STUDIES ON URENARY ENCONTINENCE AND “WET BELLY” 3N RANCH MINK (Mustela vision) Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY RICHARD JOHN AULERQCH 1967 1' H :815 This is to certify that the thesis entitled Studies on Urinary Incontinence and "Wet Belly" in Ranch Mink (Mustela vison). presented by Richard John Aulerich has been accepted towards fulfillment of the requirements for Ph.D. Poultry Science degree in g/ fl, ./ / ! » l-J/fLL-é v.’ \ 31%”: f2! (1 c ’é - gt) />" / x { )Iajor ~professor /./ Date June 30, 1967 0—169 LIBRARY bflfiChigafllSkEUQ University ABSTRACT STUDIES ON URINARY INCONTINENCE AND "WET BELLY" IN RANCH MINK (Mustela vison) by Richard John Aulerich Studies pertaining to urinary incontinence and ”wet belly" in mink were conducted at Michigan State University over a six year period. Twelve experiments dealing with various aspects of these two associated conditions were performed to investigate the nature of these disorders, their etiology and possible treatment. The conditions occur predominately in males and collectively are commonly called "wet belly" disease. It is generally assumed that urine from incontinent animals dribbles, stains and mats the fur around the urethral orifice, and causes a discoloration ("wet belly") in the pelt. Gross microscopic and histological examination of the "wet belly" defect in the pelt revealed that this condition was an area of unprimeness. A.delay in the normal pelting time, to give the fur of "wet belly" susceptible mink a longer period in which to prime, however, did not eliminate or minnmize the defect. This condition apparently involves other complicating factors in addition to normal maturation of the fur and skin. No pathological changes or lesions, however, were noted in the fur or skin of pelts affected with "wet belly". Richard John Aulerich A comparison was made between raw and dressed "wet belly" pelts with regards to the appearance of the defect and the value of the fur. In natural dark mink skins, fur dressing and finishing greatly reduced the conspicuousness of the "wet belly" area and enhanced the value of the affected pelts. In studying the cause of "wet belly", urine from incontinent and normal males was applied daily to the shaved backs of female mink in an attempt to produce "wet belly". No discoloration of the skin, how- ever, was noted when the females were pelted. Mineral oil applied daily to the inguinal region of incontinent males to prevent urine from coming in contact with the skin in that area did not stop the defect from appearing in the pelt.‘ An attempt was made to surgically produce urinary incontinence in three males by transaction of the sphincter muscle at the neck of the urinary bladder so that its effects on the pelt could be studied. urinary incontinence was experimentally produced in Only one mink, but on pelting "wet belly" was noted in both the male in which incontinence. was experimentally produced and inane of the other surgically treated males in which no incontinence was observed. Although these conditions are generally associated, these results suggest that the "wet belly" defect is not due to urine p333. Mink fed a diet containing 1.03 percent calcium with a calciumr phosphorus ratio of 2:1 did not significantly affect growth, but pro- duced a higher incidence of both urinary incontinence and "wet belly" than a diet containing 0.47 percent calcium with a calcium-phosphorus ratio of 1:1. Richard John Aulerich Diethylstilbestrol was administered to male mink affected with urinary incontinence as a possible treatment for this condition. In- jections of 0.4 ml of diethylstilbestrol on alternate days, and implan- tation of a 15 mg pellet of the synthetic estrogen resulted in increased incontinence and was toxic to the mink. Treating urinary incontinent mink with nitrofurantoin and nitro- furantoin plus neomycin did not reduce the incidence of either urinary incontinence or "wet belly", although greater weight gains were obtained in the animals that received the nitrofurantoin-neomycin supplementation. A.comparative histological examination of the urinary tracts of incontinent and normal male mink did not reveal any differences or abnormalities which might be responsible for this disorder. These studies plus bacteriological examination of preputial smears and urine of mink with symptoms of urinary incontinence failed to confirm pre- vious reports in the literature that this condition is associated with Proteus mirabilis and Streptococcus fecalis. STUDIES ON URINARY INCONTINENCE AND "WET BELLY" IN RANCH MINK CMustela vison) By Richard John Aulerich A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Poultry Science 1967 ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to Dr. Philip J. Schaible for his inspiration, guidance and encouragement throughout our association. I am also greatly indebted to Dr. Robert K. Ringer for his advice and assistance during this study. Appreciation is expressed to Dr. Howard C. Zindel, Chairman of the Poultry Science Department, and to Mflchigan State University for making available the animals and facilities to conduct this research. Sincere thanks are also extended to Dr. Richard E. Bostrom for his help in the histopathological aspects of this research, to Mrs. Marian Bennett for the bacteriological determinations, to Dr. Irwin J. Benne for analyses of the experimental rations, to Mr. Philip Amico, of Hudson's Bay Co., for evaluation of the pelts, to Mr. Sulo Hulkonen for photographic assistance and to Dr. Floyd M. Stout, of Oregon State University, for supplying some of the tissues used in these experiments. I am also grateful to Mrs. Sandra Pangborn, Mrs. Marjorie Tetzlaff, Mr. Gordon Shelts, Mr. Peter Steelman and'Mr. Bryan Larsen for their technical assistance in these experiments. Sincere acknowledgement is also made to the Mink Farmers' Research Foundation for their support and financial assistance in these studies. Finally, I particularly wish to thank my wife, Camilla, for her patience, confidence and encouragement during this period of study and research. 11 TABLE OF CONTENTS ACKNOWLEDGEMENTS . . . . . . LIST OF TABLES . . . . . . . . . . . . . LIST OF FIGURES . INTRODUCTION . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . Description of Urinary Incontinence Etiology . . . . . . . . . Nutritional Aspects . . . . . Genetic Aspects . . . . . . Physiological Aspects . Bacteriological Aspects Pathological Aspects . . . Suggested Treatments . . . . . METHODS . . . . . . . . . . . . . . . . EXPERIMENTS . . . . . . . . . . Experiment I. Observations on "we: Procedure . . . . Results . . . . . . . . . . Discussion . . . . . . . . . and Experiment II. Effect of Time of Pelting on "wet Procedure . . . . . . . . Results . . Discussion . . . . . . . . . . Experiment III. Histology of "Net Belly" . . . . . Procedure . . . . . . . . . . Results and Discussion . . . . iii Page ii vi vii 10 ll l4 l4 l7 l9 19 21 22 23 23 23 28 31 31 32 36 36 36 37 TABLE OF CONTENTS - Continued Page Experiment IV. Effect of Fur Dressing on "we: Belly" . . . 48 Procedure . . . . . . . . . . . . . . . . . . . . 48 Results . . . . . . . . . . . . . . . . . . . . 51 Discussion . . . . . . . . . . . . . . . . . 54 Experiment V. Effect of Urine on "we: Belly" . . . . . . . 55 Procedure . . . . . . . . . . . . . . . . . . . . . . 56 “8‘11 t8 0 O O O O O O O O O O O O O O 9 O O O O O O O 56 Discussion . . . . . . . . . . . . . . . . . . . . . . 56 Experiment VI. Effect of Mineral Oil on "we: Belly" . . . 57 Procedure . . . . . . . . . . . . . . . . . . . . . . 58 Results . . . . . . . . . . . . . . . . . . . . . . . 58 Discuasion O O O O O O O O O O O O O O O O O O O O O O 58 Experiment VII. Attempted Surgical Production of urinary Incontinence . . . . . . . . . . . . . . . . . 59 Procedure I O O O O O O O O O O O O O O O O O O O O Resul t8 0 O O O O O O O O O O O O O O O O O O O O 0 Discussion I O O O O O O O O O C O O O O O O O O O O O 88% Experiment VIII. Influence of Dietary Calcium on Growth, urinary Incontinence and "wet Belly" . . . . . 65 Procedure . . . . . . . . . . . . . Resul t8 0 O O O O O O O O C O O O O O O O O O O O O O 66 Discuss ion 0 O O O O O I O O O O O O O 0 O O O O O 0 7o Experiment IX. Effect of Diethylstilbestrol on Urinary Incontinence . . . . . . . . . . . . . . . . . 71 Procedure . . . . . . . . . . . . . . . . . . . . . . 72 Results 0 O O O O O O O O O O O O O O O O O O O O O O 72 Discussion . . . . O O O O O O O O O O O O O O O O O O 72 Experiment X. Bacteriological Examination of Preputial Smears and urine . . . . . . . . . . . . . . 73 Procedure . . . . . . . . . . . . . . . . . . . . . . 73 Results . . . . . . . . . . . . . . . . . . . . . . . 73 Discussion . . . . . . . . . . . . . . . . . . . . . . 74 iv TABLE OF CONTENTS - Continued Page Experiment XI. Effect of Antibacterial Agents on Growth, urinary Incontinence and "we: Belly" . . . . . 74 Procedure . . . . . . . . . . . . . . . . . . . . . . 75 Results . . . . . . . . . . . ... . . . . . . . . . . 75 Discussion . . . . . . . ... . . . . . . . . . . . . . 78 Experiment XII. Histological Observations on the urinary sys ten 0 O O O O O O O O O O O I O O O O 0 O O 79 Procedure . . . . . . . . . . . . . . . . . . . . . . 79 Resul t8 0 O O O O O O O O O - 0 O O O O O O O O O O O O 80 Discussion . . . . . . . . . . . . . . . . . . . . . . 80 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . .94 LIST OF TABLES Incidence and size of "wet belly" area in relation to primeness of pelt in minkpelted at specific dates . Estimated value of raw and dressed mink pelts with respect to "wet belly" condition . . . . . . . . Composition and analysis of control ration . . . . Effect of dietary calcium on weights .of male mink fed the control and experimental rations . . . . . . . . Observations on urinary incontinence and "wet belly" in mink fed the control and experimental rations . . Composition and analysis of basal ration . . . . . . . Average weights of mink and observations on urinary incontinence and "wet belly" on specific dates . . . vi Page 35 54 67 68 69 76 77 Figure 1. 3. 4. 5. 6. 7. 9. 10. .11. 12. 13. 14. LIST OF FIGURES Flashed pelts of natural dark male mink showing inguinalregion ................. Mical symptoms of urinary incontinence in male fink l O O O O O O O O O O I O O O O O O O O O O 0 Low power microscopic view of "wet belly" area and adjacent normal prime mink skin . . . . . . . . . Cross sections of flashed mink skin from the inguinal area of "wet belly" and normal pelts . . Representative skins from natural dark mink pelted ‘t .P.c1£1c d.t.. O O O O 0 O O O O O O O I O O 0 Cross section of normal prime mink skin . . . . . Horizontal section of normal prime mink skin at the level of the sebaceous glands . . . . . . . Cross section of inguinal skin of "wet belly" u“ 0 O O O O O O O O I O O O O O O O O I O O O 0 Horizontal section of "wet belly" skin at the level of the sebaceous glands . . . . . . . . . . Cross section of "wet belly" mink skin showing the-concentration of pigment at the base of the h.1r £0111C1.‘ O O O O O O O C O O I O O O O O O 0 Cross section of inguinal skin of mink showing "wet belly" area and adjacent normal prime skin . Comparison of matched pairs of pelts before and after dressing and finishing . . . . . . . . . . . Urinary incontinence produced surgically. by transaction of the urinary sphincter muscle . . . Inguinal region of pelt from the mink that showed experimentally produced urinary incontinence . . . vii Page 24 26 33 38 40 42 46 49 52 61 63 LIST OF FIGURES - Continued Figure 15. 16. 17. ' 18. Page Cross section of the kidney cortex showing fatty metamorphosis and a decrease in the size of ”mu. 3 space 0 O O O ‘ O O O O O O O O O O I O O O O O C 81 Cross section of epithelial lining of prostatic urethra of "affected" mink showing signs of early squamous metaplasia . . . . . . . . . . . . . . . . . . 83 Cross section of penile urethra of "affected" mink in the area of the glans penis showing no apparent inflammatory changes . . . . . . . . . . . . . . . . . . 86 Prepuce of "affected" mink showing no apparent inflammatory changes . . . . . . . . . . . . . . . . . . 88 viii INTRODUCTION Since the beginning of recorded history, furs have been among man's most cherished possessions. They were sought by primitive man for the warmth and protection they afforded, but later desired for adornment and prestige. The taking of furs probably ranks as one of the oldest industries.in the world. During the exploration and colo- nization of this continent, the fur trade was extremely important to the economy of colonial America. Although wild fur-bearers were the only source of skins at that time, enterprising trappers soon realized the potential of fur farming and began raising some of the more valu- able fur-bearers under domestic conditions. Hunk farming in the united States had its beginning in 1866 (Kellogg, gtugl,,l948). At first, ranch-raised pelts were considered inferior to the wild-trapped furs and it was not until the 1920's that mink ranching achieved much economic importance (Coombes, _e_t_:_ a}: , 1950). Prior to the 1930's the silver for was king of the fur trade, but then as fashion trends shifted from the long-haired fox to the shorter- furred animals (Travis, 1960), mink emerged as the leader of the fur industry. During recent years, the wild mink take in the united States has been under $00,000 skins annually (Anon.,.l966b), while the production of ranch-raised mink steadily increased (Anon., 1965b). In 1965, ranch mink production in this country reached 8,225,000 pelts (Henderson, 1966). The majority of these animals were produced in the Great Lakes 1 2 region. Michigan consistently ranks among the leading states in mink production, producing over 356,000 pelts in 1965 (Henderson, 1966). Mink are by nature solitary and unsociable animals. Possibly because of this, they were plagued by few diseases in the wild. How- ever, when raised on ranches, they were subjected to unnatural condi- tions. th only were they forced to cope with unconventional husbandry practices, but their food habits were also modified. These unfamiliar diets and husbandry practices soon resulted in nutritional and disease problems (Leonard, 1966). Fortunately, many of the problems that bur— dened the pioneer mink ranchers have been solved through either trial and error or research conducted by industry and public institutions. Even though much has been learned about the habits and requirements of the mink, many problems still remain and new ones continually appear as the industry progresses. One of the moat perplexing problems plaguing the mink industry today is an ailment commonly called "wet belly". Although this malady has been a constant problem for the mink rancher since the birth of the industry (Lauerman, 1964), it was not as prevalent during the pioneer days of mink ranching as it is today (Leoschke, 1958; Kennedy, 1951). Since this disease is seldom fatal (Anon., 1955) and, in most instances, affected animals possess a good appetite (Pollack, 1959; Stowe, 25 21., 1959) and healthy appearance (Stowe, 1958; Stowe, 35 31. 1959), it probably attracted less attention during early times than such dreaded mink diseases as botulism, virus enteritis and distemper. As the incidence of the disease increased and_more emphasis was placed on the production of quality pelts, greater concern was expressed in the etiology and treatment of this disorder. Presently, "wet belly" 3 is considered to be one of the most common (Anon., 1962) and costly (Hartsough and Gorham, 1967; Gunn, 1966) ailments affecting ranch- raised mink. Since this has been a pressing_prob1em of the mink industry, the studies reported in this thesis were conducted. These studies consisted of several experiments pertaining to the nutritional, physio- logical and pathological aspects of the "wet belly" problem. REVIEW OF LITERATURE Description of urinary Incontinence and "we: Belly" "we: belly" is a term used by the trade to describe a condition that is denoted by a discoloration in the inguinal region on the leather side of the skin when the animals are pelted (Figure 1). This defect of the pelt is commonly associated with urinary incontinence in the live animal (Hood, 1956; Stowe, 1958; Lauerman, 1963, 1964) (Figure 2). It is generally assumed that urine from incontinent animals wets, stains and mats the fur in the area adjacent to the urogenital orifice and causes inflammation of the skin and underlying tissues (Gorham, st 21., 1960b; Schaible, 35 31., 1962). The constant soaking of the skin and fur with urine, and its decomposition products, is presumed to be responsible for the loss of hair and discoloration that appears in the inguinal region of the pelt (Gorham, gt 51.,.1960b; Lauerman , 1964) . Due to the relationship between the wet, stained, and matted fur in the live animal and the darkened discoloration seen in the pelt, the industry sometimes fails to distinguish between the two. Ranchers .refer to the condition in the live animal, as well as the discoloration in the pelt, as "wet belly" which leads to imperspicuity. Thus, in an effort to avoid confusion, the term urinary incontinence is used in this thesis when reference is made to the condition in the live animal 4 Figure l. Fleahed pelts of natural dark male mink showing inguinal region. Left -- normal prime mink. Middle and right -- two different degrees of "wet belly". Figure 2. Typical symptoms of urinary incontinence in male mink. 9 and "wet belly" is used to denote the defect that appears in the pelt, as has been suggested by Schaible, 35 31., (1962). This disease has also been referred to as nutritional anemia (Chaddock, 1952; Gorham and Griffiths, 1952), paraphimosis (Chaddock, 1952), sheath trouble (Secord, 1936; Chaddock, 1952), disease of the sheath (Hegner,.l936), watery hide (Chaddock, 1952) and "acidosis" (Secord, 1936; wegner, 1936; Chaddock, 1952; Anon., 1955; Leoschke, 1955,71957). Although females may occasionally be affected (Gunn, 1962), this disorder occurs predominately in male mink (Schaible, £5 31., 1962; Stowe, 1958). Lauerman (1965) suggests that the anatomical structure and physiological function of the male predispose it to this condition. It is not uncommon for 10 percent or more of the males on a ranch to be affected (Gorhmm, st 31., 1960b), and incidences as high as 30 (Gunn, 1962) and even 50 percent (Anon.,.1964) have been reported. Since the discolored parts of the skin are considered useless by the furrier (Anon., 1955,.1966d) they may reduce the value of the pelts by as much as.one-third (Anon., 1955). Pelt damage attributed to "wet _belly", therefore, annually amounts to many thousands of dollars (Dederer, 1955; Leoschke, 1957). This malady occurs throughout the world (Schaible, gt al.,.1962) but is more frequently encountered in certain areas (Anon., 1963b). Although its severity varies from year to year (Lauerman, 1964), it is considered to be more prevalent during recent years (Kennedy,.1951; Leoschke, 1958). Roberts (1959) suggests that the recent increased incidence of this condition may be due to the use of higher levels of bone free packing house by—products in mink rations. 10 Etiology A.review of the literature on urinary incontinence and "wet belly" in mink indicates that this disease is a complex problem. Al- though it is generally agreed that there is a high positive correlation between urinary incontinence and "wet belly", some recent research (Stowe, £5 31., 1959; Schaible, st 51,, 1962) has failed to show a consistent relationship between these two conditions. However, exten- sive studies at Oregon State University (Anon., 1962, 1964) have demon- strated a definite relationship between the degree of urinary incon- tinence in the live animal and the severity of the "wet belly" defect in the pelt. various forms of urinary incontinence resulting from juvenile sex play, urolithrosis, paresis, hyperexcitability and faulty nutrition —have been.mentioned in the mink (Stowe, 1958), and may be predisposing factors associated with this condition. Hood (1956) infers that urinary calculi are directly responsible for incontinence and the resulting pelt damage. Schaible, gtugl, (1962), however, doubt that these types of incontinence produce pelt damage as seen in mink skins affected with "wet belly". The urinary incontinence associated with "wet belly" is IIOBt frequently noted at the onset of cold weather prior to pelting (Leoschke, 1957, 1961b,.1962a,.1962b; Stowe, 35 31., 1959; Schaible, 31531., 1962; Gunn, 1962; Anon., 1966) and during thebreeding season (Kennedy, 1951; Leoschke,.1961a,1961b, 1962a). Therefore, 1: appears that the type of incontinence responsible for "vet belly" may be dif- ferent from the causes of incontinence previously mentioned. 11 Many factors implicating nutrition, genetics, physiology and bacteriology have been suggested as a cause of the urinary incontinence that presumably produced "wet belly". Nutritional Aspects Studies by Leoschke (1957, 1959) have shown that there is a direct relationship between the amount of fat in the ration and the incidence-and severity of "wet belly". Lowering the fat level of the diet, however, did not completely cure the condition (Leoschke, 1959). Recent research at Oregon State university (Anon., 1962) has confirmed Leoschke's work, and also indicated that there-is a positive correla- tion between the final weight of the mink and the occurrence of "wet belly". Large fat males were more frequently affected with this con- dition (Anon., 1962; Lauerman, 1965), which suggested that the disorder may be more directly related to total caloric intake than to the per- centage of fat in the ration. Gunn (1966) reported that he was unable to demonstrate an increased incidence of urinary incontinence or "wet belly" with mink rations supplemented with high levels of fat (lard). It has been suggested by Pollack (1959) that urinary incontinence may be due to a specific allergy of certain mink to high fat levels in the diet. Leoschke (1959) postulated that mink affected with the dis- case were unable to metabolize fat efficiently. After noting that the surface tension of urine from affected mink was lower than in non- affected animals, he proposed that abnormal products of fat metabolism (dicarboxylic acids) may be responsible for urinary incontinence by reducing the surface tension of the urine allowing it to infiltrate the fur around the urethral orifice (Leoschke, 1961a). Leoschke (1961a), however, was unable to produce the condition in normal mink by feeding 12 dicarboxylic acids or lower the surface tension of normal mink urine by saturating it with these acids. Stowe and Travis (1960) were unable to confirm Leoschke's observation that the urine surface tension was lower in affected mink. Leoschke (1962b) later stated that there was no correlation between urinary excretion of free fatty acids or fats and the incidence and degree of urinary incontinence in the mink. The influence of calcium, and more specifically the ratio of calcium and phosphorus, on urinary incontinence was first suggested by Roberts (1959). He stated that improper calcium-phosphorus ratios enhanced the disease, and suggested that on high fat diets calcium might be lost via the feces as insoluble calcium salts of fatty acids which could lead to a calcium deficiency causing incontinence. Al- though no work has been reported on mineral excretion via the feces in mink, research by Leoschke (1962b), on the excretion of calcium, phos- phorus and magnesium via the kidneys, indicated there was no clear correlation between the percentage of these minerals excreted and the degree of urinary incontinence. Schaible, st 51. (1962) fed mink diets consisting of various amounts and ratios of calcium and phosphorus. A.high incidence (25 percent) of urinary incontinence was obtained from diets containing a high ratio of calcium to phosphorus (2:1). With calcium-phosphorus ratios of 1:1 or less than 1:1, only 13 percent of the animals showed incontinence. Other investigators (Anon., 1962, 1964) also noted an increase in the disease when the amount of calcium in the ration was high in relation to its phosphorus content, and concluded that the wider the calcium-phosphorus ratio the greater the incidence of the condition. 13 The feeding of spoiled feed to mink has been frequently assumed by ranchers.to cause this disease. To test this assumption, Schaible, 3531. (1962) held typical mink diets, as well as supplemental fat at 90° F. for three and ten days respectively before feeding it to mink. No symptoms of the disease occurred indicating that this malady prob- ably was not due to adverse conditions in storing and handling of mink feeds. Also acting on the suggestion of mink ranchers that this dis- order appeared to be associated with diets containing high levels of poultry by-products, Leoschke (1962b) fed mink a diet containing 70 percent entrails and heads from old hens. Although a 50 percent in— cidence of the disease occurred in the mink on this diet, the results appear questionable since the animals all had a previous history of urinary incontinence. Gunn (1962) reported a high incidence of the condition on rations containing high proportions of raw chicken waste, whole fish and tripe. Aulerich and Schaible (1965) noted a direct relationship between the percentage of "spent" hens in the ration and the incidence of "wet belly", but suggested and later (1966) showed that this was apparently due to the higher fat levels of the diets containing the "spent" chickens. Secord (1936) reported that this malady was common on ranches that fed diets consisting entirely of meat, fish and cereals without adequate minerals and vitamins supplied by bone meal and fresh vege- tables. He suggested that canned tomatoes and cod liver oil be in- cluded in the ration during the time of the year when fresh vegetables were unavailable. 14 Helgebostad attributed the disease to a boitin deficiency (Gunn, 1962). Stowe and Travis (1960) reported that the condition was not observed in uncomplicated vitamin A and E deficiencies. Other dietary factors that have been proposed to account for or exacerbate this ailment include, high meat rations (Wegner, 1936), high protein diets (Anon.,.1955), lack of liver in the ration (Kennedy, 1951; Gorham.and Griffiths, 1952), whole fresh-water fish (Belcher, 3331., 1959) and nutritional imbalance (Chaddock, 1952; Anon.,.1966d). Genetic Aspects Heredity has been implicated in the urinary incontinence -- "wet belly" syndrome by the fact that certain color phases (pastel, sapphire, white (Leoschke, 1961b), and natural dark (Anon., 1963b)), strains (Anon., 1963b,.1964, 1965a), and families (Lauerman, 1964) of mink have been reported to be especially prone to this disease. In experiments at Oregon State university (Anon., 1965a) pertaining to the nutritional and genetic aspects of this malady, it was shown that ... " (1) although nutrition influences extent and severity of W B (”wet belly"), genetic factors are overriding; (2) nutrition has its greatest influence in genetically-susceptible strains and (3) geneti- cally resistant strains are unaffected by nutritional variables." The pattern or mechanism of inheritance of this disorder has not yet been determined. Physiological Aspects In observing mink affected with urinary incontinence, Leoschke (1959) noted that urine flow appeared normal, although the last few drops of urine tended to infiltrate into the fur around the urethral orifice. He, however, reported that urine volume from affected animals 15 was less than in normal mink (Leoschke, 1962b). This fact was con- firmed in studies at Oregon State University (Anon., 1966c), and it was also shown that the specific gravity and dry matter content of urine from incontinent mink was greater than in normal animals indi- cating that a more concentrated urine is excreted by mink affected with this condition. Leoschke (1962b) postulated that because of a decreased water intake this disorder might be due to certain concentrated substances present in the urine. He (Leoschke, 1962b) noted that an increased water intake tended to ameliorate the condition. However, no signifi- cant differences have been found between the urine of males affected with urinary incontinence and that of normal animals with regard to urinary pH (Leoschke, 1955; Gunn, 1962; Anon.,.1966c), ammonia nitrogen (Leoschke, 1955), creatinine (Leoschke, 1955), albumin (Gunn, 1962) or glucose (Gunn, 1962; Anon., 1966c). The urine of both affected and normal males was also reported negative for acetone, bilirubin and phenylketonuria (Anon., 1966c). In an attempt to determine whether the “wet belly" discoloration was due to an accumulation of urine in the fur, Conant (1962) daily saturated the fur of normal mink with urine but was unable to demon- strate a discoloration in the pelt. Likewise, Oregon State university workers found no characteristic "wet belly" symptoms in pelts from animals that had been treated daily with either distilled water, urine collected from incontinent males or urine from males that had no his- tory of urinary_incontinence (Anon., 1964). However, in repeating the experiment and applying the treatments directly on the skin, after re- moving the fur, they noted a discoloration in the pelts of three of 16 the five mink treated with urine from affected males (Anon., 1965a). The animals that were treated with distilled water or urine from.normal males showed no evidence of "wet belly". These results suggested that the underlying cause of "wet belly" may be associated with abnormal urine composition. In more extensive trials the following year, each mink was shaved in two areas and treated daily with urine from affected males in one area and normal urine in the other (Anon., 1966c). The results of this trial were inconclusive, as all the animals showed a general unprimeness in both areas so that no differences due to the type of urine applied were evident. A.similar trial using white lab- oratory rats was conducted to determine the effects of both types of urine on rat skin. No skin irritation was noted and histological ex- amination of sections from the treated areas of rat skin revealed no differences in the epithelium, thickness of the dermis, number, size and depth of the follicles (Anon., 1966c). Blood analyses of mink affected with urinary incontinence gave the following average values, hemoglobin 18 gm per 100 m1, erythrocytes 9.7 million per cu mm, leukocytes 8700 per cu mm (neutrophils 25 per- cent, lymphocytes 67 percent, monocytes 3 percent and eosinophils 5 percent), and were not significantly different from normal mink (Schaible,.egngl.,.l962). Based on the facts that urinary incontinence is generally more prevalent in the fall, concurrent with testicular development in the males, and because during the breeding season the most aggressive males frequently exhibit this condition, Leoschke (1961a) postulated that it may be due to excessive male hormone production in certain animals. This postulate was tested by feeding mink methyl testosterone and a 17 synthetic male hormone (Leoschke, 1962a). The results indicated that there was a significant increase in both urinary incontinence and "wet belly" on diets that contained either of these hormones. A second trial the following year, however, failed to substantiate the previous find- . ings, and attempts to treat affected males with female hormone (diethylstilbestrol) were also unsuccessful (Leoschke, 1962b). Other investigators (Anon., 1964) subjected female mink to weekly injections of either five or ten mg of testosterone and reported a trend toward a direct relationship between the incidence of the disease and the level of testosterone injected. However, more extensive trials the next year gave inconclusive results (Anon., 1965a). These re- searchers (Anon.,.l965a) also tried to determine the effect of a re- duction of androgens on this disorder in the male. This was attempted through castration or estrogen injections. Their results were incon- clusive since the untreated controls were 100 percent affected, the castrated males showed an 80 percent incidence of the condition, and‘ eight out of the ten animals injected with estrogen died as a result of severe urinary tract infections. Bacteriological Aspects Bacteriological investigations by Gunn (1962) indicated the presence of Proteus organisms in the prepuce cavity in nine of ten male mink affected with urinary incontinence. no Proteus organisms were re- covered from unaffected males.a1though Staphylococcus, Streptococcus and Salmonella were occasionally found in both normal and affected mink. Agglutination tests, conducted with blood sera of mink that had recovered from the condition, showed high antibody titres (160 - 1,280) to the strain of Proteus mdrabilis taken from their prepuce cavities. 18 Similar tests carried out with the sera of normal males fed the same ration gave negative results. Proteus mirabilis was also recovered from the feces of animals that had recovered from the disease and gave similar morphological, cultural and biochemical tests, and agglutinated to the same titre with the same mink sera as Proteus mirabilis obtained from the prepuce cavities. Gunn (1962) further reported high concen- trations of Proteus mirabilis, in raw chicken waste, whole fish and tripe from the diet, which gave the same bacteriological tests and were agglutinated by the sera of mink that had recovered from the disease. The incidence of urinary incontinence was reduced 50 percent by cooking the ration containing_chicken waste, fish and tripe, 44 percent by freezing (-100 to 20° F.) it, and 50 percent by feeding a standard horsemeat ration contaminated daily with Proteus mirabilis (Gunn, 1962). These results indicated to Gunn that the disease origi- nated via the intestinal tract, was of an infectious nature and was not caused by some nutrient factor(s) pe£_§e_in the ration (Gunn, 1964). In a similar study, Lauerman (1963) observed that both Proteus mirabilis and Streptococcus fecalis were associated with urinary in- continence. Pelt damage ("wet belly"), however, was not found to be associated with Proteus, Staphylococcus or Streptococcus nor Proteus or Staphylococcus serology (Lauerman, 1963). Schaible, 35 21., (1962) reported that bacteriological examina- tions of spleen, liver and heart, of three mink showing advanced urinary incontinence, were negative when cultured on blood agar media. In connection with his bacteriological observations, Gunn (1966) postulated that the stress of cold weather lowers the mink's 19 resistance and predisposes it to infection that causes the disease. He reported that circumcision of males did not prevent the condition (Gunn, 1966). He did note, however, that housing mink at warm tempera- tures (60 - 70° C.) prevented the occurrence of the symptoms of the disorder and stated that when the same animals were subjected to temperatures of 30 to 35° F., 45 percent of them developed urinary incontinence (Gunn, 1966). Studies (Anon., 1966) conducted to determine the effect of anti- bacterial agents (Gentamicin and Mandelamine) on the incidence of urinary incontinence and "wet belly" indicated that these compounds had no effect in preventing the symptoms of the disease. Patholggieal Aspects According to Schaible_g§ugl., (1962), histological examination of tissues from.the nervous, excretory and digestive systems of six males affected with urinary incontinence revealed no infective agent. Lauerman (1963) found only local preputial inflammation in some affected animals and no pathologic lesions of the urinary system. Suggested Treatments Although many factors have been postulated to be responsible for, or influence, the incidence and severity of this disease, no satisfactory explanation has yet been found (Hartsough and Gorham, 1967). A.number of attempts have been made to treat the ailment, but only a few have had any influence on its occurrence or severity, and none have been entirely satisfactory in curing the malady. Treatment of affected mink with choline, betaine, thiamine, riboflavin, pyridoxine, niacin, calcium pantothenate, folic acid, 20 biotin, inositol, vitamins B and E, sodium bicarbonate, potassium 12 bicarbonate have been ineffective (Leoschke, 1959). Likewise, treat- ‘ment with dicalcium phosphate, magnesium sulfate, lipotrophic factor, fumarate, aspartic acid, methionine, .asparagineq, cysteine, bone meal, milk powder, bran and wheat germ, antibiotics, sulfa drugs, and genito- urinary antiseptics have not proven beneficial (Gunn, 1962). Other treatments that have been suggested include, feeding tomatoes (Secord, 1936; Smith, 1940; Lalor,_e£ual., 1952; Leonard, 1966), lemon juice (Dederer, 1955), apples (Stowe, 35 11., 1959), wheat germ (Leoschke, 1958; Stowe, et 21,, 1959), cod liver oil (Secord, 1936), liver (Kennedy, 1951; Gorham, eg 31., 1960a) and ammonium chloride (wood, 1956). Also, a complete change of diet (Stowe, 95 31., 1959), use of fresh fish and meat products (Leonard, 1966), decreased fat level and increased carbohydrate content of the diet (Leoschke, 1958; Roberts, 1959; Gorham,_e£”al,, 1960a, 1960b; Leonard, 1966), increased per— centage of horsemeat in the ration (Lalor, 35 31., 1952; Stowe, £5 51., 1959), increased water intake (Gorham, 35 21., 1960a; Anon., 1966a; Leonard, 1966), early separation of kits into individual cages (Gorham, 35 51,, 1960a), increased sanitation (Leonard, 1966), warmer housing (Gunn, 1964) and careful selection of breeding stock (Leonard, 1966) have all been recommended as a remedy for, or a method of re- ducing, the incidence and severity of this disease. METHODS Except where stated otherwise in the text, the following pro- cedures pertain to all the experiments. The animals employed in these studies consisted of natural dark, pastel and sapphire mink from the Michigan State university experimental mink ranch. Mink were confined in individual pens of uniform size and each cage was equipped with a water cup and nest box. Management pro- cedures were similar to those of a well-run commercial mink ranch. The mink.were fed twice daily starting in May, when the young were born, and continuing throughout the summer. During the fall, winter and early spring, they received one feeding daily. A11 matings were checked for the presence of live sperm. Litters were separated into individual cages at eight weeks of age. weights and observations were recorded periodically as noted in the various experiments. In alloting the mink into various groups for experimentation, littermates were divided between groups in an effort to balance genetic differences in growth, fur quality and possible susceptibility to urinary incontinence and "wet belly". . All the mink on the ranch were vaccinated each year (July) against botulism, canine distemper and virus enteritis. Casualty animals were submitted to the Pathology department for necropsy to determine the cause of death. At pelting time, the animals were killed by cervical dislocation and skinned. The pelts were fleshed and dried using routine ranch procedures. 21 and 1966. EXPERIMENTS The research reported in this thesis was conducted between 1961 Experiment Experiment Experiment Experiment Experiment Experiment Experiment Experiment Experiment Experiment Experiment Experiment II. III. IV. VII. VIII. IX. XII. It consisted of experiments as follows: Observations on "wet belly". Effect of time of pelting on "wet belly". Histology of "wet belly" skin. Effect of fur dressing on "wet belly". Effect of urine on "wet belly". Effect of mineral oil on "wet belly". Attempted surgical production of urinary incontinence. Influence of dietary calcium on growth, urinary incontinence and "wet belly". Effect of diethylstilbestrol on urinary incontinence. Bacteriological examination of preputial smears and urine. Effect of antibacterial agents on growth, urinary incontinence and "wet belly". Histological observations on the urinary system. 22 23 Experiment 1 Observations on "Wet Belly" This study was conducted to obtain basic information on the nature of the "wet belly" defect in the pelt. Procedure Skin samples were taken from the inguinal area of normal and "wet belly" pelts for gross microscopic examination and histological comparison. The tissues for histological study were fixed in ten per- cent formalin, dehydrated and embedded in paraffin. Sections were cut at six microns, mounted on slides, stained with hematoxylin and eosin and examined with a compound light microscope for any histological dif- ferences or pathological lesions. Results It was noted that after mink pelts had been fleshed and dried, the "wet belly" discoloration was very conspicuous in natural dark (mink. In the lighter colored sapphires it was less evident and appeared only slightly discolored. When the affected areas were observed through a binocular dissecting microscope it was apparent that the discolora- tion on the leather side of the pelts was due to the pigment in the hair follicles (Figure 3). Tissue sections made from the inguinal region of the "wet belly" and unaffected normal area of a natural dark mink pelt are shown in Figure 4. As shown in these photomicrographs, the hair follicles in the "wet belly" area are distributed through the dermis; whereas, in the section from the normal area the hairs originate higher in the dermis close to the fur side of the pelt. Figure 3. 24 Low-power microscope view of "wet belly" area and adjacent normal prime mink skin. unstained X—27. _ _— ‘—.*—_‘—_— 26 Figure 4. Cross sections of fleshed mink skin from the inguinal area of "wet belly" (above) and normal (below) pelts. Hematoxylin and eosin. x-19 28 Morphologically, the skin and fur of the "wet belly" area showed no changes of a pathological nature. Both structurally and functionally the tissue appeared similar to that of normal mink skin prior to priming. Discussion As noted in the results, both natural darks and sapphires are subject to the "wet belly" condition, although it is much more notice- able in the dark mink. The lightness in color of the sapphire fur made the affected area more difficult to see. Mink skin, like that of other mammals, is divided into a super- ficial epithelial layer (the epidermis), a subadjacent vascular con- nective tissue area (the dermis), and a deeper subcutaneous layer of loose connective tissue (the hypodermis) (Maximow and Bloom, 1957; Trautmann and Fiebiger, 1957; Copenhaver and thnso , 1958; Arey, 1963). The fur consists of straight and intermediate guard hairs, as well as shorter fine hairs (underfur) (Dolnick, 1959a, 1961). Guard hairs arise from the larger primary hair follicles and the underfur from the -more numerous smaller secondary follicles. Associated with each straight guard hair is a pair of intermediate guard hairs and several underfur hairs, all of which emerge through the surface of the skin from a common opening (Dolnick, 1959b). Continuous changes occur in the skin and hair during normal growth and maturation of pelage. Fur grows in cycles or waves (Chase and Eaton, 1959). The length of the follicles and the position of the hairs within the skin changes with the-life cycle (Chase, 1959; Montagna and Ellis, 1959). Follicular position in the skin depends on the state of priming. During the growing period, when the fur is unprime, the follicles are 29 located deep within the dermis. At this time, the pigment in the developing follicles can easily be seen from the leather side of the fleshed pelt. In the darker color phases this pigment imparts a very dark appearance to the skin; whereas, in the lighter colored mutations only a slight discoloration is noticeable. As maturation of the fur progresses, follicular proliferation slows and then ceases (Dolnick, 1959b; Chase and Eaton, 1959). The hair bulbs, located at the base of the follicles, kerattoize and become solid club-shaped masses. These club—shaped bulbs, along with their inner epithelial root sheaths, are then gradually forced upward by proliferation of the undifferentiated cellular matrix (Maximow and Bloom, 1957; Trautmann and Fiebiger, 1957; Copenhaver and Johnson, 1958; Arey, 1963) to a level just below the main body of the sebaceous glands. During this priming process, the hypodermis and lower portion of the dermis become richly infiltrated with fat. The resultant fatty layer is called the panniculus adiposus and is the portion of the skin that is removed during the flashing process. The koratinizcd hair follicles that have migrated into the upper regions of the dermis during the priming process are called club hairs so as to distinguish them from those that still have a well- formed papillae and are located deep within the dermis. When the club hairs have reached the upper portion of the dermis (just below the level of the main body of the sebaceous glands), the fur is considered prime. The club hairs remain at this level until they are shed or forced out by new developing hair. Meanwhile, the papillae of the primary and secondary hair follicles atrophy and the outer epithelial root sheaths collapse forming thin strands of epithelial cells extending from the 30 atrophied papillae to the lower ends of the elevated club hairs. These strands of cells shorten and draw the remnants of the papillae toward the club hairs where they remain until the latter are replaced (Maximow and Bloom, 1957; Trautmann and Fiebiger, 1957; Copenhaver and Johnson, 1958; Arey, 1963). Due to the movement of the follicles into the upper dermis and the-migration of the pigment into the periphery of the hair shaft during the maturation process, the fleshed side of a prime pelt is creamy- white in appearance. In describing the progressive development of the winter coat of the-mink, it was stated that new hairs gradually spread up the tail, progress over the hips, aides, back, shoulders, neck and head (Bassett and Llewellyn,-l949; Travis and Schaible, 1960). Changes in the fur of the inguinal area are not mentioned. Tb tell when mink are prime, ranchers pay special attention to the area between the ears and about the head since this region is believed to be the last to change. If the fur appears thick, dense, and glossy, with no dustiness, and the skin is.creamy-white, ranchers assume that a mink is prime.over its entire body. It is generally recognized that various strains of mink do not become prime at the same time; therefore, some ranchers pelt a few sample-mink before killing the rest of the herd. TWenty or 30 years ago, all pelting was completed in November -— in many instances in early November. Now, some ranchers do not even start pelting until December 1. Possibly the practice of late pelting has resulted in singling out strains of mink that are genetically late primers (Hartsough, 1961). Selection of breeders at a later date each 31 year may well have caused this to happen without the ranchers realizing the-gradual change over the years. Since no changes of a pathological nature were noted in the skin and fur and the affected area appeared to be similar to normal skin prior to priming, possibly the inguinal area primes later than the region around the head. If so, a delay in pelting "wet belly" sus- ceptible-animals would give the mink more time to prime in this area which might eliminate or minimize the defect. Experiment II Effect of Time of Pelting on "Wet Belly" It was suggested in Experiment I that possibly the fur in the inguinal region primes later than in the head area, which is generally considered by ranchers to be the last area to become prime (Travis and Schaible, 1960). If so, a delay in the time of pelting might minimize the presence of "wet belly" by allowing additional time for the fur in the inguinal area of affected animals to prime. To ascertain the validity of this theory, the following experiment was performed. Procedure Thirty-six natural dark male-mink were selected for this experi- ment. Beginning November 15 and terminating December 20, six mink were sacrificed weekly. The pelts were fleshed and dried, and the size of any "wet belly" area measured. Observations were also made on the primeness of the entire pelt. Sections for histological examination were prepared from repre- sentative tissue samples of primed and unprimed areas of normal and "wet belly" pelts. Comparative observations on the structure and 32 degree of maturation of the skin and hair follicles were made at the -various pelting dates. Results The leather side of representative skins from the 36 mink pelted at weekly intervals between Nbvember 15 and December 20 is shown in Figure 5. These pelting dates were several weeks before and after the usual.pe1ting time in this geographic vicinity. Four of the six mink pelts taken November 15 were definitely unprimed (Table l) and, when fleshed, exhibited discolored leather. The leather side of the other two pelts was lighter in appearance, and histological examination re- vealed that the hair follicles had commenced to retract into the upper layers of the skin, indicating that priming was in progress. These Islightly primed pelts were from the largest and smallest mink of the ~group. Thus, it would seem that the order of priming was not in- fluenced by body size. All six mink pelted on November 22 showed some degree of prime— ness. They appeared to be in a transitional stage, but not far enough along to be considered prime. By Nbvember 29 and thereafter, all the skins were prime and displayed the creamy-white leather indicative of primeness. It is readily apparent from Table l that_1ate pelting (December 13 and 20) had no effect in eliminating or diminishing the incidence or severity of "wet belly". The defect was as frequent in pelts taken after the usual pelting time as before it. The size of the affected area of the pelts also appeared to be uninfluenced by extending the time of pelting. Figure 5. 33 Representative skins from natural dark mink pelted at specific dates. NOV. l5 NOV. 22 NOV. 29 DEC. 6 DEC. l3 DEC. 20 35 Table l -- Incidence and size of "wet belly" area in relation to prime- ness of pelt in mink pelted at specific dates Date and Area of Condition of Date and Area of Condition pelt "wet belly" pelt pelt "wet belly" of number (sq. in.) number (sq. in.) pelt Nev. 15 Dec. 6 1 (a) Uhprime l9 0 Prime 2 (a) Uhprime 20 6.0 Prime 3 (a) Uhprime 21 1.0 Prime 4 (a) Slightly prime 22 0 Prime 5 (a) Uhprime 23 0 Prime 6 (a) Slightly prime 24 6 6 Prime Av. 4.5 Nov. 22 Dec. 13 7 0 Semi~prime 25 4.7 Prime 8 12.0 Semi-prime 26 0 Prime 9 2.0 Semi-prime 27 9.0 Prime 10 0 Semi-prime 28 3.8 Prime 11 1.9 Semi-prime 29 3.1 Prime 12 4 5 Semi-prime 30 1.0 Prime Av. S 1 Av. 4 3 NOV. 29 Dec. 20 13 O Prbme 31 0 Prime 14 10.5 Prime 32 0 Prime 15 0 Prime 33 1.2 Prime 16 4.4 Prime 34 18.0 Prime 17 1.0 Prime 35 2.5 Prime 18 2.3 Prime 36 8.3 Prime Av. 4.6 Av. 7.5 (a) Unable to determine due to unprimed condition of pelts. 36 Discussion The results of this experiment illustrate the celerity of the priming process. The majority of the pelts examined on Nevember 15 showed no evidence of priming, yet, in only two weeks all the mink appeared fully primed. Histological studies on these pelts confirmed previous observa- tions regarding the similarity of "wet belly" areas and normal unprimed skin. Pigmented hair follicles penetrating through the fleshed side of the skin were responsible for the darkened discoloration evident in both these conditions. Since neither the incidence nor size of "wet belly" was in- fluenced by the delayed pelting, it would seem that the discoloration of the defect is not a simple condition of protracted tissue and fur maturation. It probably involves other complicating factors in con— Junction with the normal mechanisms of the priming process. Experiment III Histology of "wet Belly" Skin Based on the observations made in Experiments 1 and II, this study was undertaken to more thoroughly investigate the possibility of any histopathological differences between the skin in the inguinal region of "wet belly" and normal male mink. Procedure During the normal pelting season (November 27 to December 10), samples of skin were taken from the inguinal region of five natural dark and pastel male mink that exhibited a typical "wet belly" dis- coloration and from five unaffected animals. Similar tisSues from six 37 affected and six normal males were also obtained for study from the Oregon State university mink ranch. The skin samples were fixed in ten percent buffered formalin for 24 hours or more, dehydrated in dioxane and then embedded in Paraplast under vacuum (15 mm. Hg). Tissue sections were cut at 6 to 8 microns, mounted on slides and stained with hematoxylin and eosin. Results and Discussion Microscopic examination of the skin from the inguinal region of normal prime mink showed hair follicles in the form of club hairs 'located in the upper part of the dermis near the level of the main body of the sebaceous glands (Figure 6). The papillae of the club hairs had atrophied and were situated in the upper portion of the re- ticular layer of the dermis. Most of the pigment was located in the outer portion of the hair shafts (Figure 7) and, therefore, was not visible from the leather side of the pelt. Grossly, the prime skin had a smooth, creamy-white leather. Sections from the discolored area of the "wet belly" pelts showed hair follicles located deep within the dermis (Figure 8). The follicles had not migrated into the upper regions of the dermis, as in the normal skin during priming. No atrophy of the papillae or ‘keratinization of the root sheaths had taken place, and very few club hairs were evident. Most of the pigment was still concentrated at the base and lower portion of the hair follicles (Figures 8,,9 and 10) and had not migrated to the distal portion of the hair shafts. Thus, when the hypodermis and the lower portion of the dermis were removed during the fleshing process, the base of the hair follicles was exposed to the underside of the skin. Grossly, this gave a dark appearance to the skin Figure 6. 38 Cross section of normal prime mink skin (inguinal region).' 1. Dermis; 2. Sebaceous glands; 3. Panniculus adiposus. Hematoxylin and eosin. Xr48 40 Figure 7. Horizontal section of normal prime mink skin at the level of the sebaceous glands. Hematoxylin and eosin. Xr154 Figure 8. 42 Cross section of inguinal skin of "wet belly" mink. l. Dermis; 2. Sebaceous glands. Hematoxylin and eosin. X—48 a ‘n I '\ i . u " ‘ ' “U: a ‘ . s . . ‘0 .._ Niel"... ,. 0 McDM. wt.‘t\ \ K . o . . uc’fi ,i.’ V? n.. . . .Jl.lv(.l!.§\' ......1.._.\.V»Xn.mro . . ..r.\.\..o .... I Figure 9. 44 Horizontal section of "wet belly" skin at the level of the sebaceous glands. HBmatoxylin and eosin. Xr154. 46 Figure 10. Cross section of "wet belly" mink skin showing the -concentration of pigment at the base of the hair follicles. Hematoxylin and eosin. X300 48 in the affected area because most of the pigment was still contained in the base of the hair follicles and lower portions of the hair shafts (compare Figures 6 and 7 with Figures 8, 9 and 10). Sections of skin from areas adjacent to the "wet belly" discoloration were nor- mal and fully prime as shown in Figure 11. No inflammatory changes or pathological lesions were observed in any of the "wet belly" sections, except varying degrees of hyper- keratosis in the stratum corneum of the epidermis. These observations confirmed the findings of the previous study (Experiment I) and supported the postulate that "wet belly" is an area of unprimed skin. Experiment IV Effect of Fur Dressing on "wet Belly" Mink pelts showing the "wet belly" defect represent a substan- tial portion of the pelts marketed. When sold at auction, these pelts bring considerably reduced prices (Anon., 1955). It is claimed by fur buyers that the fur around the urethral orifice of affected pelts is stained and not anchored firmly. Therefore, this area must be re- moved prior to use of the pelts by the furrier. This experiment was conducted to investigate this pretension and to obtain information on the gross appearance of the skin and fur «of "wet belly" pelts subjected to commercial dressing and finish procedures . Procedure Sixteen pelts from natural dark male mink were selected from Several hundred skins and placed in four lots. Lot Icontained normal iA _ 4‘ u. nah...“ Figure 11. 49 Cross section of the inguinal skin of mink showing “wet belly" area and adjacent normal prime skin. 1. Nermal prime skin; 2. Skin affected with "wet belly". Hematoxylin and eosin. Xrl9. 51 pelts with a darkened discoloration around the urethral orifice of 0.4 sq. in. or less. Lots 11, III and IV had "wet belly" areas in the inguinal regions of approximately 2.5, 7 and 12 sq. in., respec- tively. TWO pelts from each lot were dressed and finished by a commer- cial furrier. The 16 raw and dressed pelts were then sent to an auction company for an estimation of their value. A comparison of the value of the skins and their gross appearance was made with respect to other pelts within and between the lots. Tests were conducted on the fur in the inguinal area of the normal and affected pelts to determine if the fur from the "wet belly" area is permanently discolored and not firmly anchored. Results A comparison of the corresponding raw and dressed pelts from each lot is shown in Figure 12. It can be readily seen from the figure that the darkened "wet belly" area of the raw pelts was drastically diminished by the dressing and finishing processes. An evaluation of the four normal and 12 "wet belly" pelts by the auction company is shown in Table 2. It is evident from this table that the values placed on the pelts had no apparent relationship to the presence, absence or size of the "wet belly" area. Tests conducted on the fur in the affected region of the dressed .and finished skins showed that the hair appeared to be firmly anchored. It withstood pulling and did not slough off. Individual hairs invar- 1ab1y broke off above the skin when attempts were made to pluck them .from.the pelt. Even in the severely affected pelts, the fur did not appear to be any more brittle than that in the normal skins. hcept kw" w: $.34“ Figure 12. 52 Comparison of matched pairs of pelts before and after dressing and finishing. Lot I. Nbrmal pelts; Lots II, III, and IV. Increasing intensity of "wet belly" condition. Raw pelts are shown at the bottom of the picture; dressed pelts at top. Lot I Lot 11 I Lot 111 I Lot IV 54 in the very severe cases of "wet belly" where the fur was also badly stained, it was difficult to tell it grossly from similar fur on normal pelts after dressing and finishing. Table 2 -- Estimated value of raw and dressed mink pelts with respect to "wet belly" condition Approximate area of value in dollars Lot "wet belly" . (sq. in.) Raw pelts Dressed pelts I 0.4 25 20 0.4 20 18 Average... 22.50 19.00 II 2.5 15 34 2.5 14 24 Average... 14.50 29.00 III 7.0 23 30 7.0 17 24 Average... 20.00 27.00 IV 12.0 17 26 12.0 16 28 Average... 16.50 27.00 Discussion As described in Experiment I, there is a profound difference in the manner in which the fur is anchored in unprime and prime mink pelts. In unprima pelts, the base of the hair shafts appear bulb-like and are contained in individual follicles located deep in the skin (hypodermis). In the prime pelts, the shorter follicles are in the upper region of the skin close to the surface, and the hairs appear brush-like or club- like. Both the actively growing and mature hairs are surrounded by several sheaths and penetrate the skin on an angle (Dolnick, 1959a, 1961). 55 In live animals, actively growing hairs resist plucking. Only hairs that are about to be shed pluck easily; all others offer resist- ance. Morphologically, there was no indication that unprimed furor fur from the “wet belly" area should be less firmly held by supporting tissues than primed fur. The results of this study indicated that changes in the thickness of the skin, integrity of the elastic fibers and other factors that might be influenced by the dressing and finishing processes, did not appear to affect the anchorage of the fur in the normal or "wet belly" pelts. Whether the "wet belly" fur would wear less well over the years, however, would have to be determined through more intensive testing. Natural dark mink pelts are sold at fur auctions in an undressed or raw condition; whereas, mutation skins are sold dressed. Since dressing very substantially reduced the conspicuousness of the "wet belly" defect, less attention might be called to this condition in natural dark mink if they were sold dressed as in the-case of mutations. Experiment V Effect of urine on "we: Belly" It is generally assumed that urinary incontinence and "wet belly" in.mdnk are associated conditions. Experiments I and III have suggested that "wet belly" is a condition of unprimed fur. If these two disorders are related, possibly the unprimeness results from pro- longed wetness in this area. Thus, this experiment was conducted to determine if urine is responsible for the discoloration in the pelt. 56 Procedure The backs of three natural dark female mink were shaved weekly. urine collected from males that showed urinary incontinence was applied daily to the shaved area of one of the females. The second female was treated in a similar manner with urine from normal males, and the third mink was untreated and acted as a control. The urine was applied to the shaved area with cotton and thoroughly rubbed into the skin from September 16 until pelting (December 2). Results When the three females were pelted and fleshed, none of them showed the characteristic discoloration in the pelt indicative of "wet belly". Discussion It is generally assumed by mink ranchers that urine dribbled on the fur and skin around the urethral orifice by incontinent mink is responsible for the "wet belly" defect in the pelt. Some recent re— search (Schaible, 25 31., 1962; Conant, 1962), however, casts doubt on this assumption and indicated that there appears to be no consistent relationship between the two conditions. The results of this experiment tend to support these observations and suggest that urinary incontinence and "wet belly" are not cause and effect. However, it is possible that the daily applications of urine were not sufficient to produce a typical "wet belly" discolora- tion in the pelt and, even though the urine might tend to remain on the skin longer in the back region, the skin of the back is much thicker than that in the inguinal area. 57 In more recent studies (Anon., 1965) similar to this experiment, it was noted that a darkened discoloration indicative of "wet belly" appeared in the pelts of three of the five mink that were treated with urine fromumales that showed urinary incontinence. No "wet belly" symptoms were found in pelts from animals treated with urine from nor- mal mink. However, in more extensive trials the following year with mink and rats the results were inconclusive (Anon., 1966). The treated mink all showed a general unprimeness (discoloration) so that no dif— ferences, in the type of urine applied, could be distinguished. Urine from affected and normal male mink applied to the skin of white rats failed to cause any noticeable irritation and no histological differ- ences in the skin were noted. Experiment VI Effect of MHneral Oil on "Wet Belly" This experiment was designed to determine if there was a possi- ble relationship between urinary incontinence in the live animal and "wet belly" as seen in the pelt. It was conjectured that if the fur and skin in the inguinal area of mank.that exhibited urinary incontinence were coated with mineral oil, urine that dribbled on the pelt would be prevented from coming in contact with the skin in that area. Animals treated in this way should not show a discoloration in the skin when pelted, if urinary incontinence is responsible for the "wet belly" defect. However, if a typical "wet belly" discoloration is noted in the pelts, it would suggest that this defect is not directly associated with urine per 32. 58 Procedure During the summer and fall, natural dark male mink were checked at weekly intervals for evidence of urinary incontinence. At the first signs of incontinence (late September) the area around the urethral orifice of three males was treated with mineral oil. About a table- spoon of mineral oil was thoroughly rubbed into the fur of the inguinal area each day in an attempt to coat the skin and prevent any dribbled urine from coming in contact with it in that area. Three additional males, not showing any evidence of urinary in- continence, were also treated daily with mineral oil and served as a control. After two weeks of the mineral oil treatment, one of the control animals developed what appeared to be urinary incontinence, but the treatment was_continued. The daily application of mineral oil was applied until December when the animals were pelted, fleshed and checked for "wet belly". Results The pelts from.the three males that originally displayed symptoms of urinary incontinence and from the control animal that later developed the condition all showed a typical "wet belly" discoloration in the pelt. No evidence of "wet belly" was observed in the pelts of the two normal control mink. Discussion The results of this trial suggest that mink pelts affected with "wet belly" are not damaged by external contact with urine or its decomposition products. This, of course, presupposes that the daily application of mineral oil prevented the urine from reaching the skin in that area. This fact was not definitely determined. Although these 59 results support Conant's (1962) observations that urine is not respon- sible for the "wet belly" defect, they do not indicate that urinary incontinence and "wet belly" are unassociated. Experiment VII Attempted Surgical Production of urinary Incontinence This experiment was undertaken in an attempt to experimentally produce urinary incontinence in male mink through surgical techniques in order to ascertain the effects of this condition on "wet belly" discoloration of the pelt. Procedure Two possible surgical techniques to produce urinary incontinence were investigated. The first involved transection of the pelvic, hypogastric and colonic nerves. The pelvic nerve carries impulses that induce the act of urination and the hypogastric and colonic nerves con- vey impulses that assist in retention of urine by the bladder. Thus, it was conjectured that transection of these nerves might result in urinary incontinence. However, due to the difficulties involved in locating the appropriate nerves, this technique was abandoned in favor of the alternate method. The second procedure consisted of transection of the sphincter muscle at the neck of the urinary bladder. The transection of this muscle should allow the urine to continually flow from the bladder rather than be retained until forcefully ejected as in normal urina- tion. To test this approach, three clinically normal natural dark male mink approximately five months of age were anesthetized with 1 cc of 3 percent sodium pentobarbital solution per kg of body weight, and the 6O hair shaved from the mid-ventral pelvic region posterior and lateral to the urethral orifice. An incision was then made in this area to expose the urinary bladder, and transection of the sphincter muscle at the neck of the bladder attempted. Extreme care was exercised in severing the muscle fibers of the sphincter so that the urethral mucosa was not pierced. After transection of the muscle, several stitches were taken in the abdominal incision and a powdered antibiotic applied at the site of the wound. Results Five days after the operation, one of the three surgically treated males showed symptoms of urinary incontinence (Figure 13). This experimentally induced urinary incontinence was noticeable for approximately six weeks, where after normal urination was again evi- dent. No urinary incontinence was observed in the other two surgically treated animals. Examination of the primed skins of the surgically treated males indicated a "wet belly" discoloration was present in the inguinal region of the pelt from the mink in which urinary incontinence had been experimentally produced (Figure 14). It was noted that the area of wet, stained and matted fur observed in the live animal corresponded with the area of discoloration in the pelt. The pelt from one of the other surgically treated mink also showed a typical "wet belly" dis- coloration, but no evidence of incontinence had been observed in this animal. Discussion In the mink that failed to exhibit urinary incontinence after surgery, it is possible that not all the muscle fibers-of the bladder 61 Figure 13. urinary incontinence produced surgically by transection of the urinary sphincter muscle. 63 Figure 14. Inguinal region of pelt from the mink that showed experi- mentally produced urinary incontinence. 65 sphincter had been severed, so that the few intact fibers were capable of retaining the urine in the bladder allowing for apparent normal urination. Healing of the cut sphincter, in the mink that did show urinary incontinence, probably accounted for the cessation of the con- dition after it had been observed for a period of time. The surgical techniques employed in this experiment were only partially successful, in that urinary incontinence was experimentally produced in only one of three treated mink. And, since two of the three surgically treated animals showed "wet belly" in the pelt, the results of the trial were inconclusive and the effects of the urine and its decomposition products on the pelt could not definitely be ascertained. Experiment VIII Influence of Dietary Ca1cium.on Growth, Urinary Incontinence and "Wet Belly" Minerals were first implicated in the etiology of urinary in- continence and "wet belly" by Roberts (1959). He stated that the calciumrphosphorus ratio; influenced the availability of calcium in the ration and was related to the cause of the disease. Roberts (1969) postulated that, where high fat diets are fed, calcium might be tied-up as insoluble calcium salts of fatty acids and thus lost to the4 animal by way of the feces. If this occurs and a deficiency results as he proposes, the addition of supplemental calcium would be bene- ficial in alleviating the condition. However, in studies-by Schaible _ggugl, (1962) where both the ratio and amount of calcium and phosphorus were varied, a high incidence of urinary incontinence and "wet belly" was obtained in animals fed diets high in calcium. 66 The discrepancies in these studies suggested the need for further research on the role of calcium in the etiology of this disorder. Con- sequently, this experiment was initiated to investigate the relation- ship of different dietary levels of calcium to growth rate and the occurrence of urinary incontinence and ”wet belly". Procedure Thirty-six natural dark male mink kits 10 to 12 weeks old were allocated into two groups, each containing 18 animals. Group one served as a control and received a typical ranch ration (Table 3) which contained 0.47 percent calcium and had a calcium-phosphorus ratio of approximately 1:1. The second group was fed the same diet except that 1.5 percent ground limestone (containing 38% calcium) was added. This diet contained 1.03 percent calcium and had a calcium-phosphorus ratio of approximately 2:1. The mink were weighed periodically and examined for urinary incontinence bi-weekly using a long—handled mirror held beneath the cages so as to avoid handling and exciting them, which has been re— ported to cause urinary incontinence (Stowe, 1958). Results The individual weights of the mink in both groups are shown in Table 4 and the incidences of urinary incontinence and "wet belly" are recorded in Table 5. Statistical analysis of the data presented in Table 4 indicated there were no significant differences (P < 0.05) in weights between the experimental and control animals at corresponding dates. As shown in Table 4, the occurrence of urinary incontinence and "wet belly" was greater on the higher calcium diet. The number of 67 Table 3 —- Composition and analysis of control ration Composition Ingredients Percent Horsemeat 26.7 Tripe 26.7 Ocean whiting 26.7 Liver, (beef, inedible) 5.0 Supplemented cereal 15.0 Analysis (in_p§rcent) Ether Crude Moisture Protein extract fiber Calcium Phosphorus "As fed" basis 64.7 13.6 6.9 0.99 0.47 0.52 Moisture-free basis 0 38.5 19.5 2.80 1.33 1.47 68 Table 4 -- Effect of dietary calcium on weights of male mink fed the control and experimental rations Mink Body weight in gramm number At time of pelting Aug. 31 Sept. 28 Oct. 27 NOV. 15 - Dec. 20 CONTROL RATION 1 1220 1290 1330 1360 2 1450 1320 1380 1400 3 1390 1200 1030 1300 4 1060 1100 1180 1210 5 1580 1520 1550 1500 6 2020 2090 2270 2400 7 1500 1370 1440 1510 8 1600 1560 1720 1800 9 1580 1520 1610 1680 10 1370 1080 1000 880 11 1205 1100 1320 11200 12 1710 1630 11700 1520 -13 1590 1510 1680 1680 14 1230 1190 1120 1000 15 1400 1360 1610 1300 .16 1570 1400 1380 1210 17 1470 1380 1540 1470 18 1260 1030 1225 1150 Average 1456 1369 1449 1421 EXPERIMENTAL RATION (CONTAINING SUPPLEMENTAL CALCIUM) 19 1405 1120 1175 1220 20 1440 1440 1480 .1520 21 1580 1640 1540 1610 22 1430 1410 1505 1600 23 1410 1400 »1420 1510 24 1310 1330 -1395 1405 25 1250 1300 1315 1410 26 1460 1360 1395 1305 27 1490 1320 1305 1340 28 1450 1350 1340 950 29 1400 1300 1140 1240 - 30 1580 1420 1450 1400 31 1490 1420 1560 1500 32 1170 1120 1280 1390 33 1370 1380 1450 1420 34 1700 1610 1680 1580 35 1160 1340 1390 1350 36 1460 960 1000 960 Average 1422 1345 1379 1371 69 Table 5 -- Observations on urinary incontinence and "wet belly" in mink fed the control and experimental rations Size of Mink Urinary incontinence (UI) observations "wet belly" number Aug. Sept. Sept. Oct. Oct. Nev. area 31 14 27 12 27 6 (sq. in.) CONTROL RATION 1 -- -- UI -- -— -- (b) 2 —— —- -- —- -- -- (b) 3 -- -- -- —- —- -- (b) 4 -- -- -- -- -- -- 0 5 -- -- U1 U1 U1 U1 12.0 6 -- -- -- -- -- UI(a) 2.0 7 -- -- -- —- -- 0 8 UI (a) U1 (a) -- UI UI 10 5 9 -- -- - -- -- -- 0 10 U1 U1 (a) U1 -- -- -- o 11 -- -- -- -- -- -- 6 0 12 -- -- -- -- U1 U1 1 0 13 -- -- UI(a) -- UI(a) UI(a) 4 8 14 -- -- -- -- -- —- 0 15 -- -- UI U1 U1 -- 9 0 l6 —- -- -— -- -— -- 0 l7 -- UI -- -- —- -- 0 18 -- -- -- -- -- -- l 2 Summary of Date: Number of observations of U1 ........ .; ....... 23 Number of pelts with "wet belly" ....... . . 8 Average size of "wet belly" area (sq. in. )3 5.8 EXPERIMENTAL RATION (CONTAINING SUPPLEMENTAL CALCIUM) 19 -- UI -- -- -— -- (b) 20 -- -- -- -- -— -- (b) 21 -- -- -- -- -- —- (b) 22 -- -- -- -- -- 0 23 -- UI (a) U1 (a) U1 (a) U1 U1 (a) 1 . 9 , 24 -- -— UI(a) UI(a) -- 4.5 25 UI -- U1 U1 -- UI 4.4 26 -- UI -- -- -- -- 1.0 27 -- UI(a) UI(a) UI -- -- 2.3 28 -- -- -— -- -— -- 0 29 U1 U1 U1 -- -- -- 0 30 -- -- —- -— UI UI 6.6 31 -— -- -- -- -- -- 3.8 32 U1 U1 U1 (a) U1 (a) U1 -- 3 . 1 33 -- -- UI UI(a) -- -- 1.0 34 -- -- -- UI(a) U1 U1 18.0 35 U1 U1 U1 ‘ UI (a) U1 -- 2. 5 36 U1 U1 -- U1 U1 -- .3 Summary of Data: NUmber of observations of UI.......... ....... 40 Number of pelts with "wet belly" ............. 12 Average size of "wet belly" area (sq. in.)... 4.8 (a) Fur stained but not wet. (b) Entire pelt unprimed. 70 observations showing urinary incontinence and "wet belly" increased from 23 to 40 and from 8 to 12, respectively, for the control and high calcium.rations. Only a small difference was noted in the average "wet belly" area between the two groups, with the greater area in the control animals. Generally, the weight or size of the animals appeared to be unrelated to the incidences of urinary incontinence and "wet belly". Discussion Although a high positive correlation was noted between the .presence of urinary incontinence in the live animal and the "wet belly" defect in the pelt, the results of the present study support previous observations (Stowe, g£”§1., 1959; Schaible, 25g§1., 1962) that wet, stained and matted undersides are not necessarily indicative of "wet belly". Mink numbers 11, 18 and 31, in Table 5, did not exhibit symptoms of urinary incontinence during the bi-weekly inspections, but the presence of "wet belly" defect on the leather side of the raw pelt was quite obvious. Other males (Numbers 10, 17 and 29) showed early evidence of urinary incontinence, but did not exhibit "wet belly" when pelted. These results are a bit confusing. However, it is possible that in the mink that showed no symptoms of urinary incontinence but did show "wet belly", the condition may have been present in a tran- sient form that wasn't observed during the bi—weekly examinations. It is also possible in the males which exhibited symptoms of urinary in— continence early in the experiment that the condition might have cleared up prior to fur priming in the fall and thus, the fur in the inguinal region primed normally. 71 This study indicated that urinary incontinence and "wet belly" can occur on a relatively low calcium.diet but the incidence increases at a higher level of calcium. These results are in disagreement with the observations of Roberts (1959), but have recently been confirmed by other investigators (Anon., 1962, 1964). It is conceivable that supplemental calcium might cause-urinary incontinence through its in— fluence on acid-base balance of the blood and renal function. Although in this study, there appeared to be no apparent rela- tionship between the size of the mink and the incidence of either urinary incontinence or "wet belly", in more extensive studies (Anon., 1962; Lauerman, 1965), other researchers have reported that the disease is more frequently encountered in the larger animals. Experiment IX Effect of Diethylstilbestrol on urinary Incontinence In previous studies, Leoschke (1962b) experimentally produced urinary incontinence in mink by oral administration of testosterone and showed that the level of this hormone influenced the incidence and severity of the disorder. Since the physiological action of come pounds that exhibit estrogenic activity is in many respects antagonis- tic to that of testosterone, he attempted to cure the condition by injections of synthetic estrogen. The results were inconclusive and he stated that more research was needed on the possible therapeutic role of estrogens in this disease. It was therefore decided to investigate the effects of diethylstilbestrol (a synthetic steroid with estrogenic activity) on naturally occurring urinary incontinence. 72 Procedure Four natural dark male mink showing typical symptoms of urinary incontinence were selected for treatment with diethylstilbestrol. The animals were weighed and the area of wet stained fur around the urethral orifice recorded. Three of the males were injected with 0.4 m1 of diethylstilbestrol (60 mgms per cc) in the axial region of the hind leg every other day. The fourth mink was anesthetized with 1 cc of 3 percent sodium pentobarbital solution per kg of body weight, and a pellet containing 15 mg of diethylstilbestrol implanted just beneath the skin at the base of the neck. Results The effect of the diethylstilbestrol on the mink was readily apparent. Five days after the treatment was started, the injected males exhibited signs of increased incontinence. Therefore, the administration of the hormone was discontinued after the third injec- tion, but the severity of the condition increased and the animals died -- one on the sixth day and the others on the 11th and 14th day after the initial injection. The male implanted with the diethylstilbestrol pellet showed a continual increase in incontinence and a steady loss of weight until its death four weeks after implantation of the hormone. Discussion The effect of diethylstilbestrol on normal mink was not ascer— tained, but at the levels administered to the affected males in this trial the hormone considerably increased the degree of incontinence and was toxic to the mink. 73 Since this trial, a similar experiment was conducted by Oregon State University researchers (Anon., 1965) using weekly injections of 200 or 400 mcg of estrogen (estradiolcyclopentylpropionate) rather than diethylstilbestrol. This treatment also resulted in severe symptoms of urinary incontinence and by the fourth week, 8 of the 10 treated animals had died as a result of urinary tract infections. Experiment X Bacteriological Examination of Preputial Smears and urine In a recent study, Gunn (1962) reported that urinary inconti- nence in mink was due to an infection of Proteus organisms in the pre— puce cavity of male mink. Lauerman (1963) observed that both Proteus mirabilis and Streptococcus fecalis were associated with urinary in- continence. This experiment was conducted in cooperation with the Microbiology department in an attempt to confirm these observations. Procedure Preputial smears were taken from three natural dark male mink affected with urinary incontinence and from three clinically normal males. A urine sample from an affected and a normal mink was also collected in a sterile tube during micturition, and together with the smears were submitted for bacteriological examination and cultured for Proteus.gg. Results An analysis of the bacteriological examination revealed that no Proteus organisms were recovered from the cultures. Staphylococcus aureus and Bacillus a2, were the only bacteria isolated.from the preputial smears and these organisms were found in both the incontinent 74 and normal males. Coliform and Enterococcus were isolated from.the urine of the affected male while Staphylococcus aureus and Bacillus a2, were found in the urine of the normal mink. Discussion Although these are the results of a limited number of observa-. tions, they do not support Gunn's or Lauerman's work, but suggest that if pathogenic bacteria are responsible for urinary incontinence, organisms other than Proteus and Streptococcus may contribute to this condition. Experiment XI Effect of Antibacterial Agents on Growth, Urinary Incontinence and "Wet Belly" Recent studies on urinary incontinence in mink have suggested that this disorder is due to bacterial infection. Gunn (1962) reported that Proteus mirabilis was consistently isolated from the prepuce cavities and feces of mink with symptoms of urinary incontinence. He also noted high concentrations of this organism in the ration. According to Lauerman (1963), Streptococcus fecalis as well as Proteus mirabilis is associated with this condition. Experiment X of the present study did not support these observations. The objectives of this study were to further investigate the presumption that this disease is caused by bacterial infection and to determine if antibacterial agents (nitrofurantoina and neomycinb) aAs Furadantin, l-(S-nitrofurfurylideneamino) hydantoin. Eaton Laboratories, Division of Norwich Pharmaceutical Company, Norwich, N.Y. bAs Neomix. Michigan Agricultural Company, Richland, Michigan. 75 would be effective in lowering the incidence of urinary incontinence and/or "wet belly". Procedure On August 1, sixty natural dark male mink were allocated into three groups. The mink in Group I received a regular ranch ration, as shown in Table 6, and served as a control. The second group of mink were given the same diet but supplemented with nitrofurantoin to pro- vide 2 mg per pound of body weight per day. The basal ration, supple— mented with nitrofurantoin as in Group II, plus neomycin (added at a level to provide 4 mg per pound of body weight per day) was fed to the animals in group III. Mink were weighed and the incidences of urinary incontinence recorded at specific intervals, as shown in Table 7. The experiment was terminated at pelting (December 7), at which time the occurrence and size of "wet belly" defects in the pelts were noted (Table 7). A darkened discoloration around the urethral orifice in excess of 1 sq. in. in area was considered as "wet belly". Results It is readily apparent from the data presented in Table 7 that nitrofurantoin and nitrofurantoin-neomycin combination were not effec- tive in preventing the occurrence of either urinary incontinence or "wet belly". Although the average area of "wet belly" was greatest in the unmedicated controls, it does not seem expedient to base con- clusions on such a small number of observations. Considerably more males had "wet belly" pelts than showed symptoms of urinary inconti— nence. And, it was noted in two cases that the animals that showed signs of incontinence did not produce "wet belly" pelts. 76 Table 6 -- Composition and analysis of basal ration Composition Ingredients Percent Horsemeat -18.5 Ocean whiting 18.5 Tripe 23.0 Liver (beef, inedible) 7.0 Lungs 10.0 Supplemented cereal 23.0 Analysis (in percent) Moisture Protein Ether Crude Nitrogen- Ash Calcium Phosphorus extract fiber free extract "As fed" basis 45.2 20.7 12.3 1.3 .16.4 4.1 0.80 0.65 Moisture- free basis 0 37.8 22.4 2.4 29.9 7.5 1.46 1.19 77 .mumoomuo mm umox moans manna “mafia NH mo ommmmo .muoooouo mm umox momma oau mxcaa ma oooommmo .ooaoowucooafihumowuam m.~ on ewe N nose. m name a name. m ammo o mafia Acauxaooz mafia aHOummummouuwzv HHH e.m on man a name a some 0 meme N none o seem Annouauuamouuazv HH m.e am Nae N mama a Name a Hana N mama o gaze Amaonuaoov H 9.3 :5 c5 A33 33 A.ea .amv Aamv .H.a games; .H.: cameos .H.= unease .H.= names: .H.a names: :maaom modem can» .>< .2< .>< .2< .a< . ooz: :hHHom unwfioz macaw mo smu< use: omenmsa a .umn Ha .poz a .uuo a .uamm a .m=< mount oamwuomm do :mfiaoo mos: one moaocaoaooea humaauo no mcoaum>ummoo one acme no muswfloa omeno>< I: u wanna 78 There was a greater weight gain in the mink that received the antibiotic (neomycin) supplementation, although no significant dif- ferences were observed between the weight gains of the three groups. Discussion .Nitrofurantoin-is a systemic antibacterial agent effective in treating bacterial infections of the urinary tract. Its antibacterial spectrum covers the majority of urinary tract pathogens, including Proteus (Anon., 1961). Neomycin possesses a wide range of antibacterial activity and is effective against many gramrpositive and gram—negative bacteria including Proteus and Streptococcus organisms. It is a rela- tively stable antibiotic used primarily in treating gastrointestinal infections (Davis, 1963, 1965). If urinary incontinence and/or "wet belly" are caused by infec- tions of Proteus or Streptococcus organisms, as suggested by Gunn (1962) and Lauerman (1963), or other potential pathogenic bacteria, then the antibacterial agents used in this trial were not effective in preventing or reducing the incidence of these disorders. Similar results were obtained at Oregon State university (Anon., 1966c) in attempts to treat affected mink with Gentamdcin and Mandelamine. As shown by the data presented in Table 7, urinary incontinence also did not appear to be directly associated with "wet belly", as more "wet belly" pelts occurred than observations of urinary incon- tinence. In addition, two of the animals that had symptoms of urinary incontinence did not show "wet belly" in the pelt. It is possible, however, that the monthly examinations for symptoms of urinary incon- tinence may not have been sufficient to account for all incidences of the condition if it was of a transient nature or short duration. II‘ ‘Ill "1 I}... ll iii if. as .. III] .lilel i .10 I 1" 1 {VIII 79 Other studies (Stowe, egnal., 1959; Schaible,_e£nal., 1962) and experi- ment VIII have likewise cast doubt on the relationship between these two disorders. The greater weight gains of the mink that received the nitro— furantoin plus neomycin (Group III) might be attributable to the growth promoting effects of the neomycin. In another experiment (Aulerich and Schaible, 1966), male mink fed "spent" chickens treated with neomycin showed significantly greater growth than control animals fed diets of similar protein and fat content. Experiment XII Histological Observations on the Urinary System In previous experiments (I and III), histological examination of the skin in the "wet belly" area failed to reveal the presence of any significant pathologic lesions or an indication as to the cause of this defect. Thus, since "wet belly" is usually associated with urinary incontinence, it was decided to examine the urinary tracts of normal and affected mink for any histological differences or abnor- malities that might be responsible for this disorder. Procedure During the pelting season (November), fixed tissues from the urinary systems of six natural dark male mink affected with urinary incontinence and six clinically normal males were obtained from the Oregon State University mink ranch. The former showed typical "wet belly" discoloration in the pelt. Representative samples from the kidney, ureter, bladder, prepuce, as well as the prostatic, pelvic and penile urethrae, fixed in 10 percent buffered formalin, were 80 processed by routine procedures for histological study. The tissues were sectioned at 5 microns, mounted and stained with hematoxylin and eosin. Results Histological examination of tissue sections from the urinary systems of the control and "wet belly" mink failed to reveal any signif- icant differences. However, some abnormal changes were observed, par- ticularly a decrease in the size of Bowman's spaces in the kidney sec- tions in both groups (Figure 15), but this appeared to be due to post- mortem,changes. Sections of bladder and prostatic urethra from both lots of mink showed epithelial changes resembling early squamous metaplasia (Figure 16), but all sections from the ureters, as well as the pelvic and penile urethrae, appeared normal. Fatty metamorphosis was evident in the cortex of the kidney of all mink (Figure 15). A few solitary lymphoid nodules were present in the prepuce of both groups of animals. One affected mink had what appeared to be a mild focal subacute lymphocytic inflammation of the prepuce, as many lymphocytes and eosinophils had infiltrated the lamina propria and epithelium. Discussion None of these histological observations would seem to have a significant role in producing urinary incontinence or "wet belly". Although fatty metamorphosis occurred in all the kidney sections ex— amined, this condition has been shown to be present in clinically normal mink (Bostrom, 1966). With one exception, inflammatory changes in the glans penis, tenminal urethra and prepuce of affected mink, as yuan—Ia. Figure 15. 81 Cross section of the kidney cortex showing fatty metamor- phosis and a decrease in the size of Bowman's space. 1. Fat droplets; 2. Bowman's space. Hematoxylin and eosin. 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