s ^ 4° ^ s O £ «^ 6 July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 75 >-] »< CM '^ _ I • I CM •" E fr^ bo Q. t X 4' :2 § I lO — O^ lO — I > V 76 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 Q S ^12 6 o S 2 6 o °- o 'S "" f ? rt . I o c ^ ES 5 S ^ O 5 bo o S ' S 2 . E.a :^ e X oo |-E 2 s KM IZI M u O CJ o o o CTi I I I 1/3 M CO 04 CU 0k DIDP XX oo in lO 0> O) in lo CM CM i S-2 ° X S »i|i-tf|||i* ill »li|ii.i«^.iJ='|S:£ = | = .Ss;d-s 3*^.2 g Sis cutaneous injections of PMS at the rate of 100 lU daily for 12 days were begun in September. Many large follicles were found on the 13th day of treatment. This female was given 550 lU of PMS intra- venously on the 13 th day. Seventy-two hours later many blut punkte were ob- served in each ovary. The left ovary (826 mg) and'a piece of the uterus were removed. Histological examination re- vealed 26 corpora lutea in early stages. Most had blood in the lumina and ap- peared to be freshly ovulated. The uterus showed a fairly typical effect of estrogen, and no material was present in the uterine glands, indicating the near absence of progesterone. Eleven days later (26 days after the first injection) the female was killed and the right ovary was removed (2,147 mg) . There were 29 early-stage corpora lutea, most of them packed with luteal cells, but lumina were present in 2-4 corpora. The cytoplasm and nuclei of these luteal cells were more darkly stained and the nuclei were smaller than usual. The intracellular space exceeded the norm. Secretory material was present in the uterine glands. The third female was about 22 months old when caught, but was 53 months of age when these experiments were begun. Subcutaneous injections of PMS were begun 2 months before the breeding season, at the rate of 50 lU per day, and were continued for a total of 12 injections. On the 13th day 200 lU were injected intravenously. At that time each ovary measured 12 X 6 mm and con- tained approximately nine follicles, each about 2 mm in diameter. Two days later the ovaries and follicles had not changed in size, but one follicle was hemorrhagic and one had a thin red line across the surface at its highest point. Twenty-four hours later when the ovaries were exam- ined, blood oozed from most or all of 11 or 12 follicles in each. There were tiny holes in the highest points of most, and perhaps in all, of them. The ovu- lated follicles were partly hollow and partly filled with fluid and stringy ma- terial. The left ovary, measuring 10 X 6 mm, was removed (242 mg). When ex- amined histologically, it was found to contain six or more blood-filled, early- stage corpora lutea. Among 16 wild rac- coons the average number of corpora lutea per ovary, determined by histologi- cal examination, was 2.1. Thus, in this most nearly normal ovulation induced by exogenous hormones, the ovaries were somewhat less than twice normal weight, but the ovulation rate was approximately 5.7 times normal. The female just discussed weighed 5.35 kg and received a total of 800 lU of PMS, a dosage of about 150 lU per kg, a rate similar to that used success- fully to cause ovulation in ranch mink (Hammond 1952:219). In a third series of experiments four different hormones were used on four sexually immature female raccoons from 2 to 4 months old (the last four animals in Table 12) in an attempt to learn how immature ovaries respond to hormones and to study differential responses to the several hormones. The injection of hu- man menopausal gonadotropin (HMG- J5, largely FSH) subcutaneously twice a day for 7 days immediately after 2 days of single injections resulted in little stimu- lation of either the ovary or the uterus in one immature female. In the second young raccoon 50 international units (lU) of PMS daily for 8 days, followed by 100 lU and 250 lU on the 9th and 10th days, respectively, resulted in a slightly more stimulated uterus than did the HMG-J5 injected into the animal just discussed. In the third animal in this age group 10 Armour units of LH in- jected subcutaneously daily for 9 days, followed by 25 units on the 10th day, resulted in larger follicles than did either of the two previous treatments. In the raccoon that received only LH this hormone caused more development of the follicles than did either PMS or HMG-J5 in the other young females. PMS and HMG-J5 contain both FSH and LH and might be expected to cause greater stimulation than LH alone. The ovaries stimulated by HMG-J5 contained 78 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 more interstitial tissue than did the ovar- ies of the females that received the other hormones, and the ovaries of the raccoon that received PMS had less interstitial tissue than those of the female that re- ceived LH. The uterus of the female injected with HMG-J5 was somewhat less stimulated (endometrium 650 mi- crons) than that (endometrium 820 mi- crons) of the female that was given PMS although the differences in these uteri were slight. The uterus of the female that received LH was more stimulated (endometrium 1,275 microns) than was either of the other two. On the basis of the information ob- tained from our experiments with four female raccoons, it appears that 35-50 lU of PMS given subcutaneously each day for 1 2 days caused the development of follicles at any time of year in adult females. A dose of 200 lU given on the 13th day might be expected to cause ovulation 48-60 hours later. UTERINE MILK Uteri, and ovaries containing corpora lutea, were sectioned from 18 raccoons that had not been treated with hormones. In 17 of the 18 secretory material (uterine milk) was present in the lumina of most, but not all, of the uterine glands although it may have been present in all 18 uteri but overlooked in some of the sections. Histological sections of ovaries con- taining no corpora lutea and the corre- sponding uteri were examined from 89 raccoons collected throughout the year. February and March were each repre- sented by a single animal, but each other month was represented by three or more animals. The uterine sections from these animals, with two exceptions, contained no secretory material in the endometrial glands. Small amounts of secretory ma- terial were present in the uterine glands of one nulliparous adult killed in Septem- ber and in another, approximately 7 months of age, collected in November. Secretory material was not abundant in either one, but was definitely present. These data indicate that, in the rac- coon, secretory material (presumably uterine milk) is present when corpora lutea are present. In one female, judged to have been only 10 days prepartum, secretory material was present. Progesterone alone or in combination with estrogen was probably responsible for the secretion of uterine milk (Table 13). Progesterone alone was given for an insufficient length of time to determine whether it alone can cause the uterine glands to secrete. Two castrated females (No. 1297 and 1786) received a combi- nation af progesterone and estrogen for several days, and the endometrial glands of both contained uterine milk (Table 13). Any combination of gonadotrophic hormones that resulted in the formation of corpora lutea caused secretion by the uterine glands. Five treatments of 2.5 mg each of estradiol over periods of 10 and 20 days, respectively, did not cause secretion by the uterine glands in one castrated female. One intact female (No. 2184B) received five daily injections of 20 units each of FSH, followed on the 6th day by 50 units of LH. Three days later, when she was killed, each ovary contained approximately 20-30 follicles measuring up to 750 X 1,250 microns, but no corpora lutea. The lumina of a few endometrial glands contained small bits of secretory material. Several hor- mones, including various combinations of FSH, LH, CGH, PMS, and HMG-J5, were given to intact females. Except pos- sibly in the female just discussed, none of these hormones caused the uterine glands to secrete except indirectly by causing the formation of corpora lutea. Methods described by Pearse (1960: 265-271) and by Lillie (1954:274-299) were used in an attempt to demonstrate the nature of the secretory material. In no case did digestion with either pty- alin or diastase remove the secretory ma- terial from the endometrial glands. This finding was taken as evidence that it was not glycogen. According to the in- formation on the identification of carbo- hydrate-containing materials given by July, iy/0 SANDERSON (Kl-NALBANDOV: K.EPRODUCTIVE l^YCLE OF THE RACCOON 79 Table 13.—Presence of secretory material in the uterine glands of captive raccoons as related to njections of exogenous hormones. Raccoon Number Estimated Age in Months Hormone Number of Days After First Treatment Corpora Lutea Uterine Milk 1292 80 Illinois Natural History Survey Bulletin Vol. 31, Art. 2 conceived in the wild was April 18 (range, March 9-June 24) and for 11 litters conceived and born in captivity it was April 24 (range, March 16-June 3). 4.—The measurement of the largest external uterine swelling enabled us to estimate birth dates with a maximum error of 4 days. 5.—The sex ratios of young raccoons less than 2 months of age and of embryos and young at birth were not significantly different from 50:50, but there were more males among the young less than 2 months old than among the other group, possibly indicating some differen- tial mortality of females between birth and 2 months of age. 6.—Yearling females either bred when adults bred or did not breed until they were almost 2 years of age. If female raccoons ovulated but did not become pregnant, if they aborted or resorbed their young, or if they lost their young at or near birth, they sometimes ovulated a second time in one season. The inter- val between ovulations in five captive raccoons held in Urbana, 111., varied approximately from 80 to 140 days. Severe weather conditions (extreme cold or deep snow) interfered with the normal breeding cycle and resulted in an un- usually large number of late litters. Fe- male raccoons sometimes gave birth to two litters in one season, but they did not rear more than one litter in one season. The vaginal smear was no more specific for indicating estrus than was gross vulval swelling. 7.—Contrary to published reports, the raccoon is a spontaneous ovulator. Ovulation was followed by the formation of corpora lutea whether the animal be- came pregnant or pseudopregnant. The formation of corpora lutea always re- sulted in changes in the uteri and nipples. The nipples always enlarged; some be- came heavily pigmented, some became slightly pigmented, and others remained unpigmented. Thus, it was possible to determine whether a female raccoon had ovulated by examining her nipples. Corpora lutea in both isolated and non- isolated pseudopregnant females formed from ovulated Graafian follicles and not from luteinization of follicles. Corpora lutea persisted in pregnant females until parturition and apparently disappeared 14-16 days after parturition. 8.-Raccoons that ovulate become either pregnant or pseudopregnant, and the corpora lutea persist for about the same time in pseudopregnant raccoons as they do in those that give birth to young. Corpora in females that went at least halfway to term persisted about the same length of time whether the young were aborted, were resorbed, or were born and were removed at birth or nursed until weaned. Field evidence indicated that in Illinois about 2.5 percent of the adult females were pseudopregnant each year. 9.—Ten of 21 captive yearling females and 9 of 14 wild yearling females were sexually immature. 10.—Interstitial tissue occurred in the ovaries at some stage of the reproductive cycle, often occupying as much as 50-90 percent of the space in the ovary, but seldom occurred when corpora lutea were present. Ovaries of females less than 2 months old did not contain large amounts of interstitial tissue, but with this exception the ovaries of females less than 12 months of age contained, on the average, more interstitial tissue—both relatively and absolutely—than did those of older females. From January through June, ovaries of adults contained little interstitial tissue even when corpora lucea were not present; from July through December, ovaries from adults contained more interstitial tissue than did those collected earlier in the year. The greatest abundance of interstitial tissue was in ovaries taken from juveniles during Oc- tober and November, and the maximum ovarian weights recorded during this study were those of juvenile females in November. 1 1 .—The placenta of Procyon is de- ciduous, as in the dog, cat, fox, and seal, and is endotheliochorial. If used with caution, placental scars in raccoons are useful for estimating litter size and rate of productivity. The significance of multiple groups of scars is not clear, but July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 81 it appears that each embryo that reaches 1 month of age is represented by one scar that persists for 10 or more months. Scars in wild females with only one group of scars probably reflect implanta- tion rates for the preceding breeding season. Placental scars apparently per- sist longer in wild females than in cap- tives. 12.—The reproductive system of the male raccoon is similar to that of the dog ; seminal vesicles and Cowper's glands are lacking. 13.—The uterus of the raccoon is inter- mediate between the bicornuate and the bipartite uterus. There is a single cervix and the horns are distinct, but after they join externally to form the single uterine body, the uterine lumina remain separate to a point near the cervix. The ovoid ovary is completely surrounded by the bursa ovarii. The sac is intact except for a small slit on one side, not large enough to permit passage of the ovary, as in the mink, dog, and fox. 14.—The level of se,x hormones in the male affected the enlargement of the preputial orifice and the maturation of the penis bone but had little or no effect prior to 7 months of age. Castration in the male also apparently caused a slight delay in the closure of the epiphyseal cartilage in the radius and ulna. Re- moval of the ovaries, even after raccoons had reached sexual maturity but before the epiphyses had closed, delayed the rate of epiphyseal closure. 15.—Embryos persisted for about 19 days after castration in each of two rac- coons—to 33 days short of term in one female and 7 days short of term in the other. 16.—Limited studies indicated that in- jections of androgen did not initiate nor prolong spermatogenesis and apparently did not influence the size of the testes. 17.—A dose of 35-50 lU of pregnant mare's serum given subcutaneously each day for 12 days caused development of Graafian of follicles in adult females at any time of the year. A dose of 200 lU given on the 13th day caused ovulation 48-60 hours later; however, in all cases of successful ovulation, the ovaries were much larger—and the rates of ovulation much higher—than normal. LITERATURE CITED Allen, B. M. 1904. The embryonic develop- ment of the ovary and testis of the mam- mals. American Journal of Anatomy 3(2) : 89-146 + 7 plates. Altmann, F. 1927. Untersuchungen uber das Ovarium von Talpa europaea mit besonderer Beriicksichtigung seiner cyclischen Verander- ungen. Zeitschrift fur Anatomie und Ent- wicklungsgeschichte 82 : 482-569. .\SDELL, S. A. 1946. Patterns of mammalian reproduction. Comstock Publishing Co., Inc., Ithaca, New York. 437 pp. Berard, E. V. 1952. Evidence of a late birth for the raccoon. Journal of Mammalogy 33(2): 247-248. Brambell, F.W.R., and I. H. Mills. 1948. Studies on sterility and prenatal mortahty in wild rabbits. Part IV. The loss of em- bryos after implantation. Journal of Experi- mental Biology 25(3):241-269. Burt, W. H. 1960. Bacula of North American mammals. University of Michigan Museum of Zoology Miscellaneous Publication 113. 76 pp. -|- 25 plates. CoNAWAY, C. H. 1955. Embryo resorption and placental scar formation in the rat. Journal of Mammalogy 36(4) :516-532. Corner, G. W. 1932. Cytology of the ovum, ovary and Fallopian tube. Pages 1567-1607 in E. V. Cowdry, ed. Special cytology, 2nd ed. Vol. 3. Paul B. Hoeber, Inc., New York. Davis, D. E., and J. T. Emlen, Jr. 1948. The placental scar as a measure of fertility in rats. Journal of Wildlife Management 12(2): 162-166. Deanesly, R. 1935. XI—The reproductive processes of certain mammals. Part IX — Growth and reproduction in the stoat (Mus- tela erminea). Royal Society of London Philosophical Transactions, Series B, 225, 528:459-492 -|- 4 plates. Deng, R. A. 1937. Uterine macrophages in the mouse and their relation to involution. American Journal of Anatomy 60(3) : 433- 456 -f 8 plates. . 1941. A criterion for distinguishing between virgin and parous animals. Phar- maceutical Archives 12:12-16. DoRNEY, R. S. 1953. Some unusual juvenile raccoon weights. Journal of Mammalogy 34(1): 122-123. Elder, W. H. 1952. Failure of placental scars to reveal breeding history in mink. Journal of Wildlife Management 16(1): 110. George, J. L., and M. Stitt. 1951. March litters of raccoons (Procyon lotor) in Mich- igan. Journal of Mammalogy 32(2) :218. Goldman, E. A. 1950. Raccoons of North and Middle America. U.S. Department of the Interior, Fish and Wildlife Service, North American Fauna 60. U.S. Governmeit Printing Office, Washington, D.C. 153 pg Hammond, J., Jr. 1952. Gonadotrophii! induced ovulation in mink. Journal of Man malogy 33(2) : 218-233. Hansson, .a. 1947. The physiology of n production in mink {Mustela vison, Schreb.1 with special reference to delayed implants tion. Institute of Animal Breeding, Royj Agricultural College of Sweden, StockhoU Acta Zoologica 28. 136 pp. His, W. 1865. Beobachtungen uber den Bi^ des Saugethiereierstockes. Archiv fur Mikrt skopische Anatomie 1:151-202. Jaeger, E. C. 1947. Use of the os phallus { the racoon [sic] as ripping tool. Journal ( Mammalogy 28(3) :297. Kingsbury, B. F. 1914. The interstitial celt of the mammalian ovary: Felis domestici American Journal of Anatomy 16(1): 59-91! LiLLiE, R. D. 1954. Histopathologic technii and practical histochemistry. The Blakistoi Company, Inc., Philadelphia and Toronto 501 pp. Llewellyn, L. M. 1953. Growth rate of till raccoon fetus. Journal of Wildlife Managii ment 17(3):320-321. , and R. K. Enders. 1954a. Ovulatioi in the raccoon. Journal of Mammalojc 35(3) :440. , and . 1954b. Trans-uterine mi gration in the raccoon. Journal of Main malogy 35(3): 439. Millard, C. 1939. Raccoon experiment. Wi< consin Conservation Bulletin 4(3): 28-29." Momberg, H., and C. Conaway. 1956. TH distribution of placental scars of first aiB second pregnancies in the rat. Journal Embryology and Experimental Morpholo(( 4(4) : 376-384 + 2 plates. Montgomery, G. G. 1969. Weaning of caji live raccoons. Journal of Wildlife Managi ment 33( 1 ): 154-159. Mossman, H. W. 1937. Comparative moi phogenesis of the fetal membranes and ai. cessory uterine structures. Contributions > Embryology 158, Carnegie Institution Washington Publication 479. 129-246 + 5 plates. Nalbandov, a. V. 1958. Reproductive phjb iology. W. H. Freeman and Company, Si Francisco. 271 pp. .. Patzelt, V. 1955. Uber das Ovarium dii Karnivoren und seine Zwischenzellen. Zd/ schrift fiJr Mikroskopisch-Anatomische Fa schung 61(3): 309-359. Pearse, A. G. E. 1960. Histochemistry: tht oretical and applied, 2nd ed. Little, Brovii and Company, Boston. 998 pp. Pope, C. H. 1944. Attainment of sexual ; 82 J uly, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 83 turity in raccoons. Journal of Mammalogy 25(I):91. Iasmussen, a. T. 1918. Cyclic changes in ' the interstitial cells of the ovary and testis in the woodchuck [Marmota monax). En- dociinology 2:353-404 + 4 plates. Unker, G. C. 1944. Os clitoridis from the racoon [sic]. Journal of Mammalogy 25 (1) :91-92. lANDERSON, G. C. 1950. Methods of measur- ing productivity in raccoons. Journal of i Wildlife Management 14(4) : 389-402. I . 1961a. Techniques for determining ' age of raccoons. Illinois Natural History Survey Biological Notes 45. 16 pp. ' . 19616. The lens as an indicator of age in the raccoon. American Midland ; Naturalist 65(2) : 481-485. Inyder, R. L., and J. J. Christian. 1960. Reproductive cycle and litter size of the I woodchuck. Ecology 41 (4) ;647-656. lOOTER, C. A. 1946. Muskrats of Tule Lake Refuge, California. Journal of Wildlife Management 10(l):68-70. iTAFFORD, W. T., and H. W. Mobsman. 1945. The ovarian interstitial gland tissue and its relation to the pregnancy cycle in the guinea pig. Anatomical Record 93( 1 ) :97-107. Stains, H. J. 1956. The raccoon in Kansas: natural history, management, and economic importance. University of Kansas Museum of Natural History and State Biological Sur- vey of Kansas Miscellaneous Publication 10. 76 pp. Stockard, C. R. 1932. Cellular changes in the fluid of the mammalian vagina. Pages 1611-1629 in E. V. Cowdry, ed. Special cytology, 2nd ed. Vol. 3. Paul B. Hoeber, Inc., New York. Stuewer, F. W. 1943a. Raccoons: their habits and management in Michigan. Eco- logical Monographs 13(2) : 203-257. . 19436. Reproduction of raccoons in Michigan. Journal of Wildlife Management 7(l):60-73. U. S. Department of Agriculture, Bureau of Biological Survey. 1936. Raising raccoons. Wildlife Research and Management Leaflet BS-34. 2 pp. U. S. Weather Bureau. 1960. Climatological data: Illinois. 65(1-3) : 1-43. Watson, M. 1881. On the female organs and placentation of the racoon (Procyon lotor). Royal Society of London Proceedings 32(213) :272-298 + 4 plates. Whitney, L. F., and A. B. Underwood. 1952. The raccoon. Practical Science Publishing Company, Orange, Conn. 177 pp. Wood, J. E. 1955. Notes on reproduction and rate of increase of raccoons in the Post Oak Region of Texas. Journal of Wildlife Man- agement 19(3):409-410. INDEX Ampullae, 65 Androgen {see testosterone) B Birth date estimating, 46-47 mean, 32, 45-46, 79-80 Bulbo-urethral glands {see Cowper's glands) Bursa ovarii, 67, 81 H Histology, 33 HMG-J5 {see human menopausal gonadotro- pin) Hormones, effects of exogenous on females, 34-35, 71-78, 81 on males, 34, 70-71, 81 Human menopausal gonadotropin, 35, 76-78 Interstitial tissue, 33, 57-61, 80 Canis lupus {see wolf) Castration effects on females, 34, 50-51, 68-70, 79, 81 effects on males, 34, 68, 81 Cat domestic, 57 Cervix, 66, 81 CGH {see chorionic gonadotropin) Chorionic gonadotropin, 34, 73, 75-76, 78-79 Corpora albicantia, 55-56 Corpora lutea, 32-33, 35, 53-61, 71-75, 77-80 persistence, 53-56, 80 secretion of progesterone, 57 Cowper's glands, 65, 81 Dog, 65, 67, 80-81 LH {see luteinizing hormone) Luteinizing hormone, 34-35, 52-53, 71-72, 74-79 M Mammae pigmentation, 54, 57, 80 Marmota monax {see woodchuck) Mink, 59, 67 Muskrat, 61 Mustela erminea {see stoat) Mustela vison {see mink) N Nipples {see mammae) ECP {see estradiol) Embryos persistence after castration, 34, 63, 81 Epiphyses closure, 68-69 effects of castration on closure, 68, 81 Estradiol, 34-35, 51-53, 72, 79 Estrous cycle, 32, 48-53 Felis catus {see cat, domestic) Females percentage sexually mature as yearlings, 56-57 Fimbria, 67 Follicle-stimulating hormone, 34-35, 52-53, 71-72, 74-79 Fox, 65, 67 FSH {see follicle-stimulating hormone) Gonad (s) {see ovary and testis) Graafian follicles, 34, 72-76, 80 effect of pregnant mare's serum, 81 Ondatra zibethicus {see muskrat) Oryctolagus cuniculus {see rabbit, European) Os baculum, 66, 68 Os clitoridis, 67-68 Os penis {see os baculum) Ovary (ies), 67, 81 seasonal cycle, 30, 43-45, 79 Ovulation, 32-33, 53-55 days between, 49 effect of pregnant mare's serum, 71-74, 77,1 81 spontaneous, 54, 80 Placenta, 62, 80 Placental scars, 33, 61-65, 80-81 PMS {see pregnant mare's serum) Pregnant mare's serum, 34-35, 52-53, 60-61, 71-74, 76-79, 81 Procyon lotor {see raccoon) Progesterone, 34-35, 51-53, 57, 71, 73-74, 77-79 Prostate, 65 Pseudopregnancy, 55—56, 60—61, 80 84 July, 1973 Sanderson & Nalbandov: Reproductive Cycle of the Raccoon 85 Rabbit, European, 61 Raccoon (s) cage for captive, 29 captive, 29, 30-31 Reproductive tract (s) female, 34, 66-68, 8 male, 33, 65-66, 81 Testis (es) seasonal cycle, 30, 35-43, 79 Testosterone, 34, 70, 81 u Urethra, 65 Uterine milk, 35, 78-79 Uterus, 66-67, 81 Seminal vesicles, lacking, 65, 81 Sex ratios secondary, 32, 47-48, 80 Sexual maturity females, 55-57, 80 males, 35-36, 79 Spermatogenesis effect of testosterone, 70, 81 Stoat, 61 Vaginal biopsies, 52-53 Vaginal smears, 50-51 Vasa deferentia, 65 Vulpes fulva {see fox) w Wolf, 65 Woodchuck, 57 oome ruDlicaTions ot ine iui-imv^ij i-i/-ki wi\/-\u iii^iv^rvi juixTti BULLETIN Volume 30, Article 3.-Migrational Behavior of Mallards and Black Ducks as Determined from Banding. By Frank C. Bellrose and Robert D. Crompton. September, 1970. 68 p., frontis., 25 fig., bijjliogr., index. Volume 30, Article 4.-Fertilization of Estab- lished Trees: A Report of Field Studies. By Dan Neely, E. B. Himelick, and Webster R. Crowley, Jr. September, 1970. 32 p., fron- tis'., 8 %., bibliogr., index. Volume 30, Article 5.-A Survey of the Mussels (tlnionacea) of the Illinois River: A Pollut- ed Stream. By William C. Starrett. February, tis., 8 fig., bibliogr., index. Volume 30, Article 6.-Comparative Uptake and Biodegradability of DDT and Methoxy- chlor by Aquatic Organisms. By Keturah A. Reinbold, Inder P. Kapoor, William F. Childers, Willis N. Bruce, and Robert L. Metcalf. June, 1971. 12 p., frontis., 5 fig., bibliogr., index. Volume 30, Article 7.-A Comparative Study of Two Components of the Poinsettia Root Rot Complex. By Robert S. Perry. August, 1971. 35 p., frontis., 10 (ig., bibliogr., index. Volume 30, Article 8.-Dynamics of Condition Parameters and Organ Measurements in Pheasants. By William L. Anderson. July, 1972. 44 p., frontis., 6 fig., bibliogr., index. Volume 31, .Article l.-The Effects of Supple- mental Feeding and Fall Drawdowns on the Largemouth Bass and Bluegills at Ridge Lake, Illinois. By George W. Bennett, H. Wickliffe Adkins, and William F. Chil- ders. January, 1973. 28 p., frontis., 8 fig., bibilogr., index. BIOLOGICAL NOTES 70.-An Ecological Study of Four Darters of the Genus Percina (Percidae) in the Kaskaskia River, Illinois. By David L. Thomas. De- cember, 1970. 18 p., 11 fig., bibliogr. 71.-A Synopsis of Common and Economic Illinois Ants, with Keys to the Genera (Hymenoptera, Formicidae). By Herbert H. Ross, George L. Rotramel, and Wallace E. LaBerge. January, 1971. 22 p., 27 fig., bibliogr. 72.-The Use of Factor Analysis in Modeling Natural Communities of Plants and Ani- mals. By Robert W. Poole. February, 1971. 14 p., 14 fig., bibliogr. 73.-A Distributional .^tlas of Upper Mississip- pi River Fishes. By Philip W. Smith, Alvin C. Lopinot, and William L. Pflieger. May, 1971. 20 p., 2 fig., 107 maps, bibliogr. 74.-The Life History of the Slenderhead Dart- er, Percina phoxocephala, in the Embarras Rive.. Illinois. By Lawrence M. Page and Philip W. Smith. July, 1971. 14 p., 10 fig., bibliogr. 75.-Illinois Birds: Turdidae. By Richard R. Graber, Jean W. Graber, and Ethelyn L. Kirk. November, 1971. 44 p., 40 fig., bib- liogr. 76.-Illinois Streams: A Classification Based on Their Fishes and an Analysis of Factors Re- sponsible for Disappearance of Native Spe- cies. By Philip W. Smith. November, 1971. 14 p., 26 fig., bibliogr. 77.-The Literature of Arthropods Associated with Soybeans. I. A Bibliography of the Mexican Bean Beetle, Epilachna varivestis Mulsant (Coleoptera: Coccinellidae). By M. P. Nichols and M. Kogan. February, 1972. 20 p., 1 fig., bibliogr. 78.-The Literature of Arthropods Associated with Soybeans. II. A Bibliography of the Southern Green Stink Bug, Nezara viridula (Linneaus) (Hemiptera: Pentatomidae). By N. B DeWitt and G. L. Godfrey. March, 1972. 23 p., 1 fig., bibliogr. 79.-Combined Culture of Channel Catfish and Golden Shiners in Wading Pools. By D. Homer Buck, Richard J. Baur, Charles F. Thoits III, and C. Russell Rose. April, 1972. 12 p., 3 fig., bibliogr. 80.-Illinois Birds: Hirundinidae. By Richard R. Graber, Jean W. Graber, and Ethelyn L. Kirk. August, 1972. 36 p., 30 fig., bibliogr. 81.-Annotated Checklist of the Butterflies of Illinois. By Roderick R. Irwin and John C. Downey. May, 1973. 60 p., 3 fig., 98 maps, bibliogr. 82.-Lactate Dehydrogenase Isozymes of Dar-, ters and the Inclusiveness of the Genus Percina. By Lawrence M. Page and Greg- ory S. Whitt. May, 1973. 7 p., 5 fig., bibliogr. CIRCULAR 46.-Illinois Trees: Their Diseases. By J. Ced- ric Carter. June, 1964. (Third printing, with alterations.) 96 p., frontis., 89 fig. 49.-The Dunesland Heritage of Illinois. By Herbert H. Ross (in cooperation with Illinois Department of Conservation). August, 1963. 28 p., frontis., 16 fig., bibliogr. 51.-Illinois Trees: Selection, Planting, and Care. By J. Cedric Carter. August, 1966. 123 p., frontis., 108 fig. 52.-Fertilizing and Watering Trees. By Dan Neely and E. B. Himelick. December, 1971. (Third printing.) 20 p., 9 fig., bibliogr. " 53.-Dutrh Elm Disease in Illinois. By J. Cedric Carter. October, 1967. 19 p., frontis., 17 fig. List of available publications mailed on request No charge is made for publications of the Illinois Natural History Survey. A single copy of most publications will be sent free to anyone requesting it until the supply becomes low. Costly publications, more than one copy of a publication, and publications in short supply are subject.* for special correspondence. Such correspondence should identify the writer and explain the use to be made of the publication or publications. Address orders and correspondence to the Chief, Illinois Noturol History Survey Noturol Resources Building, Urbono, Illinois 61801