A STUDY OF CERTAIN PROPERTIES OF THE BLOOD OF
NORMAL, CARRIER, AND DY7ARF BEEF CATTIE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
RANDALL ROBERT REED, B. S., H. S
The Ohio State University 1960
Approved by
Department of Animal Science ACHTOWI3DGBJE1ITS
I wish to express my sincere appreciation to my adviser and
friend, Dr. T. M. Ludwiclc, Professor in the Department of Dairy
Science, Ohio State University for his continued inspiration, and
guidance in the preparation and editing of the manuscript.
To Dr. George R. Johnson, Chairman of the Department of Animal
Science, Ohio State University, I extend ny sincere thanks for his en
couragement, and interest in the completion of this work and also for
his assistance in editing tho manuscript.
Sincere appreciation is expressed to Dr. Walter G, Yenzke for his
cooperation and interest in editing the manuscript.
I am sincerely grateful to Dr. Ralph H. DeFalco and Dr. Elizabeth
C. Paulson, Professors in the Department of Zoology, Rutgers
University, New Brunswick, New Jersey, without whose counsel and
guidance the research could not have been conducted. I am also in
debted to Mr. Sheldon Zolna, assistant in the Department of Zoology,
Rutgers University, for his assistance with the laboratory procedure.
Also, I am greatly indebted to Otis H. Smith, owner. Bay Manor
Farm, Lewes, Delaware; ’Walter C. Zimmerer, owner, Big Brook Farm, Free hold, New Jersey; John W. Mettlar, owner, Hettlar Hereford Farms, East
Millstone, New Jersey; and Robert W. McCormick, owner, McCormick Farms,
Medina, Ohio. These persons permitted their cattle to be used repeat edly as blood donors for this work.
ii ii i
Grateful acknowledgment is also extended to Professor G. R. 7/ilson
who made blood available from the Ohio State University beef cattle
herd.
Grateful appreciation is extended to Benjamin Conner, beef cattle
herdsman, Rutgers University, who gave unsparingly of his time and
effort in assisting with blood collections and injections.
I wish also to express my appreciation to Mr. William, G. Mennen,
Morristown, hew Jersey, whose grant-in-aid made this study possible. TABLE 0? CONTENTS
Page
LIST OF T A B I E S ...... iv
LIST OF ILLUSTRATIONS ...... v
INTRODUCTION ...... 1
R3YIEW 07 LITERATURE...... 5 Dominant; Dwarfism in Cattle ...... 5 Recessive Dwarfism in Cattle...... 6 Dwarfism in Small Animals ...... 9 Inheritance of D w a r f i s m ...... 12 Physiological Factors Associated with Dwarfism ...... 20 Recent Anatomical Approaches to Dwarf Carrier Identification ...... 31 Recent Chemical Approaches to Dwarf Carrier Identification . 36
EXPERIMENTAL PROCEDURE...... 41 Total Serum Protein Analysis ...... 42 Electrophoresis Studies ...... 43 Hemagglutination with Plant Extracts ...... 44 Hemagglutination with an Enzyme Product ...... 45 Serological Investigations ...... • 46 Immunization and Bleeding S c h e d u l e ...... 49
RESULTS AND DISCUSSION...... 52 Total Serum Protein A n a l y s i s ...... 52 Electrophoresis Studies ...... 52 Hemagglutination with Plant Exbracts ...... 56 Hemagglutination with an Enzyme Product ...... 58 Serological Investigations ...... 59
SUMMARY ...... 64 Chemical Investigations...... 64 Serological Investigations ...... 65
ILLUSTRATIONS ...... 67
TABLE S ...... 73
BIBLIOGRAPHY ...... 85
AUTOBIOGRAPHY ...... 94 iV LIST OF TABLES
Table Page
1. A Comparison of the Total Serum Protein Content of Normal, Carrier, and Dwarf Beef Cattle Blood ...... 73
2. Agglutination Reaction of the Erythrocytes from Dwarf Free and Dwarf Carrier Cows by Plant E x t r a c t s ...... 74
3. A Further Investigation of the Agglutination of the Erythrocytes from Dwarf Free and Dwarf Carrier Cows by Two Plant (bean) Extracts ...... 75
4. Hemagglutination of Erythrocytes from Carrier and Normal Cattle by Armour's Technical Catalase Enzyme . . 76
5. Agglutination Reaction of the Erythrocytes from Normal and Carrier Cows with Immune Sera from Rabbits Following a Series of Three Injections ...... 77
6. Agglutination Reactions of the Erythrocytes from Normal and Carrier Cows with Immune Sera from Rabbits Following a Series of Six Injections...... 78
7. Agglutination Reactions of the Erythrocytes from Normal and Carrier Cows with Immune Sera from Rabbits Following a Series of Nine I n j e c t i o n s ...... 79
8. Agglutination Reactions of the Erythrocytes from Normal and Carrier Cows with Immune Sera from Guinea Pigs Following a Series of Three Injections ...... 80
9. Agglutination Reaction of Erythrocytes from Normal and Carrier Cows with Immune Sera from C-uinea Pigs Following a Series of Six Injections ...... 81
10. Agglutination Reaction of Erythrocytes from Normal and Carrier Cows with Immune Sera from Guinea Pigs Following a Series of Nine Injections ...... 82
11. Agglutination Reaction of Erythrocytes from Normal and Dwarf Carrier Cows to Isoimmune Sera from Normal and Dwarf Cattle Following a Series of Nine Injections . . . 83
12. Agglutination Reactions of the Erythrocytes from Normal and Carrier Cows with Immune Sera from Sheep, Induced by a Series of Nine Injections with Normal and Dwarf Red Blood Cells ...... 84 v LIST OF ILLUSTRATIONS
Figure Page
1. Starch Gel Electrophoretic Patterns of Normal and Carrier Cow Sera ...... 67
2. Starch Gel Electrophoretic Patterns of Normal Cow Sera ...... 67
3. Starch Gel Electrophoretic Patterns of Carrier Cow S e r a ...... 68
4. Starch Gel Electrophoretic Patterns of Filtered (Seitz) and "Onfiltered Dwarf Calf S e r a ...... 69
5. Starch Gel Electrophoretic Patterns of Dwarf Calf, Normal Calf, and Normal Cow S e r a ...... 69
6. Starch Gel Electrophoretic Patterns of Dwarf Calf Hemoglobin ...... 70
7. Starch Gel Electrophoretic Patterns of Normal and Carrier Cow Hemoglobin ...... 70
8. Angus Snorter Dwarf Calf ...... 71
9. Twin Hereford Bull Calves; One Normal and One Snorter D w a r f ...... 71
10. Angus X Hereford Crossbred Snorter Dwarf Calf .... 72
11. Snorter Dwarf Producing Hereford Coy/ ...... 72
Ti INTRODUCTION
The frequency of the occurrence of dwarfism in beef cattle is
high enough to constitute one of the more serious problems confronting
the beef cattle industry. Although several different types of dwarfism exist, some do not occur frequently enough to be of great economic importance. One or more types of dwarfism have been observed
in all the major beef breeds and some of the dairy breeds throughout the United States. In recent years, the kind of dwarfism that has
caused the most concern to beef cattle breeders is the so-called
"snorter" type. The term "snorter" originated because the term some what described the labored breathing condition of dwarf calves. This type of dwarfism is also characterized by a bulging forehead, undershot jaw, short legs, paunchiness, muscular weakness, and tendency to bloat.
Asphyxia is the cause of death in many snorter dwarf calves.
Even though dwarfism occurs in commercial herds, it has been of greatest concern to purebred breeders. Inbreeding programs practiced by purebred breeders in an effort to maintain some degree of homo zygosity in their herds has caused the dwarf gene to be expressed in the homozygous form with much greater frequency than has been the case with the outbreeding programs of commercial producers. There has also been some indication (although no conclusive proof) that the dwarf factor is associated with the smaller, more refined type of cattle.
1 2
This refined condition has, at times, been referred to as "show-ring" type. Bven the strongest proponents of this theory will admit that in many cases it does not hold true. Another theory that can be offered is that the two or three bulls that have been largely responsible for the spread of the dwarf gene have also sired some high quality progeny
These cattle, because of their quality, were maintained and propagated and as a result the breeders were unknowingly increasing the frequency of the dwarf gene•
The economics of dwarfism is three-folds Firstly, the dwarf calf is usually a total loss; secondly, many first calf heifers and some mature cows are lost at the time of parturition because of the abnormal thickness of many dwarf calves; and thirdly, and the most important, dwarf-free cattle that originate from dwarf carrier parents are markedly reduced in value. The research presented in the text of this dissertation was conducted primarily to develop procedures for the identification of these individuals.
Cattle breeders and purebred breed associations have recognized the dwarf problem and are making a realistic approach to its solution within the limits of current knowledge. Pedigree evaluation is of value in many of the studies which have been made. Certain herds and families are assumed, on the basis of pedigree analysis, to be free from dwarfism. If this is really true, breeders can avoid the dwarf gene by using only dwarf-free breeding stock. The classification of families and herds as dwarf-free or as dwarf-carriers will undoubtedly help but it is commonly recognized that pedigree values are dictated by the integrity of the breeders. In addition, honest mistakes may occur and many animals that are classified as carriers may be free of the dwarf gene•
Progeny testing is currently the only sure way of determining the dwarf status of a bull. &ach time a carrier bull is mated to a carrier cow the probability is l/4 that a dwarf calf will result. This means, in effect, that 3/4 of the carrier bulls would escape detection if mated to only one carrier cow. Consequently, the probability of a bull revealing his true genotype for dwarfism is increased as he sires more calves out of carrier cows. If a bull is mated to 16 known carrier cows, the chances are 99 out of 100 that he is a dwarf-free bull if all the calves are normal. The difficulties inherent in this method are obvious. The maintenance of a dwarf-carrier herd for the purpose of testing bulls requires time, space, and expense that many breeders cannot afford.
Clearly, the easiest and cheapest way to rid the beef cattle population of dwarfism is to learn to recognize normal appearing animals that are carriers of the dwarf gene. Several apparently plausible clues have been investigated. Some of the more important investigations have included studies on physical abberations and disproportions (head and vertebra), hormone activity (insulin and thyroxine), and blood constituents. It is unfortunate that none of the findings, either singly or in combination, have been demonstrated to be accurate enough for general use. Ths objectives of the research reported in this dissertation are as follows*
1. To determine whether significant variations exist in total serum protein levels between groups of normal, carrier, and dwarf beef cattle
2. To study, by electrophoresis, variations in the rate and extent of the migration of the albumens, and the alpha, beta, and gamma globulins of normal, carrier, and dwarf beef cattle sera
3. To study, by electrophoresis, variations in the rate and extent of migration of the protein fractions of hemoglobin from normal, carrier, and dwarf beef cattle
4. To test the agglutinating potential of several plant extracts against the erythrocytes from normal, carrier, and dwarf beef cattle
5. To test the agglutinating potential of an enzyme product against the erythrocytes from normal, carrier, and dwarf beef cattle
6. To determine, by serological techniques, whether a variation in antigenic response exists in rabbits, guinea pigs, cattle, or sheep when injected with erythrocytes from normal, carrier, or dwarf beef cattle. REVIEW OF LITERATURE
Dominant dwarfism in cattle» Jeffreys (60) has indicated that dwarf cattle have existed in Africa since ancient times as true species and not degenerates from larger types. Seligman (87) described the occurrence of "Cretinism" in calves as early as 1860. During recent years, dwarf types have been reported in many breeds including both dairy and beef type cattle.
Crew (24:) described "bulldog" calves in the Dexter breed in 1904.
They have short, rounded heads, depressed nostrils, bulging foreheads, projected mandibles, flabby tongues, short vertebral columns, thick loose skin, inguinal hernia, and short legs. They are about half the size of normal calves and are aborted between the sixth and eighth month of the intra-uterine period. Hutt (59) concluded that this condition is inherited as a single dominant gene. All Dexter cattle are heterozygous. Inter se matings of Dexters results in segregations in the ratio of 1:2*1 (25 per cent homozygous normal Kerry type, 50 per cent heterozygous Dexters, and 25 per cent homozygous Dexter monsters). No bulldog calf was produced from the mating of Dexters with Kerry type cattle.
Baton (33) described a similar type of lethal found in African as well as in Dexter cattle except the calves were usually aborted between the fourth and fifth month. Berger end Innes (6) and Mead et al. (76) have reported a similar type of dwarfism in British Friesians and
Jerseys respectively.
Lush (73) reported a condition in Texas cattle which he referred to as "duck-legged." No dwarf or "bulldog" calves were found among the progeny of the "duck legs." The pituitaries of these cattle were unusually small and the trait was determined to be conditioned by a
single dominant gene, although dominance was not always complete.
Woodward et al. (112) studied the performance of "comprest" Herefords.
These cattle were less malformed than the Dexters but were smaller,
shorter legged, and earlier maturing than conventional Hereford cattle. "Compact" Shorthorns, which resembled "comprest" Herefords, were described by Stonaker (97). They could be identified at birth by their short head, neck, body, and legs. Some had a tendency toward heavy shoulders and crooked legs. From available field data, it was concluded that the "compact" condition in Shorthorns is due to a single dominant gene. Washburn et al. (109) compared feed efficiency of steers of the "compact" and conventional types. They found that
(a) the "compact" type was less efficient than the "standard" type in the utilization of dry matter, (b) the compact required 70 days less time on feed to reach choice grade, and (c) the conventional-type cuts were heavier in bone.
Reoessive dwarfism in cattle. Hutt (59) described a recessive type of achondroplasia and referred to it as the "Telemark" type. Punnett
(83) reported on matings of one Telemark bull with eight Dexter cows to determine whether the two conditions were caused by the same gene. During the period 1926 to 1931, 24 calves, 11 of which were typical
Dexters, and a mummy were produced. Five long legged calves and eight others'were not classified, but no Dexter monster was produced. The hypothesis of multiple allelomorphs could not be supported since no monsters were produced. The probable explanation was that these two lethals were independent, and that each breed carried the normal factor corresponding to the lethal of the other breed. Ljutikov (71) described three achondroplastic calves in the Jaraslov breed in
Russia. All three dwarfs resulted from the mating of a champion cow
"Zolataja," to her son, grandson, and half brother respectively. It was concluded that the sires were heterozygous for the monstrosity.
Brandt (13) reported "bulldog" calves in the Guernsey, Jersey, and
Ayrshire breeds, all apparently caused by a homozygous condition of a recessive gene. Gregory (47) reported a sub-lethal type of achondroplasia as a result of the inbreeding of Jersey cattle in
California. Jersey "bulldog" calves have been reported by Sturrarrer
(104) and Becker et al. (5).
Baker et al. (3) have described a recessive achondroplasia in
Shorthorns and termed it "stumpy." This type of dwarfism w a s .dis covered following a line-breeding program in a purebred Shorthorn herd in 'Nebraska. The "stumpy" calves were characterized by curly coats and small switches, and the achondroplastic condition was more marked in the forelegs than in the hindlegs. Although the knees were enlarged and the metacarpal bones twisted the body and head seemed to be normal in size. Metabolic disturbances were always present in "stumpy" calves. These cattle lived, and reproduced but were always thin and posed a serious economic loss to breeders.
Johnson et al. (61) reported the occurrence - of several dwarf
calves observed in a South Dakota Hereford herd. This was perhaps the first investigator to describe the snorter dwarf which has been re
sponsible for much concern among cattle breeders and researchers because of the serious economic loss involved. The dwarfs were thick and blocky at birth, and most of them caused calving difficulties because of their characteristic greater width of body. This was especially noticeable with first calf heifers. Because of retarded growth, the dwarf signs became increasingly noticeable with age and most animals died before they were a year old. Most of them were chronic bloaters and some died as a result of this condition. Slightly bulging foreheads were common and the lateral ventricles of the brains of dwarf calves contained more than the normal amount of cerebrospinal fluid. Spermatogenesis was present in a two-year-old dwarf bull.
Histological examination of the endocrine glands of two dwarf calves did not reveal any abnormality. Gregory et al. (49) reported a more comprehensive study of dwarfs in the Hereford, Angus, and Shorthorn breeds. All of the dwarfs had heavy, labored breathing as if they had a respiratory disease. The mandible, with some malocclusion of the incisors with the dental pad, was larger than the maxilla. The dwarfs were low set, compact, blocky, short of neck, wide through the body, and had a broad, short head. The shortening of the long bones and the bulging of the foreheads were common characteristics of the dwarfs within the Hereford, Aberdeen Angus, and Shorthorn breeds# At two to four months of age, the dwarfs appeared to be stunted, pot-bellied, and they -were able to breathe only with difficulty.
Landauer (66) reported the Ancon mutation in sheep to be the oldest recorded mutant among domestic animals. In 1791 the first
Ancon lamb was born in Dover, Massachusetts. The owner killed the sire and used the Ancon for breeding. This program resulted in a large number of animals with the peculiar characteristics. These animals were designated as the "otter" breed because of the resemblance to the otter in shortness of its legs and length of its back. Bogart
(36) reported an occurrence of dwarf lambs from a strain of Southdown sheep. They had short legs, thick shoulders, and bulging foreheads.
The dwarfs gained in weight for a short period of time and became
"puffy" in appearance. They usually died within a few weeks after birth. The abnormality was caused by a simple recessive gene.
Dwarfism in small animals. Snell (93) studied a recessive type of dwarfism in a stock of black silver mice. The dwarf mice attained about one-fourth of the weight of their normal sibs. Their growth rates were normal up to the fourteenth day at which time their tails and noses were noticeably shortened. They were small, subnormal in vigor, and both males and females were sterile. King (62) later described a different type of dwarfism in mice called "pigmy." These mice are smaller at birth but some overlap with normals. They appear healthy and sleek and are as active as the normals but both sexes are sterile. This is conditioned by a simple recessive gene. More 10 recently, Hoeoker (56) reported a new dwarf mutation in the house mouse and designated it "Agitans." Mutants can be recognized from ten days onward. They have retarded growth, restlessness, generalized tremor, and ataxia. Many of them die at 20 to 30 days and those that live beyond this critical period are subject to digestive and parasitic diseases which apparently do not affect their normal sibs. The con dition is due to a simple recessive gene.
Lambert (64) et al. studied a dwarf mutation in the rat. The young appeared normal at birth, but on or about the twelfth day differences appeared. The hair is finer, thinner, and softer than that of the normal. The body proportions are similar to the normal, only greatly reduced in size. They are weak, less thrifty, and shorter lived than the normal.
Green (44,45) studied a dwarf mutation in the rabbit in consider able detail. The dwarfs were one-third the size of their normal litter mates. The dwarf head was malformed, giving the snout a characteristic dished appearance. The frontal and parietal bones were calcified only at their inferior borders. The character is produced by a simple recessive gene and is invariably lethal in the homozygous form.
Castle (17) described a dominant type of achondroplasia in rabbits.
The heterozygotes were reduced in body size to about two-thirds that of their normal sibs. They lived and reproduced, but mating of hoterozygotes produced one normal homozygote in every four offspring.
The homozygous dwarfs died after birth. They had an extremely short 11
upper jaw which, often resulted in death from starvation. It was
suggested that the defect was caused by hypopituitarism.
Sollas (93) reported a recessive type of dwarfism in guinea pigs.
Most of the dwarf guinea pigs died very early and those that lived vrere
sterile. The shapes of their skulls and skeletons were altered and
they were characteristically short of body and legs.
Stockard (96) made a very complete study of the various types of
dogs and their hybrids. He reported numerous cases of dwarfism in
dogs but little work has been done on the inheritance of this trait in this species. The midgets and achondroplastic dwarfs are common. Some dogs exhibit typical complete dwarfism; others have normal trunks with
achondroplastic legs; still others have normal legs with achondroplastic heads and abnormally abbreviated caudal skeletons. As a result of man's deliberate preservation of every newly merged mutation of dogs, the dog has become the most varied domestic species of animal on earth.
Cutter (25) described an achondroplastic condition in the fowl and designated it as "creeper." Their legs and wings are abnormally short but they are rather normal in other respects. The tibia of the leg is
short, bent, and not fully developed, but the fibula is over developed.
In normal birds the tibia is the main support with only a small splint to represent the fibula. Dunn et_ al. (3l) also reported genetic studies of the creeper fowl. An embryonic mortality rate of 45.5 per cent from creeper inter se matings during one to six days of incubation was found.
When creepers vrere mated with normal fowl, the mortality was 4.2 per cent. The mortality rate at other periods during incubation was about the same for both types of matings. The large difference in percentage
of eggs hatched between creeper inter se matings, and the matings of
creeper with normal birds, was due largely to the death of homozygous
creeper embryos at an early stage of development. Therefore, all
creeper chicks which hatched were heterozygous for the creeper con
dition. Landauer (65) substantiated these early views by subsequent
investigations. Mayhew (75) Upp et al. (107) have reported a recessive
type of dwarfism in the domestic fowl. The dwarf birds were not dis
tinguishable at hatching time but could be identified at about two
weeks of age. They were characterized by bulging eyes, disproportion
ate growth of the skull, "parrot-beak," enlarged tongue, dry skin, and
short tarsal and metatarsal bones. They did not live as long as normal
birds and were highly susceptible to respiratory diseases.
Inheritance of dwarfism. The Angus, Shorthorn, and Hereford
dwarfs, which have appeared in increasing frequency in recent years,
have been proved to be genetically recessive in inheritance.
Cutter (l5) points out that in such a trait as dwarfism, the gene
cannot produce the trait without the cooperation of many other genes.
A gene is one factor in a very complex chemical equation. Just as the
introduction of the same compound into different chemical equations
does not necessarily lead to the same modification of the result, so
the effect of a given gene may differ according to the genetic equation
into which it is introduced.
Johnson (6l) first described the mode of inheritance of the
snorter type dwarfism in a South Dakota Hereford herd in 1950. The 13 original sire of the herd, designated "RM" produced eighty calves, one of -which was a dwarf. The dam of this calf "RD" traced to Prince
Domino as did "RM". Two sons of "RM" wore used plus two line bred
Prince Domino bulls. Frequent dwarf calves were produced when mated to daughters of "RM". Observations led the authors to suggest that cattle could have three different genotypes. Dwarf calves supposedly were homozygous recessive (dd) for dwarfism while normal animals were homozygous dominant (DD) or contained one dominant factor for normality and one recessive factor for dwarfism (Dd), the latter being the heterozygous dwarf carrier.
Baker (3) reported a similar situation with an almost perfect 3si ratio of normal to dwarf calves in an inbrod Shorthorn herd.
Lush and Hazel (74), in 1952, offered the most extensive genetic study available at that time. The data we re collected from a large number of breeders with the cooperation of the breed association and included the complete breeding records involving hundreds of matings.
In order to remove much of the bias in such records, they eliminated from records the first dwarf calf sired by a bull and all the normal calves which preceded it. They like-wise eliminated the first dwarf calf and all normal calves which were produced by a cow. The first dwarf calf was considered the evidence that either a bull or a cow was capable of transmitting a dwarf factor to his or her offspring. They then counted all of the calves, both normal and dwarf, which were later produced by the mating of two parents, each of which had been known to have produced a dwarf calf. A summary of this study revealed that in 14 these herds a total of 266 countable offspring from such matings was
197 normal and 69 dwarf calves. These results approximate closely a ratio of three normal calves to one dwarf. These results are expected if dwarfism is caused by a simple recessive factor as previously suggested. The 69 dwarf calves would be of the genotype "dd" and would constitute 25 per cent of the offspring produced by proven carrier parents. The 197 offspring which appeared to be normal would, according to theory, consist of some (l/3 of them) which were of the genotype "DD" and therefore free of the dwarf gene and others (2/3.of them) which were of the carrier genotype "Dd". This study was confined to purebred Hereford herds of conventional type and breeding.
Pahnish et al. (8l) conducted four breeding tests to determine the mode of inheritance of "snorter" dwarfism. In the first test, 90 heterozygous X heterozygous matings produced normal and dwarf offspring in a phenotypic ratio of 3*1. A sex ratio of lil was observed in the dwarf population studied.
Test II included five dwarf X dwarf matings. The resulting progeny were dwarfs, but the abnormal characteristics were no more extreme than those exhibited by a number of dwarf offspring from heterozygous X heterozygous matings.
In Test III, a limited number of heterozygous cows were mated with dwarf bulls (six matings). The progeny appeared in a normal dwarf phenotypic ratio of about 1:1.
A dwarf sire was used on females of undetermined genotype in Test
IV (seven matings). Dwarfism h ad, not been recognized in the herd from 15 which the females we re obtained. The progeny that survived were normal. Stillbirths or early, postnatal mortality prevented the identification of a few phenotypic classifications. The need for positive methods of identifying the dwarf phenotype in the event of early death losses was pointed out.
Burris at al. (15) conducted a series of cross breeding experi ments in an effort to determine the relationship of the genes responsible for the apparent morphologically similar form of dwarfism in different breeds. Dwarfs phenotypically similar to the snorter type previously described were produced by Angus X Angus, Hereford X
Hereford, Hereford X Angus, and Angus X Hereford matings when all parents used were apparently phenotypically normal and of conventional type. Phenotypically indistinguishable dwarf calves were produced when "comprest” Hereford cows were mated to Hereford bulls and to an
Angus bull. In some instances more than one dwarf was produced by one individual cow in successive seasons. The occurrence of crossbred dwarfs from these matings indicate that the factors causing dwarfism in the two breeds act in a similar manner whether the parents in a given case are of the same or different breeds. This observation lends considerable support to the hypothesis that the recessive genes causing dwarfism in the two breeds are either the same gene or alleles with very similar effects. The apparent increased growth rate and greater vigor of the crossbred dwarfs compared with purebred dwarfs indicates that the expression of the homozygous recessive dwarf geno type may be influenced by the genetic background of the animal. 16
Gregory et al. (49) collected data from California commercial herds who used registered Angus bulls on high grade Hereford cows. The crossbred (F]_) dwarf hybrid was similar in all diagnostic features to the dwarfs that resulted from other breed crosses. Further work in this area would seem to indicate several different dwarf genotypes.
Dollahon et al. (28) reported the results from dwarf X dwarf, dwarf X carrier, and carrier X carrier matings as follows, (l) A snorter Hereford X snorter Angus produced a snorter dwarf, which indicates that the same gene is present in both breeds. (2) An atypical dwarf resulted from mating a snorter Hereford and a long headed Shorthorn. (3) When the snorter trait was introduced into
Brahma-Native-European crossbred population, the offspring produced were more compact than offspring obtained from similar dams and normal males. (4) Two types of dwarfs were observed in a herd of carrier
Herefords, a long-headed type and the conventional snorter type. The two types showed certain similarities in that each exhibited hydro cephalus and compressed vertebra. A long-headed Hereford bull from another source was mated to dwarf females of various types. These matings produced only normal offspring, indicating that this animal was a nutritional dwarf or carried a gene which was not present in the dwarf females. (5) Apparently the midget in Brahmas is inherited as an incomplete dominant or a recessive trait. The offspring from two midget X midget matings were midgets. A midget Brahma X long-headed
Angus mating produced a dwarf which was similar to the midget, but appeared less viable. The mating of a midget Brahma and a snorter 17 dwarf Hereford produced a stillborn offspring, which by anatomical classification was a snorter dwarf. (6) The guinea condition in
Florida crossbred and native cattle appears to have descended from the
Dexter. Both guinea X guinea and snorter Hereford X guinea matings produced guinea offspring.
Gregory (50) produced F]_ progeny from crossing four phenotypi cally different recessive dwarf types. The dwarf stocks used were (l)
Hereford (Johnson et al., 195l); (2) Angus (Baker et al., 1951); (0)
Shorthorn (Gregory, unpublished); and (4) another Shorthorn dwarf characterized by a heavy body with short legs (Gregory, unpublished).
Cros-ses between the Hereford and Angus dwarfs yielded two distinct types of offspring, short headed dwarfs and phenotypically normal animals that mature early and resemble "comprest” animals in size.
The Shorthorn (3 above) crossed with the Hereford produced five phenotypically normal progeny which matured early and resembled the
"comprest." When this Shorthorn was mated to an Angus dwarf, one short-headed dwarf and one phenotypically normal animal of "comprest" type was produced. The remaining Shorthorn dwarf (4 above) was mated to normal Hereford heifers, some of which were known to be heterozygous for the dwarf gene and produced dwarfs of the short-headed type, and phenotypically normal progeny that could not be classified further until maturity. The author states that the genetic relationships of the four dwarf types cannot be determined with certainty until conclusive results are obtained from other critical mating tests. The results clearly indicate that more than one pair of genes are involved in the 18 production of different morphological forms of dwarfs. The four different forms of dwarfs appear to be conditioned by the same recessive gene and further phenotypic differentiation may be con ditioned by specific genes that act as modifiers. Separation of the different dwarf types seems to be rather complete with little over lapping. These results suggest a possible relationship of "comprest" with the common type of dwarfism. Other workers have studied the inheritance of the "comprest" type of dwarfism in Hereford cattle.
According to Stonaker (97), "comprest" is incompletely dominant, and heteroaygotes can be recognized by their shorter stature. Where
"comprest" bulls are mated to normal covrs, the progeny are normal and
"comprest" in equal proportions; furthermore, when "comprest" are mated to "comprest", 25 per cent of the progeny are afflicted with a type of dwarfism, phenotypically similar to that which segregates from the mating of normal animals. This investigation also states that the dwarfism caused by two semidominant "comprest" genes is more severe and less viable, and is generally characterized by crooked legs. When the incidence of dwarfism exceeds 25 per cent by a significant amount in the "comprest" X' "comprest" matings, there must be more than one type of dwarfism segregating.
The findings of Chambers et al. (18) would somewhat substantiate the conclusions of the previous investigators. Three "comprest" bulls were bred to 25 "comprest" heifers and six dwarf calves were produced.
Bach bull sired one or more dwarfs. The following season two of the bulls were again bred to the 25 "comprest" cows and five dwarfs were produced. From 45 such matings, 37 calves were dropped, 11 of which
were dwarfs. Three of these dwarfs were of the "crooked legged" type;
five of-'them were "straight legged." In another trial, 24 of these
"comprest" cows and seven yearling heifers were bred to two known dwarf
carrier bulls of "non-comprest" breeding and one known dwarf carrier
Angus bull. Of the 27 females which calved, six produced dwarf calves,
none of which were crooked legged. One resorption or abortion occurred
during early pregnancy. Five of the dwarfs were Herefords and one was
an Angus X Hereford crossbred. These results indicate either that the
genes responsible for dwarfism in "comprest" and conventional Hereford
and Angus cattle may be allelic or that the "comprest" cattle in this
test also carried in high frequency, the recessive dwarf gene found in
"non-comprest" cattle.
In a recent report, Gregory et al. (53) present evidence to
indicate that bovine dwarfism is a complex of several components
rather than a single entity, and that each component is homozygous for
the same autosomal recessive dwarf conditioning gene. Specific
modifying genes, or combination of modifiers, are responsible for
differentiating individual dwarf types. "Comprest," and dwarfs of
brachycephalic, dolichocephalic, intermediate, and other types belong
to the complex. These workers progeny tested a Hereford bull in three ways. First, he was mated to "comprest" type cows from carrier stock.
Two brachycephalic dwarfs proved him a carrier. Next, he was progeny
tested on brachycephalic dwarf cows. Four progeny from this mating were classified as one brachycephalic dwarf, which had a complete fusion of the sphenoid and occipital bones at five weeks, and three intermediate dwarfs. Third, he was progeny tested on dwarf carrier cows of normal size. The 23 resulting progeny were classified as 15 normal, four "comprest," three intermediate dwarfs, and one unclassified dwarf. The group points out that under ranch conditions the brachyce- phalic dwarf would have been recognized. The "comprest" and inter mediate dwarfs would have been classed as normal. The unclassified stillborn dwarf would have been disregarded even though the sphenoid and occipital bones were completely fused. These tests support results from other matings which indicate that other genes present in cattle of normal or near normal size mask or modify the expression of the major dwarf conditioning gene, and thus confuse the progeny test.
Physiological factors associated with dwarfism. The fact that growth and metabolism are abnormal in the dwarfs of all species would indicate possible dysfunction in the endocrine system. Available research would strongly suggest that the pituitary, thyroid, and adrenals are more likely to be involved than the other glands of the system. Wilkens (ill) states that among endocrine disorders causing dwarfism in humans, hypothyroidism is probably the most common. The term "cretinism" was first used to designate endemic, congenital hypothyroidism, a disorder primarily caused by iodine deficiency in the mother. However, Mullinger (80) points out that in recent years the term has been used indiscriminately to refer to hypofunction in infants and young children, irrespective of the etiology. Gordon (93) concluded that the symptomatology, physical changes, and radiologic abnormalities depend, upon the degree of thyroid deficiency and upon
the age of the child when the disturbance commenced. Means (77)
further observed that in the human, hypothyroidism may exist in utero
or may develop in infancy, in childhood, or in adult life. Since the
thyroid hormone plays a role in growth and maturation, the effect of a deficiency during the growth period will be different and more devas tating than during a time of life when full maturity and growth have been attained. Fenger (37) reports that the thyroid and parathyroid
glands function very early in embryonic life. The thyrotrophic action
of the hypophysis is also exhibited very early. Apparently the
characteristic relationship between the hypophysis and the thyroid establishes itself simultaneously with histological differentiation of the glands. Studitskii (102) observed that the function of the
endocrine glands during fetal life is insignificant under optimal environmental conditions of fetal development. As soon as deviation from the optimum occurs, the endocrine system can be mobilized as one
of the most important regulators of vital functions in the organism.
Several investigators have observed that the fetal thyroid possesses a
strong affinity for iodine. Fenger (37) indicates that the thyroids of beef fetuses contain an appreciable amount of iodine as early as the third month of intra-uterine life. The iodine content increased proportionately with the age of the fetus. Seligman (87) reports and describes'crtftems in Deester-Kerry cattle. Examination of the thyroid revealed a small irregularly developed gland. The author is convinced that the "bull-dog" condition in the Dexter is produced by hypothyroid- 22
ism and is cretinoid in nature. Some workers, Ruggles (85), Symmers et al. (105), and Vfilkins (110), consider epiphyseal dysplasia as one
of the most prominent anatomic characteristics of hypothyroidism.
Wilkins (110) states that it is caused by a disorder of the cartilages
of the epiphyses and round bones leading to irregularities in their
subsequent ossification. In a study of the endocrine glands of dvrarf
•nice, Smith et al. (88) showed that the thyroids were extremely reduced in size, The granular tissue was separated by adipose and
connective tissixes. Some of the thyroid tissue was not organized into follicles and contained little or no colloid. The adrenal cortex was reduced in thickness and the characteristic zonation was absent or
indistinct. The gonads, although delayed in development, did not show profound aplasia. The anterior pituitary was markedly abnormal and no eosinophils were present. Daily implants of fresh rat anterior lobe
into the dwarfs resulted in positive improvement in all cases. In appearance, treated dwarf mice could not be distinguished from normal mice. Smith (89) showed that hereditary dwarf mice and hypophysecto- mized rats manifest similar conditions. The outstanding difference between the dwarf mice and hypophysectomized rats was the degree of development of their reproductive systems. For hereditary dwarfs, the testes and the motility of the sperm were not greatly different from normal. In the hypophysectomized rats, the testis became flabby, the
sperm did not develop and the seminal vesicles and other glands were greatly reduced in size. The ovaries of hypophysectomized rats were smaller in size than those of hereditary dwarfs. The uteri of dwarfs 23 were infantile, whereas those of the hypophysectomized rats were
thread-like. This suggested that the pituitary growth hormone of the
hereditary dwarf was suppressed without a corresponding suppression of
the gonad stimulating hormone. Mollenbach (79), in his studies on dwarf mice, observed that the livers of dwarf mice contained very
abundant quantities of glycogen. The fat content, on the other hand, was scanty in the dwarfs while normal mice had very abundant quantities
of fat in the liver. If the animals were starved, it was found that
the glycogen in the normal mice was rapidly and almost entirely mobilized; the fat, however, diminished only slightly. The dwarfs
seemed to be able to mobilize easily the, little fat they were capable
of depositing, but they were not able to mobilize the glycogen
deposits in the normal way. The dwarf mice were more sensitive to
insulin than the normal ones. Clark (19) studied hydrocephalus in mice and noted that young mice may show a swelling of the head at birth but the characteristic does not become noticeable until a week
or two later. A bombase skull was caused by the pressure of the clear
liquid which collected above the brain and distended the roof of the
skull outward. The brain tended to be pushed downward and foreward.
Hydrocephalus has also been reported in cattle. Houck (57)
reported a hydrocephalic calf born to a Jersey X Hereford cow bred to
a Durham bull. The hydrocephalus resulted from a congenital malgrowth
in the diencephalon with a constriction of the third ventricle to a
very narrow canal. Cole (20) reported hydrocephalus in Holstein calves, and Blunn (8) reported a similar condition in Duroc swine. 24
The pigs were either born dead or died shortly thereafter. This
condition is due to a simple recessive gene in this species.
Hunter et al. (58) studied the effects of thyroidectomy on
rabbits. Eighty-seven domestic rabbits vrere thyroidectomized at 13 to
21 days of age. Face and cranial development was retarded. In breadth,
the skull showed disproportionate retardation in the face and forehead.
Vertical growth of the mandible was also significantly retarded,
indicating a generally undei— developed jaw. A comparison of control
and thyroidectomized skulls showed that physiological insufficiency of
thyroid secretion was manifested within six days. Thyroidectomy in
young pups resulted in retarded growth but all parts of the body were
not affected alike. Dye et al. (32) reported a retardation of the
ossification of cartilage bone and delayed ankylosis of the sutures of
the skull bones in pups. This retardation of growth mechanically
altered the skull shape with resulting bracliycephaly. In a study of
thyroidectomized sheep, Todd (106) observed that development and
growth ’were most marked in the pre-maxilla and maxilla. Tooth develop ment is retarded but not reduced in size or shape. In another study
of thyroidectomized sheep, Liddell (60) noted skull differences. The
skulls were foreshortened, and disproportionately wide. The nasal and frontal bones were more retarded in growth than the parietal and occipital bones. Delayed dentition tos observed. Ankylosis of the basisphenoid and basioccipital was very slow in comparison to normal. 25
Spielman et al_. (94) thyroidectomized six dairy animals, five heifers and one bull, at various ages. The characteristic changes noted by these workers after 30-60 days were
1. Diminished appetites
2. Decreased activity
3. Increased susceptibility to low temperatures
4. Enlarged paunch
5. Progressively increased dry and brittle hair
6. Thickened and dry skin
7. Decreased pulse rate
8. Subnormal temperature
A heifer was thyroidectomized on the forty-sixth day of her first
gestation period. Growth was static for about 20 weeks following thyroidectomy. However, coincident with the last ten weeks of the
gestation period, a sudden resumption of height and gain in body weight occurred. Following parturition, growth again became static and little change was noted until the latter part of the subsequent
gestation period when growth was resumed. It appears that the thyroid deficiency of the mother was remedied, partially at least, either by diffusion of the thyroid hormone from the fetus into the maternal circulation or by some undefined factor related to the pregnant state.
Thyroidectomy caused complete inhibition of libido in the male but no apparent effect on spermatogenesis. Thyroidectomized cows failed to manifest normal physical signs of estrus but they could be impregnated and could carry a calf to full term. 26
Steiner (95) has postulated that dwarfism in general is the result of insufficiency of the anterior pituitary with fractional deficiencies of hormones concerned with growth and metabolism.
In a study of hereditary hypopituitary dwarf mice, Mirand et al.
(78) reported the following observations:
1. Average blood sugar values for hereditary hypopituitary dwarfs are slightly, but not significantly, lower than those of normal mice .
2. The blood sugar level of fasting dwarfs drops precipitously to a minimum of 68 mg. per cent after 96 hours of fasting.
3. Sensitiveness of the dwarf is similar to that reported for the hypophysectomized animals. This severe hypoglycemia might be attributed to the adrenal-pituitary imbalance in the dwarf.
These dwarf mice could tolerate only three per cent of the dose of insulin that produces comparable signs in normal mice.
In studies with hypophysectomized do'gs, Dandy and Reichert (26) found that they are very susceptible to disease. Distemper and pneumonia are the usual causes of death. The dogs also showed low mentality, soft puppy hair, first denture, and infantile sexual development. Skeletal growth ceased immediately after hypophysectomy.
The closure of epiphyseal lines was delayed to 19 months (they normally close between the seventh and ninth month). Thyroids, adrenals, sex glands, and pancreas exhibit cessation of growth or actual recession after the operation. 27
Ten cases of pituitary dwarfism in humans are reported by Greene
(46). He indicates that pituitary dwarfs under adequate treatment grow sporadically as do normal children, and that the periods of decline or cessation of growth are caused by a decrease in the sensi tivity of the skeletal system to the growth stimulus.
Eleven patients afflicted with pituitary dwarfism were treated by Edwards et al. (34) with pituitary growth extract. Pituitary extract containing both growth and thyrotropic- factors was given intramuscularly. .An initial dose of 0.5 cc. and 1.0 cc. on alternate days was given. A 2.0 cc. dose three times per week was continued for two to six months with control intervals without treatment. Basal metabolic rates were normal in all but three patients. One patient received thyroid extract for a short time to note any added effect on growth. There was none. Only one patient reached the minimum average height and weight for his age. The authors concluded that further studies with more potent growth hormones are needed.
Bates et al. (4) found, in their studies of hereditary pituitary dwarfism in mice, that injections caused a 30 per cent gain in body weight in 30 days.
Several investigators have studied the anatomy and physiology of hereditary dwarfism in cattle. Downs (29) in his studies of a
"bulldog" calf, found that the thyroid was distinctly abnormal. The thymus was very large although the cells appeared normal and active.
Only one parathyroid could be found and it was apparently active. The author concluded that the functions of the endocrine system could be influenced by heredity. 28
Graft and Orr (23) report their observations on an abnormal
Hereford steer at Oklahoma A & LI College. The calf came from a herd in the state and was sired by a purebred bull and out of a good grade cow. The bull calf was characterized by a general dwarf-like con dition; short irregularly curved legs, abnormally large joints, short and thickened face, and a nervous disposition. The calf was born in
March, 1928, brought to the college in November and weighed 330 pounds when it was slaughtered in January, 1929. Upon post mortum examination, it was found that the epiphyses of the long bones were enlarged and irregularly curved. The thyroid gland was about one-fifth of the normal size. The parathyroids were correspondingly small, and the pituitary was only about 50 per cent of the normal size. It was impossible to tell whether the size of the glands was caused by degen eration, or failure to develop. It was postulated, however, that the condition of the calf was the result of inadequate secretions from the endocrine glands.
Crew (24) reported his physiological study of a bulldog calf.
This author concluded that there was a pituitary dysfunction between the second and third month of intra-uterine life. Under these con ditions the proper control of cartilage bone formation is lacking. The thyroid undergoes hyperplasia and this is followed by involution.
More recently, Johnson et al. (61) autopsied calves in a study of dwarfism. Histological studies of the pituitary, adrenals, and thyroid showed no abnormality. The genital tract of a male was autopsied at about two years of age. The tubules showed little 29
spermatogenesis but spermatozoa were present in the ampullae of the
ductus deferens. Lindley (70) reported observations on what he termed
a "midget" heifer and bull. Both were about two years of age and both
showed the common physical characteristics of the true dwarf. The
female had had one calf by caesarean section and at the time of
slaughter, a fetus was removed from this cow. The fetus was thought
to be by tho dwarf bull.
Studies of the endocrine glands were made. The pituitary glands
exhibited many large cystic spaces partly filled with a reddish hyalin
material. The thyroids were widely dilated and filled with colloidal material. The adrenals and ovaries were normal.
Endocrine studies in dwarf cattle by Gregory et al. (51) showed
that the hypophyseal growth hormone is highly potent, and the thyroid
hormone is very deficient. It was suggested that probably moro than
one type of dwarfism was involved within each breed of the Hereford,
Angus, and Shorthorn.
A comparison of the physiological effect of normal and dwarf calf
pituitary extract was made by Carroll et al. (16). Dwarf calf
pituitary exbract 'was injected into day-old cockerels. The thyroids
of the chicks were recovered and weighed after four injections. The
extract from the dwarf pituitary caused some growth. However, normal
pituitary extract caused a much greater increase in thyroid weight.
This deficiency might account for the lack of growth in the dwarf.
Dwarf calf pituitary caused as much gonad stimulation as did the normal
pituitary. Small amounts of growth hormone were also present. Fransen and Andrews (4l) conducted detailed physiological studies on 66 dwarf calves. Gross studies of 44 dwarf thyroids have revealed an average thyroid weight of 10.96 gm. Twenty-six calves had glands below, and eighteen above, the average thyroid weight. Iiine animals with cystic pituitaries and 21 animals with cystic adrenals were observed at autopsy. In some instances the same animal had both cystic adrenals and a cystic pituitary, and in three instances, bilateral adrenal cysts were observed. Reproductive tracts and gonads of both sexes were apparently normal. In a large percentage of cases internal hydrocephalus with distension of the lateral ventricles was observed. Grossly, the remainder of the glands and organs were relatively normal in most instances. Histological studies also revealed a wide range of activity in most glands and organs; however, most were within the normal range. Blood cholesterol values varied greatly within the same animal and between different animals; however, in most instances the variation was within the normal range. Bio assay of dwarf calf pituitaries showed normal thyrotrophic and gonadotrophic activity. Cerebrospinal fluid pressure was high in all cases. Electrocardiographic studies revealed normal heart activity in nine of ten dwarf animals. Hormone therapy of 11 dwarf calves with thyroprotein, stilbestrol, and testosterone, singularly or in combi nation stimulated average daily gains and general behavior of the treated calves but did not significantly alter bone growth. Vari ations in blood constituents 'were very similar to those observed in normal beef calves of similar age. 31
Recent anatomical approaches to dwarf carrier identification.
Within recent years several experiment stations have conducted investi gations in search of an anatomical, physiological, or chemical factor that could be used as a means of identifying cattle which are heterozygous for the "snorter" dwarf gene.
Gregory et al. (5l), in their search for an anatomical character in the homozygous dwarf that may also be expressed in the heterozygote parents, state that "Perhaps the feature in the dwarf in greatest need of amplification concerns the forehead." These Y/orkers studied the head contour of more than sixty dwarfs and several hundred normal animals in the Hereford, Shorthorn, and Angus breeds. The most out
standing characteristic of the specific type of dwarfism studied was a brachycephalic head with a marked mid-forehead prominence present at birth and persisting throughout life. This type of contour is dis continuous and does not intergrade into the type of contour of normal homozygotes. Accompanying this anomaly is a misshapen mandible in which there is marked malocclusion of the incisors with the dental pad.
These characteristics in addition to stunted growth, pot bellies, and heavy breathing are manifestations of thyroid deficiency. These morphological features are in agreement with another study by Carroll et al. (16) in which they report that the pituitaries from snorter dwarf beef cattle to be deficient in thyrotroph!c hormone. Their results showed that pituitary tissue from dwarf cattle caused chick thyroids to increase in weight over the controls. Pituitary tissue from normal beef cattle, however, caused a much greater increase in 32
thyroid gland weight. It was postulated that if snorter dwarfism was
cretinoid in nature, the skull would be deformed accordingly.
This exploratory work suggested that an instrument which would
accurately record the head profile might be of value, not only in
\ studying the dwarfs, but in separating the homozygous normal from the heterozygous normal animals. The instrument perfected and used by this group is known as the profilometer•
The instrument is specifically designed to reproduce the contour
of an undulating surface and works extremely well on the bovine head.
A median head profile is obtained by placing the nose piece on the firm
tissue immediately above the nostrils and tho opposite end of the
instrument on the frontal eminence at the poll. The operator then moves the block carrying the contour follower and recording stylus
along the track and the profile is reproduced on the graph paper.
Gregory (52) was able to locate three critical points that have value in determining whether or not the dwarf gene has expression in the heterozygous state. The first is the parietal-frontal juncture,
(PFJ). This point occurs approximately three centimeters anterior to the origin (0), the point at which the profile begins at the poll.
The second point involves the region at the mid-forehead. This point
/ cannot always be determined directly from the configuration of the head profile because certain basic head types are of such a conformation that they tend to mask the effect of the dwarf gene. The author found it convenient to recognize several head types which are referred to as
''basic" because they do not result from the action of the dwarf gene. 33
It was found that the dwarf gene exerts its maximum effect in the mid forehead at a point one-fourth of the head length anterior to the origin 0. Thus the mid-forehead point (MFP) can be determined by the formula MFP = 0 + 4 The nasal frontal juncture can be determined satisfactorily from the configuration of the head profile. The author has divided bovine heads into three basic types and devised a key to differentiate between them. The key is further divided in such a way that within each head type the homozygous normal and the heterozygotes can be determined.
Accox'ding to the author, experienced operators can take profiles with a high repeatability. Extreme care must be exercised when profiles are taken under adverse conditions or when they are taken by inexperienced operators.
The profilometer has not been widely accepted by the beef cattle industry which perhaps results from the fact that in many instances the profile has not agreed 'with the proved genotype.
Hazel et al. (54) reported.a study in which radiographs of the thoracolumbar spinal region of young calves has been examined as a method of detecting dwarfism. All of the 40-50 snorter dwarf calves examined by this group exhibited severe longitudinal compression and irregular protrusion of the body below the usual epiphyseal-diaphyseal union. Many of the heterozygotes exhibited abnormalities of a less extreme nature. These same workers outlined a more detailed study reported by Smmerson (36), concerning the identification of the dwarf gene carrier calf. When examining the thoracolumbar spine from the lateral view, evidence of longitudinal compression of the bodies of the last four or five thoracic vertebra should be noted. Most noticeable are the undulations on the ventral profile of these vertebrae. These undulations, together with the straightness of the ventral profile of the body of the vertebra, which is arched dorsally in the dwarf gene free, individual, are thought to be evidence of the longitudinal compression of the body. This compression of the body of the vertebra occurs during the cartilagenous stage and possibly the early intramembranous stage of bone development v/hich are in the middle and latter parts of the gestation period. It is in this region, these workers point out, that the powerful longissimus dorsi and psoas major muscles, especially in the meat type animal, exert a great part of.their contractile force on these vertebrae. During the intramembranous phase of calcium deposition in the cortical part of the body of the vertebra, the undulating or folded nature of the calcium deposits creates linear areas of increased density corresponding to the position of the areas of greatest compression. There seems to be an overall shortening of the body of the lumbar vertebra with a corre sponding increase in the depth. As a result, the epiphyses do not appear to totally cover their corresponding metaphyses. Sometimes, but not always, the metaphyses seems to be wedge shaped in outline rather than rectangular. In contrast, Hazel et al. (54), point out that dairy calves have only normal vertebrae with an obvious smooth half-moon curvature of the lower body when seen in ventral profile. 35
Bovard et al. (10) reported a study in which the combined length of the six lumbar vertebrae was measured from individual radiographs of 19 snorter dwarf and 131 normal Hereford calves at birth and correlated with birth weight. The mean difference between normal and dwarf calves in lumbar length and birth weight was 1.31 era. and 13.5 pounds respectively. The regression of lumbar length on birth weight* b~0.04S, was significant (P<.Ol).
Rankin et al. (82) recently reported a study designed to determine the accuracy of the x-ray method for identifying the three genotypes for snorter dwarfism. This group made lateral lumbar radiographs of more than 1590 calves produced in several lines of Hereford and Angus cattle. Genotypic predictions were based on the degree of abnormality seen in the lumbar vertebrae. In all, 85 animals of known genotype were x-rayed, including 36 dwarf carriers and 49 dwarfs. In a Hereford herd that was presumed to be dwarf free, 317 calves were x-rayed.
Several facts became evident from the x-rays: (l) The x-ray method has an accuracy of approximately 78 per cent in identifying dwarf carriers, approximately 74 per cent in identifying dwarf free animals, and approximately 9 6 per cent in identifying dwarf calves. (2) The greatest number of errors in predictions are found in the range of normal to mildly abnormal vertebrae. (3) More males have abnormal vertebrae than females, and the abnormalities seem to be more extreme in the males. (4 ) The x-ray method is not highly accurate in identify ing carriers and non-carriers, but it is highly accurate in identifying dwarf calves. 36
This technique has not been generally accepted, by beef cattle breeders as a method of differentiating between dwarf-carrier and non carrier cattle because of the overlap of the two phenotypes.
Recent chemical approaches to dwarf carrier identification. Foley et al. (40) reported variations in the physiological response to stress in dwarf and normal beef animals in 1956. These workers used 59 animals from the Angus and Hereford breeds in a series of studies to determine possible causes of dwarfism in beef cattle, and to develop a method of identifying animals heterozygous for the dwarf gene. Normal
(pedigree-clean) animals, known carriers of the dwarf gene, and dwarfs from both the Angus and Hereford breeds were included in the study.
Insulin tolerance tests, in which insulin was injected intravenously at the rate of .36 units per pound of body weight, and the blood sugar level determined thirty minutes subsequent to the injection and at two-hour intervals for 12.5 hours, showed definite differences between dwarf and normal appearing animals. The tests indicated that the blood sugar level dropped more quickly and to a lower levol in the dwarfs and failed to return to a normal level as quickly as in normal appearing animals. This indicated a possible abnormal pituitary or adrenal hormone response to stress in dwarf animals. The increase in white cell count in the blood following insulin was then used to test for an adrenal cortical hormone response to stress. Dwarfs responded very little; pedigree-clean animals showed a rapid and extreme response, whereas known carriers of the dwarf gene were intermediate 37
in their response to this treatment* Differences between the three groups were highly significant when tested statistically.
This same group, Foley et al. (39), reported more detailed investi gations in a subsequent study where an insulin tolerance tost was conducted on 12 pedigree-clean animals, 12 known carriers, and 12 dwarf beef animals. This test involved the measurement and comparison of insulin-induced hypoglycemia and its duration following two successive different levels of insulin administration. An initial administration of 0.8 units was followed at 48 hours with 0.3 units per kilogram of body weight. Blood sugar values were determined by the Folin-Wu method (which measures other non-sugar reducing substances as well as glucose) and the ITelson-Somogyi method (which measures glucose only).
A highly significant difference existed between insulin tolerance curves for the three genotypes. Following the higher dosage, signifi cant differences were observed for the mean sugar values between all
,genotypes regardless of method of sugar determination used. Following the lower insulin dosage, dwarf animals differed significantly from those phenotypically normal; however, a significant difference was not found between clean and carrier animals (Nelson-Somogyi). A difference existed between normals and carriers at the lower dosage with the
Folin-Wu method. Correlations were observed between blood sugar read ings taken at comparable times for the two dosages. Similar toleranoe
curves were obtained on clean and dwarf cattle, but not for carriers
(Folin-Wu). This indicated that dwarf and clean pedigree animals responded comparably (within genotype) to both dosages while the 38
carriers reacted differently at the two insulin levels. A lower "area
glucose change index," derived from the up and down fluctuations above
and below the initial sugar readings following insulin administration,
indicated that the dwarf animals were the most sensitive of the
genotype s studied.
Taylor et al. (106^ recently reported a different approach to the carbohydrate metabolism studies of dwarf and non-dwarf cattle. The investigation involved hyperglycemia and its duration on fasted and non-fasted animals. Changes in the blood glucose levels of 65 non dwarf and 17 dwarf cattle of the Hereford and Angus breeds were measured after intravenous injections of epinephrine, insulin, and glucose in both fasted and non-fasted animals. Host of the animals were yearlings; however, mature cows were also used in the epinephrine studies. Both fasted and non-fasted yearling dwarfs responded to a single injection of 0.25 cc. epinephrine per 100 pounds with smaller increases in blood glucose than did non-dwarf animals. Before fasting, the per cent increase of the 20 minute sample over the initial sample was 69.7 per cent for the non-dwarf, and 35.7 per cent for the dwarf.
Differences between fasted yearlings were not significant. Epinephrine hyperglycemic differences between fasted pedigree clean and known carrier cows were very small. Glucose tolerance curves were similar for the dwarf and non-dwarf groups after the intravenous administration of 0.5 gnu glucose per kilogram of body weight. Fasting the animals for 72 hours resulted in a decreased tolerance for glucose. Dwarf animals were more sensitive than non-dwarfs to injection of 0.36 units 39
insulin per pound of body -weight when neither were fasted. Six hours
after insulin injection, the blood glucose values of the dwarf (43.0 mg. per cent) and non-dwarf (57.4 mg. per cent) were significantly different, and represented respective decreases of 30.3 per cent and
3.8 per cent from initial values. A three-day fast abolished the
significant differences which had existed before fasting.
Abbreviated studies undertaken on blood constituents of dwarfs, dwarf-carrier, and non-carrier cattle, have failed to reveal startling differences to date. Investigations by Fransen and Andrews (4l),
Andrews et al. (l), and Cornelius et al. (22), indicated that the formed elements of the blood were similar in dwarf and normal cattle.
A study by Leuchtenberger et_ al. (68) showed that desoxyribo nucleic acid (DHA) content of individual spermatogenic cells from one dwarf and two suspected carriers 'was markedly deficient when compared with cells of normal bulls.
Dollahon et al. (27) recently reported a study of certain blood constituents of dwarf-carrier and non-carrier cattle composed of four age groups. The groups consisted of (l) twenty mature Hereford females of which ten were of known dwarf-carrier and ten were of assumed non carrier genotype; (2 ) twelve three-year-old Hereford heifers which were the offspring of carrier parents or closely related individuals and seven three-year-old Hereford heifers which were assumed to be non carriers; (3) six two-year-old Hereford heifers which were the off spring of carrier parents and 14 two-year-old Angus heifers which were assumed to be non-carriers; (4) six yearling Hereford heifers whose 40 parents ’were carrier animals and six yearling Hereford heifers whose parents were assumed to be free frora the dwarf trait.
Results showed the ribonucleic- acid (RIIA.) content of the plasma protein to be significantly higher in the dwarf-carrier group. The desoxyribonucleic acid content of the plasma protein vjus higher in the non-carrier group. The difference was highly significant. HoYrever, in all cases, there were varying degrees of overlap between the two groups which prevented differentiation of the dwarf-carrier and non-carrier with an acceptable degree of accuracy.
Non-significant differences between carrier and non-carrier groups were observed for blood hemoglobin, mean corpuscular hemoglobin, hematocrit, erythrocyte number, Leucocyte number, blood cell fragility, blood serum albumen, blood globulin fractions, blood copper, and blood zinc levels. The same 16 amino acids were present in the blood plasma of both groups. Lucas and Turman (72) substantiated these results in a similar study. In addition, they reported highly significant increases in the mean values for hemoglobin, hematocrit, and erythrocyte number with increasing age from birth to three months. EXPERIMENTAL PROCEDURE
Blood samples used in this work were collected from 123 Hereford and Angus cows and 10 snorter dwarf calves originating on several different farms. The Rutgers University Angus herd was also included in this study. Blood samples were collected from the same individuals on several occasions.
Results in this study are based on the assumption that pedigree clean cows were free from the dwarf gene. In the case of the carrier cows, blood samples were drawn only from cows which had previously produced a dwarf calf. The dwarf blood was from calves diagnosed as typical snorter dwarfs. Several of them died during the course of this work.
Blood samples of 50 to 100 ml. were drawn from the jugular vein of the cattle by means of a bleeding needle. Ho anticoagulant was used with the fresh samples when the serum was to be recovered. The blood was refrigerated immediately at 36°-38° P. and the serum was collected after it separated from the cell mass. Blood to be used in other investigations was drawn into vials containing Alsever’s solution.
This material contained 2.05 per cent glucose, 0.42 per cent sodium chloride, 0.8 per cent of trisodium citrate, and 0.055 per cent of citric acid. Alsever's solution functioned as a combination anti coagulant and preservative.
41 42
All sample vials were dated and labeled with respect to cow number, genotype and breed.
Total serum protein analysis. Previous research shows hypogly cemia to be a physiological disturbance in virtually all snorter dwarf calves. This suggests a possible dysfunction of the anterior pituitary or adrenal cortex which may indirectly alter liver function with respect to serum protein levels. Therefore, an exploratory study of serum protein levels of dwarf calves, carrier cows, and normal cows was indicated. This study involved 12 normal cows, 12 carrier cows and 5 dwarf calves.
The biuret method as described by Garnall et al. (42) was used for total protein determination. The biuret reagent was composed of 1.50 grams of cupric sulphate (C^SO^ZIigO) and 6.0 grams of sodium potassium tartrate (haKC4iI4.Og.4H 2O) which was transferred to a dry 1 liter flask and dissolved in 500 ml. of water. Three-hundred ml. of
10 per cent sodium, hydroxide was added with constant swirling. It was made to volume with water, mixBd and stored in a paraffin lined bottle.
Colorimetric test tubes were used with one labeled as a blank (b ) and the remainder was labeled in accordance with the samples to be determined. Into tube B was pipetted 2.0 ml. of a 22.6 per cent sodium sulphate solution. This blank (B) served for all protein analysis being conducted at any one time. Into another centrifuge tube was measured 0.5 ml. of serum and 9.5 ml. of a 22.6 per cent sodium sulphate. The tube was stoppered, and the contents mixed thoroughly by inversion (not shaking). Two ml. of the solution was immediately 43
transferred, to each tube used for total protein determination. Bight
ml. of the biuret reagent was pipetted into each of the tubes and
mixed thoroughly by swirling. The contents were allowed to stand for
30 minutes at room temperature.
The test employed The Weston Photoelectric Colorimeter, trans
mitting maximally at 540 millimicrons. It was adjusted to 100 per
cent transmission with the "blank" in position. The "blank" was then
replaced with each tube in question and the optical density recorded.
Blectrophoresis studies. In view of the absence of such infor
mation in the literature electrophoretic patterns of the serum
proteins from 12 normal cows, 12 carrier cows and three dwarf calves were studied. The method of starch gel zone electrophoresis as de
scribed by Smithies (89) was used. Soluble starch was produced by
treatment of potato starch with acetone - IiCi at 36.1° C. for 70 minutes. Gels were prepared from this soluble starch in 0.03 M sodium
borate buffer of pH 9.05. The starch gel was prepared in a plastic
tray. The sample to be fractionated was introduced into a vertical slit
in the gel at right angles to the greatest length of the gel. Electri
cal contact was made to the ends of the gel with filter paper soaked
in the buffer solution which dip into vessels containing the same so
lution. Filter paper bridges in turn connect these vessels to the electrode chambers. Blectrophoresis was accomplished at 10° c. for 20 hours with a potential of 4.5 volts/cm.
The gel vras removed from the tray and sliced lengthwise in a horizontal plane and the slices stained with a protein dye. After 44 washing with dye solvent, the stained separated proteins were observed
and photographed.
Starch gel zone electrophoretic patterns (as described in the
previous test) of the hemoglobin of 12 normal cows, 12 dwarf carrier
cows and 3 dwarf calves were also measured. The erythrocytes from the
respective cattle groups were thrice washed in isotonic saline (0.9 per cent) solution. Complete hemolysis of the red blood cells was then induced by suspending them in hypotonic (0.4 per cent) saline.
The hemoglobin was recovered by centrifugation.
Hemagglutination with plant extracts. It has long been recognized that in seeds of certain plants proteins occur which strongly aggluti nate human as well as other red blood cells. Landsteiner (67) pointed out that some of these extracts displayed various degrees of specifici- ty, agglutinating the blood of some species strongly, and the blood of other species v.-ealcly or not at all. Boyd (ll) investigated the agglutinating action of extracts of a large number of plants and report ed some to be specific for certain blood group antigens of the same species. Other workers have shown this to be a rather widespread phenomenon. These facts prompted the investigation of the agglutin- ability of the red blood cells of carrier and normal beef cattle by plant extracts.
The plant agglutinins were prepared by first soaking the beans over night in water at room temperature. The excess water was poured off and the seeds were chopped as finely as possible with three times 45 their volume of 0.9 per cent saline solution with the use of a Waring
Blender. The resulting mixture was centrifuged and filtered to remove excess debris.
The agglutination reaction was carried out in four ml. centrifuge tubes. The thrice washed erythrocytes were suspended in a 0.9 per cent saline solution at a one per cent concentration. One-tenth ml. (-two drops) of the plant extract was transferred to the tubes and 0.2 ml.
(four drops) of the blood, corpuscle suspension to be investigated was added. The mixture was allowed to stand 10 to 15 minutes and then centrifuged. To facilitate reading, the tubes containing the corpuscles were carefully shaken. A clumping of the cells indicated a positive test. If no clumping occurred, the test was deemed to be negative.
Thirty varieties of beans were screened for their agglutinating characteristics on the erythrocytes of cattle. The initial screening indicated that twenty of these varieties could agglutinate cattle cells.
However, upon using a larger number of blood samples, the extract from two of these beans exhibited potential promise. These varieties, commercially labeled "Bountiful" and "Pencil Pod Wax," were tested for their agglutinating capacity of red blood cells against samples from 20 known carrier and 20 presumed dwarf free cows.
Hemagglutination with an enzyme product. An investigation of the agglutinability of the erythrocytes of 50 normal blood samples and 30 carrier blood samples was conducted using Armours Bovine Catalase.
When the enzyme was suspended in 0.9 per cent saline, the agglutinating character was not found in the supernatant, but in the residual 46
material. The technique employed was to thrice wash the erythrocytes
and prepare a one per cent red blood cell concentration in 0.9 per
cent saline solution. One-tenth ml. of the erythrocyte suspension and
two-tenths ml. of the enzyme preparation was transferred to each of
the 4 ml. centrifuge tubes. The mixture was allowed to stand 15-20
minutes and then centrifuged. The degree of agglutination, as
described in the previous test, was read and recorded.
Serological investigations. This phase of the work was initiated
to determine whether a demonstrable antigenic difference could be
detected between dwarf-carrier and normal cattle. The present investi
gation was largely exploratory in nature since no similar approach to the solution of identifying cattle heterozygous for the dwarf gene has been reported in the literature. Antigenic components with respect to the various blood types in dairy cattle has been investigated and
reported rather extensively. With this information at hand a study of this nature was indicated.
The procedure included the injection of rabbits, guinea pigs,
sheep, normal cattle and dwarf calves with erythrocytes and whole blood from normal cows and dwarf calves. The site of injection, substance
and quantity injected, and the time intervals are outlined in the
injection and bleeding schedule.
The samples of red blood cells used were d r a w from the jugular vein of the cattle in question into tubes one-third filled with
Alsever’s solution. The tubes were labeled and stored in the refriger ator until used. In order to prepare the erythrocytes for the 4-7
subsequent agglutination procedures 5 ml. of the blood xvere transferred to tubes containing approximately 40 ml. of isotonic (0.9 per cent)
saline solution. The contents of the tubes were well mixed and
centrifuged. The supernatant 'was removed and the packed red blood
cells were resuspended in saline to a volume of 50 ml. This procedure was repeated three times and a final 30 per cent suspension of the red blood cells was made. The preparation was injected as outlined in the injection and bleeding schedule.
The antisera was prepared following withdrawal of blood by veni puncture from the immunized animals. The cells were allowed to clot and the supernatant serum was withdrawn. The clot was then broken and
centrifuged to recover the remainder of the serum. Any remaining cells were separated from the antiserum by prolonged centrifugation.
The antisera (anti-normal, anti-carrier- and anti-dwarf) were absorbed with the heterologous red blood cells in question.
In the hemagglutination reaction the serum of an immunized animal and the thrice washed erythrocytes of the carrier cows, normal cows or dwarf calves were allowed to react together. The reaction was observed in small (4 ml.) test tubes. The antiserum was added to the tost tubes in the amount of 0.1 ml. along with 0.2 ml. of the red blood cell suspension (one por cent) of the homologous animal. The mixture was
shaken, let stand for 10-15 minutes and centrifuged. After centrifu gation the erythrocytes were found as a sediment with a clear supernatant. To facilitate reading, the sediment was carefully shaken up. It could then he seen whether the red blood cells again dispersed themselves evenly throughout the fluid. All readings were made macroscopically. 49
TIE DMimiZATIOH AND BIBEDING SCHBDUL3
Rabbits, guinea pigs, oattle, and sheep were employed as subjects for the production of immune sera in this experiment. Thrice washed erythrocytes from normal, carrier and dwarf cattle were used in the preparation of the antigen. The injectable antigen was composed of a
30 per cent erythrocyte suspension in 0.9 per cent saline solution.
Separate antigens were prepared from each of the three cattle genotypes. The injection routes were subcutaneous, intravenous, or intra-abdominal. The blood was drawn from the subjects by venipuncture in the case of rabbits, cattle and sheep, and by cardiac puncture in the guinea pigs.
The predetermined time lapse between each series of injections was to allow a systemic reaction to the injected erythrocytes with the intent of increasing antibody production sufficient to agglutinate the homologous cells.
One ml. of the antigen was used at each injection in the case of the rabbits and guinea pigs and 5 and 10 ml. were used for the sheep and cattle respectively. Whole blood was used in the third series of injections in the cattle and sheep.
Blood collections were made in this work, perhaps earlier during the series of injections, and more frequent than those commonly used in most serological work. This is justified on the basis that the work with normal, carrier, and dwarf cattle cells is exploratory and no accurate prediction could be made with respect to time or level of antibody titer. 50
Sheep - Antigens prepared from dwarf calf cells and normal cow cells were used in the sheep immunizations.
1. (a) Injected 5 ml. subcutaneously each day for three consecutive days (b) Collected blood 7 to 9 days subsequent to the last injection
2. (a) Administered 3.0 ml. of the coll suspension subcutane ously the day preceding intravenous injections to avoid anaphylactic reactions (b) Injected 5 ml. intravenously each day for three con secutive days (c) Collected blood 7 to 9 days subsequent to the last injection
3. (a) Administered 5.0 ml, of the cell suspension subcutane ously the day preceding intravenous injections to avoid anaphylactic reactions (b) Injected 5 ml. intravenously each day for three con secutive days with whole blood (c) Collected blood on 7th, 10th, and 14th day subsequent to the last injection
Cattle - Cross immunizations were conducted with the normal and dwarf cattle.
1. (a) Injected 10 ml. intravenously each day for three con secutive days (b) Collected blood on the 7th day subsequent to the last injection
2. (a) Five ml. of the cell suspension was administered subcu taneously on the day preceding intravenous injections to desensitise the cattle to anaphylactic reaction (b) Administered 10 ml. intravenous injections each dajr for three consecutive days beginning the eleventh day subsequent to the last injection (c) Collected blood seven days subsequent to the last injection
3. (a) Administered 5 ml. of the cell suspension subcutane ously on the day preceding intravenous injections to desensi tize to anaphylactic reaction (b) Administered three intravenous injections 10 ml. of whole blood on alternate days (c) Collected blood on the 7th, 10th, and 14th days subsequent to the last injection 51
Rabbits --
1. (a) Injected 1 ml. intravenously each day for three con secutive days (b) Collected blood 7 days subsequent to the last injection
2. (a) ■ Administered 0.5 ml. of the cell suspension subcutane ously the day preceding intravenous injections to avoid anaphylactic reactions (b)• Injected 1 ml. intravenously each day for three con secutive days (c) Collected blood seven days subsequent to the last injection
3. (a) Administered 0.5 ml. of the cell suspension subcutaneously the day preceding intravenous injections to avoid anaphylactic reactions (b) Injected 1 ml. intra-abdominal on three alternate days (c) Collected blood seven days subsequent to the last injection
Guinea Pigs -
1. (a) Injected 1 ml. intravenously each day for three con secutive days (b) Collected blood seven days subsequent to the last injection
2. (a) Administered 0.01 ml. of the cell suspension subcutane ously the day preceding intravenous injections to avoid anaphy lactic reactions (b) Injected 1 ml. intravenously each day for three con secutive days (c) Collected blood seven days subsequent to the last Injection
3. (a) Administered 0.01 ml. of the cell suspension subcutane ously the day preceding intravenous injections to avoid anaphy lactic reactions (b) Injected 1 ml. intra-abdominal each day for three con secutive days (c) Collected blood seven days subsequent to the last injection RESULTS AND DISCUSSION
Total serum protein analysis. Data obtained from total serum protein analysis, by use of the biuret reagent, are shoivn in Table 1.
The blood was drawn from a group of 24 Hereford cows, 12 of which were known dwarf carriers and the remaining 12 were pedigree clean. The dwarf blood samples were drawn from two Hereford and three Angus snorter dvrarf calves.
Serum protein levels indicate some variation both within and between the respective genotypes; however, all values fall within the norraal range. Mean values for the non-carrier and carrier groups we re
8.01 and 8.15 gm. per 100 ml. respectively compared with 7.44 gm. per
100 ml. for the dwarf calf group. A lesser mean value will be noted for the dwarf calf group; however, statistical analysis showed no significant differences in the serum protein levels among the various genotypes studies.
Electrophoresis studies. Electrophoresis is the migration of charged particles in an electric field. The rate of migration depends upon the size of the particle, viscosity of tho medium, and the voltage of the field. Serum proteins contain radicals (C00" and NEg*) which dissociate to give positive and negative ions. The numbers of positive and negative ions determine the sign and magnitude of the charge of
52 53 the protein particles. A negatively charged particle migrates toward the positive electrode and positively charge particles migrate to the negative electrode. The migrating faces of serum proteins form boundaries because identical molecules move at the same rate and in the same direction. The density of a front reflects the homogeneity of the protein molecules. Some of the electrophoretic components of the serum used in this work present somewhat indistinct fronts which indicates that the electric charges of the proteins grade into one another.
The several serum protein fractions may change in value inde pendently of one another, either with or without alteration in the quantity of total protein. The albumin and globulin fractions may change in opposite directions in several pathological states (7). It is now fairly well established that the liver is the site of serum protein synthesis. Carbohydrate metabolism studies with cattle suggest a disturbance in liver function of dwarf and dwarf-carrier cattle. With this information at hand, an investigation of the serum protein fractions of normal, carrier, and dwarf cattle was indicated.
Thirty-nine comparative starch gel zone electrophoretic patterns of the serum and hemoglobin protein fractions from normal cows, carrier cows, and dwarf calves are shown in Fig. 1 through 7.
The sera used in this phase of the work were from Hereford cows ranging in age from 3 to 10 years. The heterozygous cows were of known genotype in the case of dwarfism and the presumed normal cows 54 v/ere dvrarf-gene free so far as pedigree estimates could determine.
The dwarf calve s were from the Angus breed and ranged in age from one to eight months.
It is of value to note that cattle serum contains a mixture of albumins and globulins. The lattor contains several different fractions. It can be observed by a study of the electrophoretic patterns presented in Fij$. 1 through 5 that the serum proteins are separated into albumin, and alpha, beta, and gamma globulins. The most rapidly moving boundary is that of the smaller albumin molecules which occupy the most extensive zone in the anodic region of all the normal, carrier, and., dwarf sera. The alpha, beta, and gamma globulins have slower rates of migration but of the three, the alpha fractions moved the fastest and can be observed in the zone adjacent to the albumin. The gamma globulin had a slower rate of migration and is ob served as the only zone in the cathodic region to the left of the point of origin. The beta globulin fraction also migrated rather slowly and shows as a rather distinct band in the anodic region immediately to the right of the point of origin and adjacent to the gamma fraction.
Pig. 1 shows a comparison of the electrophoretic patterns ex hibited by the serum proteins from three normal and two dwarf carrier cows following 20 hours of electrophoresis. Pig. 2 exhibits four electrophoretic patterns of the sera from normal cows while Fig. 3 shows similar patterns of the sera for two groups of dwarf gene carriers with five cows in each group. 55
Fig. 4 shows comparative patterns of filtered. (Seitz filter) and unfiltered dwarf calf sera following six hours of electrophoresis. It can be noted that the gamma globulin fraction is only slightly evident in a six-hour electrophoretic pattern. This appears to be the only marked variation between the 6 and the 20 hour exposures.
Fig. 5 compares the migration rates of the various serum protein fractions of three dwarf calves, a normal calf, and a normal cow following 20 hours of electrophoresis. It is of interest to note the virtual absence of the gamma globulin fraction in the normal calf pattern. This characteristic, however, does not exist in either the normal or carrier cow patterns in Fig. 1 through 3.
Figs. 6 and 7 illustrate the comparative migration rates of the protein molecules of the hemoglobin from normal cows, carrier cows, and dwarf calves following electrophoresis for 20 hours. Hemoglobin, of the erythrocytes, is a conjugated protein and lends itself to the electrophoretic process. It can be noted that the hemoglobin migrates into the anodic region but does not fractionate or form distinct zones as is the case with serum proteins.
An evaluation of the electrophoretic patterns exhibited by the hemoglobin from the erythrocytes of the cattle used in this study would not seem to present any distinguishable variation between the cattle genotypes. Likewise, this study of the serum protein electro phoretic patterns of the previously described cattle does not provide a pronounced variation between the respective protein fractions that can serve as a method of differentiating between the genotypes studied. 56
It appears that the various fractions of tho serum proteins are not affected, by the genotype of the animal so far as the dwarfism trait is concerned.
This investigation was indicated however, since similar infor mation had not been reported in the research literature with respect to either the serum protein or the hemoglobin of normal, carrier, or dwarf cattle•
Hemagglutination with plant extracts. Observations on the ag glutination potential of lectins is fragmentary. Human blood typing has been most extensivoly investigated with nothing reported relative to cattle blood.
The agglutinins in plant extracts have been demonstrated to show some specificity for erythrocytes from normal and dwarf-carrier cattle,
(it would seem proper to refer to these plant proteins as agglutinins, since this implies an activity, but it is obvious that any reference to them as antibodies would bo incorrect.)
This work was conducted primarily as a test to differentiate between normal and dwarf carrier bloods. There appears to be a some what logical explanation for the lack of specificity as was exhibited in many instances (Tables 2-3). Since the specific configurations on the surface of the lectin molecules which enable them to react with the blood antigens have not been produced in response to an antigenic stimulus, one is forced to regard the correspondence as accidental.
It, therefore, does not seem too unusual that these accidental 57 correspondences should in some cases result in cross reaction, if the chemical structure of the blood antigens in question are similar.
The results of the trials are tabulated in Tables 2 and 3. From
Table 2 it will be noted that extracts from several different varieties of beans possess hemagglutinating activity for the erythrocytes of beef cattle blood. Of value also is the variation in the agglutin- ability between botanical species; some of which agglutinated red blood cells very strongly while others indicated little or no reaction.
The agglutinability of the erythrocytes by variety 227 was very strong whereas variety 209 was weak and varieties 247 and 226 we re negative for both the carrier and normal bloods. Varieties 1 and 2 showed definite separation of the cells from carrier and dwarf-free cattle in the initial screening work.
Table 3 shows a continuation of the work with plant extracts using only varieties 1 and 2 with red blood cells from 40 cows of known genotype. Tihen the extracts of these two varieties were added to the erythrocytes (l per cent suspension) of known origin, the normals would usually agglutinate, whereas, the carrier cells would usually remain suspended. This technique proved accurate on about 70 per cent of the known samples. These data were not significant when tested statistically. Yfhile this method does not currently provide an un disputed technique for detecting dwarf carriers, it does provide an indication of the specificity of the lectins for certain blood groups.
If the dwarf factor is associated with one of the cattle blood groups, then it is possible that one of the botanical species may be specific 58
and serve to differentiate bet1,Teen the carrier and normal red blood
cells. This area of dwarfism research should be expanded to include a
greater number of plant species along with several variations in
laboratory technique.
Hemagglutination with an enzyme product. The blood used in this
experiment was obtained from 60 Hereford cows originating on three
different farms. The 30 carriers wore of known genotype in the case
of dwarfism since all had produced a dwarf calf sometime previously,
and the 30 normal cows were pedigree clean.
The results of the agglutination reaction of the erythrocytes
from normal and carrier cattle are shown in Table 4.
Analyses of these data show that 77 per cent of the erythrocyte
samples from pedigree clean cows were positive for the agglutination
test while the erythrocytes from the known carrier cows were negative
for the same agglutination test in 73 per cent of the cases checked.
■While this test does not offer conclusive results, indications are
that the red blood cells from normal cows possess more of the aggluti
nating capacity than those of the carrier blood. In the questionable
blood samples, it was observed that agglutination in a carrier blood,
although present, was not as prominent as might be expected in a real
diagnostic test. In the same light, when a normal blood did not ag
glutinate, there were particles apparent in the centrifuge tube which were indicative of slight clumping of the cells. These were not
sufficiently distinct to differentiate the cattle genotypes. From
these data and the previously described tests, there does appear to be 59
easily detected, differences in the agglutinating capacity of the red
blood cells from different genotypes. There are, however, some
important trends in the various reactions.
Serological investigations. The serological methods employed in
this work were designed to determine whether specific antigenic factors
exist that may function to differentiate between dwarf-carrier and
normal (dwarf-free) beef cattle.
The term antigen refers to inherited entities which are usually
specific in serological reactions. These single factors are detected
by their reaction with the homologous antibodies.
If dwarf-carrier cattle possess an antigenic specificity different
from that of normal cattle, then it should be possible to induce the
formation of a specific antibody by introducing the dwarf or dwarf-
carrier erythrocytes into the bloodstream of appropriate subjects. On
the other hand, if normal (dwarf-free) cattle possess some heritable
entity not present on the erythrocytes of dwarf or dwarf-carrier
cattle, the anti-normal sera (antisera formed to normal cattle cells)
should exhibit a specificity in its agglutinating potential vfhen tested with normal, carrier, and dwarf cattle erythrocytes.
Antigens determined by genes at the same locus are said to belong
to the same blood group. At present the blood groups of cattle are
thought to appear at 11 different non-linked loci. These blood groups
control the antigenic pattern of cattle erythrocytes. Some workers
(84) suggest that the blood group factors, instead of being controlled
at one locus might be a chromosome segnent with closely linked loci. 60
Other investigators (102) prefer to think of the system as one locus with multiple alleles. As many as 164 alleles have been reported to occur at one locus.
Cattle have 30 pairs of chromosomes. It is, therefore, likely that some of the blood group genes are associated with genes governing other characters. If one considers the several morphologically different types of cattle dwarfism, and the supposition that each type is manifested by a different gene, it would seem reasonable to assume that a portion or possibly all the dwarf types are associated or linked with some of the blood groups' genes. The types of dwarfism that are indicated to bo controlled by an allelic gene series could also fit into such a pattern. The possibility of detecting such linkage, however, depends on the closeness of association and the degree of manifestation of the genes to which the blood group genes are linked.
Linkage between genes for qualitative characters with simple inheritance would seem to offer the most promise. The mode of dwarfism inheritance in beef cattle would appear to lend credence to this approach. However, no systematic studies of this kind relative to the dwarfism problem has been reported.
These facts led to the serological studies reported in this dissertation. Emphasis was placed on devising a technique which could be used to identify dwarf gene carrier cattle. To facilitate such a study, a number of subjects were injected periodically (see injection and bleeding schedule) with a prepared antigen (a 30 per cent cell suspension in 0.9 per cent saline solution) from normal, carrier, and 61 dwarf beef cattle. The subjects used in this particular study were rabbits, guinea pigs, sheep, dwarf calves, and normal cattle.
The results of the immune sera production by the various subjects
as measured by the agglutination test of the homologous red blood cells
are presented in Tables 5 through 12. The response to the production of a specific immune sera by rabbits following two series of three intravenous injections each of the prepared cattle antigen are tabu
lated in Tables 5 and 6 respectively. These data do not appear to indicate any appreciable specificity with respect to antibody formations to normal, carrier, or dwarf cattle cells. The agglutinations which did appear were comparatively weak; however, some were strong enough to be interpreted as positive.
Table 7 shows the agglutination reaction of normal and carrier
cattle cells to the immune sera produced by the same group of rabbits following a third series of three injections each (total of nine
injections). The intra-abdominal route was used for the last three injections.
The anti-normal scrum (serum from the rabbit injected with cells of normal cattle) which had been absorbed with heterologous (carrier) cells tends to indicate some specificity for the normal cattle cells
(Table 7). While these data do not show statistical significance, further investigation would seem to be indicated in this area. The immune sera to carrier and dwarf cells produced by the rabbits showed no significant discrimination between the erythrocytes from normal and carrier cattle according to the data presented in Table 7. 62
The results of antibody formation to normal, carrier, and dwarf cattle in the guinea pig as indicated by the agglutination test, are shown in Tables 8 through 10. The first series of injections (Table 8) did not appear to induce a differential in antibody production for either the normal or carrier bloods. Table 9 clearly indicates an antibody response to the injections but little or no discrimination can be detected between the carrier and normal genotypes.
The third series of injections into the guinea pigs increased the antibody titer to cattle cells as indicated by the agglutination reaction of the carrier and normal blood samples, bven though the ag glutination potential was increased, there appeared to be no pronounced differentiation between the agglutination reaction of the cells from the carrier and normal cows.
The attempt to induce production of isoimmune antibodies by cross immunization of dwarf calves and normal cows yielded inconclusive results in this particular trial (Table ll). The cow and dwarf calves received a total of 11 injections over a period of approximately 36 days. Any systemic reaction to the immunization was not indicated by the agglutination studies.
A series of immunizations were conducted in which normal and dwarf cattle cell antigens (30 per cent suspension) were injected into the jugular vein of sheep. Periodic trial bleeding was made and the anti sera tested against cattle cells. Analysis of the blood collected on the- seventh, tenth and fourteenth day subsequent to the last injection gave evidence of antibody formation. Table 12 shows the extent to which the 63 cell agglutinations occurred. There was a pronounced systemic reaction to the injections but no discrimination between the cattle cell genotypes could be detected. The carrier and normal cattle cells were agglutinated with but few exceptions.
Many variations exist in serological techniques. As a conse quence, no attempt was made in this study to investigate all the available approaches. This work was confined to agglutination studies to the exclusion of precipitin and hemolytic tests. The results presented here are inconclusive; however, certain trends would encourage a continuation of the serological approach as a method of identifying dwarf carrier cattle. SUMMARY
Blood, samples of 50-100 ml. were collected from 123 Hereford and
Angus cows and 10 dwarf calves to study certain chemical and antigenic properties.
Chemical investigations, The chemical analysis included a de termination of serum protein levels as well as starch gel zone electrophoretic patterns of sera from normal, carrier, and dwarf cattle.
The buiret method of serum protein determinations was employed to ascertain the variation in protein levels of 12 normal, 12 carrier, and 5 dwarf cattle, lie an values for the normal, carrier, and dwarf groups were 8.01, 8.15, and 7.44 grans per ml. respectively. Some variation existed, however all values were within the normal range for beef cattle sera.
The electrophoretic properties of the blood of 39 normal, carrier, and dwarf cattle were examined. The method of zone electrophoresis in starch gel was used to separate the serum protein and hemoglobin fractions. The albumin, and alpha, beta, and gamma globulins formed representative electrophoretic patterns for cattle blood sera.
However, no significant variation in rate or extent of the migration of the protein fractions could be noted. Starch gel electrophoretic 65 patterns of the hemoglobin from normal, carrier, and dwarf cattle were prepared. The hemoglobin migrated to the anodic region of the pattern. but no distinguishable differences could be observed between the normal, carrier, and dwarf hemoglobin.
The antigenic properties were studied by different techniques.
Plant proteins were prepared by suspending finely chopped beans in 0.9 per cent saline solution. The mixture was centrifuged to remove the debris and the supernatant filtered through paper. The agglutinating potential of the resulting extract was tested against erythrocytes from normal, carrier, and dwarf cattle. Variations in the agglutin- ability of the plant exbracts occurred. Two varieties proved accurate
011 approximately 70 per cent of 40 known samples tested. These results would tend to indicate some specificity of the lectins for blood of certain cattle.
Serological investigations. A study of the antigenic differences between normal, carrier, and dwarf cattle was conducted.
Rabbits, guinea pigs, cattle and sheep were immunized with prepared antigen and whole blood from normal, carrier, and dwarf beef cattle. Thrice washed erythrocytes from the three cattlo genotypes suspended in a 0.9 per cent saline solution served as the antigen.
Immunizations were administered over a period of 36-40 days and periodic bleedings of the subjects were made.
Antibody production of the subjects was measured by the hemag glutination test of the homologous red blood cells. 66
The guinea pigs and sheep indicated a positive antibody response to the injections but tended to agglutinate all cattle cells rather than differentiate between the carrier and normal blood samples. The agglutinating capacity of the immune sera, as measured by the hemag glutination test was more pronounced in all cases tested following the third series of injections.
The antisera produced by rabbits indicated more discrimination between the cattle genotypes than did the antisera from guinea pigs, sheep or cattle. The incidence of agglutination with rabbit anti normal sera and normal cattle cells (70 per cent of the known geno types) was greater than the anti-carrier or anti-dwarf sera with the homologous red blood cells.
Immune sera resulting from cross-immunizations of normal cows and dwarf calves yielded inconclusive results when measured by the hemag glutination test in this study. The normal cow and dwarf calves received a total of eleven intravenous and subcutaneous injections of the prepared antigen (a 30 per cent erythrocytes suspension in 0.9 per cent saline solution) and whole blood over a period of 36 days. Wo significant specificity to the homologous red blood cells could be detected. 67
Normal Cow
Normal Cow J I r Normal Cow
Carrier Cow
Carrier Cow
Cathodic Point of Anodic Region Origin Region
Fig. 1. Starch gel electrophoretic patterns of normal and carrier cow sera.
Normal Cow
Normal Cow
Normal Cow
Normal Cow
Normal Cow
Cathodic Point of Anodic Region Origin Region
Fig. 2. Starch gel electrophoretic patterns of normal cow sera. 68
Carrier Caw
Carrier Cow
Carrier Cow
Carrier Cow
Carrier Cow
Carrier Cow
Carrier Cow
Carrier Cow
Carrier Cow
Carrier Cow
Cathodic Point of Anodic Region Origin Region
Fig. 3. Starch gel electrophoretic patterns of carrier cow sera. 69
Dwarf Calf #1
Dwarf Calf ff2
Dwarf Calf #3
Dwarf Calf -j'fl (filtered) Dwarf Calf -ifZ (filtered)
Cathodic ‘f'Point of Anodic Region Origin Region
Pig. 4. Starch gel electrophoretic patterns of filtered (Seitz) and unfiltered dwarf calf sera.
Dwarf Calf 7^1
Dwarf Calf #2
Dwarf Calf 7f3
Noraal Calf /.
Normal Cow
Cathodic 'fVPoint of Anodic Region Origin Region
Fig. 5. Starch gel electrophoretic patterns of dwarf calf, normal calf and normal cow sera. 70
Dwarf Calf #1 I M M M M i
..'.pp * Dwarf Calf #1
* J Dwarf Calf $2
Dwarf Calf #3
Dwarf Calf #3 j
Cathodic ^Point of Anodic Region Origin Region
Fig. 6. Starch gel electrophoretic patterns of dwarf calf hemoglobin.
Normal Cow ' r - f
Normal Cow-
Normal Cow
Carrier Cow ' *
Carrier Cow
Cathodic ^Point of Anodic Region Origin Region
Fig. 7. Starch gel electrophoretic patterns of normal and carrier cow hemoglobin. 71
Fig. 8. Angus snorter dwarf calf
Fig. 9. Twin Hereford bull calves; one normal and one snorter dwarf. Pig. 10. Angus X Hereford crossbred snorter dwarf
Pig. 11. Snorter dwarf producing Hereford cow. 73
TABIE 1
A COMPARISON OF THE TOTAL SERUM PROTEIN CONTENT OF NORMAL, CARRIER, AND DWARF BEEF CATTLE BLOOD
NORMAL CARRIER DWARF Cow Grams per Cow Grams per Calf Grams per Number 100 ml. Serum Number 100 ml. Serum Number 100 ml. Serum
36 8.1 101 8.1 1 6.9
47 7.5 300 8.1 2 7.2
063 8.1 301 8.7 3 7.2
181 8.1 607 8.7 449 7.8
1111 8.1 745 8.1 458 8.1
77 8.0 806 8.5
53 7.8 50 8.1
70 7.9 277 7.6 CO • 50 8.4 40 ro
71 8.2 143 8.1
24 7.9 850 7.5
82 8.1 20 8.1
Mean 8.02 Mean 8.15 Me an . 7.44 74
TABIE 2
AGGLUTINATION REACTION OF THE ERYTHROCYTES FROM DWARF FREE AMD DWARF CARRIER COWS BY PLANT EXTRACTS
Source of Cells (by cow numbers) Bean Normal Carrier Number 47 936 383 10 49 33 2240 2035 93 89
224 +-}•-)- ++ -S-+ + 4-t- + -r4' + + + + + ++ + + + + + -f 209 ± 4- ++ + - + + + + +
203 + 4*4* + + + -5-4.+ 4. + + ++ + - L - -!■+-&--t- •{*+
211 ++ + + + + + + + + + + +• + + + + + + -M-
228 + + 4* + + + + + ■$* ++-?•+ + + + + + + + +
223 - + + + + + + + + ++ + +
227 ■f+4 + + 4- + + + + + + +++ +++ +-5--f -M-++ + +-S-
230 ++ 4-+ + + + + + + +-«-+ +4* + + + •(-•{- + + + +
247 ------
224 j. 4*4-4- > +4*4* + + ++ 4* 4*-I' ++ -t-+ ++ + 4* ’■ -f-
226 ------
+ l t t ++ + 4“ + + +,;.+ + - - --
2 i & + + +4*4* 4* ++ - ++ + -- + - - Variety "Bountiful" vF Variety "Pencil Pod Wax"
+ Positive reaction ± Partial reaction - Negative reaction 75
TABIE 3
A FURTHER INVESTIGATION OF THE AGGLUTINATION OF THE ERYTHROCYTES FROM DWARF-FREE AND DY7ARF-CARRIER. COWS BY TWO PLANT (Bean) EXTRACTS
DWARF-•FREE CELLS DWARF-•CARRIER CELLS Cow Reaction Cow Reaction Number Variety -jfz Number Variety $1
34 815 ++ 62 _ 806 - 33 - 847 75 + + 115 - 82 +•*■ 36 50 + + 149 - 35 - 794 - 7 ++++ 113 - 899 - 850 - 95 + + + + 40 - 36 +++ 30 + 78 - 160 - 33 + + 20 39 ++ 57 - 15 ++-■-+ 675 - 11 ++ 527 02 + 477 - 19 +-{• 189 - 15 - 590 + 250 504
+ Positivei reaction Negative reaction 76
TABIE 4
HEMAGGLUTINATION OF ERYTHROCYTES FROM CARRIER AND NORMAL CATTLE BY ARMOUR'S TECHNICAL CATALASE ENZYME
NORMAL CARRIER Cow Cow Cow Cow Number Reaction Number Reaction Number Reaction Number Reaction
22 + 15 + + 813 - 20 +
6 - 439 +•(• 13 - 57 -
47 + + 33 + 733 - 675 -
936 78 + 815 - 527 -
383 + + 36 + + 806 - 477 -
10 - 34 + 847 + 189 -
49 - 62 + -E- 115 J. 590 +
4-7 + 33 36 - 504 -
32 + + 75 + + 149 - 485 -
23 + 82 + 794 - 341 -
30 - 50 - 113 - 609 4"f*
15 + + 35 + 850 - 496 +
19 + + + 7 - 40 - 540 -
02 + 899 + 30 + 6 -
11 - 95 + + 160 - 130 +
+ Positive reaction - Negative reaction 77
TABIE 5
AGGLUTINATION'REACTION OP THE ERYTHROCYTES PROM NORMAL AND CARRIER COWS WITH IXIUNE SERA FRO.'i RABBITS FOLLOWING A SERIES OF TERES INJECTIONS
IXIUNB SERA ANTIGENS Normal Carrier Dwarf
Carrier Cow 33 ± - - 2240 --- 2035' ++ - _ 93 4* + 5 — - 89 - -- 458 - - T*I 449 - - + 813 - + 13 •“ “
Normal Cow
47 - i - 936 - - 383 - - - + 10 _ — 4-9 - - + 47 -- 32 _ — + 23 - - 230 --- 15 “ ■—
+ Positive reaction + - Partial reaction - Negative reaction 78
TABIE 6
AGGLUTINATION REACTIONS OF THE ERYTHROCYTES FROM NORMAL AND CARRIER -COWS WITH IMMUNE SERA FROM RABBITS FOLLOWING A SERIES OF SIX INJECTIONS
IMJUNS SERA ANTIGENS Normal Carrier Dwarf
Carrier Cow 33 + 224-0 - - - 2035 - - - 93 --- 5 _ ± 89 + ± 458 - - + 449 -- 813 - + 4* 13 + --
Normal Cow 47 -- - 936 + - ± 583 + - + 10 - - 49 - + 47 + - + + 32 - - 23 4. j. - 230 _— — •U 1 r + + l o
+ Positive reaction - Partial reaction - Negative reaction 79
TABIE 7
AGGLUTINATION REACTIONS OF THE ERYTHROCYTES FROM NORMAL AND CARRIER COWS WITH IMMUNE SERA FROM RABBITS FOLLOWING A SERIES OF NINE INJECTIONS
IMBUNE SERA ANTIGENS N ormal Carrier Dwarf
Carrier Cow 815 + *1* - 806 --- 847 - - - 115 - -- 36 - + ± 149 ++ - + 794 --- 113 --- 8 50 -- + 40 + + -
Normal Cow 34 ++ - + 62 ++ - - 33 + -- 75 ++ _ - 82 + + - 50 -- ± 35 + + ++ 7 + + - 899 -- - 95 —
+ Positive reaction * Partial reaction - Negative reaction 80
TABLE 8
AGGLUTINATION REACTIONS OF TIE ERYTHROCYTES FROM NORMAL AND CARRIER COWS WITH IMMUNE SERA FROM GUINEA PIGS FOLLOWING A SERIES OF THREE INJECTIONS
IMMUNE SERA ANTIGENS Normal Carrier Dwarf
Carrier Cow 33 + 2240 - - 2035 --- 93 - - _ ± 5 - - 89 + -f + 458 - •f + + -f. 449 _ 813 + ± 13 - -
Normal Cow 47 -s* a. + 936 - + + 383 -- 10 — 49 - ± - 47 - - - 32 + -f- — 23 ± - - 230 — 15 -
Po sitive reaction Partial reaction Negative reaction 81
TABIE 9
AGGLUTINATION REACTION OF ERYTHROCYTES FROM 1JOBMAL AND CARRIER COWS WITH IMMUNE SERA FROM GUINEA PIGS FOLLOWING A SERIES OF SIX INJECTIONS
IXTUNE SERA ANTIGENS Normal Carrier Dwarf
Carrier Cow ~33 4 + 2240 + 2035 + + 4 93 4 5 89 F* 58 + 4 4. 449 4 813 44 ++ 4 + 13
Normal Cow 47 •4* 4 4 936 4 4 383 4 4*4 4 4 10 4 4 49 4 4 47 32 4*4* 4 23 4 4 4 4 4 4 230 4 4 15
+ Positive reaction * Partial reaction - Negative reaction 82
TABIE 10
AGGLUTINATION REACTION OF ERYTHROCYTES FROM NORMAL AND CARRIER COWS WITH IMMUNE SERA FROM GUINEA PIGS FOLLOWING A SERIES OF NINE INJECTIONS
IMMUNE SERA ANTIGENS Normal Carrier Ewarf
Carrier Cow 815 + ++ 4-4- + 806 + + + + 847 - + 4-4* 115 — 4- ± 36 - ± 4* 149 4* 4-4- ± 794 + + 4* 113 - 4-4- 4* 850 + 4-4*4- + 40 4-
Normal Cow 34 + + 4* 4- ± 62 4- ± + 33 u 4-4* 75 + ± ± 82 4- 4* 4*4* 50 ++ 4-4-4* + 35 + 4* 4* 7 ± 4* + 899 ++ 4- ± 95 *£■ ± 4*
+ Positive reaction - Partial reaction - Negative reaction 83
TABIE 11
AGGLUTINATION R3ACTI0N OF ERYTHROCYTES FROM NORMAL AND DWARF -CARRIER COWS TO ISOIMMUNE SERA FROM NORMAL AMD DWARF CATTLE FOLLOWING A SERIES OF NINE INJECTIONS.
H M U N S SERA ANTIGENS Normal Cow Dwarf 1 Dwarf 2
Carrier Cow 815 -- 806 + - - 847 - + - 115 --- + 36 - + + 149 - - 794 --- 113 --- 850 ± - + 40 - ± - 30 --- 160 -- -
Normal Cow 34 - - - 62 - - 33 ± - + -«■ 75 —_ + *4* 82 - 50 - - - 35 - - 7 -- - 899 - - - 95 ——
+ Positive reaction - Partial reaction - Negative reaction 84:
TABIS 12
AGGLUTINATION HBACTION OF THE ERYTHROCYTES FROM NORMAL AND CARRIER COWS WITH IMMUNE SERA FROM SHEEP, INDUCED BY A SERIES OF NINE INJECTIONS WITH NORMAL AND DWARF RED BLOOD CELLS
Immune Se r a-Dwarf Immune Sera-Normal Sheep No. Sheep No. 654 658 666 723
Carrier Cow 675 4 ± 4 4 527 4 4 - 4 4 4 477 ± 4 4 189 4 4 4 4 4 4 4 4 590 4 - 4 4 504 4 4 4 4 4 485 4 4 ± 4 4 4 4 341 4-v ± 4 4 4 4 496 - 4 4 4 4 609 t - - 4
Normal Cow 4 54 4 4 ± 62 44 ± 4 4 4 33 ± ± - 4 75 £ 4 ± - 82 4 4 4 4 4 4 ± + 50 4 4 4 35 -f* 4 4 ++ 7 - 4*4* 4 4 ++ 899 ± - 4 + 4 95 4 + + 4
+ Positive reaction * Partial reaction - Negative reaction BIBLIOGRAPHY
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112. Woodward., R. R., Clark, R. T., and Cummings, J. H. Studies on Large and Small Type Hereford Cattle. Mont. Agr. 13 xp. Sta. Bull. 401, 1942. AUTOBIOGRAPHY
I, Randall Robert Reed, was born at Davis, West Virginia,
September 1, 1921. I was reared on a livestock farm and received my primary and secondary education in the Tucker Comity Public Schools.
Following three and one-half years in the U. S. Wavy, I enrolled at
West Virginia Universit}/, February, 1946. I was granted a Bachelor of
Science degree, June, 1949. I was employed as an associate county agricultural agent in Harrison County, West Virginiaj until September,
1950, at which time I enrolled in the Graduate School, West Virginia
University. I was granted a Waster of Science degree February, 1952.
March 1, 1952, I received an appointment as assistant professor on the research and teaching staff in the Department of Animal Husbandry,
Rutgers University, Hew Brunswick, New Jersey, a position I held until
July 1, 1959. I continued graduate work during my appointment at
Rutgers University. October 1, 1959, I was granted a one-year leave of absence from Rutgers University to accept a Graduate Assistantship in the Department of Animal Science, Ohio State University, which enabled me to continue graduate studies. On July 1, 1959, I received an appointment in the Agricultural Extension Service, Ohio State
University, as an Animal Science Extension Specialist, a position I have held during the completion of the requirements for the Doctor of
Philosophy Degree in the Department of Animal Science.