A REGIONAL COMPARISON OF COTTONTAIL RABBIT REPRODUCTION IN OHIO
DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
NORMAN CURTISS NEGUS, B* A., M. A.
s}:s);:$:s}5sfe
The Ohio State University 1956
Approved by:
" " Chapter- I - INTRODUCTION. • ...... 1 Chapter- II - MATERIALS AND METHODS, ...... 9 Chapter III - R E S U L T S ...... • ...... 53 : Chapter- IV - DISCUSSION...... 127 Chapter- V - SUMMARY AND CONCLUSIONS..,...... !1^ - Chapter VI - RECOMMENDAT IONS ...... iMf APPENDIX...... l1^ LITERATURE: CITED...... l1^ AUTOBIOGRAPHY...... 152 ii LIST OF ILLUSTRATIONS Figur© 1. Map of physiographic regions of Ohio....*. **.13 Figure 2# Map of general soil regions of Ohio...... I1* Figure 3. Map of collecting localities...... ,,,19 Figure *f. Nestling fur:;...... 25 Figure 5* Juvenal pelage-'...... 26 Figure 6 . Subadult pelage.... 27 Figure- 7. Adult summer pelage...... 28 Figure 8 , X-ray photograph of forelegs of adult and ' juvenile: Cottontail rabbits...... 32 Figure 9* Relationship of crown-rump length to age in New Zealand Whit© rabbit embryos...... 36 Figure 10. Relationship of ear pinna length to age in New Zealand White rabbit embryos...... 37 Figure: 11. A V-day embryo of New Zealand White rabbit..39 Figure: 12?. A 17-day embryo of New Zealand White rabbit...... *••••39 Figure: 13. A 20-day embryo of New Zealand White rabbit. ..•••..... •»••...... •••••••••••••39 Figure l1*. A 21+~day fetusoof New Zealand' White rabbit...... Figure 15. A 28-day fetus of New Zealand White rabbit..*,...... *+0 Figure 16. A 30-day fetus of New Zealand White rabbit...... kO Figure 17. Photomicrograph of maturing Graafian follicles in cottontail rabbit ovary.....k7 iii Figure 18, Photomicrograph of freshly formed corpus: luteum...... b7 * Figure 19* Photomicrograph of active corpus luteum of pregnancy...... *+8 Figure 20. Photomicrograph of active corpus luteum enlarged to show cellular .structure.....*1-8 Figure 21. Photomicrograph of corpus albicantia from a previous pregnancy...... ^9 Figure 22. Photomicrograph of corpus albicantia at the end of the breeding season...... 1+9 Figure 23. Frequency of estimated parturition dates for litters of cottontail rabbits...... 81 iv LIST OF TABLES Table 1. Characteristics of Known-Age Embryos from New Zealand White Rabbit Breeding Experiment...... 35 Table 2, Data on Adult Female Cottontail Rabbits from Urbana Game Farm...... 56 Table 3, Unglaciated Allegheny Plateau Data on Subadult Cottontail Rabbits...... 6 6 - 6 9 Table 4. Till Plain Data on Subadult Cottontail Rabbits...... 70-73 Table 5* The Pelage Stages of Female Cottontails Correlated with Reproduction...... 89-90 Table 6 , Data from Breeding Experiment Using New Zealand White Rabbits ..... 93 Table 7 , Frequency Distribution of Litter Sizes of Cottontails from Areas A and B.-..... 96 Table 8 , Summary of Analysis of Variance of Litter Sizes from Areas A and B...... 99 Table 9 , Summary of a Comparison of Mean Litter Sizes from Areas A and B ...... 101 Table 10, Frequency of Regional Litter Sizes of Cottontails ...... 103 Table 11, Analysis of B luegrass Samples for Calcium and Phosphorus Content...... 105 Table 12, Comparison of Calcium and Phosphorus Content in Samples of Bluegrass from Areas A and B ...... 106 v Table 13. Corpora Lutea and Albicantia in Left anddRight Ovaries of Cottontail Rabbits...... 108 Tabl6el4. Embryos and Scars in Left and Right Uterine H orns ...... 109 Table 15. Individual Litter Records from Areas A and B...... Ill Table 16. Adult Male Weights...... 119 Table 17. Summary of a Comparison of Mean Weights of Adult Male Cottontails from Areas A and B ...... 120 Table 18. Testis Length of Adult Ma les 122 Tableel9. Humeri Lengths of Adult Cottontails.... 124 Table 20, Humeri Weights of Adult Cottontails.... 125 vi - 1- Chapter I INTRODUCTION Numerous studies have been conducted on various phases of the ecology of the Mearns cottontail rabbit, Svlvilagus floridanus: mearnsii (J.A. Allen). VJhile there is no doubt that the results of these investigations have contributed much to our knowledge: of the species, there remain many gaps in the known bionomics of the cottontail which is indeed surprising in view of its great economic importance:. Often previous studies have been repetitions of work conducted in some other state or locality. The result has been a large amount of data relative to move ments, nesting sites, pre and post-hunting season popula tions, and sex ratios, whereas a. paucity of basic informa tion exists relative to reproductive physiology and envi ronmental factors. The present study was designed to evaluate the repro ductive performance of wild cottontail rabbit populations and to compare various reproductive phenomena, in areas of high and low soil fertility. It was the opinion of the in vestigator that such an approach might yield basic infor mation on cottontail reproduction as well as afford some insight into the factors which"affect population densities in various regions of Ohio. - 2 - The primary objectives of the study were* 1. To study the relative fertility and fecundity of cottontail rabbit populations with respect to good and poor soil types in Ohio* 2, To determine the length of the breeding season and number of litters produced per year in var ious regions* 3* To determine the breeding segment of the popula tions and the age at which sexual maturity is attained* if* To gain some knowledge of the factors affecting the various reproductive phenomena of the popula tions* As is frequently the case in studies which Involve the collection of data through field work, not all of the foregoing objectives were realized to their fullest extent* The writer feels, however, that a number of important con tributions to our knowledge of cottontail ecology have re sulted from this study* This study was begun in the summer of 1952 and was continued into the spring of 1955* Financial support was gained from the Ohio Cooperative Wildlife Research Unit* The Unit is supported jointly by the U.S. fish and Wildlife - 3 - Service, the Ohio Division of Wildlife, the Ohio State Uni versity, and the Wildlife Management Institute* In addition, a supplementary research grant was contributed by the Wild life Management Institute In 1953* The accumulation of data for a project of this magni tude would not have been possible were it not for the co operation of many individuals* Many of the personnel of the Ohio Division of Wildlife contributed generously of their time or facilities* In particular the writer wishes to acknowledge the assistance of Mr* E*B* Nash, Mr* Karl E* Bednarik, Mr* C.E. Khoder, Mr* James 0* Lee, the late Mr* PTank D* Bailer, Mr* Bodney J. Smith, Mr* William B* Edwards, Mr* Paul J* Moore, Dr* Ployd B* Chapman, Dr* Charles A* Dambach and Mr* William B« Bender shot* Contri butions, suggestions, advice and assistance by various per sonnel from the Ohio Cooperative Wildlife Besearch Unit have been of inestimable value in the completion of this research* Those deserving particular mention for the great amount of time so generously contributed in the collection of specimens and other tasks are Mr* Bobert 6* Sagar, Dr* E* H* Dustman, Dr* E*E* Good, Mr* Charles Cr is pens, Jr*, Mr* Bobert W* Winner, Mr* Paul A* Stewart, and Mr* Francis w* Kessler* In particular the writer wishes to acknowledge the patience, guidance and encouragement afforded him through- - If - out the study by his adviser, Dr# E.H. Dustman# Mr# John McCready rendered valuable advice in regard to the X-ray of specimens and use of the X-ray camera purchased by the Research Unit# Dr, Maurice L# Giltz assisted in the devel opment of the staining and sectioning technique used in the preparation of ovarian material and in the preparation of a large amount of the ovarian material examined during the study# Dr# Vincent W. Schultz rendered valuable advice and assistance in regard to statistical analysis of the data# The -writers wife assisted greatly in many phases of the preparation of the manuscript. Previous Work on the Cottontail and Other Species Reproductive behavior of cottontail rabbits has been studied more or less extensively in New York, Michigan, Pennsylvania, Kentucky, Virginia, Missouri and Connecticut# In the northern states there is general agreement from the results of investigations made by Trippensee (1936), Haugen (19^2), Beule (19^6), and Smith (1950) that the breeding season of cottontails may extend over a ^-0-week period from January to September# In Virginia, however, Lewellyn and Handley (19^5) report that breeding starts early in March with some exceptions# It is likewise generally considered by Beule (19^1), Haugen (191+2), Cooley (19^6), and Smith (1990) that cottontails do not breed during their first - 5 _ summer. In Pennsylvania, Beule found no indication of breed ing during the first year* Dalke et al (19^2) in an exten sive investigation of cottontails in Connecticut, found no evidence of young females breeding in their first year; how ever they collected very little data on immature rabbits and hence offer no conclusive evidence* In the reproductive data reported in the literature the writer noted several types of information consistently lacking* There appears to have been no extensive sampling and examination of females of all ages during the breeding season to definitely clarify the question of the breeding segment of the population* In addition, there has been no intensive effort made to determine the variability in the number of litters produced per female per year, nor the fac tors involved in such variability. Many general statements have been made to the effect that a cottontail produces from one to five litters per year. Studies by Laurie (19^6) of large numbers of wild house mice, Mus muscuius. from differ ent environments, indicate that the number of litters pro duced per female per year varies directly with the quality of the environment while the litter size remains quite con stant. This indicates that the number of litters produced per year may be a direct reflection of the quality of the range. Studies in Missouri on the cottontail rabbit and the raccoon (Procyon lotor^ have indicated that soil fer tility is an important factor in the overall development of the Individuals in a population* Cheatum and Seviringhaus (19**9) working on the white-tailed deer (Odocoileus virginianus virginianus) in New York State found a signifi cant correlation between range quality, as judged by land use and soil type, and the reproductive performance, growth rates and attainment of sexual maturity* As early as 1857, Owen called attention to the lux uriant growth of bluegrass on soils derived from blue lime stone marls in Kentucky, and to the remarkable development of livestock pastured in these fields* He observed that such stock was almost a year in advance in bulk, weight, and form to the stock raised on other soils* This was apparently due to the preponderance of lime, phosphoric acid and alkalies in the diet* Stewart and Moir (19*6) studying the fertility of Merino sheep in Australia, found that a low reproductive rate and light weights of the ewes in certain areas reflected changes in the level of nutrition of the grasses, etc* This was partially controlled by rain fall and condition of the ranges* Braestrup (19**D in re gard to carnivores, presents interesting evidence that in land populations of the Arctic fox (Alopex lagopus). which feed primarily on lemmings (Dircrostonyx groenlandicus), - 7 - have a much higher average litter size in years when lem mings are abundant. These he contrasts with a coastal population of Arctic foxes which feed primarily upon marine organisms. In this group the average litter size shows little variation, and is only about half the litter size found among inland fox populations during a peak lem ming year. Schwartz (194-2) described six apparent breeding per iods through the season in Missouri. This was done by estimating the parturition date of all pregnant animals collected. The highest peak of litter births occurred at the end of March, the second highest from the middle to the end of April, and smaller peaks of breeding in the latter parts of May, June, July, August, and September. Chi the basis of these six supposed breeding periods, Schwartz concluded that a female theoretically could produce six litters in one season. Hammond (1925) suggested a relationship between re productive function and molting. Hadwen (1929), after maintaining captive hares in heated rooms over an extended period, decided that environmental factors were not the cause of molting sequences. Dalke (19^2) found a relation ship between the weight of testes in adult male cottontails and the beginning of molting. Testes began to gain weight - 8 - during the non-molting period in December, and lost weight at the beginning of the molting period in the spring* It was also found that the testes weight decreased markedly with the onset of the molt in September* He noted further that pregnant females captured during the months of Septem ber to November had a mottled molt pattern, but in most of the non-pregnant females taken during the same period a sheet molting pattern was present* Chapter II MATERIALS AND METHODS As the present study was originally planned, a state wide sampling in each of the major physiographic regions of the state was to be undertaken for purposes of comparing reproductive performance and other characteristics of the populations. It soon became evident that such an approach could not be feasibly undertaken in view of the limited per sonnel, time and finances available for the study. Further more, consideration of a comparison of physiographic re gions revealed that a complexity of sroil types existed wiih- in each physiographic type which, it seemed might well serve to obscure differences which actually might be present in good and poor soil types. The magnitude of sample sizes which would be needed to demonstrate possible differences in reproductive performance in such a mixture of soil types and fertility led to the conclusion that such a comparison was impractical. Consequently it was decided that perhaps the most enlightening approach to the problem would be a comparison of populations from good and poor soil types as reflected by productivity of such crops as corn, wheat, and oats, as well as relative mineral content of the soil. On the basis of this assumption, two areas having strikingly - 9 - - 10 - dissimilar soil types were selected for intensive sampling and evaluation of reproductive performance, breeding incre ment of the population, attainment of sexual maturity, and breeding of young individuals in their first year. The soils of the till plain region of Ohio are consid ered to be generally the most productive in the State. This area includes roughly one third of the area of the State from central Ohio westward to the Indiana State line exclu sive of the old lake basin region in the northwestern part of the State. The till plain region also includes much of the prairie peninsula that extended into Ohio during a per iod of prolonged drought sometime after the retreat of the Wisconsin glacier. Evidences of invasion of prairie vege tation may be found in the presence oft rich dark prairie soils in many of the counties in the till plain region. Marion County is one of those areas richly endowed with prairie soils of high fertility and high productivity. A study area of 671 acres was established in this county, the soil of which is primarily Clyde silty clay loam. This had been formerly a magazine storage area of the Marion Engineers1 Depot of the U.S. Army Engineers. During the present study the area was being farmed by a local citizen. The cultivated fields were planted in wheat, soy beans, and corn. Several woodlots were included in the area, two of - 11 - these being less than five acres in total area, and the third measuring between 10 and 15 acres. Bluegrass (Poa pratensis) was the dominant species of grass in most of the uncultivated and unwooded areas. Prairie plants were abun dant throughout the area and locally attained dominance in some instances. Where big blue stem (Andropoeon furcatus) was well established it excluded most other species of grass es. In several locations in the area briar patches of rasp berries as large as an acre in extent constituted what might almost be considered ’‘rabbit warrens” for cottontails. Such areas invariably supported dense populations of rabbits, due perhaps to the great amount of protective cover afforded them by such vegetation. In contrast to the till plain region, the unglaciated Allegheny Plateau of Ohio is considered to be an area of generally poor soil types. This region includes the south eastern portion of the State from Columbiana, Stark, and Holmes Counties southward, and from Hoss County, eastern Fairfield, Licking, and Knox Counties eastward. This is an area of residual sandstone and shale soils of relatively low fertility. The sharp relief of the area intensifies such factors as runoff and erosion. Steep phase Muskingum silt loam is considered to be one of the more inferior soils of the region. In Muskingum County the writer was - 12 - fortunate to gain permission from the Hanna Coal Company to sample the rabbit population on areas of optioned farm land in the uplands. These areas were primarily composed of steep phase Muskingum silt loam. The total acreage in this study area is not known because the areas available for study were not in one continuous block as was the case in the Marion County study area. It may be said, however, that the acreage sampled was far in excess of 700 acres; in other words, the area was substantially larger than the Marlon County area. Seme of this land was abandoned farm land; some of it was in active use at the time of the study. Other portions were in second growth timber, and a few old orchards and many briar patches were scattered throughout the area, Bluegrass was abundant only along dirt roads and around old abandoned homesites® Boverty grass (Aristlda dichotoma) and broom sedge (Andropoeon virginicus) were abundant, and in many places they were the dominant grasses, particularly on hillsides. Figures 1 and 2 indicate the boundaries of the physiographic and general soil regions in Ohio, In addition to the selected areas chosen for Intensive sampling of the population, it was deemed advisable that a sample of rabbits throughout the State be collected in order to evaluate the variation in reproductive performance on a Figure 1. Map of physiographic regions of Ohio. Regions are denoted as; follows: No.i, glaciated Allegheny Plateau; No.2, unglaciated Allegheny Plateau; No.3, Wisconsin till plain; No A, glacial lake beds; No. 5, Illinoan till plain. Cross-hatched line shows the escarpment of the Allegheny Plateau. Figure- 2* Map of general soil regions of Ohio. Soil regions- are designated as follows: I - glacial limestone soils? II - glacial sandstone and shale soils; III - lacustrine limestone soils; IV - lacustrine sandstone and shale soils; V - residual limestone and shale soils: VI - residual sandstone and shale soils* - 15 - statewide basis. Cooperation was gained from the field per sonnel of the Ohio Division of Wildlife and from graduate students of the Ohio Cooperative Wildlife Research Unit. Each individual was furnished a jar of alcohol-formalin- acetic acid solution (A.F.A.), a series of numbered speci men tags, a pair of dissecting scissors, and a booklet of data cards on which they were to Indicate the location to the nearest town, county, and date of collection of each specimen. The source of specimens was to be those female rabbits found freshly dead on the road. At the outset of this particular program the writer was optimistic that a large sample might be obtained. However, as the breeding season advanced, from about mid-April through August, the number of road kills of females decreased markedly, result ing in a much smaller sample than had originally been ex pected from such a program. Prior to the establishment of the study areas describ ed above, preliminary sampling of rabbits was undertaken during 1953 in the fenced pheasant pens on the Urbana State Game Farm in Champaign County, on the Ohio State University farm land in Franklin County, in fenced 5-acre study pens on the Woodbury Restoration Area in Coshocton County, and in the *+O-acre fenced plot at the Olentangy Wildlife Experi ment Station, Delaware County. Collecting Methods Several methods of collecting specimens were employed in this study. As mentioned previously, specimens that had been hilled by automobiles were picked up on roads. Stand ard wooden box traps were used in sampling particular areas. The traps were not baited except during autumn collecting when bait seemed to increase trapping success. In areas that were completely fenced, such as those on the state game farm and at Woodbury Wildlife Bestoration Area, the use of an English hare net proved highly successful in captur ing large numbers of individuals. These nets were obtained in lengths of 150 feet from England at very low cost. In appearance they resemble a long seine with a mesh of 1 inch in diameter. The width of these is *+ feet. The nets are set vertical to the ground and hung on a series of stakes driven at intervals along a line where the net is to be strung. It was desirable to fasten the bottom line of the net to the ground by means of bent pieces of No. 9 wire that could be shoved 6 or 8 inches into the ground. In addi tion it was found that for maximum efficiency the net must be hung loosely with a large amount of slack, particularly in the part of the net closest to the ground. If the nets were strung too tightly with only small amounts of slack netting near the ground, more rabbits tended to leap - 17 - through the net without becoming entangled in its mesh* Once the nets were in place, the rabbits were driven toward it by a number of individuals, first from one side, then from the other* One or two individuals were stationed at the net to extract rabbits as soon as they became entangled* This was found necessary because some animals literally skinned themselves alive if they remained entangled in the net for any extended period* The size of the mesh proved to be somewhat selective in catching rabbits* The younger and smaller rabbits showed a remarkable ability to jump through the net at full speed with apparently no entangle ment whatsoever* As a result, rabbits captured by this method were either adults or subadults of adult proportions* The maximum efficiency attained by this method was the cap ture of *+0 rabbits in a 15-acre plot in approximately four hours, with the cooperation of 12 individuals. During the months of June, July, and August, trapping success decreased markedly. This has been observed by other workers who have studied the cottontail by trapping methods. In order to alleviate this situation an attempt was made to collect rabbits at night by driving down dirt roads in the Marion County area, and, with the aid of a spotlight, shooting the animals with a shotgun whenever they were observed either on the road or off to the side in fields* This proved very successful in the Marion County area* A number of old dirt roads criss-crossed the area, rendering the majority of the fields vulnerable to such a technique* This method met with little success in the hilly country of Muskingum County* Figure 3 indicates the localities where collections were made* Aging Techniques The objectives of the present study required that the investigator be able to accurately age specimens in the field. A number of aging techniques have been employed on the cottontail rabbit, all of which have their limitations and some of which are impractical to use other than in the laboratory. Methods that have been used are listed below: 1* Size of nipples, presence or absence of hymen* 2, Presence of placental scarss if present - adult. 3. Hind foot measurement. h. Degree of ossification of epiphyseal cartilage of radius and ulna by x-ray examination. 5. Degree of ossification of humerus. 6. Weight in grams: if over 1000 grams - adult. 7e Total length measurement. The above methods have all been used to age rabbits - 19 - i ! .L.J‘ ; * u * i £-j / . fir :• ■ T'l \ cHAW l0Nf. | .j—I i — j . i Figure 3. Map of collecting localities One or two in particular are probably very reliable tech niques, namely the ossification methods* The remainder are highly questionable due to the fact that they are sub ject to a large amount of variation* In 1952, at the start of the study, the writer did preliminary trapping at the Ohio State University woodlot in order to become more familiar with aging techniques and methods of evaluation which he intended to use. During July and August, 11 females were trapped from this area* Even in this small sample three or four of the above aging methods would not have correctly aged all of the rabbits. When such a small sample indicates the inadequacy of tech niques, the value of such methods is subject to doubt. Saith (1950) stated that weight alone could be used to sep arate juveniles from adults up to the 1000 gram level, but beyond this other criteria were necessary. He found that young rabbits average less than two pounds in late summer and early fall. It would seem, however, that this method would not prove accurate throughout a variety of habitats, for it has been shown in Missouri by Schwartz (19^2) that rates of growth may vary according to type of range* Petrides (19^8) described an aging technique on the basis of teat size and condition of the hymen, but some of the 11 animals collected in 1952 could not be aged accurately by - 21 - this method* Some young females had enlarged teats and per forate hymens, and two adult females had no visible teats at all* According to Petrides, young females of the year may he distinguished from older females on the basis of un developed nipples until at least January 1, and often later than that* Determination of the presence or absence of a vaginal membrane is recommended as a desirable check* Even in the preliminary sampling the development of nipples and the presence or absence of a hymen .were not re liable indicators of age* &nce it is highly unlikely that in such a small sample the writer happened to collect a num ber of rare individual variations, it appeared that some other method was needed for aging in the field* The pelage stages of cottontails have never been adequately described* Nelson (1909) in a monograph on the Leopridae described three distinct pelage stages apparently common to all North American hares and rabbits* These he designated as .luvenal, uost.luvenal and adult pelage* He described Svlvilagus as a genus having only one annual molt* Dalke (19^2) concluded that adults experienced a continuous molt over a nine-month period from February to November, during which two periods of pronounced new hair growth occurred, one from March to August and a second from - 22 - September to November. In 1952, during preliminary fieldwork, the writer de tected markedly different pelage stages in the cottontail rabbit. At first these differences bore no apparent rela tionship to age. As more rabbits were trapped and scruti nized a pelage-age relationship became evident. In 19539 notes on pelage were recorded on every rabbit that was trapped. This was done immediately upon withdrawing the rabbit from the trap before weight or reproductive data were recorded. Three definite pelage stages were at once discern ible in young cottontails. These are, as the writer has de signated them: nestling fur, .iuvenal pelage. and adult pel age, In addition, there are two molting phases that are definitely related to ages (1) the molt from nestling fur to Juvenal pelage, and (2) the molt from Juvenal to subadult pelage. Once it had become obvious that various stages were typical of rather specific weight ranges of rabbits, each pelage observation was assigned an age approximation on the basis of the weight of the rabbits compared to an age-weight curve derived from known-age cottontails by Petrides (19^8), As a result, the following age groups appear consistently well defined by specific pelage stages* Nestling fur — this stage is characterized by its fine downy texture, dense nature, and rather grey colora- - 23 - tion. Age range: 2*0 - 5.5 weeks. Weight range: 113 - 2^1 grams (Figure *f). Molt to -iuvenal - this molt occurs in a sheet pattern growing through the nestling fur. Age range: 5*6 - 7 weeks. Weight range: 2*+l - 28M- grams. Juvenal pelage - this is characterized by thinness of the pelage, salt and pepper appearance, and general dark coloration dorsally. Age range: 7*5 - 10.5 weeks. Weight range: 298 - ^96 grams (Figure 5). Molt to subadult - occurs in a sheet pattern, begin ning on rump and sides. Age range: 11 - 1^.5 weeks. Weight range: 510 - 765 grams. Subadult pelage - characterized by a lighter buffy coloration of the under fur, and a salt and pepper appearance caused by sparseness of dark outer long hairs. Age range: 15 - 30 weeks. Weight range: 780 - 12^7+grams (Figure 6), Adult summer pelage - characterized by overall darker brown coloration and glossy appearance due to greater density of long hairs. (Figure 7). Three constituents make up the dorsal pelage of the cottontail: (1) a fine short under fur usually buffy or grey; (2) a long, thinner, coarser coat of hairs, dusky at the tips, with a broader subterminal zone of buffy or grey- ish; and (3 ) an even longer, coarser, sparser set of hairs glossy and blackish in their terminal half. The distribu tion and abundance of these various constituent hairs accounts primarily for the gross differences of coloration and appearance of the various pelage stages. A few additional comments are advisable in regard to the use of this technique in aging rabbits. Aging rabbits by pelage apparently can be done only during the breeding season, for with the onset of the molt to winter pelage it appears that one can no longer distinguish young individuals from full adults. Apparently young individuals having either adult pelage or Juvenal pelage at the end of the breeding season molt directly into adult winter pelage. In most cases the molt of young individuals into winter pelage appears to occur during October and is almost complete by the first of November. The recognition of subadult pelage iS of particular value in reproductive studies since this stage has been found by the writer to be indicative of sex ual maturity. Thus, a young female cottontail in subadult pelage even as early as July was usually found to be sex ually active, that is, either pregnant or possessing per forate hymen or enlarged uterine horns and ovaries. Corre spondingly, no individual found in Juvenal pelage of either sex was sexually active. - 25 - Figure if. Nestling fur - 26 - Figure !?• Juvenal palage - 27 - Figure. 6. Subadult pelage 28 - Figure 7 • Mult summer- pelage - 29 - In adult cottontails the molt from winter to summer pelage is a gradual process beginning in the latter part of March or the fore part of April, and extending well into August. A mottled molt pattern similar to that observed by Severaid (19^5) in snowshoe rabbits is characteristic in the dorsal region, at least in adults. Tufts of winter pelage persist throughout most of the breeding season, and may be readily distinguished from the darker, finer summer pelage by the lighter color and coarser texture of the hairs. The presence of these tufts is thus a useful character in identifying some individuals as adults which, due to varia tion in color, might otherwise be difficult to age. The Juvenal and post Juvenal pelages described by Nelson in the Leporidae probably correspond to the Juvenal and subadult pelages respectively described herein. No pelage comparable to what the writer designates as nestling fur was described by Nelson* In view of the significant difference in appear ance as well as in the change in pelage composition brought about by the new growth of hair from nestling pelage to Juvenal pelage, it seems advisable to include nestling fur as an additional pelage stage in the cottontail. Actually five pelage stages occur in a cottontail from the time of birth through one full year of life: nestling fur, Juvenal pelage, subadult pelage, adult winter pelage and adult summer pelage. Although Dalke (19^2) described a continu- ous molt in the cottontail over a nine-month period from February to November, examination of a large number of Ohio specimens revealed that, actually, the molt from win ter to adult summer pelage was a distinct period which ter minated in a full or complete summer pelage sometime in August, and for a short period thereafter no apparent molting occurs* The onset of the fall molt to winter pel age is apparently a separate molt which appears to be re lated to the reproductive cycle and will be described later in this paper* In the early part of the study before the development of the previously described pelage-aging technique, it had been decided that the most accurate manner in which to age rabbits would be by taking X-ray photographs of the fore feet to determine the degree of ossification in the distal epiphyseal cartilage of the radius and ulna* This method has been described by Petrides (19^9)# The degree of ossi fication in this region when used in conjunction with the degree of ossification in the head of the humerus is pro bably one of the most reliable techniques of aging rabbits in the laboratory* An old model portable dental X-ray cam era was obtained for the purpose of X-raying forefeet. This means of aging proved to be a useful check on the pel- age-aging technique, particularly in cases where breeding females were identified as subadults according to pelage* La all cases X-ray of the forefeet in such individuals re vealed that the pelage-aging technique was highly successful in separating young individuals breeding in their first sea son from the true adults* In only one case did aging by X- ray reveal an incorrect age determination in the field* This was an individual (R-5) that was collected during the preliminary sampling on the University farm in August, and prior to the development and use of the pelage-aging tech nique by the writer* The particular individual mentioned was a female and was identified in the field at the time of trapping as an adult on the basis of enlarged teats and per forate hymen* Examination of this individual in the labora tory revealed that she was pregnant. This was her first litter of the year. There was no evidence of a previous litter during that season. This in itself was sufficient evidence to cast doubt upon the age determination of the individual. An X-ray photograph of.4 forefoot revealed it to be a young rabbit. This mistake would not have occurred had the animal been aged by pelage. Figure 8 indicates the appearance on X-ray film of the distal epiphyseal cartilage in the forefoot of an adult and a young rabbit as well as the degree of ossification in the head region of the humerus. During the course of the study a series of embryos - 32 - i / \ ! Figure 8. X-ray photograph of forelegs of adult (lower) and juvenile; (upper) cottontail rabbits, showing degrees of ossification in epiphyseal cartilage. was obtained from females collected during the breeding season. An attempt was made to determine whether peaks of litter births occurred during the breeding season. In order to do this it was desirable to know the ages of the embryos collected so that their approximate birth date or concep tion date could be ascertained. This tendency of grouping of breeding periods during the season was observed by Schwartz (19*+2) in Missouri. Ideally, a known-age series of embryos from pregnant cottontails held in captivity should have been obtained for use in aging embryos from rabbits collected in the field. However, it appears to be extremely difficult to obtain litters from cottontails in captivity unless they are maintained in extremely large en closures. The facilities for such a procedure were not available for this study. Several attempts by the writer to breed cottontails in captivity and to hold pregnant fe males in hutches until they bore litters proved unsuccess ful. The pregnant females that were held in hutches invar iably aborted their litters before they reached full term. Domestic breeds of rabbits are known to have gestation periods closely approximating that of the wild cottontail. The normal gestation period of domestic rabbits averages 29-30 days. In cottontails various writers have stated that the average gestation period is 27-30 days. On this basis it was thought that a known-age series of embryos from domestic rabbits would yield data useful in aging cottontail embryos based on the development of various structures and body proportions. Schwartz (19*+2) used this same type of approach in assigning ages to his embryos of wild cottontails. The source of his known-age data of do mestic rabbits was from Minot and Taylor (1905). Since not all age stages, particularly the more advanced ones, were well represented by this work, the writer deemed it advis able to obtain a series of known-age embryos. Pure bred New Zealand White rabbits were used in this procedure. It has been demonstrated in the laboratory that fertilization of the domestic rabbit occurs approximately 12-2^- hours following copulation. In the present study females were bred once to a male, and the time and date carefully noted. Animals were killed at the following intervals after mating: 7 days, l*f days, 17 days, 21 days, 2*+ days, 28 days, and 30 days. Various criteria appeared to be indicative of the age of the embryo or fetus. Table 1 indicates the develop ment of various characteristics according to the ages of the embryos. Figures 9 and 10 indicate the relationship of ear pinna length and crown-rump length to age of the fetus. Notably, formation of limb buds, separation of digits, and modification in the head region as well as crown-rump length proved most indicative of age in the earlier stages# Crown- Table 1. Characteristics of Known-Age Embryos from New Zealand White Rabbits Breeding Experiment Age in Cc-pmp Pinnae Vibris Hd ft Tail Days length length length length length Other characteristics mm. mm. mm. mm. mm. Ik 15 1 none 2 1+ eyes well developed, not covered, on surface 17 22 2 none k-5 5-6 eyes about 5<$ covered:, by eyelids, no claws in feet 20 35 3+ none 8 9 claws beginning to form eyes completely covered by eyelids 2k 60- 9-10- -1.0 12- 12 no hair 6*+ 13 28 71- 13 k-6 17 15- claws well formed, 1-2 mm., 75 16 no hair co H ir 1 o 30 \0 15 6-8 21 17- pigmented skin, hair over 18 entire body 36 - 9 0 . 8 0 . Crown-Rump Length 70 - of Known Age Embryos ■£ 80 ■ 1 4 0 - CO . 10 . CO tt 26 3026 Ait in day* Figure 9» Relationship of crown-rump: length to age in Hew Zealand1 White rabbit embryos. age in New Zealand White rabbit embryos. rabbit White Zealand New in pinna, age ear of Relationship to length 10. Figure 15 Ear pinnae length in mm.13 li 9 7 5 3 f nw Ae Embryos Age Known of 10 r ine Length Pinnae ar E 12 14 Age in days in Age 37- 20 22 24 9 SO 29 - 38 - rump length and ear pinna length are perhaps the best indica tors of age in the more advanced stages of development* Pig mentation of the skin, the appearance of hair on the body, and the length of vibrissae were also used as indicators of age. Following accumulation of these data, embryos of wild cottontails were compared with the New Zealand rabbit embry os on the basis of the various criteria to determine wheth er development was comparable* The criteria of development of structure and measurement indices were almost identical when embryos of the same crown-rump length were compared* This agreed very well with the data of Schwartz in his com parison of cottontail embryos with the data on known-age domestic rabbit embryos of Minot and Taylor* It seems safe, therefore, to assume that cottontail embryo development approximates that of domestic rabbits closely enough to enable aging by these criteria. Cottontail embryos falling in between the measurements of the known-age specimens of domestic rabbits were assigned an age on the basis of rate increase in crown-rump length, ear pinna length, and hind foot length as shown graphically in Figures 9 and 10. Figures 11 through .16 indicate the appearance of known-age embryos of various ages. All cottontail embryos were thus assigned an age in days on this basis, and from thdse data it was possible to determine approximate conception dates and prospective parturition dates* - 39 - Figures- 11-13. Photographs of New Zealand White rabbit embryosv Figure 11 at top is a. l^-day embryo, figure 12 in middle is' a 17-day embryo, and figure 13 on bottom is- a. 20-day embryo. — 1fO — Figures 1^-16. Photographs of advanced stages in development of New jgbaland White rabbit embryos* Figure I1* on top is a 2i+-day fetus, figure 15 in middle is a 28-day fgtus, and figure on bottom is a 30-day (full term) fetus. - »+l - Laboratory Meliliods Evaluation of reproductive performance in cottontails • was accomplished on the basis of the following criteria: 1. Litter size 2. Number of litters per year 3. Total productivity *f. Length of the breeding season 5. Breeding of subadults 6. Fecundity rates One of the methods of accumulating litter size data was by direct embryo counts from the uteri of females col lected during the breeding season. At the time of dissec tion of each pregnant specimen the following was noted: total number of embryos, the number and location of embryos in each uterine horn, their size, and evidence of resorp tion. Placental scar counts of previous litters also yielded some data in regard to litter size. However it was found that placental scars could only be utilized with ac curacy for litter size counts during a relatively short period following the birth of a particular litter. Exper iments conducted to test the reliability of placental scar counts will be described later in this paper. - k-Z - Evaluation of reproductive performance by examination of ovaries has been developed and used successfully by Cheatum (19^9) > and Cheatum and Seviringhaus (1950) on white-tailed deer in New York, This method involves the identification and counting of corpora lutea and corpora albicantia of pregnancy. Of course this does not take into account resorption rates. Thus in order to increase the accuracy of such data one must establish corpora lutea and corpora albicantia-litter size ratios from pregnant animals before making final evaluation on all counts from animals that were not pregnant. Thus if a pregnant animal is examined and found to have six actively developing embryos and six corpora lutea in the ovaries, one may say that the animal has a one to one ratio of corpora lutea to litter size, Erom a sample of pregnant animals one may thus establish a mean ratio which may be applied to corpora albicantia counts from non-pregnant animals that have borne litters. This, then, makes allowance for early resorptions as well as ova released but not fertilized. Such counts also enable one to evaluate fecundity in individuals, actually constituting another means of evaluation of repro ductive performance in the form of the ratio itself. It must be remembered that the white-tailed deer is a mammal having only one litter per year. Prior to the - *+3 - present study there was no information in the literature regarding the application of such a technique to evaluation of reproductive performance in mammals having multiple litters per year. Correspondence in 1952 with Mr. Joseph Dell of the New York Conservation Department, indicated that preliminary studies on the varying hare (Lepus americanus) suggested that the technique might prove useful on multiple litter animals. As such a means of evaluation appeared to be the only way in which certain types of data might be obtained, the writer attempted to adapt the tech nique for use on cottontail rabbits. 'While it is possible to determine the number of corpora lutea in an ovary of a white-tailed deer by macroscopic examination, it was found that the size of rabbit ovaries was such that sectioning, staining, and microscopic examination was essential for accurate evaluation. It was immediately obvious that a great amount of time would be consumed in the preparation of ovarian material for examination microscopically. The procedure involved dehydration, inpregnation and imbedding in paraffin, sectioning, staining, and mounting of the material on slides. In view of the anticipated large number of ovaries to be examined, it seemed advisable to devise methods by which the time consumed in the prepara tion of tissues could be shortened. Consequently, Dr. Maurice L. Giltz and the writer attempted various "short - bb - cuts” in the standard method of preparation* The resulting procedure (See Appendix) will probably evoke disapproval on the part of histologists, but it proved adequate for the purposes of evaluation in this study. As indicated in the Appendix, a double stain was employed in the technique. Delafield’s hematoxylin is more specific as a nuclear stain, while eosin red in more specifically a cell-wall stain. The advantage of a double stain over a single stain such as Delafield’s hematoxylin in this case appears to be in rendering differences in the development or degeneration of lutein bodies more easily discernible. Note should be made here regarding the thickness at which ovaries were sectioned. Following preliminary sec tioning, all ovaries were cut at *+5-50 microns. This was governed by the maximum thickness at which the microtomes used would cut. All active corpora lutea are of sufficient size that they could be discerned readily in sections much thicker than 50 microns. Older corpora lutea and albican tia, jwhile smaller than active lutein bodies, are never theless of sufficient size to be discerned in sections of much greater thickness than 50 microns. It should be noted that had a freezing microtome been available it might have shortened the time consumed in the sectioning procedure greatly. It was a relatively simple matter to differentiate active corpora lutea from degenerating corpora lutea or corpora albicantia of previous pregnancies. Corpora lutea may be differentiated from corpora albicantia on the basis of several characteristics. The corpora lutea of a pregnan cy had a more granular structure than corpora albicantia when viewed under a lower power of the microscope. Corpora lutea of pregnancy or immediate post partus seem to absorb a greater amount of stain than corpora albicantia, and hence are discernible from albicantia on the basis of darker col oration. This may be due to the greater abundance of secre tive cells in active corpora lutea than in corpora albican tia and the greater abundance of interstitial tissue in corpora albicantia. Size too is a criterion when an ovary is examined having more than one litter represented. Active corpora lutea are appreciably larger in size than the albi cantia of the previous litter. Similarly two series of corpora Albicantia in the same ovary may be differentiated primarily on the basis of size since they progressively decrease in size following partirition. At the end of the breeding season when ovaries have begun to retrogress rap idly, it becomes impossible to obtain anything more than a total count of corpora albicantia due to the fact that all of the corpora approach the same small size at that season, m several cases active corpora lutea were noted in early post partum females which were not accompanied by the visible presence of embryos. However, examination of these specimens showed a pronounced plug or scar at the edge of the ovary at each lutein site where the ova had recently erupted from the follicle. Figures 17 through 22 are photomicrographs showing the appearance of typical ovarian structures in the adult cottontail ovary. Corpora lutea-litter size ratios were determined from pregnant animals. Counts were made of embryos and corpora lutea and ratios established for each individual. In addition, those specimens having fresh placental scars were utilized. In such individuals the corpora albicantia count of the last pregnancy was compared to the placental scar count and a ratio established. From this type of data mean ratios were established for application to counts of corpora albicantia which could not be verified by embryos or placental scars. By this method it was possible to determine litter sizes as well as the number of litters a female had borne up to the time of her collection. It also enabled an approximation of the date of onset of breeding. It seemed highly desirable that some sort of check be made to ascertain reliability and persistence of placental scars as indicators of litter size as well as the reliabil- Figure 17-top* Photomicrograph of maturing Graafian follicles in cottontail ovary. Figure 18-hottom. Photomicrograph of freshly formed corpus luteum. Note plug a£ site of ovum release. - lf-8 - mmmiM .vrtV-v£^XYft.v -x- ^4 „:>>»uv. m m m m m m m m m-fmMMmmi Figure 19-top. Photomicrograph of an active corpus luteum of pregnancy. Figure 20-bottom. Photomicrograph of an active luteum enlarged to show cellular structure. *+9 - Figure 21-top. Photomicrograph of a corpus albicantia from a previous pregnancy. Figure 22-bottom. Photomicrograph of a. corpus albican tia at the end of the breeding season. - 50 - ity and persistence of corpora lutea and albicantia. Since it was not practical to attempt to conduct controlled studies on cottontails in captivity, it was decided that a controlled breeding program would be conducted using pure bred New Zealand White rabbits. Initially 15 rabbits were obtained for the study. Of these 12 were virgin females between 5 and 7 months of age. The other three were males of varying ages. The original plan was to kill two rabbits for examination after they had produced one litter, two after producing two litters, and so on through four litters. One control animal which had not been bred at all was to be killed with each group. Each rabbit was marked with a numbered ear tag. Selection of control animals as well as those to be used for one, two, three, and four litter production was done in a random fash ion using a table of random numbers (Snedecor, 19*+6). In addition, animals were randomly selected as to the male with which they would be mated. Selection for mating how ever was not rigidly adhered to since two of the males proved to be far more successful at mating than did the youngest and smallest male. All animals were maintained in wire hutches outdoors in the University mink yard. Standard rabbit pellets vtfre used throughout the study, and an average ration of three ounces per day was given to -51- all animals except during pregnancy when the quantity of food was increased to approximately five or six ounces. The data derived from this experiment are described in Chapter III of this paper. Bone measurements. In most of the specimens collected by the writer one entire forearm was saved from each animal. These were X-rayed for checking on age determinations, after which the humerus was detached and cleaned. These were then placed in a drying oven for a period of 48 hours at a temperature of about 103 C. Weights and measurements were then made. Originally it was planned to analyze the humeri for ash content and to compare this value in rabbits from selected soil types. HEowever facilities for such analysis were not available at the time the writer was ready to ana lyze the bones, and as a result this objective was not realized. Grass analysis. It was thought that differences in repro ductive performance in areas of high and low soil fertility might be correlated with mineral content in the vegetation. On the basis of this, grass samples were collected in Marion County and Muskingum County periodically throughout the sea son in 1954. The writer had planned to analyze these sam ples himself and undertook preliminary analyses of a few 52 - samples. However, time did not allow the completion of this work, and, as a result, samples were sent to the Ohio State Experimental Laboratories in Reynoldsburg, Ohio, for analysis. The results of several samples taken at intervals through the season revealed quite a large amount of varia tion in mineral content from one sample to another even in the same area. Consequently in 1955 ten samples were col lected on the same date from Area A as well as ten from Area B during the same month. Samples were taken only of blue- grass (Poa pratensis). Sample sites were selected randomly, and actual samples were obtained from the site wherever bluegrass was present. An effort was made to sample only the younger bluegrass which is more heavily utilized by rabbits for food than the older tougher grass. An analysis of ash content was also made on the 195^ sample, but not on those collected in 1955* All analyses were made on the basis of dry weight of the samples which were dried in an oven at constant temperatures for *+8 hours. Chapter III RESULTS The data recorded in the following pages have been acquired from various sources and methods. During no two years were the actual methods of data collection uniform from the standpoint of procedure. In 1952, preliminary collections and examinations were made. These contributed a certain amount of data to all phases of the study. In 1953, data were collected primarily from collections made in fenced areas from hunter-bag checks of specimens. In 1954, specimens were obtained primarily through inten sive collecting In selected areas and from specimens picked up dead bn the road throughout the State. While this would at first seem a rather variable manner in which to conduct an investigation, it actually representa a development of ideas throughout the problem. In 1952, collections and studies of data gave some indication of the problems in volved and approaches which should be made to resolve the objectives. In 1953, more Intensive collecting in specific areas was undertaken which led to the final procedure fol lowed in 1954, of intensive collecting in unfenced selected areas on the basis of soil types within specific physio graphic regions of the State. 53 - Breeding Season The length of the breeding season of the cottontail rabbit in the northern states has been estimated by various workers to extend over a *+0-week period from January to September. One of the objectives of the present study was to obtain data on the length of the breeding season in varl ous parts of Ohio* In 1953? as stated earlier, fieldwork was confined largely to fenced areas on State-owned land. By fenced areas, the writer refers to plots on game farms and research areas that were enclosed by wire fences. The fences were buried in the ground to render entrance and exit impossible by rabbits and other animals. Two such plots were made available to the writer in 1953 on the Urbana State Game Farm in Champaign County. One of these (Area No. 5) was a 15-acre enclosure planted to alfalfa. The other (Area 9) was a l^t-acre plot which was dominated primarily by bluegrass. A grove of hawthorne tfcees (Crataegus) was located in the central part of the area. On April 12, 1953, a drive was conducted in Area No. 5 in an effort to catch all the rabbits present on the area at the time. Twelve rabbits were captured in this area, six adult females, four adult males, and two immature females. Of the adult females, three were pregnant for the first time that season as determined by palpation and condition - 55 - of the teats, one was pregnant for the second time, and one was not pregnant and had not produced a litter that season (Table 2). On April 26, 1953» a drive was conducted on Area $o. 9. English hare nets were employed on this area as described under Materials and Methods, This drive resulted in the capture of 37 adult rabbits. The sex ratio of this sample was 18 males and 19 females. Of these 19 adult females, 12 showed no signs of being pregnant or having produced a litter that season, one was pregnant for the first time, five had produced one litter but were not pregnant for the second time as determined by palpation, and one had pro duced one litter and was pregnant for the second time (Table 2). The animals captured in both areas were examin ed, ear tagged and released. One adult female in Area 5 (A313-A31^) was injured during the drive and was killed for examination. She possessed five embryos that were approxi mately 50 mm, in crown-rump length. Of the entire sample of 25 adult females, from both areas, Ik or 56$ had not produced a litter that season and showed no signs of being pregnant. Pour or 16$ were preg nant for the first time, five (20$$ had produced one litter but were not pregnant for the second time, and two (8$) had 5 6 ^ Table 2. Data on Adult Female Cottontail Rabbits from the Urbana Game Farm April, 1953 No. Pregnant 1st 2nd Lactation litter litter A-^+56 - A-J+76 « M » > A-^75 - A-U-71 - - A - 1+ o H' - A-^+68 — A-^78 X x A-483 - A-M3 8 - A-M-29 — A - ^ 2 h> •M* X & - b 0 9 - A-Jf27 - X A-*+01 - A-J4-13 - X A-M l 5 A-ML7 X X A-*+22 - A-301 X x A-305 - A-307 X X A-3 09 X X A-313 X X A-3X5 - -57- produced one litter and were pregnant for the second time. From these data it appears as if the earliest mating pro bably occurred early in March. The three animals trapped on April 12 that were pregnant for the first time must have mated sometime after the middle of March. The five individ uals in Area 9 which had produced on e litter and were not noticeably pregnant had probably mated for the first time between March 15 and 25. It should be noted that while scattered breeding apparently occurred from about the be ginning of March on, that as late as April 26 over half of the sample was not yet pregnant for the first time. Although the areas on the Urbana Game Farm were sam pled again in the fall of 1953 to determine, among other things, the length of the breeding season, they were not sampled in the sprin g of the followln g year. Instead, in tensive sampling was undertaken in the selected areas men tioned previously in Marion and Muskingum Counties. Sam pling in these areas was begun in April. It is interest ing to note the results of the April sampling in these areas and to compare them with the results of sampling during the identical period of the previous year in the fenced plots on the Game Farm. In the Marion County area the sampling was begun on April 22, 1954. A total of seven adult females were collected in April from this area. In addition, eight other adult females were collected on the random sample program throughout the till plain region. Of these 15 adult females collected during April in the till plain region in 195*+, seven were pregnant and lac- tating, indicating that they had produced one litter and were pregnant with their second, six were pregnant and apparently not lactating. However, the data on lactation was not certain regarding four of these females. Two fe males were found to he lactating and not noticeably preg nant with second litters. Thus, of a group of 15 animals, all had begun breeding; and almost half were pregnant with their second litter. By aging embryos from the tracts collected, it was possible to approximate the time at which the first mating occurred in the majority of the sample. In two females the first mating had occurred within the first week of March, In six of the animals mating had occurred approx imately the second week of March, and in three during the third week. Three of the remaining four had apparently mated within the last week of March, and one during the first week of April, Comparison of these data with those from the Urbana Game Farm in 1953 (See Table 23) suggests that breeding in 195^ was in full swing several weeks to a month earlier than in 1953 in the till plain region. -59- All of these samples are admittedly rather small, but the differences are so great that it seems likely that the on set of breeding in the populations began earlier in 1954 than in 19 53. A sample of twelve adult females was obtained in April and May, 1954, from the unglaciated Allegheny Plateau region. Nine of these were obtained in or near the Muskingum County study area. Two of these were not yet pregnant with their first litter* Six were pregnant with their first litters; three were pregnant with their second litters; one, which was captured in Muskingum County study area on May 10, was pregnant with her third litter. The ages of the embryos in the ten animals that were breeding indicates that one individual probably mated for the first time during the first week of March, two individuals mated about March 7-10, three during the last week of March, and five between April 1 and 10. Comparison of this sample with that taken from the Marion County area reveals that the breeding season had on the average begun earlier in the till plain region than in the unglaciated Allegheny Plateau. Based on calculated first matings of the breeding animals, the mean date of first mating for the till plain sample is March 13. The mean date of first mating for the sample taken from the unglaciated Allegheny Plateau is March 27. - 60 - A mean difference of two weeks in first breeding dates exists between the two samples. Due to the relatively small size of the samples these data were not subjected to statistical analysis. However, there is evidence which suggests that the onset of breeding differed in the till plain region in 1953 and 195**# Also, there appears to have been a marked regional difference between the till plain and the unglaciated Allegheny Plateau in 195^* Actually this can have a profound effect upon the total production for the year since it is more than likely that, if the breeding season begins several weeks later than in a pre vious year, one less litter may be produced during the late season. Possible factors affecting the onset of breeding will be discudsed in Chapter IV. In 1953, Area No. 9 at the Urbana Game Farm was again driven on October 25. Thirty-seven rabbits were captured using the English hare net. Of these, only 13 were adults which had been tagged in April. Eleven of the 19 females that had been originally tagged were recaptured. All 11 animals were killed and their reproductive tractsexamined. All of these animals had ceased breeding for the season and were not lactating* In 3 of the 11, placental scars were still evident. Since lactation usually continues for three to four weeks following parturition, it may be assumed 61 - that all of the adult animals captured in October 1953 had ceased breeding by the end of September* Sight subadult;* females were captured in this sample, and were killed for examination of breeding in their first season. None of the subadults showed any indication of having produced litters during 1953, even though all had attained adult proportions. From September 19 - 30, 1953, 72 box traps were main tained in the *+0-acre south pen at the Olentangy Wildlife Experiment Station in Delaware County. Ten adult females were trapped between September 20 add 28, Nine of these rabbits had ceased breeding, were not lactating, and were in the process of molting to winter pelage. One individual was judged to be pregnant and was held in captivity for several days. October 1, she aborted five embryos which were judged to be within several days of full term. This litter probably would have been born on October or 5, and represents the latest record of adult breeding in 1953* Of six adult males collected during this period, five possessed testes that were greatly reduced in size. In one the testes measured only 19mm. in length. Of eight first litter subadult females collected, all but one show ed no signs of being pregnant or having produced a litter during their first season. One appeared to be sexually active on the basis of enlarged teats, perforate hymen, and the suspected presence of small embryos determined by palpation. On the basis of samples taken in September and October in Champaign and Delaware Counties the breeding season in 1953 in the till plain region terminated between September 15 and September 30, with few exceptions. Sampling throughout the breeding season in selected areas in 195** indicated that all breeding ceased between the middle and the end of September, since ho animals were found pregnant or with early post partus characteristics after the end of September. Thus it would seem that al though the breeding season started somewhat later in 1953 than in 195*+, breeding terminated about the same time in both years on the basis of the samples examined. Number of Litters per Year information regarding the number of litters produced per year by a given female was gained by microscopic exam ination of serially sectioned ovaries taken from animals collected throughout the study. Of course, only those ani mals that were collected late in the breeding season yield ed data which revealed the entire breeding history of the - 63 - animal during that season* Females collected earlier in the season did yield information on the number of litters they had produced up to the time of capture* In rabbits collected near the end of the breeding season the number of litters was ascertained by counting the number of differ ent series of corpora lutea and albicantia in the ovaries* At this season series of degenerating lutein bodies were still fairly distinct from one another* In specimens collected shortly after the breeding season had terminated, i.e. from three to four weeks following the termination of all breeding activities, the degenerated lutein bodies had become so similar in size and appearance that it was possible to obtain only a total count rather than differ entiate each litter* m such specimens an approximation of the number of litters produced was gained by dividing the total number of degenerated corpora albicantia by the mean litter size established for that region. This, it was felt, gave a rather good estimate of the number of litters produced in such cases* Eleven rabbits were obtained during 195**- at the end of the summer when their entire breeding history for the season could be ascertained. The maximum number of lit ters produced by any of these animals was four and the - 6b - minimum was two. From this small sample, of which the majority were collected from the Muskingum County area, it is impossible to make an adequate comparison between Marion County and Muskingum County so far as the total num ber of litters produced per year by the individuals is con cerned. It should be noted that of nine rabbits collected in Muskingum County for which a complete record of produc tion was gained from the ovaries, only one of these had produced four litters during the season. Seven of these i had produced three litters, and one had produced only two litters at the time it was collected on September 18. They showed no further signs of sexual activity. Of the three rabbits collected in Marion County for which total season records were available, one had produced four litters and the other two had produced three litters. It should be further noted that one female collected in Marion County on July 31, had already produced three litters and was pregnant with her fourth. It is likely that she might have produced a fifth litter before the termination of the breeding season. Along this same line, a specimen col lected on May 10 in Muskingum County had already produced two litters and was pregnant with her third litter. Thus it was possible for this animal to have produced five lit ters in a complete breeding season. From the data at 1 hand, it appears that cottontails in Ohio rarely produce less than three litters per year; and many may produce four litters, judging by the number of litters certain animals had produced at the time they were examined ear lier in the season. Breeding of Subadults The consensus of many workers in the past has been that young cottontails do not breed during the season in which they were born. There are few recorded exceptions to this in the literature. Cooley (19^6) summarizing six years of intensive work on the cottontail in Michigan, cited three observations of young rabbits breeding or sex ually active in their first season. Hendrickson (19^7) and Bruna (1951) each cite one record of apparent sexual activity in a young female. During the present study much emphasis was placed upon scrutinizing young rabbits for evidences of sexual activity during their first summer. As a result, a rather substantial amount of information on this subject has been accumulated. Tables and £ summarize the data on young rabbits collected from 1952 to 1955. Table 3-. Unglaclated Allegheny Plateau Data on Subadult; Cottontail Rabbits Spec. Date Location Weight Age as of Pregnancy Evidence Sexually Uterus No. (Grams) Nov. 1 status of Active & (months) lactation Ovaries r -i"6 id-21-£2 Coshocton ^30^* - -;- MM prom, teats u.-3,0mm. Co. 2,8 mm. o.-8,*fmm. R-15 10-21-52 II 1361* -- X U.-2 mm, o,-7 mm. R-l^ 10-21-52 II 935 1+.6 - -- R-13 10-21-52 2 1106 6 - -- R-l 7 10-21-52 ti 1219 7.7 - prom, teats X u.-2.3mm. 1.5 mm. o.-5.'+mm. R-l8 10-23-52 ti 12^7 7.7 - - - - R-135 11-20-52 11 1616* - prom.teats X u.-2 mm, 2.5 mm. plac, sc. R-13 6 11-20-52 11 l^J+6* - - prom.teats X u.-3.5mm. 2,5 mm. 0.-9.0mm. R-13 7 10-20-52 11 1502* - prom.teats X u.-3.8mm. 2.2 mm. a-12,5mm, plac. sc. * These were known to be subadults since they were tagged at an early age. Table 3-. (Continued) Spec, Date Location Weight Age as of Pregnancy Evidence Sexually Uterus No, (Grams) Nov, 1 status of active & (months)______lactation ovaries R-80 11-18-52 Coshoc- 1530 promjrteats x ton Co, 2,5 mm. 0.-7 mm, 3-*+ plac, sc, R-81 11-18-52 » 1020 5 . -- - u.-1 .8mm. 0.-6.Qmm. R-82 11-18-52 « 963 5 -- - u.-1 .5mm. 0.-7*7mm. R-l*+0 11-2*+-52 " 1190 6 - - - u.-1 ,5mm. 0,-6,5mm. R-l*+l ll-2lf-52 '» 130*+* 10 -- - u.-1.9mm. o,-6 .H-mm, R-1^2 11-2*+-52 » l*+03* - prom.teats X u.-^.Qmm. 2.3 mm. ©,-6 mm. 3037 8-7-53 " 96^ % 7 - -m 3279 8-7-53 " 1276* X teats enlr. X pea-sized embryos 3053 8-7-53 " 113^ 9 2nd X X preg. with litter 2nd litter Table 3., (Continued) Spec. Date Location Weight Age as of Pregnancy Evidence Sexually Uterus No. ' (Grams) Nov. 1 status of active & (months) lactation ovaries 3078 8-7-53 Coshocton 879 7 X teats X pea smzed Co. enlrg. embryos 3051 8-7-53 ti 907 7 ? - -? App. impf. hyman B-7 7-30-5br Muskingum 765 7.5 3 embryos teats X Co. x enlrg. I CT\ B-9 8-2b-5k it 113^ ; 8 - - - _ O) 1 B-10 8-2k-$k tt 850 6 - mm - u.-not dist o.-5 mm. B-ll 9-16-51+ it 1077 7 - - - B-12 9-16-5^ 11 907 6 - — X u.-dist, <■ 0-S. 7-30-5^ tt 1020 7.5 x(5) teatg X enlrg, f 0-3 7-29-5^ 11 850 7 -- — _ 0-5 8-23-5^ 11 907 6.5 x(k) X X 3 scars Table 3 . (Continued) Spec, Date Location Weight Age as of Pregnancy Evidence Sexually Uterus No. (Grams) Nov. 1 status of active & (month) lactation ovaries C-6 8-2*+- 5*+Muskingum 79*+ 6 -- -- Co. C-7 9-13-5*+ » 907 - 6 teats X u.-dist. 2 mm. 0.-8.6mm, B-15 10-26-5*+ " 1190 7 - - - - B-16 1219 8 — teats X 10-29-5*+ " On enlrg. n£> lac. B-17 10-29-5*+ " 992 5 - - - perf. hymen B-2*+ 8-6-55 ” 850 6 — teats X u.-dist. enlrg *+.5 mm. 5 mm. 0.-6-8mm. B-26 8-8-55 11 907 7 - — ? u.-2,5mm. Table U. Till Plain Data on Subadult Cottontail Rabbits Spec, Date Location Weight Age as of Pregnancy JfeidaiiBa/:: Sexually Uterus No, (Grams) Nov, 1 status of active & ______'______(months)______lactating______ovaries R-7 8-8-52 Franklin 1CA-8 7 — —— — Co, R-8 8-10-52 ti 975 7 5 emb. — - - X R-20 8-20-52 it 935 8 ? teats X enlrg. 1 R-20 7-2^f-52 H m $.5 mm u.-l,6 mm, 0 o.-9,1+ mm. 1 R-3 7-29-52 ti 765 7.5 - teats X u.-dist. enlrg. 5c.1. in 0. R-lf 7-30-52 it 623 5.5 - teats X? u.-dist, enlrg. 2.7 mm. A 10-25-53 Champaign 10 - - - - Co. 1332 B 10-25-53 ti 1360 ---- ! Table It. (Continued) Spec. Date Location Weight Age as of Pregnancy Evidence Sexually Uterus No. (Grams) Nov. 1 status of active & (months) lactation ovaries C 10-25-53 Champaign m m Co. 1 W D 10-25-53 ti 130^ - -- E 10-25-53 ti 1389 - -- F 10-25-53 ii 1389 - 1mm - G 10-25-53 it 1389 - - - H 10-25-53 n 1*+17 -- 9-20-53 Delaware 1275 X - Co. 9-22-53- » 1020 - - 9-23-53 ii 130^ X - 9-23-53 ii 1105 6.5 - - 9-26-53 ii 1077 6.5 -- Table U. (Continued) Spec. Date Location Weight Age a.s of Pregnancy Evidence Sexually Uterus No. (Grams) Nov. 1 status of active & (months) lactation ovaries 9-27-53 Delaware 1219 9 ? - Co. 9-28-53 11 1020 6 -- 9-28-53 11 1105 6.5 - - Ib6 1-5-53 Champaign 13 89 - -- Co. ~o !lO 9-28-53 Delaware 992 - - Co. lh5 1-5-53 Champaign 1275 - - - Co. lh2 1-5-53 " 1389 Teats X o.-9.lfmm. l.1* mm, perf hymen width Ibh 1-5-53 » 12^7 _ X perf, hymen 65 11-17-52 Delaware - - X perf.hymen Co. Table !w (Continued) Spec. Date Location Weight Age of Pregnancy Evidence Sexually Uterus No. (Grams) Nov. 1 status of active & ______(months) lactation ovaries A-25 11-11-5*+ Marion 12^7 - x x Co. A-28 11-12-5^ » 1360 enlrg. x Sixty-seven young rabbits were examined during the study that were judged to be first litter females on the basis of pelage and size. Those animals placed in the subadult category were in complete or nearly complete sub adult pelage. A few such individuals were quite possibly second litter females produced early enough in the season to attain subadult status prior to the end of the breeding season. Not all of the subadults examined were killed for laboratory examination, but an entire fore foot from each animal killed was saved for X-ray examination. This was done in order to make a double check on the age determina tion of the animals. In all cases where fore feet were saved, X-ray examination revealed that they were, without doubt, young of the year on the basis of ossification of the distal epiphyseal cartilage of the radius and ulna. Erom 1952 to 1955, 36 subadult females were examined from the unglaciated Allegheny Plateau region of Ohio. Exactly 50$ (18) of this sample were found to be sexually active during the same season in which they were born on the basis of observed pregnancy, placental scars, lacta tion, or distension of the uterus and presence of mature follicles in the ovaries. Through the cooperation of the Ohio Division of Wildlife the writer was able to examine a number of specimens in 1952 and 1953 that were trapped in the five-acre fenced enclosures maintained as part of a rabbit study on the Woodbury Wildlife Restoration Area in Coshocton County. It was not possible to kill all the rabbits examined from this source, but the approximate age for all was known since they weie ear-tagged at an early age in the spring, and most of them had been recaptured and weighed several times during the summer. This sample from the unglaciated region of Ohio might be divided into two parts on the basis of the source of the specimens. Of the animals examined in 1952 and 1953> all were obtained from the fenced five-acre plots on the Woodbury Wildlife Restoration Area in Coshocton County, Ohio; those examined in 195*+ were animals trapped in the study areas in Muskingum County. Thus the quality of the environment of the animals examined from Woodbury might be considered to be somewhat superior to that of the spec imens obtained from Muskingum County. Enclosures were lo cated on typically poor quality soil in the area, but the soil within these enclosures had been subjected to inten sive fertilization practices. Examination of the data from the two samples reveals that 11 of the 20 subadults from Woodbury were sexually active, while 7 of the 16 an- - 76 - imals examined from Muskingum County were sexually active. Considering the small sample size, there seems to be rath er close agreement of the percent of subadults breeding in both samples. Each rabbit was assigned an approximate age in months on the basis of weight and pelage at the time of capture. Erom these data each animal was assigned an approximate age as of November 1, in order to make all individuals in the sample of comparable age. "While these figures are by no means exact, it is interesting to note that on the basis of such age determinations no subadults which were judged to be less than six months of age by November 1 were found to be sexually active. This suggests that no rabbits born much later than the end of April might be expected to breed during their first summer. However it should be noted that some individuals which were judged to be much in excess of 6 months of age by November 1 were not sexually active. Apparently Individual variation is great in the age at which sexual maturity is attained. Individual vari ation is further evident when we consider the data of two individuals which were known to have produced two litters in their first season. One of these (305-30*+) from the Woodbury Area was judged to be 9 months old as of November 1, - 7-7 - and was found to "be pregnant with the second litter on August 7, 1953. Another individual, captured on August 23, 195^ in Muskingum County, was pregnant with the second litter at the time of capture. This animal was judged to be 6.5 months old as of November 1. By aging of second litter embryos and back dating, a conservative estimate of the age at which the first fertile mating occurred is 2.5 monthsJ A total of 30 subadult females were examined from the till plain region during the study. Sources of spec imens were as follows: University Woodlot, Franklin County, Olentangy Wildlife Experiment Station, Delaware County, 10; Urbana Game Farm, Champaign County, 12; Marion County area, 2. In this sample 10 (33$) of the rabbits were found to have been sexually active during their first sum mer. Again it is of interest to inspect the sample crit ically. Eleven .subadults were obtained in the 1952 breed ing season, six from the University Woodlot, four from the Urbana Game Farm and one from the Olentangy Wildlife Experiment Station. Of these, six (55%) were found to be sexually active. During 1953 > a total of 17 aubadults were obtained for study from the till plain region. Eight were from the Urbana Game Farm, and nine from the - 78 - Olentangy Wildlife Experiment Station. The animals com prising the Urbana sample were all taken on October 25, and killed for examination. None of these animals showed any indication of having been active reproductively. The sample from the Olentangy Wildlife Experiment Station was collected from September 20 to the 28. Only two of these rabbits showed definite signs of sexual activity. Unfor tunately the writer was able to obtain only two sdhAdults during trapping operations in the Marion County area in 195^. Both of these individuals showed evidences of having produced a litter.• The low rate of subadult breed ing in the 1953 sample is interesting in view of the record ed data on the onset of breeding of adults in the spring of that year. It will be recalled that as late as April 26, over half of the adult population from the sample taken at the Urbana Game Farm was not yet pregnant with their first litter. This could very possibly have a pro nounced influence upon the number of subadults reaching maturity and breeding in their first summer. Comparison of the unglaciated plateau sample of subadults with the till plain sample reveals that a higher incidence of sexual activity is recorded in the unglaciated Allegheny Plateau. Since sample sizes from each region - 79 - are very nearly comparable, it must be concluded that over a three»year period a significantly higher rate of breed ing in subadults occurred in the unglaciated region than in the till plain region. When both samples are combined into one, the percent of subadults breeding in both regions as a whole is k2fo» The mean litter size for all subadult females based on k counts of embryos and one set of fresh placental scars is ^.O, with a range of 3 to 5 (Table #). These data indicate that a large proportion of first litter females and possibly some second litter females do breed in their first season. Breeding Periods The information on breeding derived from this study is based on 51 pregnant Sdult females collected during 195^. Specimens examined include those collected from March to September. Parturition dates were estimated on the basis of the age of embryos of pregnant animals. In those rabbits pregnant with their second &r third litter, the approximate dates of birth of the previous litters were determined by back dating from the time of conception of the current litter. The basis $&r this is the work done by Hammond and Marshall (1925) on the domestic rabbit which indicates that mating occurs usually within **8 hours following parturition. That this is also probably true in the cottontail is evidenced by some specimens which were collected within a day or so after they had given birth to a litter. These were found to have mated and ovulated, and undoubtedly were pregnant. On this basis, the approximate birth dates of 75 litters were determined. The frequency curves and individual distribution are in dicated in Figure 23, On the basis of this sample, four major peaks occur throughout the breeding season. The first of these is the period from April 1 to 15, which is composed solely of the birth of first litters. The second peak, which is the highest breeding peak so far as births are concerned, occurs between May 1 and 15. It is composed of 73$ first litters and 27% second litters. The third peak occurs from June 1 to 15, and although it is a much smaller peak th$n the two preceding, it is nevertheless represented by 12 litter births. The third peak is composed of 66$ first litters and 33$ second litters. The fourth peak, which is smaller than any of the three preceding peaks, is composed almost wholly (86$) of second litters. It should be noted in Figure 23^ that, although first litters decline sharply for litters forof cottontail rabbits* Figur© No. of Utters 5 2 0 3 5 3 23;. Apr. Apr. Apr. Apr. Frequencyof estimated parturition dates rqec o Estimated of Frequency 94 reig Season Breeding 1954 atrto Dates Parturition o My u. u. u. u. u. Aug. Aug. Jul. Jul. Jun. Jun. May Moy - 1 8 - Combined Curve 1st litters n litters2nd 82 ~ after the middle of June, they do occur as late as July 16 to 31. The most remarkable feature of these data is that the first three breeding periods are composed mainly of first litter births, which indicates a high degree of individual variation in the onset of breeding in the spring. This may well be related to the age of the females and to regional differences. Actually, the data included in Figure 23 do not give a true representation of the en tire breeding season since most of the specimens collected were taken from March through June or July. This is re flected in the very few records of third litter births included in the data. Undoubtedly, if more material were available from August and September, there would be a high er late peak consisting primarily of third litters and some second litters. This would occur probably from the last week in July to the 10th of August.. The estimated birth dates in three of five third litter pregnancies were within ten days of each other from July 25 to August 1+. One early third litter pregnancy was estimated to have a parturition date of June 11 to 12, and one was estimated to have a parturition date of September 20 to 21, Only one fourth litter pregnancy was recorded although other evidence of four litters being produced was obtained. But parturi tion dates could not be determined, due to lack of preg - 83 - nancy, from which dating might he based. One fourth litter pregnancy was estimated to have a parturition date of August 27 to 28, It appears that if more data had been ob tained on pregnant animals toward the end of the season, two minor peaks would have been evident: one from around August 1 to the 15, and a second from about the end of August to about the 10th of September, It appears that the peak of breeding in 195*+ occurred from about April 1-15, as evidenced by the peak of estimated parturition dates from May 1 to 15, The breeding season must have begun about March and ended in September, It is important to note that, due to the great amount of variation of the date at which first litters are produced, one cannot des ignate a litter as first, second, third, or fourth merely on the basis of the month in which the animal is found pregnant (Figure 23), Molts in Relation to the Breeding Cycle The molting and pelage stages in immature rabbits have been described earlier in this paper. It seems worth while to set forth here the information collected on the( molting of adults in relation to the breeding cycle, Dalke et al (19^2) described molting in the adult cotton- - 84 - tail as a continuous growth of hair extending over a 9- month period from February to November, and having two distinct phases. One he called the spring and summer molt, which occurred from March to August; and the fall molt occurred from September to November. The writer prefers to consider these as two distinct and separate molts since, in Ohio of the specimens examined, it was found that the molt from winter to summer pelage was nearly always completed by early August. For a short period, once the complete adult summer pelage had grown in, no apparent molting occurred. The molt from summer to winter pelage begins usually in September, and is complete early in November. Thus the molt from winter to summer pelage takes approximately 5 months, whereas the fall molt is completed in about 2,5 months at the most. Information on molting during the present study in dicates a rather close relationship to reproductive activ ity. As mentioned previously, special attention was paid to pelage of all animals obtained during 1953 and 195^. It was not until 195*+, however, when rabbits were collect ed on the selected areas from April through December, that the relationship of molting to stage of reproductive activ ity was strikingly evident in the adult animals examined. In those animals collected in April which were preg nant with their first litters, molting to summer pelage from winter pelage had just begun in the head region, par ticularly around the eyes. Of the animals caught in April, those individuals having; more advanced embryos of either first or second litters were found to be more advanced in the stage of molting from winter to summer pelage. This was true in both Marion and Muskingum County speci mens. May specimens were characterized by a profuse growth of summer pelage all over the body and persistence of tufts of winter pelage in the dorsal region. The ear liest complete adult summer pelage was noted in an adult female (A-11) collected on June b in the Marion County area. At the time of collection, this animal was in com plete adult summer pelage. Subsequent examination of the reproductive tract and ovaries revealed that this animal was well along in her third pregnancy, far advanced so far as numbers of litters is concerned at that time of the breeding season. There appears to have been a definite relationship between the early attainment of complete adult summer pelage and the fact that the animal had un doubtedly begun breeding much earlier than the average adult in 195^. This was the only specimen collected throughout the month of June which did not have remnants - 86 - of the winter pelage persisting. Not until the end of July were more specimens of adult females observed that were in complete summer pelage. The molting of adult males seems to follow approx imately the same sequence as that of the females. Molt ing was observed to have just begun in April, and was progressing all over the dorsal region of the body at least throughout May. Mottled molt patterns persisted through the month of June. From about the 10th of July to the end of July males were in adult summer pelage with a few traces of winter pelage still persisting in most animals. "What at first appeared to be a great amount of in dividual variation in regard to the time of molt in adult cottontails, appears now, after further laboratory analy sis of the reproductive tracts of specimens collected, to be far more consistent:then was formerly supposed, at least in relation to the stage of reproductive activity. For example, an adult female from Knox County collected on May 21 was still in complete winter pelage with no signs of molting to summer pelage. This individual, pregnant with her first litter, had begun breeding later then the average cottontail in 195*+* An adult male trapped in Muskingum County in June was observed to have partly undergone molt toward summer pelage at the time of his capture. He was retained in a rabbit hutch outdoors throughout the summer and observed from time to time in regard to pelage. This individual never completed the molt ftom winter pelage but passed the season in much the same condition as he was trapped, namely with a large area of winter pelage persisting dorsally and laterally. Ex amination at intervals throughout the summer revealed that the testes of this animal remained very small, and at no time were they observed to be scrotal. Although more data of this type is needed,it is suggested that completion of the summer molt in this case was dependent upon the repro ductive condition of the male. Similarly, the stage of molt in which adult females were found from March through July seems to be somewhat closely related to the number of litters they had produced and hence to the date on which they had begun breeding in the spring, Erom spec imens examined in 1953 and 195^, it appeared that molting from summer to winter pelage in adults began within the first ten days of September, Again this was subject to quite a large amount of variation which appeared related to the stage of the reproductive cycle of each specimen. These observations agree rather closely with those made by Dalke et al in regard to fall molting. Thus an adult fe male captured on September 28, 1953 at the Olentangy Wildlife Experiment Station was pregnant with five embryos which were later aborted in captivity. This individual had not yet begun molting toward winter pelage. An adult female caught on the same day with enlarged teats but not lactating or pregnant, in other words, having had her last litter of the season, was molting profusely to winter pel age. The fall molt occurs more in a sheet pattern, usually beginning in the region of the rump and sides and progress ing dorsally. This type of fall molt pattern has been ob- served by Dalke dub O&nnecticut. Most adults examined had attained full winter pelage by the third or fourth week of October. In no instances was winter pelage observed to be incomplete by the first week of November. Table 5 summar izes the observational data on pelage from adult females collected in 1953 and 195^, as well as the reproductive data from those animals which were killed. With the accu mulation of additional data on adults throughout the year, it might be possible to utilize the stage of molt, partic ularly in females, as an indicator of the stage in the - 89 - Table 5. The Pelage Stages of’ Female Cottontails Correlated with Reproduction Date Reproductive Pelage stage collected condition 5-22-5% Pregnant (2nd of season) winter, molting slightly %-23~5% Pregnant (early) 2nd of’ winter, molting season around face 5-23-5% Pregnant it tt winter, molting around eyes 5-23- 5% Pregnant- winter, molting on rump and back 5-25-55 Pregnant (2nd of season) winter, molting slightly Pregnant winter, molting to summer dorsally 5-25-5% Pregnant winter, molting to summer dor sally 5-8-5 if Pregnant (1st litter) winter, molting in patches dorsally 5-8-5% Not preg. (had one litter)winter, molting in patches dorsally 5-10-5% Pregnant (3rd of season) mottled molt pattern dorsally to summer 5-23-5% Pregnant (2nd of season) summer pel. in all over1 dorsally 5-25-5% Pregnant (2nd of season) molting profusely to sximmer 5-29-5% Pregnant (1st litter) winter, shedding sum. growing in 5-30-5% Pregnant (2nd of season) summer coming in all over 6-3-5% Not preg. (had two litter)summer almost com plete 6—%—5% Pregnant (3rd of season) complete summer 6- 11- 5% Not preg. (Had two litter»)molting to summer 6-15-5% Not preg. ” M summer complete dorsally 66-15-5% Pregnant (2nd of season) summer almost com plete dorsally 6-i%-5% Pregnant (2nd of season) mottled, molting all over ~ o G - Table $, (Continued) Date Reproductive Pelage Stage collected______condition______.. 6-16-5*+ Pregnant (3rd' of season) summer almost complete 6-16-5*+ Pregnant (2nd of season) 3/*+ molted to summer 6-l6-5*+ Not preg. (had one litter) summer almost complete 7-10-5*+ Pregnant (2nd of season) mottled molt to summer 7-30-5*+ Not preg. (had two litter?)summer complete except for tuits 7-.3i-.5i4, Pregnant (2nd of season) summer complete except for tufts 7-31-5lf Pregnant (*+th of season) summer complete 7-31-5^ Pregnant (3rd of season) summer complete except for- tufts 8-23-5ft- Not preg. (Had 3 litters) summer' complete 9-18-5*+ Not preg. (had two litters) summer, molting to winter all over 10-26-5*+ Not preg. (had 3 litters) winter growing in on rump 100-26-5*+ Not preg. winter complete 11-1-5k Not preg. (had *+ litter) winter growing in 11-11-5*+ Not preg. winter complete - 91 - breeding cycle without having to kill the animal. This would require a much more detailed study of the pelage in relation to reproduction than was undertaken in the present investigation. Hew Zealand White Rabbit Breeding Experiment One of the objectives of the present study was to obtain comparative data on litter sizes in various parts of Ohio. As mentioned previously, information on litter sizes was obtained from embryo counts from the tracts of pregnant animals, from the counts of fresh placental scars, and from counts of corpora lutea and corpora albicantia by microscopic examination of serially sectioned ovaries from adult rabbits. Since the evaluation of reproduction on the basis of ovarian analysis has not previously been used extensively in rabbits, it was thought advisable to con duct a controlled experiment to investigate the reliability of corpora lutea and corpora albicantia as indicators of litter size, as well as to ascertain the reliability of placental scar counts. It was important also to gain some idea of the persistence of placental scars in the uteri as well as the corpora lutea and corpora albicantia in the ovaries. The procedhfi involved in setting up this exper- - 92 - iment has been reviewed in Chapter II. A summary of the data derived from this breeding program is presented in Table 6. Placental scar counts were found to be unreliable as indicators of litter size after three or four weeks follow ing parturition. Furthermore, scars are not readily discernible immediately after an animal parturiates due to distension of the uterine horns. A fairly accurate pla cental scar count for one litter may be obtained only dur ing a period of several weeks following parturition. When multiple litters are involved, placental scar counts be come increasingly inaccurate. The following data are pre sented as an example of the inaccuracy of placental scar counts in determining the breeding history of a sample of females. This includes a group of five females used in the breeding experiment, all of which possessed at least some placental scars and had produced a total of 53 young. The scar counts indicated a total of only 35 young, and ovarian analyses of this same group without the application of a corpora lutea-albicantia-litter size ratio factor in dicated a total of 57 young produced. The persistence of corpora albicantia in the ovary Table 6'. Bata from Breeding Experiment Using New Zealand White Rabbits Animal No, litters Size & Size & Size & Total Total No. C.l.-C.a,- No. produced date of date of date of all c.l. & 1itter 1st lit. 2nd lit. 3rd lit. litters ratio A-l+37 1 (7)5/2/5^ - 7 7 1.00:1,00 k-bb6 1 (8 )V 9 /5 ^ - 8 11 1.3 7:1 .0 0 A-^32 2 (6)V10/ (6)5/31/ 12 13 1 .0 8 :1 .0 0 5b 5^ A-M+8 2 (#) V 9/ (7)5/12/ lb 1^- 1 .0 0 :1 .0 0 5b 5b A-b3b 2 (6)5/3/ (6)6/10/ 12 12 1 .0 0:1 .0 0 5b 5^ A-i+35 3 (7)5/3/ (5)6/10/ (6)10/31 18 2b 1 . 30: 1.00 5b 5b 5b a-^5o Control - 0 0 - A-bb5 Control ~ _ 0 0 Totals and mean ratio 71 81 1 ,1 :1,0 9 4 - appears to extend over a period well in excess of two months following parturition. One female (A-446) was kill ed for examination approximately 60 days after giving birth to one litter. Corpora albicantia were still dis cernible in sections stained with Delafield*s hematoxylin and eosin red stains. It appeared that in animals having multiple litters the persistence and visibility of corpora albicantia was not greatly decreased. On the basis of the data obtained, it was concluded that corjfora lutea add albicantia would provide the best means of deriving estimates of litter size and number of litters produced. It seemed likely that corpora albican tia might be expected to persist long enough in wild cottontail females to enable evaluation of an entire sea sons breeding history from animals collected at the end of the breeding season. A mean corpora lutea-corpora albicantia-litter size ratio was derived for all animals in the breeding experi ment. This ratio equaled 1.1 corpora lutea and/or albi cantia per single young produced. This ratio was not employed in the evaluation of wild cottontail reproductive tracts. Rather, using wild cottontails, a similar ratio - 95 - was determined on the basis of corpora lutea-corpora albi- cantia counts made from ovaries of pregnant animals where the number of developing embryos was known. This ratio was 1,03s1.00 for Marion County rabbits, and 1.00s1.00 for Muskingum County. Litter Sizes The determination of cottontail rabbit litter sizes from various parts of the State was an important phase of the present study. Mean litter sizes were compared from samples taken in Marion and Muskingum Counties. Mean lit ter sizes were compared on a regional basis by combining the samples of specimens picked up dead on public highways with the selected area samples. Differences in quality of range have been found to be related to differences in litter size in the white-tailed deer in New York State by Cheatum and Seviringhaus (19^9)• It seemed likely that this might also be the case with the cottontail rabbit. With this in mind, the reproduc tive tracts of more than 150 adult cottontails were ex amined between 1952 and 1955. Not all of these tracts yielded data on litter size since some were collected - 96 - after breeding had ceased in the fall and winter months. Eighty-nine of the adult females yielded at least some data on litter size# In some eases more than one litter size was recorded for an individual on the basis of fresh pla cental scars and corpora lutea and albicantia counts in sectioned ovaries# A grand total of 12^ litter sizes were obtained from all adult specimens examined throughout the study. Not all of these litter sizes were included in making comparisons between regions and selected areas since some of the specimens collected dead on the road were pick ed up at localities which were so close to the boundary between two particular regions that it was difficult to assign them to a specific sample# Litter size data collect ed in the till plain region were compared to data collected in the unglaciated Allegheny Plateau and to a small sample from the old lake basin region# Litter sizes recorded from the Marion County area were compared to those recorded from the Muskingum County area. In addition, comparisons were made of litter sizes of first, second, and third litters from rabbits collected in the Marion and Muskingum County areas# Comparisons of litter sizes between the two areas were made on the basis of the analysis of variance# Twenty-six adult females from the Marion County area - 97 - (Area A) during 195*+ yielded some data on litter size; 19 adult females from the Muskingum County area (Area B) during the same period yielded some litter size data. The frequency distribution of litter sizes from both areas is indicated in Table fy. It is immediately obvious by visual inspection of the frequency distribution that the two pop ulations are apparently distinct in regard to litter size. For Area A cottontails a total mean litter size of 6,1* was established. The total mean litter size of Area B rabbits was determined to be W,7* The Area A mean is bas ed on a total of b-9 separate litter sizes; that of Area B is based on 37 separate litter sizes. Visual examination of the frequency distribution and mean litter sizes of the two areas indicates that they are significantly different. Analysis of variance of the two samples indicates that the differences in litter size are highly significant at the 1;% level of significance (Table g). The mean litter size of 21 first litters from Area A was 6.3, for 19 second lit ters it was 6,5, and the mean for five third litters was 6,0. The mean for 16 first litters from Area B was ^.6, for 12 second litters it was ^.9, and for nine third lit ters it was In both areas there appears to be con sistent increase in second litter sizes compared to first and third litter sizes. Comparison of means of first Table ? Frequency Distribution of Litter Sizes of Cottontails in Areas A and B "Area A. 1st 2nd 3rd ifth 1st Sid" 3rd ifth! Size litter litter litter litter litter litter litter litter 1 0 0 0 0 0 0 0 0 2 0 0 0 0 1 0 0 0 3 1 0 0 0 1 0 2 1 if 0 0 1 0 if 5 6 0 VO (XX 5 if 3 1 1 7 if 1 1 6 7 8 2 0 2 2 0 0 7 if 5 0 0 1 1 1 0 8 5 2 0 0 0 0 0 0 9 0 1 1 0 0 0 0 0 Table 8. Summary of Analysis of Variance of Litter Sizes from Areas A and B Source d.f. Sums of Mean F of variance squares square ♦Between areas 1 60.92 60.92 k6.15 Between litter order 2 12.37 6.18 if.68 Within cells 78 102.61 1.32 ♦This analysis may not be justified since some animals supplied more than 1 litter size and the assumption of independence is not satisfied. - 100 - second and third litter sizes between Area A and Area B was highly significant in all cases at the 1% level of significance (Table 9 ). A comparison of litter sizes was also made on the basis of region. All of the litter sizes collected in the till plain region, regardless of the site of collection of the females, were incorporated into a sample representing the entire region. From the unglaciated Allegheny Plateau all litter sizes from the Muskingum County area as well as any others obtained from random sampling also were incor porated into a sample representing the entire region. A small sample of 12 litter sizes was obtained from the gla cial lake basin physiographic region which is an area of intermediate to high fertility compared to the till plain and the unglaciated plateau. The frequency distributions of litter sizes from these three samples are represented in Table 10.. Again, even in this more general large sample, it is immediately obvious that a distinct difference exists in the distribution of litter sizes in the till plain re gion and the unglaciated Allegheny Plateau even with sample sizes closely approximating each other. The mean litter size for 53 litter sizes from the till plain region is 6,1 . The mean litter size for 50 litter sizes in the unglaciated Table ,9 • Summary of a Comparison of Mean Litter Sizes § f First, Second, and Third Litters from Areas A and B 1st Sample n d.f. Mean Sums of litters litter squares A 21 20 6.33 877 B 16 15 *+.750 382 t = 3.783 t#0i(35d.f.) = 2*72b 2nd Sample n d.f Mean Sums of litters litter squares A 18 17 6.55 792 B 12 11 k ,92 301 t - b .29b t.ei(28d.f.) = 2.763 3rd Sample n d.f. Mean Sums of litters litter squares A 5 b 6.0 19b B 10 9 ^.2 188 t = 2 .32^2 t #05(13d.f.) = 2 . 1 6 0 ■"] P 1 "". -‘JL'J1"*"-- - 102- Allegheny Plateau region is 4.6. The mean litter size for the 13 litters from the old lake basin is 5.4. (See Table 10) Grass Analysis On the basis of evidence in the literature it seemed that regional variations in the litter size of the cotton tail rabbit could be expected to be related to variations in certain constituents of the vegetation. During the present study samples of bluegrass (Poa pratensis) were collected from the Marion and Muskingum County areas to be analyzed quantitatively for calcium, phosphorus, and ash content. Bluegrass was selected for analysis on the basis of DusiTs observations (1952) that it was an impor tant part of the diet of Ohio cottontails during the breed ing season. Several samples were taken from each area throughout the breeding season in 1954. Analysis of these few samples revealed that apparently quite a bit of varia tion was to be expected from one sample to another as well as from one month to another. Consequently, it was decided to collect ten random samples from each area in August, 1955, in order to have a better basis for comparison. - 105- - Table 10. Frequency of Regional Litter Sizes of Cottontails 3 2 0 1+ If 18 2 5 9 18 7 6 19 if 1 7 10 if 2 8 7 0 1 9 2 0 0 - 104 - The content of calcium, phosphorus, and ash were determined as percent of dry weight in the grass samples. Ash content was determined only for the samples collected in 1954. Even in the ten samples taken from each area in August, quite a large amount of variation was found to exist (See Table 11). The mean calcium content for Area A grass samples was 0.72$, that for Area B grass samples was 0.60$. Mean phosphorus content for Area A was 0.30$, for Area B it was 0.35$. Th e phosphorus content was not significantly different in the samples from the two areas, but the calcium content was found to be significantly higher in Area A than in Area B (Table 12). The calcium contents of the samples taken earlier in. the season in 1954 are almost double the average calcium contents of the samples taken in late August, 1955. It appears that the calcium content declines late in the summer, and it sug gests that it may have a relationship to the decline in breeding activity and cessation of breeding. However, a much larger sample of grasses should be analyzed in order to more clearly define seasonal differences in calcium con tent . -105- Tahle. 11. Analysis of Bluegrass Samples for Calcium and Phosphorus Content (Percent of dry weight) Area A Area B Ca P Ca P .64 .25 .64 .30 .80 .24 .62 .55 ,72 .33 .50 .11 .82 .28 .68 .48 .70 .34 .60 .55 .67 .25 .66 .44 .60 .30 .60 .29 .69 .30 .61 .28 .71 .40 .72 .35 .80 .33 .40 .10 x = . 7.15 3.02 6.03 3.45 N = 10 10 10 10 x = .715 .302 .603 .345 -106- Table 12. Comparison of Calcium and Phosphorus Content in Samples of Bluegrass from Areas A and B Sample n d.f. Mean Sums Value of Sauares A 10 9 .715 5.1595 B 10 9 .603 3.7125 t = 3.019 t.01 (18 d.f.) = 2. 878 : Sample n d.f. Mean Sums Value of Squares A 10 9 .302 0.9344 B 10 9 .345 1.4261 t = 0.802 t .05 (18 d.f.) = 2.101 - 107 - Activity of Left and Right Ovaries The analysis of litter size by means of corpora lutea and albicantia counts provided an opportunity to evaluate the activity of the left and right ovaries, From a sample of 51 pairs of ovaries the writer recorded 558 corpora lutea and albicantia. The left ovaries contained 305 (55$) of the corpora lutea and albicantia (Table ; while the right ovary contained 253 (*+5$)* Similarly, from a sample of 22 uteri, a total embryo and placental scar count of 209 was obtained (Table 14), The left uterine horns con tained 125 (59*8$); the right contained 84 (40$). In both the uterine horn evaluation and the ovarian analysis there was a definite tendency for greater activity in the left side of the reproductive tract. The fact that the activity of the left uterine horn, as indicated by embryos and scars, is 2 ($ higher than the right hossn, whereas it is only 10$ higher on the basis of ovarian analysis is not without explanation. Three examples of ova crossing over to the opposite side of the tract from which they were released were recorded during the study. Strangely, the records of cross over observed were all from the right to the left side of the tract i.e., the ova had 108 - Table 1%, Corpora Lutea and Albicantia in Left and Right Ovaries of Cottontail Rabbits No. of corpora lutea and albicantia Left Ovary Right Ovary if b 3 0 7 6 1 6 10 if 6 8 6 6 if 8 11 13 7 9 5 8 9 6 9 if 6 if 3 5 11 if 9 9 2 5 9 5 5 8 4- 1 1 3 10 3 8 8 6 6 6 12 3 9 2 1 1 1 5 if 1 if 3 7 3 b If 13 2 13 3 9 2 ? 3 8 3 k 7; 8 5 k 6 k 6 2 6 5 2 if 3 6 5 9 3 Totals 305 253 Percents 55% b% Table !&'. Embryos and Scars in Left and Right Uterine Horns Left Right ..... 5 2 7 1 10 6 O£ k 6 3 5 8 7 Jf 3 8 9~ 2 9 6 b 1 3 1 5 5 b 5 5 b J■y £ 2 12 1 8 5 6 £ 2 2 Totals 125 8*+ Percent 59 • 8$ b0.2% been produced in the right ovary but had become Implanted in the left uterine horn after fertilization. Individual Productivity The writer accumulated very little data on the total number of young produced per individual female for the en tire season. However, a certain amount of interesting data has been obtained in regard to the number of young produced by females having one or more litters. In 16 rabbits from Area A which had produced two litters each, a total of 200 young were produced on the basis of embryo counts and cor pora lutea and placental scar counts. This yielded a mean of 12.5 young produced per female (Table 15 ). Eleven fe males from Area B that had produced two litters had appar ently given birth to 103 young. The mean total number of young produced per female was 9*3* On the basis of the mean litter sizes of these groups, the total number of young produced per female is about what one would expect it to be. One animal collected from Area A was pregnant with her fourth litter with evidences of 26 young produced in the four litters. A female from Area B which had produced four litters showed indications of having given birth to 22 young. In five rabbits from Area B for which three lit- Table 1$. Individual Litter Records from Areas A and B, 195*+ Area A Area B Date Litter Sizes Date Litter Sizes Collected 1st 2nd 3rd ifth Collected 1st 2nd 3rd 4 th V22/51* 6 V27/51* V23/5^ I 5 V 2 7/9+ V 2 3 M 6 h/29/9+ 6 I 5/1/54 5 « 7 6 5/8/9+ 5 if/2 if/5 if 8 7 5/18/54 7 V2V5^ 8 9 5/18/5^ 5 1 5/29/5^- 6 5/10/5^ 3 H 5/30/9+ 8 5/23/5^- MM 6/3/9+ 5 7 5/25/g* 6A-/9+ 6 6 7/30/9+ I 6/11/9 + 6 8/5/54 6/lV5^ I 8 8/23/9+ if 6 /lk /5 k 7 6 9/l8/5\ if 6/1b/9+ 8 7 10/28/9+ 5 if 6/16/9+ 5 6 10/29 /9+ 5 if 6/16/9+ 6 5 11/ 1/ 9 + 6 6/16/9+ 6 11/5/9+ if 7/10/9+ 7 11/6/9+ if 7/31/9+ I 7/31/9+ S 7 6 7/31/9+ 7 8 7/31/9+ - 112- ter sizes were available, the mean number of young per female was 12.8. In three females from Area A for which three litter sizes were available the mean number of young per female was 18.3. Trapping Success A few comments are in order regarding trapping tech niques and trapping success during the present study. Most of the intensive trapping was undertaken during 1954. Standard wooden box traps were used for this purpose, and, generally, at least 100 traps were set in a given area. In April, when trapping was begun, success seemed to run slightly under 10$ per night in Area A. This means that almost 10$ of the traps caught rabbits each night. Success in Area B was at first slightly below that of Area A. Prom about May 15 through most of August, trapping success declined markedly and remained at a rather low point until September. The use of various baits seemed to have little effect upon the success of trapping during the period from May to the end of August. When a comparison was made in April of using baited traps on e night and unbaited the next, there was no noticeable difference in trapping success. Apparently rabbits entered the unbaited traps 113 - equally as readily as those 'which were baited* The site of the trap proved to be an exceedingly influential factor in regard to trapping success, particularly during the summer months when cover was heavy* When trapping was first initiated, the writer was of the opinion that traps set in heavy cover where a rabbit would most likely be found, should be the most successful trap sites* This did not prove to be the case, Of course, those traps set in heavy cover along or across a well worn rabbit path did prove as successful as any other location, but such sites were not available for all traps* Rather than place traps in heavy cover where it was most likely that cottontails were situated during the day, it was found that by placing traps in the most obvious locations possible a noticeably higher degree of success in trapping was attained. Follow ing this procedure, the choicest trap sites for rabbits proved to be along the edges of plowed or freshly planted fields where traps would be obvious for a maximum distance, or along dirt roads with the trap placed well out in the road so as to be perfectly obvious to any rabbit which ven tured out onto it during the evening hours. 131 some in stances it was found that traps placed on flat rocks in open pastures were successful; those set in heavier cover nearby were not visited by rabbits. - 114. The -writer attempted to test this apparent differ ence in trapping success based on location. In the Marion County area on two successive nights, one line of 15 traps ■was plaeed in heavy cover just off a dirt road with the openings of the traps visible from the road. On an adja cent dirt road of equal length 15 traps were placed on the road away from heavy vegetation so that maximum visibility was afforded for each trap. None of the traps was baited. On the first night, the 15 traps set in the middle of the road captured 6 rabbits, four of which were juveniles and two adults. The 15 traps set back off the road in heavy cover captured 2 rabbits, both small juveniles. On the second night, the row of traps which were off the road captured three young rabbits; the traps in the middle of the road captured six rabbits. No further tests were made of the effectiveness of trap sites, but throughout the season*s trapping in Area A and Area B the advantage of setting traps in obvious locations not concealed by cover was apparent, A rather remarkable increase in trapping success be came apparent in late September, At this season much of the vegetation was drying up and turning brown, with the result that a great amount of the natural cover and food - 115 - was not nearly so abundant as earlier in the season. Ac companying these changes was the onset of extensive corn picking in Ohio, An increase in rabbit movement must have occurred at this time, Judging by the great increase in numbers of rabbits killed on the road and the increased trapping success. Attempts to use baits at this season proved to be highly successful particularly when dried kernels of corn or slices of fresh apple were scattered about in front of a trap, 3n Area B where trapping success during the main part of the summer was as low as 1$ and 2$, it increased to 20$ or more. On the basis of trapping success in Areas A and B during comparable periods of the season, it would appear that the population was considerably higher in Area A than in Area B, During the spring, for example, trapping success in Area A averaged 6 to 7% and ran as high as 10$, Trapping success in Area B during the spring averaged 2 to 3% and never was greater than 6$, Comparisons cannot be made during the later part of the season since trapping was largely discontinued in Area A when it was found that adequate samples could be obtained by shooting at night. In Area B, however, trapping success dwindled to almost nothing in the summer and increased 20 to 30$ in the fall. -116' Sex and Age Ratios No special effort was made during the present study to determine the sex and age ratios in populations where trapping was carried on. A certain amount of information was accumulated, however, merely as a result of tabula tions made during collecting operations in the various areas. At least some data of this sort were obtained from the Urbana Game Farm, the Olentangy Wildlife Experiment Station, the Marion County area and the Muskingum County area. Samples from the Urbana Game Farm in April 1953 yielded 22 males to 27 females captured. In October of the same year another sampling from the same areas yielded 18 males to 15 females. During September 1953, sampling in the 40-acre south pen of the Olentangy Wildlife Experi ment Station resulted in a ratio of 15 males to 22 females in the sample of rabbits that were sexed. Some of the very young animals caught in this area were examined and weighed but were not sexed. A ratio o f 31 juveniles to 14 adults was obtained during the sampling from this area. Nine of the 14 adults were females and five were males. During 1954, 21 adult males and 28 adult females were captured - 11? - in the Marion County area. A ratio of *+0 juveniles to *+9 adults was recorded from all sampling in the Marion County (A) area. Much more intensive and prolonged trapping in the Muskingum County (B) area yielded a ratio of 18 adult males to 19 adult females, and a ratio of *f8 juveniles to 37 adults. While these age ratios might lead one to be lieve that the population in Area B was greater than that in Area A, based on the findings of Petrides (1951), the manner in which some of the Marion sample was taken undoubtedly accounts for the low ratio of juveniles to adults, From June to the end of the breeding season rabbits were collected from Marion County primarily by hunting at night from a car as described earlier in this report. This sampling technique yielded a much higher proportion of adults than did trapping techniques in the same area. There appear to be two possible explanations to this: (1) juvenile rabbits were undoubtedly more easily overlooked during the night hunting with the use of a spot light than were the larger and more conspicuous adults rabbits; (2) it is possible that the younger rabbits, par ticularly the very small ones, remained in heavier cover more than did the adults which came out onto the roads at night and into the freshly cut fields and were thus more easily collected. As a result, the juvenile to adult 118 - ratios of Marion and Muskingum Counties are probably not truly comparable. Adult Weights Although not a part of the study from the standpoint of objectives, a certain amount of information on adult weights was obtained. Although the weights of a number of adult females were obtained, the writer does not con sider this material worthy of comparison from one area to another since the condition of adult females in various stages of pregnancy has a profound effect upon their weights. The mean weight of 21 adult males from Area A was *t^,9 ounces (1273 grams). In Area B, the mean weight of 19 adult males was ^3,8 ounces (12*fl grams). Thus, Area A CTabte It) males averaged 2.7$ heavier than Area B males. Analysis of these data using a t-test indicated that there was no significant difference between the areas. This, however, does not mean that no difference exists between the two areas as far as weight is concerned. (Table 17). Adult Male Weights (Grams) Area A Area B 1219 1247 1262 1276 1417 1021 1339 1219 1332 1247 1191 1304 1332 1162 1304 1219 1219 1361 1191 1361 1531 1276 1191 1361 1177 1205 1049 1162 1162 1304 1219 1247 1162 1361 1191 1162 1247 1077 1417 1531 x = 1273 gms. x = 1241 gms. - 120 - Table 17• Summary of a Comparison of Mean Weights of Adult Male Cottontails from Areas A and B Sample n d.f. Mean X(x-x) 2 Wt. A 21 20 1273.1 317,031881 B 19 18 12^0.6 16^,376.^2 t = 0.29 t.05(^0 d.f.) = 2.021 t#05(35 d.f.) = 2.030 Size of Testes Comparison of testicular size is made doubly diffi cult by the fact that the organs are changing almost con stantly in dimensions throughout the breeding season. Therefore it is difficult to obtain comparable samples from any two areas from adult males. However, so far as the interval of the season during which the sample was taken is concerned, a sample of 1^ adult males taken from Area A from April to the end of July appears to be roughly comparable to a sample of 11 adult males trapped from Area B from April through July (See Table 18). These data are not analyzed statistically particularly since the size of the testes is not completely independent of the date on which the rabbit is captured. Examination of the two small samples from the same general period suggests that perhaps the testes of adult males from Area A average somewhat larger than the testes from males in Area B, Total length of testes seems to decline gradually from June on through tfte season and in many cases they are minute and no longer scrotal by the end of August or early in September. On the bails of all observations of testicular size during the study, the peak of testis enlargement occurs between April 15 and May 15-31* This appears to be related to the - 122 - Table 18, Testis length of Adult Males (left testis) AprII-July 195^ Date Area A Date Area B b/2b 39*0 mm. l*/28 35.0 mm. h/2b 39.6 « k/29 3^.0 « 5/28 1*6.8 *• 5/7 35.0 " 5/28 36.^ " 5/25 36.5 " 5/29 1*0.0 » 5/26 25.0 « 5/30 1*2.3 H 7/13 31.8 »* 5/30 1+5*8 »* 7/16 28.0 » 6A 35.0 « 7/16 1+0.0 « 6Ao 36.5 " 7/21 32.9 " 6/10 3 5 A " 7/ 2b 32.0 «* 7/10 38.2 " 7/30 23.0 » 7/10 3^.0 » 7/10 36.5 " 7/31 31.5 " N = ii* N =S- I1* Zx = 537.0 mm. £ x ss— 353.2 mm. X m 38.!+ mm. x ■« 32.1 mm. mmKSCss------123 - estimated peak of frequency of parturition dates in 195^* Humeri Lengths and Weights A small sample of adult rahbit humeri from Area A and Area 5 were cleaned, dried in an oven for *+8 hours, measured, and weighed to the nearest tenth of a milligram. The range of weights and lengths in these samples is given in Tables 19 and 20, Although neither of these comparisons was subjected to statistical analysis, the mean length and weight of the samples indicate quite clearly that no significant difference existed between the samples. If a significant difference does exist between bone weight and length between the two areas, the differ ence is so small that it would require a much larger sample to render it discernible by statistical analysis. Tick and Cuterebra Infestations During the 195*+ samplings of Area A and Area B, the writer noticed a marked difference in the degree and rate of ectoparasite infestation in the rabbits of the two areas. This was first noticed toward the end of May and the first part of June when ticks became extremely abun- Table 19. Bhmeri lengths of Adult Cottontail® Afda A ' ' Are a B Spec. Fo. ' Length Spec, No. length _____ (mm.)______(mm.) &~1 6%.0 B-l 6%*7 A-2 66.34- B-2 6%.0 A-3 66.9 B^3 65.0 A J ^ 65.2 BJ + 66.6 A-5 60.% b -5 60.6 A-6 63+. 5 B^6 63.1 Mm? 66.0 B-7 66.5 A-8 61.0 B+9 62.8 A^9 65.0 B-10 60.0 A-ll 65.0 B-13 6 3 . 6 A-12 65*0 B-1% 67.0 A-13 65*> B-16 61.2 A~l% 6 3 .% B-19 66.0 A -15 60.6 B-20 71.0 A-lo 62.5 B-21 62.9 A - 1 7 66.0 A-18 6%.0 A-19 66.0 A~20 6?.o A-21 66*0 A~2% 66.0 A-25 62.3 SfX'r lb27»6 mm Iis956*0 mm. N - 22 IT - 1? X = 6%.86 x ~ 63.73 - 12 5 - Table 20. Humeri Weights of Adult Cottontails Area A Area B Spec. No. Weight Spec. No. Weight (gms.) (gms.) A-5 1.5252 B-l 2.0310 A-9 1.9302 B-3 2.0510 A-11 2.0658 B-1+ 2.1775 A-12 1.9526 B-5 1.903^ A-13 2.0011 b -6 1.8517 A-l^ 1.8186 B-13 1.71+97 A-15 1.872^ B-l^ 2 .01+98 A-18 1.7816 B-15 1.5831 k-2b 2.3282 B-16 1.9702 A-25 1.7287 B-19 2.1553 B-20 2.0951 B-21 1.9825 I X = 19.0051+ gms. SX ■ 23.6003 gms. N B 10 N = 12 X B 1.9005 gms. x = 1.9667 gms. - 126 - dant on rabbits captured In Area 5. Those rabbits examined from Area A were found practically devoid of ticks, or, when they did occur, only one or two were usually present* In Area B, as many as 30 to 50 ticks were counted on one rabbit* They were especially abundant around the neck and ears. Many of these of course were young larval stages. Juvenile rabbits in Area B during this period were found to be particularly heavily infested. From about the first week in July into August a very high incidence of Cuterebra infestations was recorded in rabbits from Area B. Only one rabbit was collected in Area A which had a single Cuterebra in the neck. Many of the rabbits in Area B which were found to have Cuterebra infestations had two or three of these parasites in the neck region. One adult male caught July 21 possessed four large Cuterebra in the neck region. It was also heavily infested with ticks. Such double in festations of both Cuterebra and ticks were not unoommbnly encountered in specimens from Area B. There is no doubt that infestations of these ectoparasites were far greater in Area B than in Area A. Chapter IV DISCUSSION It has been the •uniform opinion of previous workers that cottontails do not produce young during their first summer of life. Few exceptions to this have been noted in the literature. On the basis of 67 young subadult females examined during the study, h2% were found to be sexually active' in their first summer-. It appears that the repro ductive contribution mad© by this- segment of the popula tion has been underestimated. This may well be attributed to the manner in which age determinations of rabbits have been made in previous studies. Many of the subadult fe males found sexually active in this study could easily have been incorrectly called adults by various aging cri teria. which have been employed in other studies'. Weight, condition of hymen, and teat size are aging methods that would have placed many of the subadults in an adult cate gory, The writer is inclined to think that the comparison of subadult breeding between the till plain region and the unglaciated plateau is not an adequate one, and he would prefer to have examined a much larger sample of subadults from the Marion County area, during 1 9 It is well to recognize that the breeding subadult segment of the pop- lation in previous studies might well have had an effect -127 - - 128 - upon the calculated mean litter size for certain areas since breeding subadults in the present study were found to have rather small litter sizes. On the basis of esti mated age, no subadult females born much later than the end of April were found to be sexually active during their first summer. Thus the percentage of subadults breeding may very well vary from year to year depending upon the onset of breeding in late winter and early spring. Appar ently the breeding season started somewhat later in 1953 than in 195*+ in the till plain region of Ohio. This may account for the lower rate of subadult sexual activity ob served in Urbana Game Farm specimens in 1953# This may have a profound effect upon the total productivity of a particular season. The writer prefers to consider that the breeding season is fully underway at the time when most of the adult females have become sexually active, i.e,, either they are pregnant or they have already had a litter at any particular sampling time in the spring. There is variation in the physiological processes of any population. The full onset of the breeding season should not be ascertained by the observations of one or two individuals found to be pregnant or nesting perhaps in ftebruary, but rather a determination of the percent of - 129 - females found to have begun breeding in any given sample taken from February into April. The determination of the onset of breeding of the entire adult population appears to the writer to be highly important information in regard to the management of populations from year to year. Apparently the full onset of the breeding season may vary from year to year in a particular region, whereas the cessation of breeding appears to be more stable from the results of the present study. The explanation for this is not clear. The onset of breeding as defined above appears to have begun at least two weeks earlier in 195^ than in 1953* The late winter and spring months of 1953 were characterized by below normal precipitation and above normal temperatures. This was not true in 195*+• The writer cannot conceive what affect this might have upon initiation of breeding in the cottontail. However, Crespo (19^) correlated the increases in field mice (Hesperomys) with extremely rainy summers in Argentina. Collett (1895) found that European lemmings had very prolific years, usually occurring after a cold wet summer. It may well be that the factors involved may be resolved through investi gations in the field of plant physiology. -130- Admittedly, the following discussion is presented with a high degree of speculation, although not without some basis. Friedman and Friedman (1934) obtained an extract from plant juice which proved to stimulate ovula tion in rabbits when injected intra-peritoneally or intra venously. From these results they decided that there were plant gonadotropins present in plants. Friedman and Mitchell (1941) carrying this work still further found a strong seasonal variation in the yield of gonadotropic activity, such juices being apparently quite inactive in fall, winter, and spring. There also appeared to be vary ing degrees of potency in plants. Borasky and Bradbury (1942) using a refined technique of extraction and storage, found that a gonadotropic factor was present at all times of the year but that the responsiveness of female rabbits to the ovulating factor was somewhat less in the late sum mer and fall months of the year. By analysis of pituitary glands they concluded that the substance in plants acted as a neuro-toxin, stimulating secretion of the pituitary gonadotropins via the midbrain. At the risk of being over speculative, the writer suggests the possibility of cli matic factors influencing neurotoxic substances in plants. Such relationships if they exist, might well be influential in regard to the onset of breeding in cottontails as well - 131 - as other species. Controlled experiments with animals in the laboratory should be conducted in order to determine the validity of such a concept. It becomes obvious from the data gathered in Marion and Muskingum Counties that productivity in cottontails apparently varies somewhat in relation to the quality of the soil. The very significant difference in litter sizes from the two areas leaves little room for speculation in this regard. Exactly what the relationship is of rabbit reproductive capacity to soil cannot be clearly defined from the results of this study. However, the very signif icantly higher calcium content in Marion County bluegrass samples (.72$) than in Muskingum County samples (.60$) suggests that a nutritive relationship may be an influ ential factor. While the data collected in this study do not clearly demonstrate that calcium content in the veg etation is influential upon reproductive capacity, or success, it does suggest the existence of such a relation ship. Since the calcium thresholds in the diet affecting reproduction have not been established for the cottontail, it is impossible to state whether the differences observed between the two areas might account in part, at least, for the observed differences in litter size. However, there - 132 - is evidence in the literature that calcium intake does have an influence upon reproductive activity. Dale (1955) cites evidence which suggests that the amount of calcium in the diet is an important factor regulating ovulation in the ring-necked pheasant (Phasianus colchicus). Prom the work of Baldini and Zarrow (1952) it seems probable that the level of calcium in the blood may be related in some manner to hormone secretion and ovulation. The data of the present study can do no more than suggest the possible influence of calcium intake in cotton tails via the vegetation as a factor affecting the rate of ovulation and hence litter size. The rate of ovulation in Area A cottontails was even higher than was indicated by the larger mean litter size compared to Area B cottontails. The ratio of released ova to actual litter size as deter mined from pregnant animals captured in Area A was 1.03 ova per 1 young. This would have been slightly higher were it not for the fact that in one animal (A-6) there were more embryos present than there were corpora lutea present for the second pregnancy of the animal. This is apparently a record of twinning from one of the ovum re leased. The ratio of released ova to the number of young in Area B as determined from ten pregnant animals and - 133 - animals with fresh placental scars, was 1.00 ovum per 1 young. Two cases of cross-over of ova from the side on which they were released to the other side of the uterus were recorded during the examination of ovaries. It would appear that, while there is less wastage of ova in the sample from Muskingum County, the rate of ovulation as well as litter size is considerably greater in the Marion County area even though the ratio of released ova to litter size is higher. Three cases of resorption of a single embryo were recorded from Marion County reproductive tracts; none were recorded from the Muskingum County rabbits. It would seem that perhaps the rate of ovulation in the Marion County rabbits in some cases exceeded the physiological capacity of the uterus to support embryos; thus resorption as well as the higher ratio of released ova to number of young was observed. The writer does not wish to give the impression that he believes only one factor may be responsible for rabbit productivity and population density in various regions of Ohio. Undoubtedly there are many* The apparent differ ence in the rate of parasitism by ectoparasites during the summer months in the two areas may conceivably affect survival, health, disease incidence, and possibly repro- < - 13*+ - ductive performance. Topographic and local climatic dif ferences may have some effect upon populations. It is not probable that all factors affecting productivity are equal ly influential. More than likely there is a primary fac tor affecting reproductive capacity, with other less impor tant influencing factors being present. The writer submits that soil quality, as reflected in the nutritive value and mineral eontent of the vegetation, is the primary factor influencing reproduction and hence, to a large degree, population density of the cottontail rabbit in Ohio, Ex periments with rabbits raised in five-acre fenced plots on the Woodbury Wildlife Restoration Area by the Ohio Division of Wildlife seem to substantiate this theory. The high reproductive success of adults and the large percentage of subadults breeding were recorded from fenced plots on poor soil which had been fertilized. Of course, reduced preda tion in such plots must have some effect upon population density, but not upon reproductive and growth phenomena. Controlled studies with breeding populations in fenced plots on various soil types and employing different fer tilization practices should prove enlightening. The composition of the peaks of parturition observed in the present study suggest that some factor such as age 135 - may strongly influence reproductive activity of females. On the basis of mean litter sizes from the Marion and Muskingum County samples, females having three litters per year in Marion County produce an average of five more young per adult than Muskingum County females having the same number of litters per year. Thus, a sample of 20 adult females from Marion County might produce an average of 100 more young in a season than the same number of fe males from Muskingum County. If we consider that 32 of these were first litter young, of which 16 were females, seven of the females might be expected to breed in their first summer and produce an average of 28 young based on a mean litter size of *f.0 for subadult litters, A total of 128 more young might thus be produced in Marion County than Muskingum County starting with the same number of breeding females. This provides a rough indication of the density differences which might exist between the two areas. Observations and trapping results certainly indicate that populations were much higher in Marion County. One-sided activity of female reproductive tracts is well known in avian species, but is less well known In mammals. Asdell (19^6) cites the following information - 136 - concerning the activity of the left and right sides of the reproductive tracts of mammals. In Eotesicus fuscus 53$ of the corpora lutea have been observed in the left ovary. In the little brown bat (Myotis lucifueus) embryos are al most always found in the right horn of the uterus. However, in first season females there is a preponderance of ripe follicles in the left ovary. In the European little brown bat fMyotis myotis) embryos are nearly always in the right horn of the uterus, but the corpora lutea are found in the right ovary in only 55*7$ of the cases. In domestic cattle, records indicate that 60.2$ of the corpora lutea are found in the right ovary, 60.5$ of the embryos are in the right horn of the uterus. In guinea pigs, embryos are found in the left horn of the uterus 51$ of the timej while in horses the left ovary is more active as judged by 6l$ of the corpora lutea occurring in the left ovary. In the rhesus monkey activity appears to be approximately equal since 50.8$ of the corpora lutea were observed in the right ovary. In l1* sets of corpora;.lutea from the domestic pig, 51*9$ were from the left ovary. In larger samples, 55.3$ were from the left ovary, showing a definite tendency for greater activity on the left side of the tract. In the domestic rabbit the data of Hammond and Marshall (1925) indicates that activity is approximately equal since they - 137 - ' found 51 of the corpora lutea were from the left ovary. In the rat, 51$ of the corpora lutea were found in the left ovary. Sheep on the other hand have been found to have apparently greater activity in the right ovary on the basis of three separate samples; 57%, 5*$, and 58.6$ of the corpora lutea being in the right ovary. PTom a small sample of I1* pregnant females of the brush rabbit C Svlvilagus bachmani) Mossman (1955) in California found that 2*+ to 53 embryos were found in the right horn of the uterus and 29 in the left horn, while 25 corpora lutea were in the right ovary and 30 in the left ovary. On the basis of this there appears to be a slight difference in the activity of the two ovaries. The data on the cottontail rabbit presented in this study rather clearly indicates greater activity in the left ovary, and an even greater tendency for implantation of fertilized ova in the left horn of the uterus. It would seem that, on the basis of the comparatively greater success when traps were set in the most obvious locations, there is an element of curiosity involved in the attraction of a rabbit to a trap. A box trap set in the middle of a dirt road presents a dark object, and perhaps nothing more, to a rabbit which may be 50 t o 60 feet away. - 138, - Apparently, on the basis of the increased yield of such trap sites, this may stimulate a reaction on the part of a rabbit which results in the animal investigating the dark object* The fact that this object ultimately proves to be a dark hollow, for which rabbits apparently have a predi lection, is the culminating factor which results in the capture of the rabbit even in unbaited traps. Thus from the standpoint of behavior, it is suggested that differ ential attraction to traps on the part of rabbits as re ported by Geis (1955), may in part be a reflection of the attractiveness of the various types on the basis of their location. It would seem virtually impossible to the writ er to set out a substantial series of rabbit traps and safely assume that each trap was equally attractive to rabbits for an equally large radius around each trap. Thus, if several traps were particularly attractive within the home range of an individual rabbit, i.e., if they were especially prominent then one might expect that this rabbit would be captured more frequently than a rabbit in another home range where perhaps the traps were set in less obvious locations. This, however, is more speculation on the basis of the writer’s observations regarding the effectiveness of trap location. - 139 - The results of the present study provide evidence that stocking and habitat improvement alone are not suffie:'.': cient to maintain high rabbit populations in some areas where essential elements in the soil and plants are defi cient. Only by improving basic deficiencies in the soil can we hope to attain high stable populations by stocking and habitat improvement. It is the writer*s conviction from the results of the present study that an intimate relationship exists among soil, vegetation, and animal pop ulations. Elucidation of this relationship should lead us into a new era of wildlife management. Chapter V SUMMARY AND CONCLUSIONS Prom 1952 to 1955, data were gathered on reproduction, maturation, and pelage stages of cottontail rabbits in Ohio. Specimens were collected from selected areas as well as randomly in the State. Methods were devised for the eval uation of reproduction by ovarian analysis and for the aging of young pelage stages. A controlled breeding experiment was conducted, using New Zealand White rabbits, to determine the persistence of placental scars and corpora lutea, and to ascertain their reliability as indicators of litter size and breeding history of a female. A series of known age embryos were obtained from New Zealand White rabbits for use in aging cottontail embryos. Regional comparisons were made of litter sizes, length of breeding season, breeding of subadult, body measurements, and cal cium and phosphorus content in bluegrass. The following conclusions have been drawn from the results of this study. 1. Prom 40 to 50 percent of the first litter females become sexually active in their first summer. 2. Litter sizes of subadults average 4.0 young per - 140 - - 1^1 - litter# 3# Placental scars are inadequate as indicators of litter size after two or three weeks following parturition. b. Corpora lutea and corpora albicantia provide an accurate means of determining litter sizes and number of litters produced. 5. The rate of resorption of embryos is negligible in cottontails in Ohio. 6. The left ovary averages 10 percent greater activ ity than the right ovary in cottontails. 7. Crossover of ova from one horn of the uterus to the other occurs in cottontails. 8. Litter sizes of cottontail rabbits average appre- iably larger in populations living on areas of high fertility soils than those of rabbits living on low fertility soils. 9. The calcium content in the vegetation may very possibly be related to reproductive performance in cottontail rabbits. 10. Adult male cottontails from Marion County are not significantly larger than those from Muskingum county. 11. On the basis of the significance of the differ- - I k 2 - ence in mean litter sizes between Marion and Muskingum County areas, it is suggested that lit ter sizes of cottontails in the till plain re gion of Ohio are appreciably larger than litter sizes in the unglaciated Allegheny Plateau* 12. Four main peaks of litter production occurred in Ohio in 195*+, from April 1-15, May 1-15, June 1- 15, July 1-15. 13. Three to four litters are produced per year by most adult female cottontails in Ohio, with a few producing five litters. I1*, First litters of the season are produced as late as the July peak, and constitute 50 percent or more of the first three peaks. A large amount of individual variation apparently exists in the onset of breeding of females. This may be due to various age groups in the female population. 15. The full onset of breeding in a population may vary considerably from one year to another, but the cessation of breeding in the fall appears to remain quite constant. 16. Breeding begins sporadically in February, and the latest litters are born in October. 17. Five pelage stages are discernible in the cotton- 1^3 - tail rabbits nestling fur, Juvenal pelage, sub adult pelage, adult summer pelage, and adult winter pelage. Nestling fur, Juvenal pelage and subadult pelage together with the molt stages from one pelage to another are indicative of particular age ranges. Chapter VI RECOMMENDATIONS On the "basis of the present study, the following suggestions for further study are made: 1. Further investigation of the molting stages of adult pelage in order to determine whether it may be used as an indicator of stage of breeding activity in females. 2. Investigation of the possibility of using pelage age groups at the end of the summer as an indica tor of productivity and population level. 3. A long term investigation of the variability of subadult cottontails breeding in their first sum mer. b, A 10-year study of the reproductive performance of rabbit populations in selected areas of high and low soil quality. The present study was lim ited in time and did not gain adequate data regard ing multiannual population fluctuations which may occur in an area. 5. An extended investigation of seasonal and annual variation of various substances in plants in dif- - iMp - I h 5 - ferent areas. This should he carried on in rela tion to studies of rabbit reproduction and popula tion levels. 6. It is recommended that other investigators contem plate the use of the pelage-aging technique in future reproductive studies of the cottontail rabbit. Errors in the identification of subadults should be reduced if this method is employed. 7. Controlled experimentation with rabbits in fenced plots on differing soil types and under varying fertilization practices. This should help to clarify the nutritional-reproductive relationships suggested in the present study. 8. An investigation of the feasibility of applying extensive fertilization methods to deficient soil areas in Ohio. 9. An investigation of the feasibility of maintaining limited fertilized areas as reservoirs of high rabbit productivity in areas of low quality soils. 10* Controlled laboratory experiments with gonadotro pic extracts from plant juices in order to deter mine seasonal variation, effectiveness, and respon siveness of rabbits to the active factor in plants. APPENDIX Preparation of Ovaries for Analysis 1. Ovaries are fixed in alcohol-formalin-acetic acid (A.F.A.). They need not he changed from this solution hut may he stored indefinitely, 2. Remove ovaries to 9% ethyl alcohol for at least 30 minutes, 3. Remove ovaries to 10($ ethyl alcohol for at least 30 minutes. b, Remove ovaries to xylene at room temperature for at least one hour. Place in oven at 55-56 degrees Centigrade for at least one hour. 5. Remove ovaries to melted paraffin (Fisher Tiaauemat 52-5^ degrees Centigrade gives hest results). Leave ovaries in paraffin in oven for 12-2*+ hours. 6. Imhed ovaries in paraffin block. 7. Cool blocks in ice water. 8. Section ovaries (at least lf5-50 microns thickness). 9. Prepare slides with albumin fixative and mount sec tions by floating with distilled water. 10. Dry slides for several hours depending upon heat source. 11. Place slides in xylene for 15-30 minutes. - IMS - I k ? - 12. Remove to 100$ alcohol for 5 minutes. 13. Remove to 95$ alcohol for 5 minutes. Ik. Remove to 70$ alcohol for 5 minutes. 15. Remove to 50$ alcohol for 5 minutes. 16. Remove to Delafields hematoxylin for 5-10 seconds. 17. Remove to distilled water wash. 18. Remove to 50$ alcohol for 1 minute. 19. Remove to 70$ alcohol for 1 minute. 20. Remove to eosin red stain for less than 1 minute. 21. Remove to 95$ alcohol for 1 minute. 22. Remove to 100$ alcohol for several minutes, 23. Remove to fresh change of 100$ alcohol for several minutes. 2kt , Remove to xylene for 5-10 minutes. 25. Mount cover glasses with piccolyte or other standard mounting medium. LITERATURE CITED Allen, Durward L. 1938. Breeding of the cottontail rabbit in southern Michigan. Amer. Midi, Nat. 20sU-61+-l+69 Asdell, S. A. 19*+6. Pattern of mammalian reproduction. Comstock Pub. Co. Baldini, J. T. and M. X. Zarrow. 1952. Estrogen and serum calcium levels in the bobwhite quail. Poultry Sci., 31:800-80**. Beule, John D. 19*+1, Cottontail nesting study in Pennsylvania, Trans. N. Amer. Wildlife Conf., 5:320-328. Bodenheimer, F. S. 19^9. Problems of vole populations in the Middle East. Report of the population dynamics of the levant vole. Research Council of Israel, Azriel Printing Works. Bradbury, J. T. 19^+. The rabbit ovulatory factor of plant juice. Amer, Jour, Physiology, 1**2:**87. Braestrup, F. W. 19*+1. A study on the arctic fox in Greenlands immigrations, fluctuations in numbers based mainly on trading statistics, Meddelelser om Gronland, 131:1-101. Bruna, Joe. 1951. Kentucky rabbit investigations. Paper pres, at 5th Southeast Wildlife Conf. Cheatum, E. L. 19**9. The use of corpora lutea for deter mining ovulation incidence and variations in fertil ity of white-tailed deer, Cornell Vet. 39s282-291. Cheatum. E. L. and C. W. Seviringhaus. 1950. Variations m fertility of white-tailed deer related to range conditions. Trans. 15th N. Amer. Wildlife Conf., pp 170-189. Ciba Foundation. 1953, Mammalian germ cells, pp 1-302. Cooley, Marvin E. 19*+6. Cottontails breeding in their first summer. Jour. Mammal. 27(3):273-27*+. - 1**8 - 1^9 - Dale, Fred H. 1955. The role of calcitun in reproduction of the ring-necked pheasant. Jour. Wildlife Mgt., 19:325-331. Dalke, Paul D., et al, 19*+2. The cottontail rabbits in Connecticut. Conn. Pub. Doc. b?, pp 1-93. Denney, A. H. 19*+*+. Wildlife relationships to soil types. Trans. 9th N. Amer. Wildlife Conf., pp 316-323. Dice, L. R. 1929. An attempt to breed cottontail rabbits in captivity. Jour. Mammal., 10:225-229. Dusi, Julian L. 1952. The food habits of several popula tions of cottontail rabbits in Ohio. Jour. Wildlife Mgt., 16:180-186. Ecke, Dean H. 19^8. Reproduction in Illinois cottontails. Paper pres, at 10th Midwest Wildlife Conf. (Mimeo,). Elder, W. H. and L. K. Sowls, 191+2. Body weight and sex ratio of cottontail rabbits. Jour. Wildlife Mgt., 6:203. Friedman, M. H, and G. S. Friedman. 1939. Gonadotropic extracts from green leaves. Amer. Jour. Physiology, 125:^86. Geis, Aelred D, 1955. Trap response of the cottontail rabbit and its effect on censusing. Jour. Wildlife Mgt., 19:^+66-1+72. Gertsell, Richard. 1937. Management of the cottontail in Pennsylvania. Pa. Game News, 7:6-7. Hadwen, Seymour. 1929. Color changes in Lepus americanus and other animals. Canad. Jour. Research, 1:189-200. Hamilton, W. J. Jr. 19^+0. Breeding habits of the cotton tail rabbit in New York State. Jour. Mammal., 21: p8. Hammond, John and F. G, H. Marshall. 1925. Reproduction in the rabbit. Oliver and Boyd, Edinburgh. 210pp. Haugen, Arnold 0. 19^2. Home range of the cottontail rabbit. Ecology 23:35*+-367. - 150 - Hendrickson. George 0, 19**7, Cottontail breeding in its first summer. Jour, Mammal,, 28:63, Laurie, E. M, 0, 19*+6. The reproduction of the house mouse (Mus muscuius) living in different environ ments, Proc, Roy, Soc, Ser, B, 133:2*+2-28l. Lewellyn, L,M. and C, 0. Handley, 19^5. The cottontail rabbits of Virginia. Jour. Mammal., 26:379-390. Minot, C. S. and E. Taylor. 1905. ' Normal plates of the development of the rabbit (Lepus cuniculus L.). Normentafeln zur Entwicklung sge schichte der Wirbeltiere, Keibel, Heft 5, Gustav Fischer, PP 1-27. Mart on G, H. and E. L. Cheatum. 19*+6. Regional differ ences in breeding potential of the white-tailed deer in New York. Jour. Wildlife Mgt., 10:2*+2-2*+8. Mossman, Archie S. 1955# Reproduction in the brush rabbit in California. Jour. Wildlife Mgt., 19:177-18*+. Nelson, E. W. 1909. The rabbits of North America. North Amer. Fauna No. 29, 31*+ pp. Owen, David D. 1857, Second report of the geological survey of Kentucky made during the years 1856 and 1857. Ky. Geol. Sur. Rept. 2:1-11*+. Petrides, George A. 1951. The determination of sex and age ratios in the cottontail rabbit. Amer. Midi. Nat., *+6:312-336. Schwartz, C. W. 19*+2. Breeding season of the cottontail rabbit in central Missouri. Jour. Mammal., 23:1-16. Severaid, Joye Harold, 19*+5« Pelage changes in the snow- shoe hare. Jour. Mammal., 26:*+1-63. Severaid, Joye Harold. 19*+2. The snowshoe hare - its life history and artificial propagation. Contr. Maine Coop, Wildlife Res. Unit. \ anith, Ralph' H. 1950. Cottontail rabbit investigations. Final Rept, Pittman-Robertson Pro;). 1-R, pp 1-8*+. - i?i - Stewart, A, M. and P. S. Moir. 19*+3* Fertility of Merino sheep in northwestern Australia, Australian Vet, Jour* 19:152-161*. Trippensee, R. R, 1936, Reproductive function in the cottontail rabbit, Svlvilagus floridanus mearnsii. in southern Michigan, Proc. N, Amer, WilcEIife Conf,, p 3¥+, AUTOBIOGRAPHY I, Norman Curtiss Negus, was' born in Portland, Oregon, on September 20, 1926.. I attended public schools in Cleveland Heights, Ohio. My undergraduate training was received from Western Reserve University and Miami University, Oxford, Ohio. I received the Bachelor of Arts degree from Miami University in 19^8, and the Master of Arts degree in 1950. From 19M3 to 1950, I held the appointment of graduate assistant in the Department of Zoology. During the suwmers of 1 9 ^ and 19^9, I conducted studies on mammalian ecology in Wyoming, supported hy research grants-in-aid from the Jackson Hole Biological Research Station. From 1951-55, I held the position of research fellow in the Ohio Cooperative Wildlife R e s e a rc h Unit, Ohio State University, while completing the require ments for the degree Doctor of Philosophy in the Depart ment of Zoology and Entomology.