Problem of Fertility 9781400886876

Table of contents :
PREFACE
CONTENTS
PATTERNS OF ESTROUS CYCLES
OVULATION AND ESTRUS IN SHEEP AND GOATS
INDUCTION OF OVULATION AND SUBSEQUENT FERTILITY IN DOMESTIC ANIMALS
THE INDUCTION OF OVULATION IN DOMESTIC ANIMALS
THE OVART AT THE TIME OF OVULATION
HORMONAL CONTROL OF OVULATION
CERVICAL MUCUS AND THE MENSTRUAL CYCLE
SPERMATOZOA AND CERVICAL MUCUS
THE GLYCOLYSIS, LIVABILITY, AND FERTILITY OF BOVINE SPERMATOZOA AS INFLUENCED BY THEIR CONCENTRATION
METABOLISM AND MOTILITY OF HUMAN SPERMATOZOA
FERTILIZING CAPACITY OF RABBIT SPERMATOZOA
THE BIOLOGY OF EQUINE SPERMATOZOA
ARTIFICIAL INSEMINATION OF DAIRY CATTLE
THE CERVIX UTERI IN INFERTILE MATINGS
THE EFFECT OF SYNTHETIC THYROPROTEIN ON STERILITY IN BULLS
METHODS FOR DETERMINING THE TIME OF OVULATION IN DOMESTIC ANIMALS
MEMBERS OF THE CONFERENCE ON FERTILITY

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THE PROBLEM OF FERTILITY

THE PROBLEM

OF F E R T I L I T Y PROCEEDINGS OF THE CONFERENCE ON FERTILITY HELD UNDER THE AUSPICES OF THE NATIONAL COMMITTEE ON MATERNAL HEALTH

EDITED Br EARL T. ENGLE

PRINCETON NEW JERSEY PRINCETON UNIVERSITY PRESS 1946

Copyright, 7946, by Princeton University Press London: Geoffrey Cumberlege, Oxford University Press

Princeton Legacy Library edition 2017 Paperback ISBN: 978-0-691-62764-9 Hardcover ISBN: 978-0-691-62863-9

Printed in the United States of America The Maple Press Company, York, Pa.

PREFACE

THE National Committee on Maternal Health presents in

this book the proceedings of the recent Conference on Fertil­ ity. Research on various aspects of reproduction has ramified widely in the last two decades. Investigation using the smaller laboratory animals has been most fruitful in revealing the fundamental biological factors in the physiology of reproduc­ tion. In many respects, the successful application of these basic principles to clinical therapy leaves much to be desired. No area of this field has been more successfully developed and applied to practical purposes than that occupied by Animal Husbandry. The purpose of this Conference was to reveal the successes of investigations on the processes of reproduction in domestic animals. The results of these investigations are of utmost importance and practical value in themselves. They should also serve to reactivate investigations on the induction of ovulation in woman and to encourage further detailed investigations in the biology of human spermatozoa. This book is made up of the papers which were read at the Conference and the discussions which took place. Each paper is followed by a Discussion in which various members of the conference contribute their knowledge of the subject under consideration. The National Committee on Maternal Health wishes to thank all those who participated in the Conference, with a word of special gratitude to Drs. Corner, Phillips, Hammond, and Taylor, who presided so skillfully at the several sessions. The Conference was made possible by a generous grant from the Ortho Research Foundation, through the sympa­ thetic interest and scientific spirit of its officers, Dr. Clair E. Folsome, Director, and Mr. Philip B. Hofmann, President. EARL T. ENGLE

CONTENTS

Preface

ν

BY EARL T. ENGLE

Patterns of Estrous Cycles

1

BY S, A. ASDELL

Ovulation and Estrus in Sheep and Goats

11

BY R. W. PHILLIPS, R. M. FRAPS, AND A. H. FRANK

Induction of Ovulation and Subsequent Fertility in Domestic Animals 49 BY L. E. CASIDA

The Induction of Ovulation in Domestic Animals

60

BY JOHN HAMMOND

The Ovary at the Time of Ovulation

67

BY GEORGE W. CORNER

Hormonal Control of Ovulation

74

BY H. H. COLE

Cervical Mucus and the Menstrual Cycle

102

BY W. T. POMMERENKE AND ELLENMAE VIERGIVER

Spermatozoa and Cervical Mucus

119

BY A. R. ABARBANEL

Glycolysis, Livability, and Fertility of Bovine Sper­ matozoa 134 BY G. W. SALISBURY

Metabolism and Motility of Human Spermatozoa 154 BY JOHN MACLEOD

Fertilizing Capacity of Rabbit Spermatozoa BY M. C. CHANG

169

viii

Contents

Biology of Equine Spermatozoa

187

BY VICTOR R. BERLINER

Artificial Insemination of Dairy Cattle

206

BY J. W. BARTLETT

The Cervix Uteri in Sterile Matings

218

BY FRED A. SIMMONS

The Effect of Synthetic Thyroprotein on Sterility in Bulls 233 BY E. P. REINEKE

Methods for Determining the Time of Ovulation in Domestic Animals 243 BY JOHN HAMMOND

List of Members of the Conference on Fertility

251

THE PROBLEM OF FERTILITY

PATTERNS OF ESTROUS CYCLES S. A. ASDELL

DURING the past twenty years a great deal of knowledge has

been gained concerning the patterns of reproduction in mammals and the factors which determine these patterns. Much of this knowledge has recently been brought together1 and this is an appropriate time to make a general survey of what we know, if only to decide the gaps to be filled before we can describe with any certainty the changes which have occurred in the reproductive mechanisms during the evolu­ tionary process. A broad division may be made by considering species in which the central nervous system has a marked measure of control separately from those in which no apparent control exists. One of the features of recent work is the demonstration that such nervous control is more widespread, usually through the anterior pituitary, than had been anticipated. It is pos­ sible that this control is never very remote, but it can only be seen with certainty in relatively few mechanisms at the present time. The first type of nervous control to be considered is that in which the development of the graafian follicle is regulated by a nervous mechanism. This is shown by the fact that in cer­ tain species the final enlargement of the follicle, secretion of liquor folliculi in quantity, and ovulation, depend upon sexual excitement, either actual coitus or an intense nervous stimulation resembling coitus in its results. There is ample evidence that the nervous stimulus causes a sudden release of anterior pituitary hormones. The classic three species with this type of mechanism, the rabbit, cat, and ferret, have now been extended to include the mink, the thirteen-lined ground squirrel and the short-tailed shrew. It may be considered premature to speculate upon the evolutionary distribution of

2

S. A. Asdell

the condition on the bases of only six species, but from the reproductive behavior of allied species it may be inferred that the condition will be found in many forms closely allied to those already known. Also we have good knowledge of many families in which the condition is not found and in which it probably will not be found. Putting these lines of evidence, positive and negative, together, one may infer that this form of control is a primitive one amongst mammals. The rabbit, a lagomorph, is a primitive mammal, an early branch of the evolutionary tree. Then it is found in the shrews, an early family of the insectivores; and in the squirrels, an early rodent family. Two families of the carnivores show the condition, the mustelids and the felids. This is the only order in which the condition breaks through from primitive families tt) recent ones, for the cats are highly advanced mammals. Spontaneous ovulation seems to be the rule in the other advanced orders and families. It is interesting to note that in the latest addi­ tion to the group, the short-tailed or mole shrew, one copu­ lation is insufficient to induce ovulation, several are necessary2. Another form of nervous control appears to be later in evolution and to be more highly specialized. This is the con­ dition in which the activation of the corpus luteum, so that it secretes progesterone, depends upon the transmission of a nervous impulse. Stimulation of the cervix uteri is necessary to start the chain of reactions and it determines whether the estrous cycle shall consist merely of a succession of waves of ripening follicles, or whether the diestrous interval, in which the corpus luteum is active, shall separate these waves. It is obviously a mechanism resulting in greater opportunity for fertilization and economy of hormone synthesis. This con­ dition has been found only in the cricetids and murids, (hamsters, rats, and mice) both fairly recent rodent families. Another type of nervous control has been found, but it is of an entirely different type as it does not seem to be depend­ ent upon the anterior pituitary but upon the length of time in which the central nervous system can respond to estrogens. This condition is found in the cow, a species with a relative short heat period, averaging about 13¾ hours, When thresh­ old doses of estrogens, not much more than the amounts needed to induce heat, are injected daily into ovariectomized cows, the resulting estrus is not continuous but ceases at the

Estrous Cycles

3

usual time in spite of the continued injection3. Evidently the length of the heat period is curtailed by the induction of a refractory period in the brain which causes sexual desire to cease. This is, in a sense, parallel to the brief but rhythmic response of the vaginal epithelium of the rat to threshold doses of estrogens and the periodic response of the monkey endometrium to similar doses, both of which have been de­ scribed by Zuckerman4. This phenomenon in the cow has an interesting conse­ quence. The cow is unique in that ovulation occurs after she has gone out of heat, usually about 13 hours afterwards. The average threshold dose of estrogens necessary to bring a cow in heat is very low, about 600 r.u.* This is apparently reached early in the development of the graafian follicle and the re­ fractory period sets in before the follicle is sufficiently grown for it to rupture. Thus there is an interval, in which heat has ceased, before ovulation occurs. The question of seasonal reproduction has been investigated by Marshall5 and others, mainly in England. It is clear that climatic factors influence the condition since the transfer of seasonal breeders from one side of the equator to the other Soon results in an adjustment of the breeding season to the appropriate time of the year. Temperature appears to be a minor factor in the control of reproduction, length of day­ light and the amount of feed are of greater importance. Some polyestrous rodents which reproduce all the year round in the laboratory experience a pause of varying length in winter, and others, especially under desert or semi-desert conditions have an additional pause during the summer. More experi­ mental work is needed upon these particular patterns before much can be said about them. In most species investigated, a seasonal fluctuation in the potency of anterior pituitary tissue has been found; but this is believed not to occur in the sheep6. This subject also, needs further investigation. * The following symbols will be used throughout this book: FSH follicle stimulating hormone LH luteinizing hormone PMS pregnant mare serum hormone PU chorionic gonadotropin from human urine of pregnancy AP extract of anterior pituitary gland Rab. u. rabbit unit r.u. rat unit

4

S. A. Asdell

The polyestrous condition as opposed to the monestrous state has been ascribed to the effects of climatic factors. Polyestrous species are believed to be more frequent in trop­ ical regions than in temperate regions, but our knowledge of the details of the cycle in tropical species is very fragmentary. It seems to me that the polyestrous type of behavior is more frequent in groups and species which have been evolved com­ paratively recently. Also it is an obvious advantage to species which are the prey of others and it occurs more frequently amongst such types. Perhaps they would be extinguished too rapidly if they did not have this advantage in reproduction. The burden of continuous reproduction might also be a handi­ cap to active carnivores and other hunters, so that seasonal breeders are more frequent in this type of mammal. Turning now to the structure of the estrous cycle in detail, several factors may be discussed. Proestrum has a different value in different species. In monestrous forms it is purely an estrogen effect due to the developing follicle. In polyestrous forms it is a period of mixed causes and effects since the decline of the corpus luteum and the growth of the follicle overlap. This is well brought out in the cow. In this species the uterine muscle, like that of the cat and dog, responds differently to epinephrin depending upon whether the female is under the influence of estrogen or of progesterone. If she is under the influence of estrogens the muscle is inhibited by epinephrin; if she is under the influence of progesterone it contracts. During proestrum the muscle shows a diphasic response, contraction and inhibition. The same diphasic con­ dition can be brought about in the ovariectomized cow by the injection of 250 r.u. daily of estrogens together with 18 Rab. U. of progesterone. This clearly indicates the dual nature of the proestrous period in polyestrous species. Even • in seasonally polyestrous forms, such as the sheep, it is doubtful if a purely estrogenic proestrum is found at the beginning of the season, since the first marked heat is usually preceded by a few subdued heat periods. The onset of the breeding season is not sudden. In monestrous species proestrum is longer than in polyestrous species since the gradual subdued growth is not cyclical but is continuous. In polyestrous species it usually takes four to five days for the follicles to reach the stage at which heat occurs. This is the

Estrous Cycles

5

length of the interval in murids and is the length of time re­ quired by the cow to come in heat after the corpus luteurnis extirpated. In the guinea pig the time is somewhat longer, probably about ten days. This species is notoriously slow and uncertain in its response to anterior pituitary hormones. The length of the heat period depends upon several factors. The possible existence of a refractory condition in the central nervous system leading to a heat period of a definite length has already been mentioned. Another major factor is related to the activity of the anterior pituitary. In the domestic animals an interesting chain has been found. The length of the heat period is directly related to the FSH content of the anterior pituitary; the more FSH content per unit weight of pituitary, the longer is the average heat period for the species. The threshold of estrogens necessary to bring the females into heat follows the same pattern, and so does the level of estrogen excretion. The series in descending order is horse, pig, sheep, and cow. Man fits into the series quite well at about the high level of the horse. If the relationship should hold in a general way for other species, when the* necessary information has been gained, it will be a most important generalization. There also seems to be a relationship between the FSH content of the anterior pituitary and the brain-body size ratio which may be significant from the point of view of evolution. The length of life of the corpus luteum in the absence of pregnancy is from ten to fifteen days in most mammals. The cow is again exceptional, with a length of about eighteen days. This greater length appears to be correlated with a high prolactin content of the anterior pituitary, but the extent of knowledge in this field is very small for most species. It may also be significant that the prolactin level is high in the rat and mouse when the life of the corpus luteum is prolonged by lactation, a fact that points in the direction of a definite relationship. The responses of individual organs to hormones are varied and depend not only upon dose levels but also upon response thresholds. The clear cut vaginal smears, cornified only during heat and terminated by a heavy invasion of leucocytes, ap­ pear in species in which the estrogen level is moderate. In species such as the cow and the sheep, in which it is low, cornification is not nearly so definite a landmark. In fact, the

6

S. A. Asdell

cow, with its exceptionally low level, does not cornify except to a limited extent in the vestibule. Instead, an intense mucification occurs in proestrum and estrus. In species with a high estrogen level, such as the monkeys, man, and the horse, there is always a certain amount of vaginal cornification which confuses the vaginal smear picture. The uterus, also, has a variable response to progesterone In the uteri of murids there is very little glandular develop­ ment during the life of the corpus luteum (diestrum). In the guinea pig, cow, and pig the response is moderate; while in the rabbit, cat, and the higher primates, it is intense. Our knowledge of this condition is still too inadequate to enable us to make any correlations with other factors, but differences in reaction should be borne in mind so that further progress in the classification of the conditions under which uterine responses are elicited may be made. The primate biologist is mainly interested in the develop­ ment of the uterine reaction which follows the cessation of a growth stimulus to the endometrium. This degenerative re­ action, menstruation, normally follows the cessation of corpus luteum activity and it appears to be quite distinct from the hemorrhagic proestrous reaction of the dog and the similar metestrous reaction of the cow, or (occasionally) of the guinea pig. In the first species mentioned it is accompanied by cir­ cumstances, i.e. the increase in estrogen secretion, which should lead to active growth7. In the other two species it has not yet been evoked experimentally. Unfortunately we still know very little concerning reproduc­ tion in the lower primates and in the insectivores closest to the primate evolutionary tree. Work on these forms is greatly to be desired so that we may gain some idea of the evolution of the primate reactions. The most significant recent work is that by van der Horst and Gillman8, which shows how much we may expect to gain by an exploration of the field. They used the elephant shrew, a member of the family of Macroscelididae. This family is now usually put in the insectivores, but it has been ascribed to the primates by some taxonomists, and by others it has been put in a separate order. One thing seems to be certain, that it is an early offshoot of the branch which leads to the higher primates. At the end of the life of the corpus luteum a polyp-like formation, part of the endo-

Estrous Cycles

7

metrium, degenerates and is shed in a reaction resembling menstruation, thus recalling, or perhaps anticipating, the old world monkeys. The corpora lutea, themselves, are, at one stage of their growth, distinguished with difficulty from the ovarian stroma, thus providing a link with the new world monkeys. A further peculiarity is that the species studied by van der Horst and Gillman sheds an enormous number of eggs at a time, a characteristic of many of the marsupials. These resemblances are remarkable and suggest that this elephant shrew is indeed a physiological "missing link." The fact that certain other species of the genus do not shed large numbers of eggs at each heat period suggests the idea that further exploration of the family might lead to interesting results, especially if attention were paid to the anterior pitui­ tary hormones and their levels. REFERENCES 1. Asdeil, S.A.I 946. Patterns of Mammalian Reproduction, Ithaca, Ν. Y.

2. Pearson, O. P. P. 1944. Am. J. Anat. 75: 39-93. 3. Asdell, S. A., J. deAlba, and S.J. Roberts. 1945. J. Animal Sci., 4: 277-284. 4. Zuckerman, S. 1938. Proc. Physiol. Soc., J. Physiol., 92: 128; 1940-1. J. Endoerin., 2: 263-267. 5. Marshall, F. H. A. 1937. Proc. Roy. Soc., London, 122B: 413-428. 6. Cole, H. H., and R. F. Miller. 1935. Am. J. Anat. 57: 39-97. 7. Meyer, R. K., and S. Saike. 1931. Proc. Soc. Exp. Biol, and Med. 29: 301-303. 8. van der Horst, C. J., and J. Gillman. 1944. Anat. Rec., 90: 101— 106.

DISCUSSION, LED BY G. W. CORNER DR. CASIDA : I would like to know more about the hog and the sheep placement in your series. I do not know of aily data that shows that the hog has a markedly higher FSH. content than the sheep. DR. ASDELL: As far as I can recall at the moment, I am relying on the evidence brought forth by Witschi chiefly on that point. There is also some work by R. T. Hill on the same subject.

8

S. A. Asdell

DR. ATKINSON: Dr. Asdell subscribes to the common theory that the corpus luteum is not functional in the cycle of the rat and mouse. This has been due mainly, I believe, to the fact that there has been no adequate criterion of luteal activity. Dr. Hooker at Yale has recently published a criterion of luteal activity based on morphological changes in the stromal nuclei in the uterus. He claims, and I have seen his material and believe he is correct, that the corpusaluteum is functional for about a twenty-four hour period. DR. REECE : The lactogen content of the hypophysis of the cow is extremely high. We don't know what the blood levels are, or whether the lactogen maintains the corpus luteum or any other functional activity. It seems to me extremely diffi­ cult to determine how a cow maintains estrous cycles when she is lactating heavily. We know that the prolactin must be necessary for lactation to continue. DR. ASDELL: The only suggestion which I can make along those lines is that certain organs have priority in hormone use. That may perhaps be true in this instance but it is a subject about which we know nothing. DR. ENGLE: Has it been demonstrated that prolactin is essential for the continuance of lactation in the cow and the other heavy milk producers? I thought it was essential only for the initiation of lactation, and that mechanical stimulus or nervous activation might continue it. I concede you could have a low prolactin level. Is there any data to that effect? DR. REESE: There are no experiments on the cow, but there was one goat hypophysectomized. In my previous comment I was drawing liirgely on the observations of laboratory animals. DR. CORNER: I should like to ask Dr. Asdell about his ob­ servation that in the cow there seems to be a block which prevents the continuance of estrous behavior. I may have missed the evidence which led you to ascribe it offhand to the central nervous system. Is there evidence which points directly to the central nervous system as the source of the block as opposed, for example, to some possible explanation of an endocrine character? DR. ASDELL: I simply say it is central nervous system, the heat itself. Cyclic heat is a central nervous system phenom-

Estrous Cycles

9

enon. There is something in the central nervous system which cuts off the impulse which causes sex desire. It seems a logical inference. DR. CORNER: The block has to evince itself through the nervous system physiology at any rate? DR. ASDELL: Yes. Although I do not have enough evidence on the subject yet, I think that the relationship between the dose and the length of the heat period follows a hyperbolic curve. I believe that is a chronaxic phenomenon which repre­ sents the type of reaction one gets at the synapse, but we really do not have enough observations to say that the curve is hyperbolic. I will just say that the present evidence sug­ gests that it looks hyperbolic. I am going to tackle that problem as soon as I have enough help to do it, because it means the animals have to be kept for fairly continuous ob­ servation for a long period. DR. LEATHEM: I was interested in Dr. Asdell's comments on the correlation of the FSH content of the pituitary and the length of the estrous cycle. Do we have available infor­ mation on the pituitary content of all species? Have a long series of pituitary glands, extracted in the same way, been tested on hypophysectomized rats only for the FSH content? If they were testing on normal animals, they were testing FSH and LH, in which there is augmentation. Is there a correlation in the nature of milligram response, that is, the weight of tissue per unit response? So many of the older investigations, particularly with pituitaries of the rabbit, dog, and cat, etcetera, have been dealing with the content of the gland as a whole. If you study rabbit pituitaries under certain circumstances you find that the con­ tent of tissue per whole gland is very little as compared with the very marked response that you can get in the minute mouse pituitary. In the human pituitary, published evidence shows it to be very high in its content of FSH. In some of our own recent investigations with pooled human pituitaries we find, in contrast, that there is a considerable amount of LH in the human pituitary. DR. ASDELL: YOU mean that you think the evidence is in­ sufficient to draw any real conclusions at the present time? DR. LEATHEM: Yes. DR. ASDELL: My own opinion is that we shall not solve

10

S. A. Asdell

these things with any degree of certainty until we have pure hormones to inject and reproduce the results. That is what we have been trying to do with estrogens and progesterone in the cow. I have tried to prepare pure anterior pituitary hormones for the purposes suggested but, unfortunately, by the time I have made these hormones and managed to test them to show that they are entirely free from FSH and LH, there is nothing left for me to work with experimentally.

OVULATION AND ESTRUS IN SHEEP AND GOATS RALPH W. PHILLIPS, RICHARD M. FRAPS, AND ARCHIE H. FRANK*

MOST female sheep and goats exhibit estrus only during a limited breeding season, in the fall and early winter. Many attempts have been made in recent years to stimulate estrus and ovulation outside this natural breeding season. The chief incentives for these attempts have been the potential eco­ nomic advantages of having lambs to market when the supply is normally low, of obtaining two crops of lambs per year, and of partially equalizing the supply of goats' milk in differ­ ent seasons by having a portion of the does bred in the spring and early summer. Ewes and does are similar in the anatomy of their repro­ ductive tracts and in various reproductive processes. They differ markedly from the human female in that they have a rather definite breeding season, exhibit estrus, and do not menstruate; and it is possible that they differ in other phases of their reproductive physiology. Economic considerations are of major importance in sheep and goats, as they must be in all farm animals. An understanding of the reproductive phe­ nomena in these species, including the hormonal stimulations which induce natural estrus and ovulation, should be useful in treating sterility, increasing fertility, and controlling the time of mating in these species. Obtaining such fundamental information in the ewe and doe inevitably should yield results that shed light on problems encountered in other species. * The authors are, respectively, senior animal husbandman in charge of genetic investigations, senior physiologist, and associate veterinarian, Bureau of Animal Industry, Washington, D. C., and Beltsviile, Md. They are indebted to Ralph G. Schott and Victor L. Simmons for assistance in several phases of the work reported.

12

Phillips, FrapSi and Prank REPRODUCTIVE PHENOMENA IN EWES AND DOES

Considerable work has been done with sheep, and a limited amount with goats, to determine the extent of the breeding season, length of estrus and the estrous cycle, and time of ovulation. Knowledge of these more obvious reproductive phenomena, and others such as activity of the ovary outside the natural breeding season, speed of travel and time of sur­ vival of spermatozoa in the female tract, and optimum time of mating in relation to estrus and ovulation, is essential if the problems of improving fertility, or of obtaining fertility outside the natural breeding season by the use of hormone therapy, are to be successfully attacked. Sufficient data on the above points are reviewed below to indicate the general pattern of these reproductive processes, but no attempt has been made to present a comprehensive review of all the pertinent literature. 7 he breeding season. Data collected at the U. S. Morgan Horse Farm (Schott, Phillips, and Spencer, 1939) illustrate the possibilities and limitations of breeding ewes during the summer months. The ewes were field-mated, beginning at the end of April or early in May, in order that they would have every opportunity to conceive and produce lambs in the fall and early winter. Rams remained with the ewes until the end of September, except that the rams in one of two groups were removed at the end of August and in the other they re­ mained with the ewes through October. In some seasons additional unmated ewes were brought into the flocks during the breeding periods described. The approximate date at which each ewe must have mated effectively was calculated from the lambing date, by subtracting 147 days (the approxi­ mate average duration of pregnancy) from the lambing date. The dates of conception thus calculated give the proportions of ewes settling in each month (calculated in terms of the non­ pregnant ewes available for breeding in each month), which are presented in Table 1. Examination of these data indicates that in some groups a number of ewes setded during the early portion of the period during which they were exposed to breeding, after which there was a quiescent period during which few ewes conceived. This was followed by active breed­ ing during the fall.

13

Ovulation and Estrus

TABLE 1. Number of ewes in matings and per cents that conceived, in field breeding at the U. S. Morgan Horse Farm, Middlebury, Vermont. NUMBER IN MATING Month of mating Type of ewe

Corriedale Dorset Tasmanian Merino Dorset χ Tasmanian Merino Dorset χ Delaine Merino

May

June

J«iy

Aug.

Sept.

Oct.

62 79 18 58 29

75 60 30 21 29

80 73 26 25 29

69 57 26 15 14

36 34

7

10 12

PER CENT THAT CONCEIVED Corriedale Dorset Tasmanian Merino Dorset χ Tasmanian Merino Dorset χ Delaine Merino

17.7 35.4 44.4 63.8 13.8

9.3 5.0 46.7 0 0

15 2.7 0 4 13.8

4.3 10.5 46.2 33.3 14.3

44.4 44.1

57.1

70 75

Data collected in 1938 on the actual occurrence of estrus in flocks at the Agricultural Research Center, Beltsville, Maryland (Schott, Phillips, and Spencer, 1939) serve further to indicate the seasonal nature of breeding in sheep. Ewes were checked daily for estrus, beginning as soon as they were removed from lambing pens (usually within three days after lambing) and continuing until breeding ended in the fall. The distributions of dates of first estrus are shown in Table 2. The preceding lambing season extended from January through May, but no ewes were observed in estrus prior to July 25. Inspection of the data in Table 2 shows that there are dif­ ferences among breeds in the time of first estrus. This is brought out further by the tabulation in Table 3, which shows the average week in which estrus first occurred in each breed group. A difference is also observed between mature and yearling ewes within the breed groups, the yearlings gen­ erally tending to come in estrus somewhat later than the mature ewes.

14

Phillips, Fraps, and Frank

The Corriedale is the only breed group represented in both sets of data described in Tables 1-3, At Beltsville, Maryland, no ewes of this breed came in estrus during the spring and 2. The frequency distribution of time of occurrence offirstestrus in mature and yearling ewes in theflockat the Agricultural Research Center, BeltsvUle, Maryland, 1938.

TABLE

Distributions by breeds

Periods by weeks

Ckirriedale

Hampshire

Shropshire Southdown

Karakul purebred

Karakul x Karakul x Black-faced Highland Corriedale

Mat. Yrig. Mat. Yrlg. Mat. Yrlg. Mat. Yrig. Mat. Yrlg. Mat. Yrlg. Mat. Yrlg. July 25-31 Aug. ! - 7 Aug. 8-14 Aug. 15-21 Aug. 22-28 Aug. 28-Sept. 4 Sept. 5-11 Sept. 12-18 Sept. 19-25 Sept. 26-Oct. 2 Oct. 3-9 Oct. 10-16 Oct. 17-23 Oct. 24-30

1 2 6 3 8 9 7 1 1

1 5 2

3 12 1 1 3 3

4 1

1 2 2 6 3 5 4

1 3 1 3 1 1

5 1 5 3 2 1

5 1 1

6 3 4 7 6 1 1

3 3 2 3

2 1 8 9 5

2 2

1 1 1 1 3 3 5

1 1

3. Average weeks in which first estrus occurredj in the groups for which distribution data are given in Table 2.

TABLE

Week of first estrus Breed of ewes Mature ewes

Corriedale Hampshire Shropshire Southdown Karakul Karakul x Blackfaced Highland Kiarakul

Aug. Sept. Sept. Sept. Sept. Sept. Sept.

29-Sept. 4 12-18 12-18 19-25 5-11 12-18 5-11

Yearling ewes

Sept. Sept. Sept. Sept. Sept. Sept. Sept.

5-11 5-11 19-25 12-18 12-18 19-25 12-18

summer months (up to July 25) of 1938, while a small portion of the ewes observed at Middlebury, Vermont, over a period of five years conceived during May, June, and July. The two

2

15

OvulationandEstrus

groups of ewes were of similar breeding, those at Middlebury having been taken from the flock at Beltsville. There is an interesting indication here that differences in light (and pos­ sibly temperature and other factors) between the two points might have been sufficient to result in this difference in sexual activity during the summer months. Little is known of the effects of light on estrus in sheep, but it has been demonstrated (Marshall, 1936) that ewes shipped from the northern to the southern hemisphere adjust to the seasonal rhythm of that hemisphere. Bissonnette (1941) has shown that estrous cycles may be induced in does by reducing length of daylight and inhibited by lengthening the period of light. ABLE 4.

Lambing results in the Karakul flock at Beltsville, Maryland, for the period October 1, 1942, to September 30, 1944. Month of lambing

rear

)42-3 )43-4 wo years

Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June

J«iy

Aug. Sept.

0 1 1

2 5 7

5 1 6

0 0 0

3.2

2.7

0

0 0 0

:rcent of ewes conceived 0.4 0 Conth of breeding May June

15 11 26

27 26 53

24 26 50

13 27 40

20 3 23

3 2 5

11.7 23.9 22.5 18.0 10.4 2 . 2

July

3 8 11

5.0

Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr.

Further data have been accumulated on the breeding season of Karakul sheep at the Agricultural Research Center, Belts­ ville, Maryland (Phillips, Fraps, and Frank, 1945). This flock was last bred for regular seasonal lambing, beginning in August, 1941. After July, 1942, the ewes were kept with a ram at all times. Lambing results for a two-year period beginning October 1, 1942, and ending September 30, 1944, are sum­ marized in Table 4. Although Karakul breeders frequently claim that ewes of this breed can usually be bred twice during a year, these data indicate that breeding is rather highly seasonal in nature. Of the 222 lambings included in this study, only 21 resulted from conceptions which occurred soon after the end of pregnancy, and in all these cases the ewes

16

Phillips, Fraps1 and. Frank

were not nursing lambs, the lambs having been sacrificed for pelting within a few days after birth. Kelley and Shaw (1939) have reported observations on the occurrence of estrus in Australian Merino ewes and in a group of first cross Border Leicester χ Merino ewes. Their data show that few ewes came in heat during the summer months, that 40

30

TOGGENBURG

20

ω 30

20

30

NUBIAN

20

JAN.

FE8.

MAR.

APR.

MAY

JUNE

JULY

AUG.

SEPT.

OCT.

NOV.

OEC.

MONTH GESTATION BEGAN FIG .

1. The season at which goats are bred. Distributions show the time of conception for 100 does in each of the three breeds. (From Phillips, Simmons and Schott, 1943.)

during the fall and winter months a high percentage of the ewes were in estrus each month, and that the occurrence of estrus diminished rapidly during the spring months. Of the three strains of Merinos observed, ewes in one showed a higher incidence of estrus during the summer months than did ewes in the other two strains, indicating that there may be con­ siderable variability in this characteristic within breeds.

17

Ovulation and Estrus

It is often stated that ewes of some breeds have two breed­ ing seasons, fall and spring. The available data indicate that such ewes have only one breeding season, a somewhat more extended one than ewes of other breeds. When these ewes have their sexual cycles interrupted by conception in the fall, the cycles may begin after pregnancy ends and in some ewes may continue throughout the following summer. The extent of the breeding season in goats is similar to that in sheep. This is illustrated by the distribution of month of birth for 100 does in each of the three different breeds shown in Figure 1. It is also illustrated by the following data from Turner (1936), showing the frequency of conception by months for 37,047 kids of four breeds of milk goats: CALCULATED MONTH OF CONCEPTION

January February March April May Tune July August September October November December

PER CENT OF KIDS CONCEIVED

5.20 3.03 1.17 .39 .18

.27 .86

5.08 19.66 30.43 20.11

13.47

Does of breeding age or approaching breeding age were observed daily for estrus from October 20,1941, until Decem­ ber 13, 1942, with the exception that observations on does in the milking herd did not begin until those does freshened in the spring of 1942. The results are summarized in Figure 2. The data were arranged by 21-day periods in order to show the relative number of does showing estrus at different seasons. The total column for each 21-day period indicates the number of individuals actually under observation during that period. The solid portion of each column indicates the number that actually showed estrus during the period. If a doe came in estrus twice during the period she was included only once in the tabulation. The observations on each group of does are discussed below.

18

Phillips, Fraps, and Frank

The milking does began kidding in February, and the number under observation for estrus increased from that point as the season advanced until kidding was over. No

FIG. 2. The breeding season in goats. Shaded columns indicate mimber of females under observation in each 21-day period. Black columns show the number of females that came in estrus in each period. (From Phillips, Simons, and Schott, 1943.)

estrus was observed until July 15, this being the one individual recorded in Figure 2 as showing estrus between June 29 and July 19. In the making of the tabulations in Figure 2, each

Ovulation and Estrus

19

doe that was bred was removed from the tabulation after the period in which she conceived, hence the number under ob­ servation gradually decreased in the milking group and in the 1941 kids during the 1942 breeding season. An occasional animal was also removed because of death. The majority of the does did not begin coming in estrus until the latter part of September. The data on mature does indicate that a few does could be bred during late July and August, but that most breeding in the herd studied would need to be done between midSeptember and mid-December, a period of three months, thus limiting the extent to which the time of freshening can be spread by breeding some does early and some late in the breeding season. The kids born in 1941 were coming into estrus at about the same rate as were the dry does when observations began, but the breeding season did not extend so far into the winter. The first occurrence of estrus in the following fall was con­ siderably later that the first estrus among the milking does. These observations indicate that the anestrous period after the kid's first breeding season may be considerably longer than in older does. The kids born in 1942 differed markedly from those born in 1941, in that only two of the former group had exhibited estrus up to December 13. They were born at about the same time and had been given comparable care, hence no reason­ able explanation can be given for the difference in the two groups. The 1942 kids were placed under observation at 4 months of age. Some observations by Quinlan, Steyn and De Vos (1941) indicate that the first estrus of the breeding season in ewes is shorter than later estrous periods. The estimated average length of the first estrus in the Merino ewes they studied was 14.6 hours. Hsenmond (1944) found that frequency of twin­ ning increased from the beginning of the breeding season to a peak in November, then declined during the remainder of the season. He also found that the beginning and end of the breeding season were spaced about evenly on either side of the shortest day. These observations indicate that the estrous phenomena rise gradually to a peak, then decline. No data are available in the literature on goats to indicate the length

20

Phillips, Fraps, and Frank

of estrus or level of fertility at different times in the breeding season. Estrous cycle. Data on 1,038 estrous cycles were obtained in cooperative work conducted by the Bureau of Animal Indus­ try and the Missouri Agricultural Experiment Station. Some of these data were reported by McKenzie and Phillips (1930) and Terrill (1935), and all are summarized by McKenzie and Terrill (1937). They are shown graphically in Figure 3. Most 400

350

300

3 250

>ο ο 0 zoo fle tai ω

1ζ 150 IOO

50

5

10

15

20

25

30

35

40

45

LENGTH OF ESTROUS CYCLE (OAYS)

50

55

60

65

FIG .

3. The distribution of length of the estrous cycle (1,038 cycles). (From data summarized by McKenzie and Terrilly 1937.)

of the cycles (938) fall within the range of 14-19 days, and the average of these is 16.72. Many of the 100 cycles falling outside the 14-19 day range appear to be multiples of the normal or 16-17 day cycle, and may have resulted from ovu­ lations which were not accompanied by estrus. Further in­ vestigation is necessary, however, before all the apparently abnormal cycles can be clearly explained. m The estrous cycle in does is somewhat longer than in ewes. Data obtained by Phillips, Simmons, and Schott (1943) are summarized in the graph in Figure 4. The average duration of the estrous cycle in the dry does, milking does and kids were 22.8, 23.0 and 16.6 days, respectively. These figures may be misleading unless considered together with the distribu­ tions shown in Figure 4. Most of the cycles in mature does

21

Ovulation and Estrus

are around 21 days, and between 18 and 24 days. Most of those that are longer appear to be multiples of 21-day cycles. The majority of the cycles observed in kids also are in the 21-day and adjacent classes. But there was a high proportion of short cycles among those observed in kids, and a consider­ able number among those observed on mature does. R

J I I B

6 4

Ut

2 0-1 4 til ..J y O >O 0-1 ο Ifi Φ

I I I ι HI

194

•

Kl DS

I. I

I MILKING DOES

I

K

I

I

I DR

IU 4 GO S I? 3 Z 10

DC)ES

8 6 4

J• JI

2 0-1

Il

I

,,,I JU I

Imi

IO 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 LENGTH OF CYCLE (IN DAYS)

FIG. 4. Length of the estrous cycle in does. Distributions show the length of cycles observed in each of three groups of females. (From Phillips, Simmons, and Schott, 1943.)

No completely satisfactory explanation for these short cycles is available. McKenzie and TerriIl (1937) suggest that short cycles may be due to failure of ovulation and subsequent luteinization. They report two cases in which ewes came in estrus at the normal time but failed to ovulate. These ewes came in estrus again after 4 and 4.5 days when ovulation in each did occur, and McKenzie and Terrill point out that similar short cycles are observed after the ovary containing the only ripe follicle is removed. Removal of the ovary with the only corpus luteum was found to result in a short cycle also. Warbritton (1934) found a few cases of early degener-

22

Phillips, Fraps, and Frank

ation of the corpus luteum (7.5, 9 and 11 days after onset of estrus, compared with normal time of 14 days) which might also account for shortened cycles. Estrus. Extensive data on the length of estrus were accumu­ lated in cooperative work by the Bureau of Animal Industry and the Missouri Agricultural Experiment Station. These were reported by McKenzie and Terrill (1937) and a graph showing the distribution of 1,235 estrous periods in 344 ewes 400 •! Il

350 1

I

• ι

—

.

•

1

1

1

J

J

1

J

ρ

J

L

1

1

1

1

1

300 w O 250 £bl £200 CC ω CD

Z 150

D

Tt

100 50 0 LENGTH OFESTRUS (HOURS)

FIG. 5. Distribution of length of estrus (1,235 periods) in ewes. (From data sum­ marized by McKenzie and Terrills 1937.)

appears in Figure 5. The average of all these periods was 29.3 hours. Many other data have been reported on estrus in ewes, some indicating somewhat longer cycles, but these are suffi­ cient to indicate the approximate average duration. No comparable data are available on does. In observations on the length of the estrous cycle made thus far at the Agri­ cultural Research Center, Beltsville, Maryland it was pos­ sible to observe the does only once daily, at a uniform time. In the majority of the cases does were found in estrus on two consecutive days, and in a few cases on three days. Longer periods were rare. These observations indicate that the aver­ age length of estrus is similar to that of the ewe. Time of ovulation. Various workers have studied the time of ovulation in relation to beginning of estrus. Their results are

Ovulation and Estrus

23

in rather close agreement, with one exception, and the ranges observed in time of ovulation (expressed in hours after begin­ ning of estrus) are as follows: McKenzie, et al (1933), earliest at 23.7 hours; Grant (1934) 18 to 24 hours; Clark (1934), 2¾ to 60 hours; Cole and Miller (1935), 22 to 30 hours; McKenzie and Terrill (1937), 24 to 36 hours in most cases, but it occurred as early as 12 and as late as 41 hours; and Schott and Phillips (1941), 24 to 36 hours. In general, ovu­ lation occurs near the end of estrus or shortly after the end. Ovulation without estrus apparently occurs frequently just before and just after the normal breeding season.McKenzie and Terrill (1937) observed this in 16 of 17 cases approxi­ mately one cycle prior to the first estrus of the breeding season, and in 9 of 15 cases approximately one cycle after the last estrus of the breeding season. Grant (1933, 1934) and Cole and Miller (1935) also observed similar ovulations, with­ out estrus. Hammond (1944) found that isolated ovulation occurred after the end of the breeding season and right into the summer months, and Panyseva (1939) also reports that ovulation occurs during the summer months without external signs of estrus. Survival arid speed of travel of spermatozoa in the female tract. The length of time spermatozoa can survive in the reproductive tract of the ewe is rather limited. Quinlan, Mare, and Roux (1932, 1932a) found that spermatozoa were mostly nonmotile after 12 hours in the vagina, although they remained motile for 48 hours in the cervix. Spermatozoa deposited directly in the uterine horns did not survive over 9 to 12 hours. Green and Winters (1935) estimated that spermatozoa did not survive over 24 hours in the genital tract of the ewe, and Kelley (1937) found that spermatozoa had lost their ferti­ lizing power in 34 hours. Warbritton, et al (1937) found motile spermatozoa in the fallopian tubes of all ewes slaughtered at 2, 12 and 22 hours after insemination. They were present in greater numbers and in a better state of preservation at 12 than at 22 hours, indicating that many had degenerated after 12 hours. The power of fertilization may be lost before loss of motility, according to reports by Fuchs (1915), Wolf (1921), Hammond and Asdell (1926) and Hammond (1930). The time required after service for spermatozoa to reach the upper portion of the fallopian tube is about 18 to 20

24

Phillips, Fraps, and Frank

minutes in most ewes, according to Schott and Phillips (1941). Earlier workers reported times varying from 2 to 5 hours. Zavadovskii et al (1939) found that spermatozoa did not penetrate the uterus and oviducts in a normal manner in ewes that ovulated under the influence of gonadotroph»: agents, but which did not come in estrus. They offer this as an explanation of the low percentage of conceptions observed in such ewes after force-mating or artificial insemination. Polovcova and Judovic (1939) also studied this problem and found that the cervix was not permeable to spermatozoa in 85 per cent of the cases of silent heat (ovulation without estrus). These results are in contrast with those of Green and Winters (1935) who state that the speed of travel of spermatozoa is not affected materially by the stage of the estrous cycle. This point is of sufficient importance to justify further study. Optimum time to breed. The facts reviewed briefly above indi­ cate that the optimum time to breed should be towards the end of estrus or near the time of ovulation. This problem has been studied in actual breeding trials with sheep by several workers. McKenzie and Phillips (1930) obtained more con­ ceptions in ewes bred 14 hours or more after coming in estrus, than in ewes bred earlier. Kardymovic, et al (1934) obtained the highest percentage of conceptions in ewes bred 18 to 26 hours after estrus began. Zajac (1935) inseminated ewes at 8, 16, 24, 32, 40, 48 and 56 hours after the beginning of estrus and obtained the highest percentage of conceptions at 24 hours, followed closely by the 32- and 16-hour times of insemi­ nation. Phillips, et al (1940) obtained the highest percentage of conceptions in ewes, following inseminations with trans­ ported semen, between 20 and 30 hours after estrus began. Some previously unpublished data that were obtained at the Utah Agricultural Experiment Station (Phillips and Bennett, 1941) are summarized in Table 5, which indicate that ewes bred at 14 and 28 hours after the beginning of estrus are more apt to conceive than those mated at the beginning of estrus. Responses of ewes and does to pregnant mare serum in consecutive seasons. The data presented in Table 5 indicate the importance of proper coordination of estrus, ovulation, and mating if conception is to result. In the many attempts to stimulate estrus and ovulation in ewes and does with pregnant mare serum and other gonadotrophins, follicular growth and ovula-

25

Ovulation and Estrus

tion have been induced in a high proportion of the animals; but induction of estrus in conjunction with ovulation has been quite erratic, and no satisfactory explanation has yet been found for the inconsistent results. Fertility, as measured by percentage of conceptions, has been lower generally in animals in which estrus and ovulation have been induced, or in those force-mated after induction of ovulation, than in animals bred during natural estrus; but satisfactory fertility has been reported in some cases. Literature dealing with this problem is reviewed by Phillips, Fraps, and Frank (1945), and Cole, Hart, and Miller (1945). TABLE

5. The relation of time of breeding during the estrus to fertility of ewes. Time of breeding after beginning of estrus (in hours) Items compared

No. ewes bred No. ewes settled Per cent ewes settled Services per conception No. of pairs of twins born

0

14

28

38 20 52.6 1.90 7

35 28 80.0 1.25 13

33 27 81.8 1.22 17

Examples from our work with sheep and goats (Phillips, Simmons, and Schott, 1943; Frank and Appleby, 1943; Frank, Schott, and Simmons, 1945) will serve to illustrate the inconsistent nature of results of attempts to stimulate extraseasonal breeding with pregnant mare serum. Results of tests with does during 1942 and 1943 are sum­ marized in Table 6. The animals injected during 1942 fall into three groups (A, B, and C) depending upon initial level of injection, and a number of sub-groups representing animals subjected to differing subsequent treatments. Thus group A consisted of 3 does injected with 150 r.u. on alternate days until estrus occurred; this required 3 injections for 1 doe, 4 for a second, and 6 for the third. Group B consisted of 12 does, none of which came in estrus following the initial injection of 200 r.u. PMS; following a 20-day interval 9 does (group B-I) were injected with 400 r.u. and the remaining 3 with 200 r.u.

26

Phillips, Fraps, and Frank

PMS (group B-2). All the does receiving 400 r.u. (group B-1) came in estrus, thus terminating the tests. Since none of the does receiving a second injection of 200 r.u. came in 6. Comparison of the incidence of estrus induced in anestrous does by PMS during two consecutive seasons (1942,1943). TABLE

TESTS OF 1 9 4 2

Group

A-1 A-2 A.3 B-1 B-2 C-1 C-2 C-3

First injection (r.u.)

150 X 3» 150 X 4 ' 150 X 6' 200 200 400 400 400

Second® Third" Fourth" jNuniDer injection injection injection of docs (r.u.) (r.u.) (r.u.)

400 200

400

600 600

600

Does in estrus

Number Per cent

1 1 1 9 3 2 1 2

1 1 1 9 3 2 1 1

100 100 100 100 100 100 100 50

20

19

95

3 9 3 1 15 6 11

67

8

2 0 0 1 0 1 1 1

56

6

TESTS OF 1 9 4 3

D E-1 E-2 E-3 F-1 F-2 G-1 G-2

50 X 150 X 150 X 150 X 200 200 400 400

3° 3» 3» 3»

400 400 400 400 50 X 3«

50 X 3" 400

50 X 3"

.. . 100

... 17 9 13 11

« The injections were made at 18 or 20 day intervals. ' Multiple injections were given on alternate days. ' Multiple injections were given on consecutive days.

estrus following this injection, they were given a third injection of 400 r.u. and all came in estrus. Group C, consisting of 5 animals, was broken down into its sub-groups by similar procedures, except that one animal in group C-3 did not come in estrus following her third injection, at which time the tests were terminated.

Ovulation and Estrus

27

The injection of does during 1943 differed from the pro­ cedures of 1942 mainly in that injections were terminated before all animals came in estrus (group E-3 being a minor exception). Nevertheless, a number of direct comparisons can be made between the results of the two years. Thus of the 13 does composing group E, only one came in estrus following the first injections (150 X 3), compared with one out of three animals similarly injected during 1942 (group A). Comparison of the 9 does of group B-I with the 21 animals of group F through the interval of second injection shows that all the does came in estrus during the 1942 tests, none during the 1943 tests. Groups C and G may be similarly compared following initial injections; 2 out of 5 does came in estrus in 1942, one out of 19 during 1943. One point of interest in connection with the tests of 1943 is that 5 of the 6 animals exhibiting estrus did so following the injection of 50 r.u. to each ewe daily for three consecutive days, in 2 does as first injections, in the other 3 as later injections. However, 18 does were injected in this manner, of which the 5 just noted represent only 28 per cent. The experiments with ewes for 1943 were outlined to determine the minimum and optimum dosage of PMS that would induce ovulation and to determine the possibilities of inducing estrus with ovulation. The results of 1942 and 1943 tests with ewes are summarized in Table 7. The ewes that were injected in 1942 (with the exclusion of animals killed 48 hours after injection) fall into two groups; those that were given single or multiple daily injections (group A), and those that received injections at 16-day intervals (group B). Each group was divided into sub­ groups according to subsequent treatment. When examined after PMS injections, 13 of the ewes had ovulated, and another became pregnant when bred at this time. Two ewes out of 14 came in estrus following initial injec­ tions, and 4 ewes out of 6 came in estrus after a second injec­ tion after a 16-day interval. The tests with ewes during 1943 differed from those of 1942 principally in that in some instances both slightly smaller initial doses and sub-minimal doses of PMS were given. These ewes fell into two groups, those receiving sub-minimal doses required to induce ovulation (group C), and those

28

Phillips, Fraps, and Frank

receiving doses considered adequate to induce ovulation (group D). A few direct comparisons may be made. After comparable initial injections all three ewes in groups A-1 and A-7 (in 1942) had ovulated, in comparison with 16 out of 25 in groups D-l, 2 and 3 (in 1943). A similar comparison may be made between groups B and D following a second TABLE 7.

Comparison of the incidence of ovulation induced in anestrous ewe by PMS during two consecutive seasons (1942, 1943). TESTS OF First injecdon

Group

Second vnjettion''

Number ewes r.u.

r.u.

250 500 250 X 2* 250 + 1000 500 X 2 125 -(- 50X6 125 -1- 50X3 250 250

2 1 1 1 1 1 1

25 50 250 250 300

6 5 12 6 7

Lapar.

Ovul.

3 3

0 0 8 2 6

25X5 50 250 250 300

6 4 12 5 8

No.

Ovul.

3 3

Percei

2 1 1 1 1 1 1 3' 3

2 1 1 1 1 1 1 3 3

100 100 100 100 100 100 100 100 100

14

14

100

12 9 24 17 15

0 0 13 6 10

0 0 54 35 67

77.

29

38

1943

TESTS OF C-1 2 D-l 2 3

Lapar.

2 1 1 1 1 1 1 50 250

Ovulating

Number ewes r.u.

Lapar.

Total

Third injecdon"

Number ewes

Lapar.' Ovul.«

A-1 2 3 4 5 6 7 B-1 2

1942

0 0 5 0 4

250X2

6

4

« The injections were made at 16 day intervals. *Lapar. Laparotomized. •Ovul. - Ovulating. ' Muldple injecdons were given on consecutive days. * One ewe not examined conceived.

injection made at a 16-day interval, where all 6 animals had ovulated in 1942 and only 9 out of 25 had ovulated in 1943. Following initial injections, 2 ewes out of 14 came in estrus in 1942, while none out of 26 came in estrus in 1943. On a similar basis 4 out of 6 ewes came in estrus following a second injection made at a 16-day interval in 1942, as compared with 2 out of 26 in 1943.

Ovulation and Estrus

29

If1Another point of interest in the tests of 1943 was the occur­ rence of estrus in 3, and ovulation in 4, out of 6 ewes, following a third 16-day interval, with injection of 250 r.u. of PMS on consecutive days. This was the largest dose of PMS given at one interval and the only group of ewes injected at three periods (group D-2). The main point of interest in the tests summarized here lies in the contrasting results of 1942 and 1943, a contrast involving both ovulation in the ewes and estrus in the does. It does not seem reasonable to attribute the differing responses to differences in the PMS preparations nor to the manner of their administration. The composition of the flocks used also rules out both genetic and age factors as the basis for the different responses in the two years' tests. The possibility of antihormone effects likewise appears remote. If we may admit that the factors just noted cannot account for the differing results in 1942 and 1943, we must seek an explanation in physiological, nutritional, or environmental variables. The existence of physiological differentials is self-evident in the observed responses, but the existence of such differences dur­ ing the interim of PMS injections is probably the result of antecedent influences of nutritional or environmental origins. Zavadovskii and Margulis (1939) obtained conflicting results during two consecutive seasons with the use of PMS to induce estrus and ovulation in ewes. During the first year a high percentage of the animals came in estrus and conceived, while practically none came in estrus or conceived the follow­ ing year. They attributed the erratic results to a difference in nutrition as influenced by prevailing weather conditions. During the present work the spring of 1942 was marked by good rainfall, which assured an abundance of grass during most of the season; spring rainfall was light during 1943 and the growth of grass was somewhat below normal. While such differences cannot be accurately assessed, there seems little doubt that the 1942 season was more favorable in point of green feed, and possibly with respect also to dried feeds. Con­ ditions of light, temperature, and humidity also differed during the two years. Without attempting to evaluate these variables in any quantitative sense, their roles in the enhancement or retardation of PMS action in the induction of ovulation or estrus seems probable.

30

Phillips, Frapsi and Frank

Other extraneous factors, such as temperature or change in the length of light day, may also be involved in the variable response to injection of PMS. As previously stated, Bissonnette (1941) has shown that breeding cycles in goats may be in­ duced by artificially reducing length of day, or inhibited by lengthened days; under the conditions of his experiments he concluded further that the annual temperature cycle "is not a major factor in environmental control of sexual or breeding cycles in goats." It is at least conceivable that differences in the light cycle from season to season may predispose ewes and does to greater or lesser reactivity to PMS. Under normal conditions it would be difficult to rule out similar effects which might be attributed to temperature differences from year to year, although Bissonnette's results indicate little influence from this source. Results with ewes of feeding thyroprotein and treating with pregnant mare serum. Work with poultry (Turner, Irwin, and Reineke, 1945; Turner, Kempster, Hall, and Reineke, 1945) has indi­ cated that the feeding of thyroprotein at the rate of 5 to 10 grams per 100 pounds of feed resulted in maintenance of a higher level of egg production during the summer months than was obtained from control birds. Thyroprotein feeding had no effect on egg production during the winter months. Berliner and Warbritton (1937) studied the relation of the thyroid to sperm production in rams. Since thyroid activity varies with the season, and since thyroidectomy is usually fol­ lowed by involution of the sex organs they reasoned that the low fertility frequently observed in rams during the hot sum­ mer months might be due to a low level of activity of the thyroid gland. They concluded that rams with a high level of thyroid activity were better able to withstand high tempera­ tures which prevail in warmer portions of the country before and during the early part of the breeding season, and that thyroxine treatment in relatively normal animals could pre­ vent the summer decline in sperm production or could restore sperm production early in the fall. Findings of the type described above suggested that a low level of thyroid activity might be at least partially responsible for the anestrous period through which most ewes pass during the summer months, and that feeding of thyroxine might en­ able ewes to come into estrus during this period or make them

Ovulation and Estrus

31

more responsive to gonadotrophic hormone therapy. A pre­ liminary trial has been conducted along these lines, and the results are summarized in Table 8. Two groups of ewes were fed thyroprotein (2.7 per cent thyroxine potency) at different levels. At three intervals during the feeding period, uniform TABLE 8.

Date

Summary of work on feeding ewes with thyroprotein and treating with pregnant mare serum.

Item compared

I (Low level)

II (High level)

III Control

No. of animals 22 21 21 Average weights of ewes (lbs.) 97.3 94.0 91.8 86.3 Average weights of ewes (lbs.) 91.4 89.9 Thyroprotein feeding began (per ewe daily) 1 gm. 2 gms. Thyroprotein dosage increased to 2 gms. 4 gms. 4 PMS per ewe 19 5 c.c. 5 c.c. 5 c.c. 0 19 Estrus (no. ewes) 0 0 19 Thyroprotein dosage decreased to 1 gm. 2 gms. 4 PMS per ewe 5 c.c. 5 c.c. 5 c.c. 6-9 Estrus (no. ewes) 7 4 3 20 PMS per ewe 5 c.c. 5 c.c. 5 c.c. 23-25 Estrus (no. ewes) 3 6 4 25 Average weight of ewes (lbs.) 106.7 103.5 100

April 25 25 May 9 10 June

July July July July

Average gain in weight per ewe (lbs.) Total ewes in estrus Total ewes in estrus

9.4 13"

6.0

7

11.7 7

20s

" The proportion is significantly higher (P = .04) than lots II and III. h The proportion is not significantly higher (P - .40) than lot III.

doses of 5 cc. (250 r.u.) of PMS were injected. A significantly higher number of ewes came in estrus in lot I than in lots II and III. The combined number of ewes showing estrus in lots I and II was not significantly higher than the number showing estrus in the control group. These results are difficult to interpret. Perhaps the level of feeding in lot II was above the optimum. Turner, Kempster, Hall and, Reineke (1945) found that when thyroprotein feeding was slightly above the optimum level in poultry, egg production was below that of the controls. It is also possible that the high incidence of estrus

32

Phillips, Fraps, and Frank

in lot I was a chance variation, not actually associated with thyroprotein feeding. The results are, therefore, not conclusive, but indicate the desirability of further work. INDUCTION OF ESTRUS IN OVARIECTOMIZED SHEEP AND GOATS

It has previously been demonstrated that stilbestrol or estradiol benzoate induces estrus in ovariectomized sheep. The limiting conditions under which estrus is induced have not, however, been established. The possible role of prog­ esterone in the estrual reaction seems also to have received little attention, although Bell, Casida, and Darlow (1941) have reported results which indicate that progesterone pro­ longs the duration of estrus induced by estradiol benzoate in ovariectomized ewes. It has been shown that the full exhibition of estrus in the ovariectomized guinea pig (Dempsey, Hertz, and Young, 1936), rat (Boling and Blandeau, 1939), mouse (Ring, 1944), and the golden hamster (Frank and Fraps, 1945) is infre­ quently attained following injection of estrogen alone. In all these species the administration of appropriate dosages of progesterone following pretreatment with estrogens is neces­ sary for exhibitions of estrus. It seemed reasonable therefore to inquire if the administration of progesterone following estrogen pretreatment materially alters the incidence of estrus in ovariectomized sheep and goats. The possible action of progesterone in terminating estrus induced by estrogens was also a matter of interest. Materials and procedures. Twenty-one Karakul sheep and 17 goats (16 Toggenburg and 1 Saanen) were availablefor the pre­ sent experiments. The does were ovariectomized in May and June, 1944; the sheep in August and September, 1944. First in­ jections following operations were made some six weeks later. The does were housed during the first year following oper­ ation in rather limited quarters, while the sheep had at all times access to a considerable area. Conditions of maintenance and rations were otherwise approximately the same for both groups of animals. The estrogens and progesterone were dissolved in corn oil (Mazola). All injections were made subcutaneously, some­ what posterior to the shoulder. The quantity of oil injected varied in different series but no effect of injected substances could be attributed to this source.

Ovulation and Estrus

33

Following injection, the animals were tested at intervals for exhibition of estrus. Acceptance of the ram or buck was taken in all cases as the index of positive response to the injected hormones. The usual procedure was to inject the estrogen at about 4:00 p.m., and to test the animals at 8:00 a.m. and 4:00 p.m. of the next and succeeding days until it was certain that no further effects of injection could be expected. In some cases the animals were also tested at about noon; such tests were usually made only after the injection of progesterone in order to determine possible short-time effects. In a few other tests the injection of estrogens was made at about 10:00 p.m. in order to shorten the interval between injection and test of the animals for first evidence of estrus. In order to test for possible seasonal differentials in response to estradiol benzoate, both the ovariectomized sheep and goats were injected with 0.1 mg. estradiol benzoate at inter­ vals extending from the season of normal estrous periodicities (October or November) into the season of practically com­ plete anestrus (June-July). Results. The first tests were made with stilbestrol. It was found during the preliminary experiments that estradiol ben­ zoate induced estrus for considerably longer periods at equiva­ lent dosages than did stilbestrol. Estradiol benzoate was accordingly used in all later tests, and only results based upon the administration of this estrogen are reported in this paper. In this connection, Frank and Appleby (1943) report that two milligrams of diethylstilbestrol on the first day and one milli­ gram on the second day was sufficient to induce estrus in 11 of 12 intact ewes. Smaller doses were ineffective unless several consecutive doses were administered. Quin and Van Der Wath (1943) treated 43 ewes with 1 to 5 mg. of stilbestrol and ob­ served estrus in 8 animals and doubtful signs of estrus in 8 others. The data based upon injection of estradiol benzoate into ovariectomized ewes are summarized in Table 9 and for ovariectomized does in Table 10. In general, the duration of estrus in both ewes and does decreases with decreasing dosages. The interval between injection and first display of estrus (latent period of the tables) shows an opposite trend, although data are not altogether in agreement on this point. It seems clear from the data of Table 9 that the duration of estrus at a constant level of injection of estradiol benzoate

34

Phillips, Fraps, and, Frank

(0.1 mg.) is at a maximum (57.1 hrs.) at about the season sheep normally exhibit most regular estrous behavior. The duration of estrus decreases gradually from this maximum to about 18 hours during the early part of June, and possibly beyond this. The latent period (the interval from injection to first observed estrus) increases from about 29 hours in early November to almost 43 hours at the end of July. TABLE 9. Induction of estrus in ovariectomized ewes with estradiol benzoate. Reaction of ewes Date of injection

Estradiol benzoate mg.

Ewes injected No.

Estrus

No.

Per cent

Duration, hrs.

Latent period"

10/2/44 10/2/44 10/2/44 4/30/45

1.60 0.80 0.40 0.20

7 7 7 19

7 7 7 18

100 100 100 94.7

138.3 121.1 113,1 43.3

24.0 25.1 24.0 44.9

11/6/44 1/3/45 4/2/45 6/4/45 7/30/45

0.10 0.10 0.10 0.10 0.10

7 20 19 19 19

7 20 14 10 9

100 100 73.7 52.6 47.3

57.1 42.0 35.4 18.4 24.0

29.1 29.2 32.0 39.2 42.7

11/6/44 11/26/45 11/6/44

0.05 0.05 0.025

7 18 7

7 13 6

100 72.2 85.7

40.0 29.5 19.3

32.0 25.2 29.3

a

Time from injection of estradiol benzoate to first appearance of estrus.

It is possible that the time elapsing from ovariectomy to time of injection of estradiol benzoate at the 0.1 mg. level may account in part for the results just described. This con­ clusion does not seem probable, however, in view of the fact that administration of as little as 0.05 mg. estradiol benzoate approximately one year after the initial injection of 0.1 mg. gave a result in good agreement with those of the earlier injections. Results similar to those shown in Table 9 are given in Table 10 for ovariectomized does. The decrease in duration

35

Ovulation and Estrus

of estrus with decreasing dosages of estradiol benzoate is notably evident, the average duration at the 1.60 mg, level being 112 hours, and not more than about 57 hours at the injection level of 0.20 mg. The interval between injection and first observed estrus (latent period in the table) is not quite so uniform, although the general relation seems clear. TABLE 10. Induction of estrus in ovariectomized does with estradiol benzoate. Reaction of does Date of injection

Estradiol benzoate mg.

Does injected No.

Estrus

No.

Per cent Duration, hrs.

9/18/44 9/18/44 9/18/44 10/17/44 10/17/44 4/30/45

1.60 0.80 0.40 0.40 0.20 0.20

5 6 6 5 6 17

5 6 6 5 6 17

100 100 100 100 100 100

10/17/44 11/14/44 4/2/45 6/4/45 7/30/45

0.10 0.10 0.10 0.10 0.10

6 4 17 17 15

6 2 11 16 15

11/14/44 11/26/45 11/14/44 11/14/44

0.05 0.05 0.025 0.0125

4 14 4 5

3 12 0 1

Latent period»

112.0 90.7 58.7 67.2 57.3 31.1

22.4 20.0 35.3 15.2 18.7 31.5

100 50 64.7 94 100

33.3 16.0 31.3 34.4 17.3

20.7 24.0 27.6 32.5 51.2

75 85.7

13.3 20.7

32.0 29.3

20

24.0

8.0

• Time from injection of estradiol benzoate to first appearance of estrus.

As with the ewes, a number of injections were made at the 0.1 mg. level between mid-October and the end of July. The duration of estrus does not follow the consistent decrease with advancing season recorded for ewes. The latent period, how­ ever, shows a definite and consistent increase. It is obviously difficult from our data to conclude whether the duration of estrus or the latent period is a 'better measure of reactivity of the injected animal, but it does not seem probable that either

36

Phillips, Fraps, and Frank

index of estrual response should vary so uniformly except on the basis of some seasonal variant. It seems worth noting that certain individual ovariectomized ewes and does responded to extremely low dosages of estradiol benzoate. Six or seven injected ewes gave a positive response of considerable duration to 0.025 mg. of estradiol benzoate and at least one injected doe responded to a dosage of only 0.0125 mg., duration of estrus being 24 hours. TABLE 11.

Reaction of ovariectomized ewes and does to estrogen and progesterone injections. OVARIECTOMIZED EWES Reactions (estrus)

Dosages

Estradiol benzoate

first dose

mg.

Second dose

Time"

0.025 0.025 0.0125 0.0125 0.00625 0.00625

0.1 0.1 0.1 0.1 0.1

40 40 40

mg.

o.i QA 0.1

Progesterone

First dose

Time®

mg.

48

25

48

25

48

25

48 48

2.5 25

40

15

Second dose

Time®

64 64

Num­ ber in­ jected

mg.

2.5 25

In estrus

No.

4 3 3 4 4 3 6 7 7

Per cent

Dura­ Latent tion period*

Hours Hours

0

1 0 1 0 0

10

6 7 7 7

10

8

4

32*'

*25*

24*'

32 '

ioo

90*" 84 37.1 29.1 15.5

36 !"7 33.7 32 40 40

83.3 22.4 9.3 50 9 80 7 50 0 0 62.5 15.2 66.7 8.7

24.8 21.3 20 42

33.3

100 100 70 80

OVARIECTOMIZED DOES 0.1 0.1 0.1

16

5 25

24 24

5** 25

0.05 0.05«

16

5

24«

5

0.1 0.1

40

5 *

16

6 6

5 8

9β 8

9

5 3 4 4

0

5 6

0

40.8 40.7

ο Interval in hours from first injection of estradiol benzoate. & Hours from first injection of estradiol benzoate to first appearance of estrus. • This group of doses received a third injection of progesterone (5 mg. to each animal) 40 hours following injection of estradiol benzoate.

In comparing the reaction of ewes and does at comparable injection levels, the ewes appear on the whole somewhat more sensitive to the action of estradiol benzoate than the does. The differences, however, are not great and it may be concluded

Ovulation and Estrus

37

that the response in both species is essentially alike. It is pos­ sible that differing conditions under which the two groups of animals were maintained may have contributed to the differ­ ences in response to injections of estradiol benzoate. The reactions of ovariectomized ewes and does to prog­ esterone, following priming with estradiol benzoate, are re­ corded in Table 11. There is little or no evidence to indicate that progesterone synergizes the reaction to estradiol benzoate under any of the conditions recorded in this table. There is, on the contrary, some evidence that progesterone tends to shorten the duration of estrus induced by the estrogen. The reactions of ewes at 0.1 mg. estradiol benzoate level, followed by a second injection 40 hours later, and by progesterone 48 and 64 hours following the initial injection of the estradiol benzoate are of particular interest in this connection. The duration of estrus in those animals receiving the two injections of estrogen and no progesterone was 90 hours, in those re­ ceiving the estrogen and two injections of only 2.5 mg. of progesterone the duration was 84 hours, while those animals receiving estrogen in the same dosages, but two injections of progesterone at the 25 mg. level, were in estrus only 37.1 hours. Similar relations are exhibited in other groups of the ewes as well as in the ovariectomized does. On the basis of the foregoing results it seemed that a critical evaluation of the effect of progesterone in synergizing the action of previously injected estrogen might be determined if relatively high doses of progesterone followed the administra­ tion of estradiol benzoate at minimum levels. If progesterone accentuated the response to estrogens, the effect might become apparent only when estrogens were administered at too low a level to induce estrus on their own account. The results of injections based upon these considerations are recorded in Table 12 for both ovariectomized ewes and does. In order to test the possibility that the interval between injection of the estrogen and progesterone may be a factor in determining the estrous reaction, progesterone was injected 16, 40, and 64 hours following the administration of estradiol benzoate. It cannot be concluded from the data of Table 12 that any difference was exhibited in the response of control (estradiol benzoate injected) ewes and those subsequently re­ ceiving progesterone. Injection levels were apparently almost

38

Phillips, Fraps, and Frank

subminimal for the does, no reactions being recorded in the first two groups and only one progesterone-injected animal responding in the 64-hour group; this doe, however, showed a latent period of only 40 hours and was completely out of estrus before progesterone was injected. Reaction of ewes and does to progesterone (25 mg.), following administration of alpha-estradiol benzoate (0.02mg.). Both hormones were administered subcutaneously.

TABLE 12.

EWES Reactions of ewes and does

Treatment

Pg. injected Controls Pg. injected Controls Pg. injected Controls

No. of ewes

9 9 9 9 8 9

Hours between estradiol-benzoate and progesterone injections

16 40 64

Estrus

No.

Per cent

Duration, hrs.

1 3 4(2)6 1(1) 1(1) 3(3)

11.1 33.3 44.4 11.1 12.5 33.3

4 36 12 8 4 20

14.3

4

Latent period"

24 34.7 34 16 40 34.7

DOES Pg. injected Controls Pg. injected Controls Pg. injected Controls

7 7 7 7 7 7

16

None

40

None

64

1