Citation
The effects of a vitamin A deficiency on the reproductive capacity of rams

Material Information

Title:
The effects of a vitamin A deficiency on the reproductive capacity of rams
Creator:
Sosa, Enrique, 1940- ( Dissertant )
Warnick, Alvin C. ( Thesis advisor )
Cunha, T. J. ( Reviewer )
Shirley, R. L. ( Reviewer )
Wallace, H. D. ( Reviewer )
Callahan, W. P. ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1964
Language:
English
Physical Description:
ix, 156 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Animals ( jstor )
Death ( jstor )
Hormones ( jstor )
Irradiation ( jstor )
Liver ( jstor )
Semen ( jstor )
Spermatozoa ( jstor )
Testosterone ( jstor )
Vitamin A deficiency ( jstor )
Vitamins ( jstor )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Rams -- Feeding and feeds ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
Efficient reproduction in his animals is one of the goals that any producer must achieve in order to successfully compete in today's livestock business. This underlines the importance of research in any aspect of reproductive physiology. In the literature one finds that efforts in this area of research have been directed mostly towards studying various effects of environmental and genetic factors on female reproduction, somewhat underestimating the importance of the male; consequently the basic aspects of the male reproductive physiology have been overlooked to some extent in the past. This study Is part of a broad project being undertaken at the University of Florida to investigate the effects of various nutritional factors on the male reproductive capacity. Other studies in this project have been the work reported by Meacham (1962) and Til ton (1962) in which protein and energy have been investigated. Lack of vitamin A is the only specific dietary deficiency that has been shown with any certainty to affect semen production in farm animals. It is also true however that the studies from which this knowledge was obtained are to some extent outdated in the light of today's improved research methods and techniques. The use of purified diets is indeed a tool of great value in animal research for It facilitates complete knowledge of the nutrients present in the diet, thus allowing a more precise characterization of the role that a specific dietary factor has in the physiology of the organism in question. It is with this in mind that the present study was undertaken to further investigate the effects of avitaninosis A on the reproductive capacity of the ram. Due to the ever increasing need of information related to radiation and its effects on biological systems, a study on the tolerance of vitamin A deprived animals to gamma irradiation from a Co^60 source was included. Also attempt was made in this study to counteract the harmful effects of vitamin A deficiency by administering Testosterone, pregnant mare serum and Thyroprotein.
Thesis:
Thesis (Ph. D.)--University of Florida, 1964.
Bibliography:
Bibliography: leaves 92-96.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Enrique Sosa.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
029870799 ( AlephBibNum )
24680729 ( OCLC )
ACF2774 ( NOTIS )

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THE EFFECTS OF A VITAMIN A DEFICIENCY

ON THE REPRODUCTIVE CAPACITY OF RAMS














By
ENRIQUE SOSA


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA
August, 1964














ACKNOWLEDGMENT


The writer wishes to express his sincere appreciation to

Dr. A. C. Warnick, Chairman of his Graduate Supervisory Committee and

Dr. T. J. Cunha and Mr. R. E. Deese under whose guidance and supervision

this study was made.

Grateful acknowledgment Is extended to Dr. R. L. Shirley,

Mr. P. E. Loggins, Dr. H. D. Wallace, Dr. F. E. Neal and Dr. W. P.

Callahan for their efforts and counsel extended freely throughout this

study.

The writer expresses his appreciation to Mr. R. C. Dees, Herdsman,

for his assistance in the feeding and handling of the experimental ani-

mals.

The assistance of his fellow graduate students, Mr. J. Easley,

Mr. C. Piedra and other personnel at the Nutrition Laboratory is also

appreciated.

The advise of Dr. A. E. Brandt in the statistical analysis is

gratefully acknowledged.

The writer is indebted to his wife Irene for her patience and en-

couragement during his graduate study.


















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31 Analysis of Variance Mean Squares for Semen Volume,
Motility, Abnormal Cells and Total Sperm Cell
Production--Week 2 . . . . . . .... 144

32 Analysis of Variance Mean Squares for Semen Volume,
Motility, Abnormal Cells, and Total Sperm Cell
Production--Week 3 . . . . . . .... 145

33 Analysis of Variance Mean Squares for Semen Volume,
Motility, Abnormal Cells, and Total Sperm Cell
Production--Week 4 . . . . . . . 146

34 Analysis of Variance Mean Squares for Semen Volume,
Motility, Abnormal Cells, and Total Sperm Cell
Production--Week 5 . . . . . .. . 147

35 Analysis of Variance Mean Squares for Semen Volume,
Motility, Abnormal Cells, and Total Sperm Cell
Production--Week 6 .. .. .. .... ... .. 148

36 Analysis of Variance Mean Squares for Semen Volume,
Motility, Abnormal Cells, and Total Sperm Cell
Production--Week 7 . . . . . . . 149

37 Analysis of Variance Mean Squares for Semen Volume,
Motility, Abnormal Cells, and Total Sperm Cell
Production--Week 8 ................ 150

38 Analysis of Variance Mean Squares for Semen Volume,
Motility, Abnormal Cells, and Total Sperm Cell
Production--Week 9. . . . . . . .. .151

39 Analysis of Variance Mean Squares for Semen Volume,
Motility, Abnormal Cells, and Total Sperm Cell
Production--Week 10 . . . ... ..... 152

40 Analysis of Variance for the Difference in Mean
Size of the Testes . . . ... . . . 153

41 Analysis of Variance for the Difference in Mean
Size of the Epidydlmis . . . . . . . 153

42 Analysis of Variance for the Difference in Mean
Size of the Adrenal Glands . . . . .... 154

43 Analysis of Variance for the Difference in Mean
Size of the Seminal Vesicles . . . .... 154

44 Analysis of Variance for the Difference In Mean
Size of the Cowper's Glands .. .. .. .. .. 155











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LIST OF FIGURES


Figures Page

I Cloudy Condition of the Cornea Present in a
Vitamin A Deficient Ram . .. . . . . 30

2 A Vitamin A Deficient Ram Showing Extreme
Weakness . . . . . . . . . . 30

3 Effect of Vitamin A Supplementation on Body
Weight Changes of Rams .. . . . . . . 43

4 Effect of Various Hormone Treatments on Body
Weight Changes of Vitamin A Deficient Rams . . 44

5 Effect of Irradiation on Body Weight Changes
of Vitamin A Deficient and Normal Rams . . .. 45

6 Effect of Vitamin A Supplementation on Mean
Semen Volume of Rams . .. .. .. .. ... 54

7 Effect of Various Hormone Treatments on Mean
Semen Volume of Vitamin A Deficient Rams .... 55

8 Effect of Irradiation on Mean Semen Volume of
Vitamin A Deficient and Normal Rams . . ... 56

9 Effect of Vitamin A Supplementation on Mean
Sperm Motility of Rams ............. 59

10 Effect of Various Hormone Treatments on Mean
Sperm Motility of Vitamin A Deficient Rams . . 61

11 Effect of Irradiation on Mean Sperm Motllity of
Vitamin A Deficient and Normal Rams . . ... 63

12 Effect of Vitamin A Supplementation on Per Cent
Abnormal Sperm of Rams. . . . . . .. 65

13 Effect of Various Hormone Treatments on Per Cent
Abnormal Sperm of Vitamin A Deficient Rams . . 67

14 Effect of Irradiation on Per Cent Abnormal Sperm
of Vitamin A Deficient and Normal Rams . . .. 69














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INTRODUCTION


Efficient reproduction in his animals is one of the goals that

any producer must achieve in order to successfully compete in today

livestock business. This underlines the importance of research in any

aspect of reproductive physiology.

In the literature one finds that efforts in this area of research

have been directed mostly towards studying various effects of environmental

and genetic factors on female reproduction, somewhat underestimating the

importance of the male; consequently the basic aspects of the male repro-

ductive physiology have been overlooked to some extent in the past.

This study Is part of a broad project being undertaken at the Uni-

versity of Florida to Investigate the effects of various nutritional fac-

tors on the male reproductive capacity. Other studies in this project

have been the work reported by Meacham (1962) and Tilton (1962) in which

protein and energy have been investigated.

Lack of vitamin A is the only specific dietary deficiency that has

been shown with any certainty to affect semen production in farm animals.

It is also true however that the studies from which this knowledge was

obtained are to some extent outdated in the light of today improved re-

search methods and techniques. The use of purified diets is indeed a tool

of great value in animal research for It facilitates complete knowledge

of the nutrients present in the diet, thus allowing a more precise charac-

terization of the role that a specific dietary factor has in the physiology











of the organism in question. It Is with this in mind that t-*- p ,reer

study was undertaken to further Investigate tr'e ii'fcril of a,,raci.r..r,.

A on the reproductive capacity of the ram.

Due to the ever increasing need of information related to rjdrlal:--.

and its effects on biological systems, a study on the tolerance of ., a.Tii.

A deprived animals to gamma irradiation from a Co source was inclJdEJ.

Also attempt was made in this study to counteract the harmful

effects of vitamin A deficiency by administering Testosterone, preg-..a..

mare serum and Thyroprotein.














LITERATURE REVIEW


Vitamin A and Reproduction in the Male

As early as 1924 it was found in experiments with rats by Gross (1924)

and Wolbach and Howe (1925) that vitamin A deficiency caused pro-

nounced atrophy of the testes. Since then extensive investigations have

been carried out to elucidate the influence of this deficiency on the

male germinal epithelium in laboratory and farm animals. In addition to

atrophy of the testes this deficiency will also cause impaired develop-

ment of the epididymis, seminal vesicles, and prostate in young rats.

Injection of testosterone can obviate this atrophy in vitamin A deficient

rats as shown by Mayer and Truant (1949). Further injections of gonado-

trophic hormone will stimulate the development of the accessory sex glands

In vitamin A deficient rats according to Mayer and Goddard (1951). This

suggests that this deficiency causes a lowered excretion of pitituary gona-

dotrophic hormone or a lowered response to this hormone by the interstitial

cells of the testes.

Recent work by Howell et L. (1963) showed that male rats main-

tained on a diet in which the vitamin A alcohol had been replaced by

vitamin A acid developed lesions in the reproductive tract. The testicular

changes were a sloughing of the cells of the germinal epithelium followed

by an obliteration of the lumen of the tubule by sertoll cells. Testicular

regeneration was produced by the administration of vitamin A alcohol. The

lesions were comparable to those of vitamin A deficiency as described by












aio.- (9I I: tuL- ir*CF unciOCpLilC3ted bt the i conddr .ani bnfetati ;cn. Of

u t rir.I h d ielc ienc Tr.i paper I t r-e r lr t I0 dc cr .e le I r.s .n

Ih. tIc : IC heal in., )rOic ..i' j pra pubtFrt I r it fe. (., jit 3a.ln A acid

diet It I1 kid I cccipted Etat vr.ti -Tin A acid car. perfor.c. all .ce

Su-ci c 1C.... c r i A Iclil-n l -; CcePpf ior tT part 1, 1 .. .,. DI r l.1o 9

anrd wald li9c0l :ujuar iz; d arie rrle or vlwiArin A acid in a recer.r a,.T|-

p.:..16 i . itI a-Ir A. A Tr.e general i, uc ru-.cilur. .:.i li.litr.r, A that iup-

port growth an.d c.iratr,l.5c.i c n thee rat are itrafd dl ;.:. 1 l.a.nin A .30.d.

bul ;ACte t 6c E a EduC hi. t rd i t. :u I .anc t i or. r. ti t [er ite

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luter tcr':En .r ihe I ; ual c I det er ior are. r, i.:.l d ry the ir: ; u

a' ll L a cr a i. :1 trc ;ci r. t .cil: i t ir. 3r. ti he ,ri c c pcarir.d ret in .

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thu a I&.er rg c.f :pert0 pFr duct u C nm Qut .In, Ir I has I:e, nhon, r ,r.

Irt att r. et ai (i ..i t Erbt et a (i 4.) a.iJ tL i.t-Ert arij M.art I i 35).

b atg .n [ a i 1 1 .S1 studied the af riec of r nation |.:.. .r caruter.e or.

ihe hreedji.-j ber3,ior 5 eraI. t:.ye.-c s: : an-id Ee.Tr. prd.c ior. of *-.atu'e

Jair, bulli. Laec I o 2 1to "cy o* .i ra,.i,. A pE r i0i .TI. .:.f bl,:.yj

nere recoridcd th-f.:.re ar., ut the Jef icienc, i,.7>p e. appeared










Incoordination and loss of the ability to mount without loss of libido

were the first signs to appear and they occurred before any marked impair-

ment on semen quality was evident. Percentage of motile spermatozoa was

decreased. The per cent of abnormal spermatozoa was increased. Histolo-

gical examination of the testes showed degeneration of the germinal epi-

thelium of the seminiferous tubules. Hogdson et l. (1946) also found

that vitamin A deficiency apparently does not cause severe disturbance

in spermatogenesis until the animals are in advanced stages of deficiency.

Erb e al. (1947), Hodgson et al. (1946), Jones etal. (1946)

and Madsen etal. (1942) have reported that pituitaries are often cystic

In the vitamin A deficient bovine. These findings suggested the possi-

bility that vitamin A deficiency may exert its deleterious effect on re-

production indirectly through the anterior pituitary gland. Gunnet al.

(1942) improved the semen quality of a ram deficient in vitamin A by

injections of pregnant mare serum. Hogdson et al. (1946) obtained similar

results with a vitamin A deficient bull. However Lindley et al. (1949)

reported that testosterone propionate injected semiweekly in dosages of

12.5 mg. before and during the deficient condition or of 37.5 mg. after

vitamin A deficiency symptoms began to appear resulted in no improvement

on semen quality of vitamin A deficient rams. Similar results were ob-

tained from Injections of pregnant mare serum. This work suggests dif-

ferences between rat and sheep since Mayer and Truant (1949) were success-

ful in preventing testicular atrophy in the rat resulting from a vitamin

A deficiency by administration of testosterone propionate.











Pathology of Vitamin A Deficiency

Moore (1960) described at least three basic lesions occurring in

avitamlnosis A: (1) Lack of vitamin A, required in the form of its alde-

hyde for the formation of rhodopsin, causes defective dark adaptation

unless complications have ensued this lesion may be regarded as "bioche-

mical" rather than "structural." Thus the response to treatment with

vitamin A is rapid and dramatic. (2) Lack of vitamin A causes xerosis

or keratinization of membranes in many parts of the body, the well-known

xerophthalmia belongs to this group of lesions. Epithelia tends to

become dry and excessively thick and horny. Those membranes having a

columnar structure, often associated with secretions of mucus give place

to thick layers of stratified epithelia as found in the outerpart of the

epidermis. The most common secondary effects of avitaminosis A arise

from the bacterial infection of the abnormal membranes. In some sites

such as the urinary bladder, the injury to the membrane may sometimes

induce calcification with the formation of stones. (3) During growth,

lack of vitamin A can cause defective modeling of the bones. As a result

the bones are not compact, strong and well shaped, but cancellous, weak

and excessively thick.

Lesions of more than a single type are often super-imposed, and

the possibility of further types cannot always be regarded as secondary

effects of the bone lesions. Another abnormality Is an increase in the

pressure of spinal fluid sometimes associated with hydrocephalus. These

lesions might well be regarded as secondary effects of malformation of

the skull bones, but a rival theory suggests that deficiency of the vitamin

causes the chloroid plexus to secrete unduly large amounts of fluid.











Vitamin A generall)

Vitamin A is an alcohol and forms esters with fatty acids. Most

of the vitamin A in the livers of both mammals and fish is present in

the sterified form (White et al. 1959 and Harper 1963). Conversion of

provitamin A (carotene) takes place in the Intestinal wall in rats, pigs,

goats, rabbits, chickens and sheep. According to Barnett and Reid (1961)

carotene may be found in the liver of animals suffering from a vitamin A

deficiency and does not appear to be capable of utilization for vitamin

A synthesis. Dowiing (1960) summarized today'sviews on vitamin A metabo-

lism as follows: Vitamin A alcohol, the transport form of the vitamin

and the storage form as the ester, is oxidized to retinene because of the

rapid removal of the aldehyde in its combination with opsin to form rho-

dopsin. The alcohol is oxidized to the acid in the liver and it is rapidly

used in its function of growth and tissue maintenance. Acid is the im-

portant form for all functions other than visual, it must be stored as

the alcohol and cannot be reconverted to either aldehyde or alcohol.

Most of the information on the concentration of vitamin A in the

various organs has been gained in studies on rats. Moore (1931) used

the antimony trichloride reaction, with the aid of a Lovibond Tintometer,

In studies of vitamin A in the tissues of rats which had been given liberal

amounts of carotene. By this treatment total stores of up to 50,000 I.U.

of vitamin A were accumulated in the liver. Apart from the intestine

which contained much yellow pigment the rest of the carcass contributed

100 I.U. of vitamin A. When the various tissues were examined separately

the intraperitoneal fat contained 0.5, 5 and 5 I.U. in four experiments,

and the kidneys and lungs each 0,0 and 5 I.U. in three experiments.













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such low plasma values for two consecutive weeks were shown to be depleted

of liver stores of vitamin A. Unpublished data by Dr. R. L. Shirley and

co-workers showed that cattle receiving 30,000 I.U. per day and those

receiving no supplemental vitamin A had blood plasma levels of 54 and 34

respectively. Another trial conducted during the summer showed levels

of 46 and 18 mcg. of vitamin A per 100 ml. of plasma for groups receiving

the supplemental vitamin A and no vitamin A respectively. Vitamin A in

liver expressed as mcg. per gram of liver dry weight was found to be 119

and 78 for the supplemented group during the first and second trial res-

pectively and 17 and 7 for the second group during the first and second

trial respectively. In the second trial vitamin A level in the heart was

found to be 0.17 and .05 mcg. per gram of heart for the supplemented and

non-supplemented groups respectively.

In sheep vitamin A storage has not been as extensively studied

as it has been in rats. Harm (1942) reported the average vitamin A con-

tent in a gram of liver as 503 l.U. in 19 animals. Moore ad Payne (1942) reported

levels of 460 I.U. per gram of liver in 20 animals. The above data were

expressed in terms of grams of dry liver. Recent data in the literature

indicate the levels of vitamin A in the liver of sheep to be within a

range from 25 to 54 mcg. per gram of fresh liver, Cline (1962), Goodrich

(1962) and Cline et l. (1963). Data on the vitamin A content of tissues

other than blood and liver are lacking for sheep.

The Thyroid and its Role in Reproduction
and Vitamin A MetabolIsm

The role of thyroxine in reproduction has been most extensively

studied among the hormones that may directly or indirectly influence the











secretion rate of hormones influencing repr jj.ucC ac I. i t I :utci a: iLh

gonadotrophs and the hormces of the ovary an'd ic .ti.

Berliner and Warbrltton (1937) reported trat sth/irtodct:.., oi

rams produced a decrease in semen volume, scerr....alz:..,-, c..r.'fl
an increase in the relative numbers of abncrreai :pr... r..,r ., 'n ad..i-

nistered to the thyroidectomized animal re!ulrEa .r. tic :r.A .uci; -. cf

semen with an increased number of sperm and djcra:ced perier.r le .:.r jb,,.r-

mal spermatozoa. Bogart and Mayer (1946) rp..:rted tii.a trEac.c.ient ..lt.

thyroxine and thyroprotein alleviated sympr.iE-: 1 .j,-Tr.er tie' ll, re',il-

ting from impaired spermatogenic activity. Tr,e reeJi.g r Lt;.r.u.a i

during the fall breeding season maintained ,.-h'e.. tjractCeris [.:; t,i~iCl

of the summer months. It was concluded in tr,,-i :ud, I[r. the E[.,r:.,d

gland is of major importance in the reproduciii e 1 h, ,i.:.3, :r Er_, ra,.

Bhatnagar (1955) studied the seasonal variar ..i-. in th, rist:log, ,r te

thyroid and the testes of Indian buffalo bull- TI.- tiouL.J :..,l eir ,jL E

seasonal variations in the histological str. ucture (r.e (.:. jlai.n.

they also observed that during the period thenr the ,ual., ..,' ie.Tn jeri-

riorates in buffalo the secretary activities: ..4i r.e t .. jl j..d: j rh,..ar.

by their histological studies appears to decre.:. GC:-,.. %1 i 2 ,-

ducted an experiment to study the effect of tr.,rc.,n: and cr- -,ant ..are

serum (PMS) hormones on reaction time and .i r,-. qualir, :.r tui,i5 bl ll:.

The experiment was conducted in India from Ju-,r. ij Febr-t,, One tru 5-3..j

I.U. of PMS were administered by weekly subcutla. IIeu: Irnjecr *..: n

daily dosage of 100 mg. of thyroxine was ac..,;nisterid orallr Trca. t.e,-,

with thyroxine and PMS did not influence the reaction t'.,, .:.r t, ar.I,,,aTi.














: r I :.: l r : L lh, r ri.r,- r, .












i., thyroprotein in rams. Ten mg, of testosterone and 1 gm. thyroprotein

.-:re .Jd.... ri red daily by subcutaneous Injections and orally respectively.

O.ne ,.. of thyroprotein per hundred lbs. of body weight was given orally

t:* a second group. A third group received both testosterone and thyropro-

ten at the same level administered to groups one and two. Thyroprotein

reduced weight, decreased libido and had no effect on sperm motility,

.iume, concentration or number of ejaculates. The testosterone treated

gr:;up showed a significant decrease in semen concentration. They also

repr.rr. that testosterone could prevent the loss of libido caused by

crain.ern. with thyroprotein.


Recent studies by Brooks (1961), do not support the obser-

vations of Berliner and Warbritton (1937), and Bogart (1946) Brooks

(1961) injected thyroxine at levels of 2, 3, or 4 mg. per hundred Ibs.

of body weight to rams. Considering the fact that 1 mg. of thyroxine

injected equals approximately I gm. of thyroprotein fed (Cole and Cupps

1959) the dosages used in the experiments of Brooks (1961) are compar-

able to dosagA% used in previous work in this field. The results

obtained by Brooks (1961) indicated that injections of












Er,r:. rair.c t hi)h [e.T-.pcr6 Eulc w C.Ec.-.E[u ittd JI Ic d1eE r i nT r i I er'rir : r,.,

-e ] r I 'i L . I apa :.l ..a: l., iEt .l 3'.- r ue pr c.*uuCd

a h,p r .ld C., -.11 j". but t.li t i j r... r f'e : .:.r. i,-, .. Ha,. e lcr ..-.e

r.uit L C C.u luu -....dal *. , y.46-eI l Iz tiO at hiT h .....I ;r'tCE [hE

S rl .:. aI r rep:r [ .E .'.i j ,-r Ja-'iJer i de r -.je .j1 ... r .- .r,[ 1 :. ,, i -

(i.:.r .ih r, ..:.. i ar.,ut,(E.I h .. ei- :( r. t1-' cl,rf, a3 C cidl 0'

the. ? .ri..aIl:. .utC. u, r..:r- a: Tur. n r \Ilcl'. LI'. a.d Lupp 1 J591 a..J

MaleZ (IrI l I .) r..? t ai [i It., ui pl 1.c . ;.-..I d j r. itr. repr.,:ucti.-

i: r .e: tut i. i p. .rI, u.-dc r [ *.-- B N. ,,

,-.i[i lc i:.:.-,rI u'.de r:[ c r.-.c- ti.r, .:.r I t. L r .,r, 3 ,d E-i j[ .

re li[ *. -,h;p I i [ ,,,-, ..E b. ,He. LrI, ( i 4. I re .i e.- d [ : I- 5 :, ,-

j C( rC ..d r 5C ed i e rai .. 1 v F ;I<.o I ,: j I I The lait .L f [ ...i,, A I r.-

asc ihr i.c i. i at- d I.,p r r...T..ir.:. r .. i [t.t opp:: ct h t C .

1 I r l ;C r.:, 1i1- t L-r E' i 11 pir J .- r. p rc r opr., r the [ t e : .j

I ;1 F c r c I 3.,-, rl t,p rt.,r :.i 1 r;s. ir, rcr .'-: e ui .-.,i : r : r O it i,- A .

14I I -. (1-. Ercc t, t.-,r r .d Car.I.-.cE c. C.u l LE U .... c iei [., ..[ Et .. r

v ; t theic ..-"E .-: r > i r. ha tb.v c.:..-.r r.- d E j rk .j..e :...c

Ir i I I I ;-' ,' ,c3 : rC'. I.. -. h :,r ,-,% vc. .l j b u r.a.-l i i, u C 1 [i.ar 1,J t r

i.,rc..- .c .-cre : *.: pe., t ur .:r, ji r .t da ,-, d r.r r ar c...ever

hc p .-.i. .Su(t t.r.i [t1-6 r r culti ru C).-ipl ; : ic l tb acC I.r trc .d r.Si.th

L.t:er...:d Ei n t. [h ., r.:, i,.. r c ratI ;rn,. hi , .i uI,,, ra. i r.:v CrEa: iC.

[I.E '. T.5 j ,-.i: ra. rutr'-c'.ti i.Ii:I d.j i t. .n I. R J a-.d TrI .3]-


(l ti l -..ri *'.; ..i h cai-ac rat ,t, I i-J L,. .,I rEp.: rIt 1 [.tr.a I. h ,p:,[, h

r1.5 : C.:.ri ;.,r of cJ it. ia la.T.i, A S- jr.C'J. c.d but *u -C.aIiI':dr ;r.

pI r C r. ,rt .idi :*T t. Ei r i.c r tu'I.r-t .l -.u.7.Ler ir ai-.,ITaI i r.c E I ;.ri.i'J..











Cline et al. (1963) found that feeding 150 mg. of tapazole per day or 100

mcg. of triiodo-l-thyronlne did not affect liver storage of vitamin A.

Any conclusion drawn from work where histological examination of the thyroid

glands were not made to confirm thyroidal status should be made with

caution.

Moore (1957) in his book of vitamin A reviewed the interrelation-

ships of thyroid and vitamin A metabolism. He concluded at the time

that the usual effect of thyroxine will be to accelerate any prevailing

trend In vitamin A metabolism. If conditions are conductive to expendi-

tures the rate of expenditure Is increased. If dietary conditions are

conductive to the storage of vitamin A the amounts stored will be increased

by thyroxine, however the why and how of this remains unsolved today.


Histology

Warbritten and McKenzie (1937) described the pituitary glands of

ewes in various phases of reproduction. They found the pituitary body

of the ewe to be very complex. They were unable to classify the cells

of the pituitary into the conventional acidophils, and chromophobes.

They found 9 morphologically different types of cells which they didn't

think necessarily represented distinct functional types. In this study

the different regions of the pituitary were found to be conspicuously

different, that counts made at one level would not be valid for another

or for the whole gland. At the time of the studies by Warbritton and

McKenzie (1937) present refined histological techniques were not avail-

able.













i-.i :. t cE cr r.i a r.ji .jre ..l Ir. .- t.:rct:,r if Cr, dui( : L F it he

L..r I: pl au ary ll. :. aric ( ~I5,': E n, w ,,I .. `n.i Ear.-. i1 35 '1 td.

r .p.: *d di Ec r e. .; i : l r. ,I j Kr., ar. rni r F r., :I Trhe, peri dic

?Cid .cr-.r i t c i. u E f A l: :f 1:- r : F I fl'jI .a- *., ed b, Jb.,ut nd FcEn.-e1

I I F, I tc .L t ., f 'jh I-c. ,.:, r Tp. .tc T, r r t I: I : CI c n : ,i '

a .-rul t':rC i-n..r, ce ll : r it Ic b A I. Br a nrd F. F I 3 E Ope ,n

-r I.p i r. F p : I: n ai T..r pr.,i, c nd unrc t iur i d c r-r'.Ce rft Fa

f.1r I ..cr Sc r i ted : r p.:.: r t ne r .r.ula It d jn-.J wi.t

lI e tr.il Ct pi ., M .:-. I r. Th.,r r r .-,r: C:r.l rre ('red : t ', ,rd l r

sI :c : 1 CI c itcreJ in t r. C:.r c* .4, rhe a. r '.C :.r i:.. The r uf ncr.j i

r Ilt i :..- :r-. 1 r t I.:e t 6 Cc l: id:C ii, Cr.C. i,[ h r.,r ., tr.:ci c h rT.rc-C .nc

pr-ducC F I :... T i:. : i F: 1 ,r eFr.1F r d icyr r.. IF SEd Si cu- .> trJ ..

Ih r..ud dcF i ic ''C T b. LIc t [ : I : crc iTrC I. l r I I r C r .idcd nail ih.rpi

-*u l *i* let" j. :-r r I tu c' a *icf rc r r 'u ar i Ei ; And r pe

Tt.e C- F I: I I c. Cr.. :e. -.t I pjrt C tFhe dCe ..)F- .5I5p C .h C.F C:., f -Ccr,-

Sr aicE j i . .ecui : 1 L5I.. .-Eritt or iJ bc. Thc.: E I r ec identi Il i iblc

.I Ch y;.rISad r : p. h iC aCt .I Ezp C i FI L T.. ere u- L lc tSL .. i Id-

tI r., c i6.ia 1 Pur..: 5s.-id CrI.. t-cr. (9i 5.. I 1I Jq Cr itCf .cd t c i.ate ..r.:

C.f : or.- i d-t-Irop t r e I ia c l I. tfhe r u -'.4 rec Po'. w' i-.OC r.. E Pe

I he, tI urI re : C(edCe C Cl p r Fi Ir .. re icti.r: ran r r-c cif.cr C.: .t r 1a S rtaF

t he- g9i,.ir. ir. addi tr, E CC r. r,djr.. rhoa. dirFr.;l Ic JF ti r i but l.nr.

r-.cE r.0.' :-atcd rhA the h c ;icph .ras .Crn..J.CrcfpFi C.. .5 c.i. ar ar* uira-

F C. arcd yF .. .7-T,rE .I.erI.t reSC li.o .. -ir. Pi. rh h.: -L F c lv c

nCtur rriri .' C-c': L.' I C geb: Ct f ," d l .r.phr CtC[c Ft CE[C lCer,.r, trcSalenr

ind aCf tr ir r. pEr i cD-I C f 0>-.adeCtC..M, E c .e .:r krr S uyc Ied CI8C











their "peripheral" gonadotrophs produced FSH and their "central" gona-

dotrophs produced LH.

Wilson and Ezrln (1954) described a modified PAS method in which

the Schiff reaction is followed by a methyl blue counterstain by which

some but not all of the PAS reactive cells changed from red color to

purple color. These workers concluded that the "PAS-red" cell represented

the Beta cell or thyrotroph and the "PAS-purple" cell the Delta cell or

gonadotroph. Rennels (1957) used a modification of the PAS-methyl blue

method which in conjunction with aldehyde fuchsin, enables'the tinctorial

differentiation of two types of gonadotrophs in the pituitary gland of

the rat. These two cell types were both gonadotrophic in function as

indicated by their development into distinct PAS-red and PAS-purple cas-

tration cells following gonadectomy. Thyroidectomy cells were found to

be PAS-negative, aldehyde fuchsin-negative and to have an affinity for

orange G. Sanders and Rennels (1959) found the predominant chromophilic

cell type In grafts producing luteotrophin (LTH) to be an elongated acido-

phil staining selectively with orange G when the azan stain is used.

Pearse (1960), Humason (1962) and Ham and Leeson (1961) were also used

as references for proper histological techniques, in this dissertation.


Effects of Irradiation on Reproduction

Schubert and Lapp (1958) described the spermatogonia as the most

radiosensitive cell in the human body and indicated that 50 roentgens

inhibits their development, permanent sterility for the human male re-

quires about 500 to 600 roentgens while 250 roentgens may produce steri-

lity for one to two years. Seminal fluid production was not affected











tu -n* I .4 .rr .-r; t. r c i T t rr. Ci E l I .-a e pr., d jd:.i* de-



CFj ..; F- I 41 C.C. Kti r-c uI rec.. .* ft 1 1 r r . ti rr E ., .n (lit

fertility of the male r i an.Jd mouse by t '[.ic ;.: c l;- matings 01' tr.e

treated animals. In both species irradiation with 300 roentgens p.i:-duce

a short period of sterility associated with oligospermia. This occurred

about 45 days after treatment in the mouse and 65 days in the rat.

Histological examination showed that the main effect is on the sper-.atL:-

gonial stages but there are obvious differences in the timing and e.ctr-.

of these irradiation effects. The time from treatment to the onset iC.

sterility is a measure of the overall duration of spermatogenesis. Tri-

highest dose level used 500 roentgens did not cause a rapid onset :,i

sterility in contrast to the action of some alkylating agents, whicir r4-

pidly induce infertility. in the cock X-ray doses of up to 10,000 r.-n-

gens has been shown by Kosin (1944) to have no detectable effect o- .*r-.

motility but the fertilizing capacity of spermatozoa was markedly rijlus-:t

after the exposure to 200 -i.enlqtge and was destroyed altogether :r'.tr

a dose of 500, supposedly due to damaged chr, -sai.;. Mann (1954) f..u.d

that rams exposed to luu.0'j0 r,:.t.-rr. and examined immediately af:.r C.-

posure had normal motil :.perT.altz.:. in their semen, fructolysis a-d a -

nosine triphosphate c.c..:-' [ ,cre :1:: normal. This would indicate ri.at

,I an damage -a: 3.:-r, Ir, l-.. ram semen it must be ascertained thrc.ugi.

ucr.,.r cr;terl. hM.r; i-re ad i- rish (1956) reported that when mature t.be

.ull *.-r.- p'P-J [* ; a ingr.ji- .ose of 100-400 roentgens whole-bod-, ga..aa

.'r.r.diari.on, Er, I rlru :.t.:, r ed s.',,-n changes were an increase in p.r

;nr. stIb ,r.Tsl _IPr iT[ .- I c,c after irradiation and a decrease i., ipcri.












.:-ne nt ra.:( --.r- alt *.gh.[ iEt ar Ertr r r i. [ ;.:.r. Frrc .j-r. 1 a i luifrC.r i -

( IL I t f ju..J r. [t '.I. L, 1 T, a Cn r r d ..:i.r. '*1 r.ure He Hcr rurj I ll:

S.tr, a :;i .: ,-,j, ., Li'J, r..ciL ,e,'. ir;T. t .: l : i[ l .:,,Jr .*i i d I**-.[ 'rd : i(

,r. p.1 ap Cr ..r h r..n e .. -cr.. char act r I ti C; I E r. c.5.Cc. r-. ..E ...d

r. .[ l I r uC : I E c a l :.r p. r... C t l t.-. 5 l ......l ..J i L ,-

I c .(c l >1 urn I n e r ee p. e- rr ad .* .-,r. p.-r c '.d F Lc

I I' : :. d Erh, it raCt .' .i ;zarl~.. p .r Ilj l ;( r... .. 'I Lul : .3 ,j-

. Cn;c .[I ,d rc .. d jlu.Il y1 [ ''r[. T i l a.iu I 1 Ee l.. r : I I ... r. 4 r..

b.;d, ija,r, r r .; 3 ,. i h 4lj ''' 1 e.-. j e r.a .

T I-. fi-ccts of irradiation on semen characteristics of animals

,-.dr ;.j[ r. t ..rn l stress of any kind ..ere not available.


Errr: .-r irradiation Compounded with Vitamin A Deficiency

Tr.e :-,, ,ork found in which irradiation was involved with vitamin

; *.t e,,ic...: clone by Meacham et al. (1962) working with swine. Sixty

cr...tr .d .... c.re allowed to two groups of 30 each. One received a

L .i- r ;..., c:.-r ii ning only traces of vitamin A, the other group received

rr,. :.;.c r t.I:.-. pplemented with 2,000 I.U. of vitamin A acetate per

.ur.-d 1cr leJ. .rter 13 weeks on the ration, 10 animals of each lot were

..:.. i r,-,..... ior irradiation. These pigs were subjected to 350 roent-

*j;.-n i.: ti.-.e-.-.d gamma irradiation from a 6,000 curie cobalt 60 facility.

i, ti--. lid : i IS ays, all of the control and nine of the 10 vitamin A

deprived animals had died. The average survival periods for control pigs

and those fed the low vitamin A diet were 234 and 286 hours respectively.

Other than this no variation in irradiation response could be attributed

E.., vitamin A intake of the pigs. The major :,"..;: Ts of irradiation damage








18


were: depression, skin hemorrhaging from eyes and mouth, increased res-

piration, mild muscular spasms just prior to death, extensive hemorrhage

of lymph nodes plus varying amounts of hemorrhage in the kidneys, heart,

urinary bladder, intestine and stomach. Shirley et a. (1962) in conjunc-

tion with Meacham on the above experiment found that vitamin A supple-

mentation decreased the deposition of copper in the liver while copper

supplementation increased the level of vitamin A in the liver. Gamma

irradiation was shown to increase the deposition of copper in the liver,

but had no significant effect on the deposition of vitamin A in the

tissue.














EXPERIMENTAL PROCEDURE


Materials

Animals

The animals used in this study were 48, 20-months old Florida

native rams from Mr. L. A. Maxie's ranch in Frostproof, Florida. These

rams were brought as yearlings to the University in June 1962. At this

time they were placed In a preliminary experiment in which 12 received

5,000 I.U. of vitamin A per head per day. The remaining 36 received no

supplemental vitamin A. The rams were fed rations consisting of 25 per

cent cottonseed meal and 75 per cent white corn meal. All rams had

access to coastal bermuda hay at all times. This preliminary experiment

was continued until January, 1963. At this time two rams were brought

in as replacements, from the University Sheep Unit for two that died.

From January to February the rams were given a period of adjustment to

a new purified diet to be used in the study here reported. On February

2, 1963, the experiment described henceforth was initiated. The average

weight of the animals at the start was 87 lbs. with a range from 115 to

74 lbs. The twelve rams that had previously been receiving vitamin A

were put on the control diet consisting of a purified ration shown in

Table 2 and 3, containing 3,000 I.U. of vitamin A per Ib. of feed. The

remaining 36 rams were placed on the vitamin A free diet.

Assignment to treatments was accomplished in the following manner.

Vitamin A deprived animals were not assigned to a treatment group until










blood analysis showed 15 mcg. or less of vitamin A per 100 ml. of pi:-i,,

A liver biopsy was also performed when such levels were reached. W-,.

12 animals were a.:cu.-rji.l td -hich met the above criteria they were their .

assigned at random, two to each of 6 treatments. Treatments consistiJ :i

an irradiated group (A Irradiated), a second group received 60 R.U i

PMS/100 Ibs. body weight administered daily by subcutaneous injecti:.

(A + PMS). A third group received daily intramuscular injections ,r .ci-

tosterone propionate at a rate of 10 mg./100 lbs. body weight (A + 7-:-

tosterone). A fourth group was given daily capsules of thyroproteli-

I gram/100 lbs. body weight (A + Thyroprotein). A fifth group was l:Ica.c

on the vitamin A containing diet (returned) and the sixth group was cc..-

tinued on the deficient diet without further treatment (deficient).

At the same time rams that had been receiving vitamin A wer. ..i,

randomly divided, two to each group, a control (control) and a grouu cp.:.:c

to irradiation (control irradiated). In this manner eight treatment jr.:..pi

were formed. This procedure was repeated three times so as to alloi rjhe

experimental animals in their totality. The experimental procedure ;i i:r-

lined in Table I. One of the animals assigned to the deficient group j-e.j

in the biopsy operation and was e-,.ludEd from the experimental data.

The experiment 1w. conducted it the Physiology Barn at the .ii.r.er-

sity of Florida. T'. ara .:.i the bj.,, used in this experiment cont in l

8 pens, 12 feet wii anij 4?8 rat i.iy and all pens had concrete fl :r:.

A metal rof covered tre fe-. arca j,.J 12 feet of thp pen. The peni .-re

ij j celr I pl, d n.l -:i.. ..-.r t ar. served two pens.











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Sc aIe. and ...i:.. k r,. g rea *-' r, .. I. 1 6, Ie i r. h: 1. i 3 r. dl'E (re

, p:.r E t, I *ct- c c r. t tC'a u ; J.1 I rr- E h.E






The composition of the experimental rations is recorded in TaIte

2. Solka-floc was used to supply fiber in the rations. Corn oil was

included in the ration to supply fat, to improve palatability and to .r.-

sure a uniform blending of the fine texture ingredients with the coar:er

materials in the rations. Casein was the protein source and corn sugar

and starch were included for energy.

The mineral, mineral premix and .it-;in mixtures used in the ra-

tions are shown in Table 3. The mixtures containing 13 essential min:ral

elements was essentially the same as reported by Meacham (1962) and T.It.:r.

(1962).

Vitamins A, D, E and choline, in a corn sugar carrier were ad-

ded to each ration except that the vitamin A was deleted from that of Ir.

vitamin A deprived sheep.




Management and Feeding

The rams were fed dal., t t''u a.m. all the feed they would cliar.

up by the next feeding. Feed refusal was weighed back daily. Fresh .at.r

was available at all times. The rams were ,nrit r..d in the pens at all

I; r.i: -e epc r.X r v....F c to collect experimental data and for daily ,ir-

T-r. tr r.eC,.t T.er:- was one pen for each treatment group and six r.ja,,


























Ingredier


Cellulose


Casein (9


Corn Star


Corn Sug;


TABLE 2

COMPOSITION OF EXPERIMENTAL RATION


It Per cent in Ration


S(Solka-floc) 20


90 per cent protein) 20


ch 23

ir 25


Corn Oil


Trace Mineral Premix


Vitamin Pre-mix


Minerals 6.5


100.00


aControl group received 3,000 I.U. of vitamin A per
lb. of feed.










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*%jJJ ?i y., *r u ar [: [ I,, . .C. ..r... -. Jhir, ed r-.l .. r


_ I __ ~











y. I .:. ... r cr. pc... T .e r .. a .* .. .ed -: r I -e i : dJur ..

ti-c c*p'r: r -:,al v-ru d

TI,- th.r r c.c: I' .e 1 Jililu -r.'d 1 4 r.v ,11 r :.[I r r r.I T.,1 a rI:I ,-


























Semen was collected using an electroejaculator. A monthly col-
u j I r I E Ir.c hr1 v I .: -d1 1 E r ,I r
























election was made on all animals prior to allotment to the eight treatments.

A week prior to assignment to a treatment semen was collected eight times,

four times in each of two consecutive days. During the first week fol-

lowing initiation of the various treatments, s.en was collected four times

in one day. For the remainder of the experiment semen was collected once


a week.

Volume of semen was determined directly by collecting the semen

in graduated centrifuge tubes. Motility was evaluated to the nearest


5 per cent by microscopic examination (X430).of the cells with progressive

forward movement. A blood hemocytometer was used to determine the number

of sperm per cubic millimeter. From assignment of the various groups to











their respective treatments until slaughter a per.'.j ;r .r.,-.:r, ,-

elapsed, during this time semen collected was evel,..j- d F:r .j.jra .j il

data on percentage of abnormal cells present at ej,:- -. -i1 .:: ..


Blood Evaluation

Blood samples were taken initially and at .-rcrr 1. .:.i 8 o ,.

until the biopsy was performed. Blood was evaluar:.. r,,,- .,r ,, Sc .:,r-

ding to the method of Kimble (1939) based on the ,: r-. ... r- ,. ,,:


Liver Biopsy

In order to have a more reliable knowledge I I-- cr- r..-r..

of vitamin A in the liver of the animals to be as *.-.:.l r.: in. ar

treatments a liver biopsy was performed prior to r. : u, .-,, .,,

was done by Dr. Fred Neal from the Veterinary Sci,-n- ['p -f-r 1 :.rt [I-,r

University of Florida.

Essentially the technique consisted of thr. i .: I j

teral incision was made in the area immediately b:-.*-j [r5 i :- r r.-

right side of the body. A piece of liver was rer.:.-.: L, i.i :u ,. -

size as to allow vitamin A determinations on it. '.: .. : .. i r,

closed and 10 cc. of penicillin were injected to i--.*. r .,-r ,-, -: ,-,


Slaughter Data

On the twenty-first week from assignment E. i aC.t C u* :.. -..

rams that had survived were slaughtered. Of the i- r,. ii :ll :E C d I.. he

first group during the last week of April, 10 sur.. -~ .1r.3 m ir.; :mIu lrld

in the second week of September.













.it [I .eC,.,oJ Or.,,: r F r ,; ,u....- It., i':. h*j d t't.-,'. Il ,: IrEd


i tr. 1 1.1 11.r ..r .. rr. i n [ r-.r i I fcr .. -F C : .r.0 *..c i t : ---


tr Tr, r j Cr,..u r 1.j Lieo E1 3 : II 1- [ i :.C-:r. E 5 ..I JuI a .jk .Ej:


:Ijui"i c l r. [tI. t r. : D. E rec t r r rl ,, dl . I:-i. c .u '. ' J


u.L Il Fl r .T i C. ut [ .. rjcr .c. r

i- c,[t. mtilk i. t ,.j f. I, i C [ r r .- ..-. .. I .l c


per* 1:I [ rI c .r r ig.J dy r. .i- e re Ee'c ake.


.1. Ij ,.- (cr .i. i ir. rl u . LI 'J "e" J r-r r',r .-






Ti, i I.. S. e l. I 1 J i r .r I I *.: I LI r. jrI:.e : .:

























,., ,rCI .,2 u I I I.l i,, I ,l, :i *r I, :E : i.r i, .u Ii Ic- ,c j.[. i .
[ 'r., : i -d C[ j .[ l r ic r -e e F 1 F I r t. I:.. .:5i : i. : t.1. [ C..
r i.. i [ hdl .c r IF .i-F :'-[ ir. c t tcr dI i.



H EC c I C al rc n r I : -i-4 i i


T .: rI.[; I r r* [ r :E[ : L t .* =1 p C :v ttu E I E re F E E .













Tl.: :.r : C^ re ju[l l CJ L '.l. .. Er.1 t r. IIl : '.1 .0 t l J [C Z uJ,





tll r . -p* : C alu i .- r .v. f A (Ii rI f 3 -, al r al I E i ,-E- h-h1


*-rt t i .: 2 r.1 [E IE e I :p.,T,4 ,r : r r .i :AC rc r :.r, r .. uc .E r b..-': E .[* E ..-j


[ f. r :.i T ..- J r 1 r [ 0E I I r .. [r.4 .: . r. pc pi". 1 -r.u J .r


E I *J,.*C jE C j r .r. l il :.r ..j . Co : [ : m.













' I. -i.L As 1, j.

The Jd .I ..- JEl : ,, r : u'.'. 1. .-1 .*r,[r.. E1-. I p .1.1 P .,, : iI


.-..1 lF.. -:c I f -..C II h- rc .TL.r..r J r.:. r. 3: I (c(. c.:fI 6iCI,


Pr' s I c *E l-.Ji t. F .. [t.. 1- E .: l aCi.u...6I .i [.,EI ...(. ,lrc *u 1r a I 1 .




.1 tr _,-. F,c c.- .1 Prj:, I ti r. tec, ..3' .-n .- r [I., .- -, e.'.r i rF( r _1.*








I, rhe a ,. I. i :.r. ...e ..r. i., : ..._r, jar a. ..i,Z .J I..r *-eri




1.11 I 5 -,.1 I. [ 1 I c r.-.. -I I1 I:.* l. in l cr. 1 [E .. r- .r : II ., .. k : ..I


It,.Cc I' -:*c zr,, ,- .J .i rI : I. r, -,lu : r, t u Lt.r .pr.-:.:r ,,, .. I- .*-.,u


CLe r.I1 It,' I .I l : 1 J .c, : .I -rI[ E r. at : F C C I ..51 ,., .c *. , C S C -

I, : ..a.. r 5 C I.: r .. u r u.1- ar.. t c '1....c .. i. .


t :r.,,.: I 1,rt ..,,'( r, ., j L L, II. nE-... r. r ': -










I r. r. .. [ I .' E l r.,' .-..), L t lcr, l-cc -. c r ..r <


LE [.,,:r, [rcur ...,r. .re r .1 .,,'.co L, [ .: .[ r . rcC TK pr.: JuC r i ..1














RESULTS AND DISCUSSION


General Symptoms of Vitamin A Deficiency

Approximately two and a half months after assignment to the puri-

fied diet night blindness was observed in some of the experimental animals

on the vitamin A deficient diet. The rams were checked for night blindness

by placing a panel across an alley after dark and checking the rams indi-

vidually for their ability to avoid it when driven through the alley. All

animals on the deficient diet eventually showed symptoms of night blind-

ness.

There was considerable variation in time of onset and severity of

symptoms. Some rams showed a characteristic cocking of the head to one

side similar to that described by Lindley et a. (1949). A number of rams

showed a characteristic cloudy cornea. In some,this cloudiness disap-

peared but in most it persisted. Figure I illustrates this cornea con-

dition in one of the vitamin A deficient rams. Most rams on the deficient

diet eventually exhibited an uncoordinated gait and progressive weakness

which became so severe in some cases that they were unable to stand as shown

in Figure 2. Several rams walked on their knees, apparently as a result

of loss of control over the lower part of the leg. This condition was ob-

served in only 3 of the 35 vitamin A deficient rams. Other symptoms exhi-

bited by the deficient rams included loss of appetite and evidence of pain.

It is interesting to note that some animals did not show any degree

of incoordination even when the liver stores of vitamin A had been nearly

depleted.










'4


Fig. 1--Cloudy condition of the cornea present
in a vitamin A deficient ram.









A mr w
a---
.. ... r... .


., .' i s .. - -

Fig. 2--A vitamin A deficient ram showing extreme
weakness.


A 9, _OAW' J00











The deficient rams that were returned to a vitamin A containing

diet recovered from all lesions. However even though the vitamin A liver

stores of these animals had been drastically reduced no severe symptoms

were evident in most of the rams in this group at the time that these

began receiving vitamin A. One of the rams receiving PMS treatment exhi-

bited a protruding eye with evident infection. At autopsy the veterinarian

diagnosed purulent pleuritis as the immediate cause of death. Another

ram on the same treatment developed an abcess in the sheath area. This

same ram showed bloody urine before dying.

In general the rams on thyroprotein treatment showed less severe

symptoms than did other deficient animals. The appetite and growth of these

rams was higher than in other deficient rams. Rams in the A + Testos-

terone group exhibited symptoms similar in nature to those of other rams

on the vitamin A deficient diet.

At autopsy no striking gross pathological lesions were evident in

rams subjected to the vitamin A deprivation.

This author believes that some of the deficiency symptoms observed

were in part caused by multiple nutritional deficiencies resulting from

the decrease in feed consumption observed in the vitamin A deficient rams.

The A- + Thyroprotein group had more appetite as well as less severity in

their symptoms. It should be stated however that the deficient ration

would have supplied the required amounts of nutritients had they been

consumed at the same level as the control ration. The basic cause of this

multiple deficiency is therefore an absence of vitamin A.









Entv i f'CL i or C jr. irrsJ;Er;.r,

Rams exposed to irradiation suffered a gradual loss in appetite

until death. Those rams surviving the irradiation treatment eventually

recovered from the loss of appetite.

No signs of external hemorrhaging were evident in any of the rams

exposed to irradiation. Several rams exhibited limited anal bleeding

prior to death. Most of the rams would lie down, shiver and show signs

of pain prior to death. At autopsy multiple hemorrhages were present in

the lungs and chest cavity areas. Limited hemorrhagic spots were observed

in the cortical region of the kidneys and adrenals and in the urinary ...lj-

der of some rams. No characteristic histological lesion was observed ,r.

the testes of animals exposed to irradiation.

The multiple external and internal hemorrhaging described by

Meacham et al. (1962) in pigs exposed to 350 roentgens of whole body

gamma irradiation from a Co60 source were not observed in these rams

suggesting different mode of action of Irradiation in these two species.



Plasma and Liver Vitamin A Content

Table 4 shows plasma vitamin A values for rams in the various all,.-t-

ment groups (Group i, II and III) and treatments, a summary of all treat-

ment groups is presented in Table 5. It should be noted that the various

treatments appearing in Table 4 were not started until after the biopsy.

However the rams appear grouped into the various treatments so that compai-

isons can be made on the vitamin A reserves of rams assigned to a given

treatment. Liver storage of vitamin A determined on biopsy samples is

also shown in Table 5.























- I C C N -





















4
- r I .



2I 4 C -. 2 7


Cr




















- II -l: ~ 7' - .







-3 -
















-Z -.r~


-T C S


Zr' -Zr







C-
\I J





9




'' ''
















4 i


i







r


b-





_1


.i .n











r






c




















i


-


























1


z


? i


'-

















TABLE 5

AVERAGE VITAMIN A PLASMA AND LIVER VALUES FOR THE
VARIOUS TREATMENTS AT TIME OF REALLOTMENT


Control'

Deficier

Return

A + PMS

A + Thy

A + Tes

A + Irr

Control

All vit;

All vita


Number Plasma
of mcg./lOO ml.
Treatment Rams of plasma

6 36

nta 5 3

6 9

a 6 5
a
6 3

t.a 6 3
a
ad. 6 8

Irrad.a 6 57
b
amin A normal 12 39

amin A deficient 35 5


Liver
mcg./g. of
fresh 1iver

154.0

1.4

17.8

2.3

1.9

2.4

7.7

157.8

156.0

5.6


aValues represent an average of all three groups for
a given treatment.

bValues represent an average of all three groups for
all animals receiving vitamin A in their diets.

CValues represent an average of all three groups for
all animals deprived of vitamin A in their diets.













T ; I I.-. i re . vr. jII r1.- .. :; E h C J I't-r- E r. II 4i .-.. I

Ti irv c: r peered IIu n ..: r [t-. r. -r r .:.&- E -, E[ IT.. J i ..Cr -I

' I I r -. i...-. [ .1-. II: I ,r l t . : C E I I C: J K T - e j.,iu

I 1f h*'.. v r E t i : >1 -: ." ,- 'I.:...- -. DC.k p I *-.: it *I I I E f r. r. ..

T i.e J I r I fi rr r.r .r. r r [ a ..I. i ij r E .r. [ e r .,..: a J.:., t. C j .:. rr.;

.:tr ..-. t I t : .ir t aL i .

T ti I I:. rt-..[ E .: r i ..I I, '~ .. .r ,...,,-, I .tturrin j

from month to month ir. 3 ji --. ia r f tas . : c*.:. icrat rcr.: ii -V.Ecr,

the plasma vitamin A c.e I : .a. tee... r ri c.C j I i .i r. i ri. ., i i. r

stage a plateau is reachej. It E as at L L :,-, [ .E rr.h c t :.p ., p Cr-

formed to ascertain the deficiency conditi-:r.. I .i er.ral ..rI ...ir. li.:-r

reserves were at abnormally low levels at *--: 1 t ,v jnr.r :Fr n- re".

on the deficient diet to the various treat-.Ei : .- ;r,.. I Tre 3.7:

S : r. c.j [... r.. ri. tire rc r..r . cr-:.p III : e.1 K E. 1.r .i S. .. -

*r Ie .jr, :,[- I r ..r ii.3 ri T but C**. J. o t, I I .i [E h r. i- :.:E re : .-.

-r, f.. *...-.L i ; rn:riana l rr l. .T. Ir. j .er l ..*r.e Iea 51 r. .rate I l

a I S r.-Is iC i.r r-.C C.:..d r, .5 r -r.[ :. r t.. I. 3- C1.. ..T.-.t I.

[tEh r .; C:... [i. JC T *ci r.r .J E[ (.:. [Cl., ..... c rca ie.s[ r p- a r II. I I

: "- Ij kc= .; 1 nr .E, tE6 -nE t. t.. (i L E .E :. E E - r,v .cr I i r

A 1 .I : r. 1l. Tabiie \- Fr- r a h.rEj 1r. Eh.ese raT uk.1i .-i .I -C tC E

* riL I j re, j T 11..3 :. p or .:I :.r:Isr *r 1 :ir. ii[. ti [i -e.. ;i n.ia (.-.

r. Ir ir .i A ..u e The- . l pI : i .I a, r=: b:C r .. 11 r..-. ir., i-...

I r I .' i.aS Et -. i .ufld i[ r.i .:.t rm ll.SE-rnu .,ert- rEc he .'C I tu d

r.acje teen. r-:. pEri :...- i r :I ,-s :c T .: c E i jr. tLi cad.c i ; i. I al r* .cr l

l-rc-ugh-":'ui c- pir r. E TID .: .: .-: r,a: bie c ( i r *: .j..? (rJ












. : [ I i r.ke e .,. I (. 'IL-d ; l h.L a . JA=5 r. I : .e ,. It I 31 :*:. i: : : b0 t

IJughttr rf 3.J.. ip- .i l dj t c trerit per;ud: and ta ad :.; r, [r.e amu l

nrc r i aTa, r t.t atu [. ift .pula., t, 5 c -, 's,3 .1,e t iG t Hi; : .. r E r. [E l r. :

e p-.rir...i ,, ( t h I t 3 [ er .T ths t uI1 &r, I .: I low.. : rc or .I/ j i t.Tn ot-.

nriter .- r 1 m- were ca. Ia lat.lI

Tartl. 1 ,: r Lt tap, ,, ci n .., i ,r i ,, J duC u r I IT.T- 1 31 -.3 ai. i l i . r

..[a.T.i r, r .= E ra?. [ ..C f r.-a I .i trr.rr.. a , Tr tl, e i ,n- or. ir -

%1 a l t I .I 3 . .[3. 'I. .i .- ;3 I r .-r .. ,,-| _r. r ,: : ,_ r, I. : r r,-

a. 3 . thn C4 the pe .nr ,.c The p l3T- .13..,. 1 .E1u: ; it..ui*d pr c E:r

*r r, .,-,: .. (.he .J, ,. -, It C n c 1 t r t Ir t .:. i e, ,, .:r. Jr I t-a..

E .: :c i r Eh, 5,up ar.i In I- ir re ur .e r up r.a ra r. ( 1 1 .7.

.Ia -i Iu- 3 r lab I e tab thn C u.l r.tr.I:.



Fed. IE 'c r

Fted a .( e ca j .; r i.-1.i dual r a I r er. :t p.: L tcE t- 3Fc -r

L I-u.c c i i i uI E .- E id C l c. er 0 ii0 .I ; I.j r.-r i.t J uCid.l

3111 [r..C, E Dur lr ] i tgra- II Itrd .n j m tt Jji j rt I r rt:-r .:.rir..:..Erdi E. tc -

ca [ n a *:.4n- nt r vn. :-. n, .:. the i t T.--n e : .13. ; d..r. ii-.. .ret

lr :c. r, r.3 C cKr. ejr' held 1 -.rcna. l, I .r. r I r.uT.CItr C i a-. ur iI

thr. 4,7 >er a .-n eC 3 c' c .-- Itu. : hlt wae In.- -.t.er --.f ra- 7 ma: .-

Sre cn[ ,. l [ ..I E p r..':-:.. t .:.r .r e .: It h c t re a .. F: r

thic- r- a: i : cal l an ,-al -1 i E I rI t .k ,e .I a t t c M*A C : t-I

h er .. r -.E -ir i-I c -Ac ;f. J* r. Jt CL .: rhic : j Cu.c c I u : : i. an t dr n..-

Fr Th [t.c 343i I it Jat a

1 3tbi. :U h' W c fc- .J I :.t djaE a r...r (t.t .ar ,;.u tre -at.rn. t jr,-up

*ur ilj prd-6: I .1 11 I pha :i I r a,-: .*r [.t-c E ..C ,I 31, j A[c i;,1-,











J
P


- 4 -


0 CO 0 0
r --


- - 4 -


0

0


-~ '7 0 7 ') 7 3


44 0 0 0 4






-. 3) O4*~ 4


'3


4-4 4. '


44 '


*2 3




'3 '


- o


3T


-, -


3) '3
C LI







o C

1'1 4-1
.._3


0)
o
.D



ow o
o .3


S



( a )
C 0 -

C O 4
m 0
c- m
4i .4- 0


3) '




4< C 3,
S o S




4 3







*0 0 4-
C a3 C
O c








o< 3, 3,
i*-






3)3




7 3,
70 0 .
^


c u
34. 4) 4- 4 O

'3 0 3)










deficient diet consumed an average of 3.0 and 1.8 Ibs. per head daily,

respectively. During phase I no widespread occurrence of vitamin A defi-

ciency symptoms was observed in the rams on the vitamin A deficient diet

indicating that the detrimental effect of vitamin A deprivation on feed

intake was manifested prior to other external signs of a vitamin A defi-

cient condition.

During phase It average daily feed intake per head was 2.1, .85,

1.90, .92, .71, 1.59, .67 and 1.54 Ibs. for the control, deficient, re-

turned, A" + PMS, A + Testosterone, A" + Thyroprotein, A + Irradiated

and Control Irradiated groups respectively. Rams receiving the vitamin

A containing diet during phase 11 (control, returned and control irra-

diated groups) consumed more feed than rams on the other treatments, ex-

cept for the A" + Thyroprotein group. It is interesting to note that the

control group consumed less feed during phase II. This resulted from the

increased stress to which all rams were subjected during this phase. Thus

the decrease in feed consumption evident in rams on the vitamin A deficient

diet can be attributed partially to the increased stress. The A" + Thyro-

protein group consumed more feed than other rams on the vitamin A deficient

diet. PMS or testosterone treatment did not have any beneficial effect

on the appetite of vitamin A deficient rams. Irradiation appeared to have

had a depressing effect on feed intake on all rams irradiated.

Feed consumption in the control group was comparable to that re-

ported by Meacham (1962) but somewhat less than that of Tilton (1962).

The latter used corn cobs as a source of fiber in a diet very similar to

that used In this experiment. Meacham (1962) used Solka-Floc as the source

of fiber which is similar to that used in this experiment. At slaughter,











extensive amount of wool was found in the rumen of rams in all trcT.--rr.-r

This was to be expected from the frequent "wool eating" obser..i.r ;rn r.t

experimental rams. This suggests that the amount or the source :r r,.cr

provided or both were not adequate. The possibility also exi:t: tr..t

some other nutritional factor was not adequately provided.


Body Weight Changes

Table 6 shows average daily gains during phases 1, II a 1.d I .J.1

II combined. In phase I rams had not been assigned to the vas..,ire tr -

ments; however for the purposes of this discussion rams that ,irc .js-ij-i.e

to a treatment during phase 1I appear in this treatment in ph.:= I. :

that it is possible to follow growth rate for rams in a grou: riir E.: jnd

during treatment.

During phase I average daily gains were .24, .06, .10 ... .

.06 and .23 Ibs. for the control, deficient, returned, A- + PrI.. A- Ti:-

tosterone, A" + Thyroprotein, A- + Irradiated and Control Irradl;aed

groups respectively. Table 7 shows the ranked average daily .:.-.: Fr re.

various treatment groups. Rams on the vitamin A supplemented diet jaiwd

significantly more than those on the vitamin A deficient diet P: .,a- E...

be expected the various groups on the vitamin A deficient dire dJd .-:.

differ significantly between them. Individual daily gains arnd .,irgt

during phase I are shown In Tables 19 and 20.

Average daily gains for the various treatment groups d.urnr. prasi

II as shown in Table 6 were 0, -.10, 11, -.04, -.27, -.04, -.2:, 23 Ib:

for the control, deficient, returned, A" + PMS, A + Testoster.:rn *

Thyroprotein, A" Irradiated and Control Irradiated groups, respecii..l,








































~I















U U.





I-








CU


O
L
Y
E
O
u






o
P


+
s










+ u






o
< 0

c






u

I








<0
0











k









a
I-
c


0
0






+0
a
e


















o









LOC
t -3


LTT











Tl.1i 7 :":h o (rc r if 3.Led j.ir g ja jI .3 -. r.:. t hie :a.c grr..u : I.e

r. -tur. ,d 3' .ul c .; dl rl d ll C n I, r C [ P.1 ai I .[r.[ r r r. =i r.. L r..JP:

? *C pl t r.,r he c-, ,.rFrol Tr-. crnt i cI : d j -.it -g a ; :.gn i r c ar r,,,r

[h 3.1 Et.r. iM T ,rc.pr.. r.; r, ari.j et ic .-.( r Tu r.T, ? I 3 ,-r c

da I I a'. 6 re C ..: tr*.'l r ,m,-, : I v r)1 l lut, l [t -e Jrla : ight reJuc -

1 1.0r. r T t* 3 .h.:, i. .i i I b dur r.g ph e II ia. C.,: :cd .:

r r ail i i t! J -c r .a I. C cri a ci Jal i .arn fr, lw fcrt I. 1i3 ci ICt A I

irradiation on average daily gains was equivalent on bo:r. c.:r. rcl Ir.,d "

Irradiated jr ,>p:. Individual "..ei l. ; and average daily' gairi ir. : .:-r.

in Tables 19 and 20 of the Appendix. The combined phasv I anj II a.c.r n

daily gains are shown in Table 6. Table 7 shows the ra-.ed a..rge j~1 l ,

gain for the various treatment groups during phase I ani II c :it..-,.d. Ml

rA.7: .: .- lhe ; l 3>7. r, :upp't..'etret d I5 l .-. i l*.-illcautl rC.C re 1,I..I,

r ,i: s ,-, hF e rdi ,FC i il ,i rat li I ii r .- Ipni l* thl : i e l .idu l




t -,.,I : : r s r i l. [r te [rC b.:. -, 9rg t cr.mr- .a3 ir r. T.i. ,.r,

[r,. ar-tiu ; tueat.T.e i v t..ri d i :,u: I.4 thci : r i uri e .[ :h,...lId be *.':leJ

tLI [ trE e r.....L r oir r 6rr. pct (rea[r..cr-,i ., :,, ll frLrJ.-. the a. (it, p.sr gr.:.up

a i. Cr.s rf., d ,r, I.1:- .r,rti tce.j I, r. T...I A L, C d rici.cn Jr : tica l,

r.*jd-,C J tie r.'11: p rr 4r-K p :-- Tha* -I r, .r.j ir- air.ce5 il.,- 5 ,e p [lcted

ir .3 rre I E .-. J( rr re t:r.( [t.E t.od, ofe.oI ch ari gce 0t ad .er, I *.i te d

nfat.Er .EI rA .-..: I Lr r -re.., cr C d3c r. i[aI [ d.d L t C.i.e Lr.-.:r.A r,

,:t c. e-.,aer r..r t :I In., .r.. .ad ,.., rty,. ne ll-.e rcu t r :.,[ -.'. i 1. t

Cu r. t t I I tie-pe .-j Ci.i p clrl .j I rme *.,.,ld har- Eari :I[eep r I.ad [ C,

'ur i .Lj.


















PHASE I





6




5



./^


PHASE II









/
..^/-, "


3s'


2-








DEFICIENT ------------
RETURNED ------



-40
3 6 9 1 4 7 10

2-WEEK PERIODS C

Fig. 3--Effect of vitamin A supplementation on body
weight changes of rams.

aphase I extended from the start of experiment to assignment to the
various treatments. From periods I to 6 there were 6 rams per group. From
periods 7 to 8 there were 4 and from periods 9 to 11, 2 rams were present per
group.
bphase 11 extended from assignment to the various treatments to termi-
nation of the experiment.

cNumbers adjacent to plotted values represent animals remaining in each
group for a given two week period. Differences in numbers between periods in
a given group resulted from death loss.


















40-








20-






o
L.)
I
0
I- -


Lu




-20-








-40-


PHASE 1I






6




S ,, 6




----
6












CONTROL
A-* PMS
A-* THYROPROTEIN --
A- TESTOSTERONE- ----



3 6 9


C
2 .,EL.- ER ODS


Fig. 4.--Effect of various hormone treatments on body
weight changes of vitamin A deficient rams.
aPhase I extended from the start of experiment to assignment to the
various treatments. From :er';.d: 1 to 6 there were 6 rams per group. From
periods 7 to 8 there were L ,ni Irom periods 9 to 11, 2 rams were present per gr;uo.
bPhase 11 extended from assignment to the various treatments to termlna-
tion of the experiment.

cf..-,Lir: aJ;acent to plotted values represent animals remaining in each
group for 0lr. nc..-week period. Differences in numbers between periods in a
given group r.:ultEJ from death loss.


PHASE IIb












5


\ ,










4

\ \



2\.
\ I

4 7 10



















PHASE I PHASE II b
40-








20- ----





5 .
m 7 ,
u 2



0
2 EEK PE










FgI 5.--Eect of irradiation on y ight h es o Gtmn
-20-
CONTROL
CONTROL IRRADIATED ------
DEFICIENT IRRADIATED -- --



40 1--- i -------,- ------------
3 6 1I 4 7 0
C
2 WEEK PERIODS


Fig. 5.--Etrrect of irradiation on body welgst changes of vitamin
A deficient and normal rams.

aphase I extended from the start of experiment to assignment to the
various treatments. From periods I to 6 there were 6 rams per group. From
periods 7 to 8 there were 4 and from periods 9 to 11, 2 rams were present per group.
bphase II extended from assignment to the various treatments to termi-
nation of the experiment.

CNumbers adjacent to plotted values represent animals remaining In each
group for a given two week period. Differences in numbers between periods in a
given group resulted from death loss.












F. ut. .1 1 u tr .,5 i the -e .-. ,.i ht Ch_.nc) i: th-I C.,. r ) Jcti -

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part Ial ly -jj., t5 a iiT1 it :n in apijIr i prduCvd bL, i.t i(.,rricprn- C n

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C. air-, :. It CI ,,r I r.at al rI I a E p .d [: rr aj i I ., : v Iul"rri r e3 uert

-r i t In- -eL. Tr: :..rrel Ate .,11 .inr tr.e Jecrcadi : n .e c .n:.J n ,)j ...

Lie .ed Ir, [.r arE,: r.iT, Tr,'.e rI : ;-, ithe ..- r. I rr ad;atle3 r up .v

:ur, i rir .3 i a t rec : .rE ,J

T1 I.' re i .1 e c t r. g r..ut.l' ra i Ar,-a I i c j -e th ;t ..-.in

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ip -. in jled. PM: ar..- t -:lt ):.erC.n. t r.Ar -A.r-t Jid rt.r pr e riE r Fturirh r ., i j h

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0iuatI nj CM' L unr. rpp- t iE c re mitlr l arP rt l i r. I c :c re rc- .l t.t











losses, as compared to other rams on the same vitamin A deficient diet

without thyroprotein. This finding would appear to contradict the widely

accepted theory that an increase in appetite which would normally sti-

mulate growth, if the diet in question is deficient in a nutrient would

accelerate needs for the nutrient in question. This would in turn increase

severity of deficiency symptoms. Irradiation affected feed intake almost

immediately. This was accompanied by a severe weight loss until death.

The appetite of rams surviving irradiation returned to normal and did not

undergo further weight reduction.

Analysis of variance for differences in daily gains between the

various treatments in phases I, II and I and II combined are shown in the

Appendix Tables 26, 27, and 28 respectively.


Survival

Average survival time expressed in days as shown in Table 8 was

141, 82, 141, 85, 97, 125, 46 and 63 for rams in the control, deficient,

returned, A" + PMS, A" + Testosterone, A" + Thyroprotein, A' irradiated

and control irradiated groups respectively. Survival time for rams in

the various allotment groups (I. II and III) was also shown in Table 8.

Per cent death loss for the same groups as shown in Table 9 was 0, 80,

0, 84, 66, 50, 84, and 66. Table 10 shows the ranked average survival

time in days for rams in the various treatment groups.

Rams in the control and returned groups suffered no death loss.

They survived a significantly larger number of days than rams on the other

treatments except for those on thyroprotein. This latter group survived

significantly more days than all remaining groups except for rams in the




















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PE- L(ElT UL-frI Lit.. Ful-F -Mi IN THE
Vit lO '. Ti.E.IMEILt


II ..r . r. l : 9- 4C ..-
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A + Testosterone group. There was no difference in tolerance to gamma

irradiation between the control and deficient groups exposed to irra-

diation as measured by survival time. Differences in survival time be-

tween allotment groups (I, II and III) were not significant.

Most of the rams whose vitamin A reserves had been depleted even-

tually died. Treatment with either testosterone or PMS was not effective

in preventing death losses resulting from a vitamin A deficiency. Daily

capsules of thyroprotein were effective in reducing death loss of vitamin

A deficient rams. Rams whose vitamin A reserves had been drastically

reduced but were replenished by feeding 3,000 I.U. of vitamin A per lb. of

feed daily suffered no death loss. The vitamin A status of rams exposed

to irradiation did not affect significantly the survival time of these

animal s.

Individual survival time for all rams is shown in Appendix Table

21. Analysis of variance for differences in survival time between the

various treatment groups is shown in the Appendix Table 28.


Semen Characteristics

Semen data %ere statistically analyzed during the first ten weeks

of phase II. Beyond this point total number of rams per group was too

limited for statistical consideration of the data.

A series of figures are included which illustrate semen charac-

teristics of rams in the various treatments during all of phase II. It

should be noted that in some instances the number of rams per treatment,

as Indicated by the numbers in parentheses adjacent to the plotted values,

is small resulting fr:e. h-a ,' death losses in those groups. It is also











true however that death losses in treatment groups on the vitamin A defi-

cient diet occurred when these rams were in advanced stages of avitaminosis

A. At this time changes in semen characteristics affected by vitamin A

deficiency had taken place. So that in many instances plotted lines even

though based on data from a reduced number of rams, do reflect the trend

of the initial population. For example Figure 10 illustrates per cent

sperm motility for several treatments. In looking at the A- + Testosterone

group one sees that from weeks II to 12, 2 rams died. After week !2 a

drop in per cent motility was observed. Appendix Table 24 shows individual

semen characteristics for all weeks. One sees that the 2 rams that died

were not showing high sperm motility, so that the drop in per cent sperm

motility at the twelveth week was not due to death of rams with high sperm

motility values but rather to a drop in values in surviving rams. Had

the rams survived the change from weeks 12 to 13 the slope of the line would

have been more pronounced. This should be kept in mind in drawing conclu-

sions from the experimental data available.

A summary of semen data collected monthly during phase I is shown

in Table 11. Per cent abnormal sperm cells was not determined during

phase I nor was semendata from phase I analyzed statistically.


Vol ume

Average semen volume for rams in all treatment groups during phases

I and II as shown in Table 11 were 2.2, 1.4; .8, .9; .6, 1.1; 1.0, 1.0; 8,

.5; 1.1, .7; 1.9, .9; and 1.0, .8 ml. for the control,deficient returned,

A + Thyroprotein, A' + PMS, A- + Testosterone, control irradiated and
























C CI ~I















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. E I I Ti -,. iI .-C t j l dI J D E ** dr.... .. c a. r il .*Clui

*lur ir., plr I 1 II rir r. :t ur ,' 1 jr, Jp.C j, l : .. d :T,-r. ,lu '.; J ur i p. :. a-

I .. ufCr a.;ige c*...ld be a1Cr *itu d C-. j..T.cri..tf r. i. .. .. 1 1pp -

C E, at. :r.. i r 6 .II :t r et :e ner. I n lu7c r..r.ar : w. r r r.c r3 jrrCu

du.' I r.j t I I i ll i e r.,:. he :*-. rul .. d,: i a .T ..; r. d .d r.

crl- .iu:. b E-... [h .. tr :r .r uP ir,..: gr rua ... \ *j. I iet.- ri

t urrneJ r.ui C rlE har : h r, ICi (i. tC.:r, r,,,-r., : upr..li.E.rl, ed .j. r upi LF. V

PM' .,id 3 c: I ir .:.. t E I d r e Tc.; :ul 6 Cr d a. I ur j .r I e u it. n i' a e r a.r

.Cie -. C.lu dur. r. n t. .E II. Th-, *rcpr,: (e Ir, ren:.i.;. di d -..;.-: r. [ Ir

riarqr. .us c E Or- ,:-I Eilu.Tn;. i ur: Iu:rr i : -.-cE r. .I.lu.T.c Cr--3r46

r,.r the hormone treated groups compared to the controls. Semen volume

in the testosterone group was quite variable from weeks 12 to 19 but it

remained lower than in the controls. The PMS treated group had low semen

volume throughout phase II. The semen volume values for the thyroprotein

groups did not drop below their initial value in any of the 20 weeks.

Both groups of irradiated rams showed reduced average semen volume during

phase II; this change was more drastic in the control irradiated group.

Figure 8 shows this change during phase II.

It should be kept in mind that semen volume differences between

all treatments were not of statistical significance when analyzed. Tables

30 through 39 show analysis of variance for differences in semen volume





















































I. .




I...

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during the first 10 weeks of phase II. However some trends were evident

enough to permit certain conclusions. Vitamin A deficiency appeared to

have had a depressing effect on average semen volume. This effect could

not be overcome with thyroprotein treatment. Treatment with PMS or tes-

tosterone resulted in a more pronounced drop in semen volume of the vitamin

A deficient rams, Irradiation appeared to have had a harmful effect on

average semen volume from normal and vitamin A deficient rams. The reduc-

tion in semen volume resulting from a vitamin A deficiency found in this

experiment is in agreement with the findings of Lindley et al. (1949).

They also found no beneficial or harmful influence on semen volume of vi-

tamin A deficient rams from treatment with PMS or testosterone. Goswami

(1962) reported a reduction in semen volume of bulls treated with thyro-

protein during the summer months in India. This effect on semen volume was

not evident in thyroprotein treated vitamin A deficient rams in the study

reported. Black et al. (1950) reported that thyroprotein had no effect on

semen volume in rams which is in agreement with our findings. However it

is very difficult to compare our data with those reported elsewhere because

of dissimilarities in climate, diets, hormones dosage and age of rams

present.

The harmful effects of Irradiation on semen volume apparent in

this study have not been reported elsewhere.

Individual semen volume values for all rams during phase II are

shown in the Appendix Table 24.



Average per cent motile sperm cells during phases I and II as

shown in Table II were 71. 72; 29, 22; 66, 68; 84, 48; 57, 19; 88, 18;











;8., l* and b61 46 r r the c...r, tr, d nc i; r.[ retur r.d, d Tr., r.:r.:-r [ein

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, rh- t .il a n rA der.C I E n I e I c .r c C:rp.ar at 16u t t re c .3r. r:I .cf3

r..: III .alu.: t.:.r tI'e C.nt r l r ~;. ere iiilJar ;n pra:: I ar.1 I. The

Jet i ic n grup .u red urer rrtr : n r tir I I., ale jr. ng phjie I

.EraI c ..:-.ti i i .:,r the ret t rr..:-d grjup ii.j r.,C Le r, artEcred Juri.nq phaue

I :. It P il 1 i t l h.: t,e r.,er = dr..p 1 iotil I i r.. .I[E.& i r.

E t I ,C .E.c:/ C .. L :.be erc*.-1 C tv y raT.I r % 4 : upliceit. at3 IO.1.

Fi.Jur 9 i luiirarJ i cat ; ig n per cent rf..:.t le : pe... dJ.r iny Dh

II. ir. it.c c'.tr.[r.. d ffi i ;cr.it r, returned *gr.up: tscept ror C -F k f I

rt lit i alue r the j: l. :i gro up i re i:.c r ti-. a ( :-.: : f r e co, -

tr:-1 3.I c ri tur d 9r.:.up: Lr i l ierer.ce i i. per cent uoi i e Eperri tie ev r,.

tihe c..*n r:. i,-d dei ~fl er.t qr.-up : h -i.r. ir. T bt e I2 .. re : ; r,.rica.Ir in

, es: d. 9 and 0 or .1 n l;,.I: or ..arl arce t.:.r d;ileren.ce! per cent

m..;Ile :peir. during all 1 r I ie rir:i 1I'0 e.kz .:.t pha:e II are :r..-:.-. in the

Appenro.ii T I e 31) tr.rougr. 39. il>erage per crit iclit. [e iperac. aluu a

:i.0,,n i r TathI- II ,.erv loirc e Jurin3y pna:c II ini tre i'ori.,-,e trrealed rai..

TI hi r ..pru etei .-. Ireatr.Ent .:,jerc *Me r hir-.a t tihe dep re : ingy crr ctl: t ;-n ac-t i I lity

IrciT. i t.e .r I. le A lef I er.c, F igurc I1 il I trite. aer .ge p,:r cent

,tile :pErii r.cr tt., r..:,r.Tonl treated anri c)rtr l group: during pha'e 11

Frc.,i uEek: i ti: 4 a drop ,n j. Eraye per cint m.ic;le .pcr cell. a.: oterred

in all r r:..,r-ritn treated ygr,'uip rp iT ..eL.; 4 to II taer:e 47..:.till ue

.arit lr iJr. the A" TE: ic.t er jane 'ruup but ej,'itual l dr:-pp d lt. O r i T,

jceek: 12 Ct 21. Fro.i. -.;6i : to 6I jrer j.3 per cer.t -,.-t i L 'peri reiT.ained
























































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qr.u m I 'ere C. [ i ati :Lica ll, iynq il caf nt ir. -ir v o r r... I i .- II) u k .:Q

phr.:e Ii 4 I ,-,a of a i rnc I i iferenflce r. per ce-i : ri7 r .: i i1t,

duri'Cy p-:at I I are -.o.r, I r. it-? App .-J Table: 31 Et.rO.a r. 19

SIul : *tL. A l ined in Ir.i neris.-eni -.nd:cate ithat :-cre., c: E; 1t,

e .c.rE.rd s ct C eC J bl .i iE a r. H f I C E.- ,. Tr,. I C- rej m per-. ..C. t -

I I E c uld rl-C DE :- ed ic It re Ei-.C .E 1;h i. h Mi O [ : ct ti r.-.e T-.,rc.-

!r.:r et c l4ji i te [ht dtepre- ing crIec of %.ii ar. r. je ic c..c .v :per.7

. i1 liE T. I :lu, p-Crit. [t hI nirj ;,,.1; 5d LioIle, cil (I 3 1 The, .-,re

ufluC:CC ui iin pre.enc g r I.Tipr. s n, p r. |prr 1T.D ix E, a ....r, P

dcr icient r a.-.- t E re a5 4&t.-.E -iEh to -<.:re r.nt r PM_. Gu e' c f (1942;



























o- ,C'







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i.ptr:-.Ed 'per.". :.'i i I t ll v o il a.. n 4 deri .c ic r .-. t I r. [ c ; :r. m,

r'1J: J i-,.. e.L i (I'l'I t c-tti ;.'.c i i ar re.,ui irn .1 I, ;-. d f ; c .e'-

bull l Th. d repar.C Ir. re,: ui : tt ..eer, thC re: t .-:. it-.e l at ter t -,

.jul h .r r (h, hJ E C:.r LI .II t d I TI ar.d .ur Ca r. r, c, t .- vF 'tr. Zedj

i nce c.r.,l a i r I I .T.;t.: r.,j T, t r ..f f e 'pc, r r..e i- ,l a.-,I.rl wE rre u:.:.I t.,

Lufr. c L ( I1421 a.d-J Ic..; .;:r. t A 01 94 I Th. e t(r. i.e t 1 c fli C C

, r tadI rl r. Cr. :;C r. ..r. c ilit, equ, e 1 ii t :. r. t) L h .rI l r.d ., a.T,,;r.

14 dI f c i- .t r a..- H.:, .c r r .rT, ,, i lii ....a rc uf i Cle tl rcduc: d It.:

:u :e j : r u : i pairi r.:r, [.; the rEpr ..(: I ; c C api :.r .hei ra n .

I, ..i ..du i tper ca.'. r.oti .1 i: r 1 I r F I r: uur.Cut t.a:E Ii ci

the tEc.per i..i. i ii,...r., i (he rpperdi* Tl e 2.


Ptr (tc.c M.Pncr ajl frer,T.

A5 :r..:.,.. ;r. Table II per c .c aL t.1,-r.T i cr. .Ma l, ..: re 9. F c ,

1. 5,, j. t. r. t,. .. t.-r t .e :-.itr .i . icie -, returr d r r.. -

r :.ic r. a '* M ;, M" :-: [CF c'.:. :.a c.. r.Ir.I i rr d. lced .-d o. irr adi itEd

9rour re ec i .cI,. I t aprarer.t rr.:mi rtee jima ir.1! )r.:.up cr. tie

.It ,.. r. A dj ic .cer.'t lie[ :r.c,:., a t.g q ..r perc:-.i.. te pe atr..r.T.. 1 .pcr., i..

[t ei r :ectr. F.gcQure I i I l, tr Ei chand e: .-. per cent 3,r.:.r.,val c-rm.

E alu fc-r 1 c .ntr .: *I d t i c, ,id rct.urr.cd jro.,up d.urli .-.q hi .-.

The c .-tr l qr.-.up T .-a' i d per cer.t Ltr,.:.r,,..,l .: r.T,. at .. le e tihrc, hn-

.ut [i-c .1 wei. .:.f p;.,a : II I, it- i(...urrh ..ee ra.: .. rr-c .leje Ci ,

gr .up begjarn :..- .,i-, ir.crc~ ni l Ir arer cr cer.t .ae :,-' ,'t,:.r.cal pCr,

t,. I li :-.p.. ire .c: c urred ir. the returned .Jr :-u, r;l r'et,-,ce: in percern-

al 4 :. r -.br..;:r .l :1 nT. bel Ec tC [te:0 .r ul: .re rI-.i[ i .j.-. ca.r :[.lt

; :al11 tuI t.e1 pp -'rejd t.. t I' I I...I...g.c.lI a mp,ort : .




















































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As shown in Table II it appeared that treatment with testosterone,

PMS or thyroprotein was not effective In overcoming the harmful effect

of vitamin A deficiency on sperm abnormality in rams, rather it appeared

that hormone treatment had a detrimental effect. Figure 13 illustrates

changes in percentage of abnormal sperm during phase 11 in the hormone

treated and control groups. The A + PMS group had a higher percentage of

abnormal sperm than the control throughout phase 11. The thyroprotein

treated group showed a temporary improvement in sperm abnormality percen-

tages from weeks 3 to 10, from then on an increase in sperm abnormality

was evident. Testosterone treatment produced a gradual decline in sperm

abnormality from weeks 4 to 7. From weeks 7 to 12 values were variable.

After week 12 no further determination of per cent abnormal sperm was pos-

sible because of the reduced sperm concentration present in the semen of

these rams. These phenomena occurred In the A + PMS group from weeks 18

to 21. Differences in per cent abnormal sperm between the returned group

(with comparable values to the control group) and the hormone treated

groups were not of statistical significance. Irradiation appeared to have

increased sperm abnormality. As shown in Table 13 this effect was of sta-

tistical significance during weeks 6, 7 and 8 of phase II. However only

2 rams were present in each of the Irradiated groups at this time. Table

13 also shows that the effects of Irradiation on sperm abnormality were

more drastic during weeks 6 and 8 on the control irradiated rams. Figure

14 illustrates changes in per cent abnormal sperm values for the control

and both irradiated groups throughout phase II.

Analyses of variance for differences between treatments in per cent
abnormal sperm during the first 10 weeks of phase II are shown in the

Appendix Tables 30 through 37.





















II~


Ill


K'










-rI -

t.. .
































O


v


1




























i
I












0 0 U
o Q
00


69















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0
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C.













0 0


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0
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I' O:C





N ,-.- .- 83 d

-.- -
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; ~ ~ ~ p ~~ *-*:^ ." ^r





























fD (










PrEulirt ct-tieJ in this experiment indicate that vitamir A dcrI -

c c.C, ..-crciEE.d (,. presence of *.,Ljr.,,l *pci i in the semen of r.-,,-.n

.II .-I r, h jer,,:,c t diet. This elrr .-:t c.:.ul, J ot be overcome witr. treat-

..ci.t o.r rei t. er.:-.e. PMS or thyroprotein. Figure 15 shows a brca en. r .'

:.ind;i.:,n i' ,cJ rai eri tlc of .per-, r.,.. vitamin A deficient rams. uilti

obtained in this study are i-i, ajree.T'r. with those reported by Lind,'e,

al. (1949) in rams, and Bratton et al. (1948), Erb et al. (19L41 ar.

Gilbert and Hart (1935) in bulls.




As shown in Table II total sperm per ejaculate (xl06) durir. pl.=.e-

I and II were 2945, 1723; 637, 461; 605, 1184; 1627, 1163; 1025, 2.s, 1':.,

313; 3036, 1346; and 1421, 670 for the control, deficient, returrea '

Thyroprotein. A" + PMS, A" + Testosterone, control irradiated and A- .rrj-

diated groups respectively. During phase I total sperm production a.i

somewhat less in rams on the vitamin A deficient diet. The defic-i,,a r,:,,p

had a further drop in sperm production during phase II. A similar Ercr-d

was observed in the controls. However the returned group experie-i:ci 3

recovery during phase II. Figure 16 illustrates changes in total Ipcr.Tr

proOuct l: ':i.n .r [rC cnrol. i i.~ ,cinrt d ri'~urred groups. The alT .r :'Tri-

diate irnr. ;: c r, t :, l :~-: r., per eiac.1 i i, -. the returned group :::urrred

fror, ix.c tr..r.d eek )1. irt *e f,:.i rt ,r :up ra r n-. ed a low spermi pr.-

uCtLl.r. (hr ,u i :,ur[ ih 1 .I ei of pria. II.

A: hou-. ;,r. T,=t. II [hE Pf h : .rj TE:r ): uraC- treated rar: under-

r.nt a3 frcrih r drop I r. perT roduct i.. dl, inrg pr- :e II. The total .per-,.

,cr elcul ate :t ri-.: ini thr. e :? .j e ri..up: .6 ijhuI / lower t-j'. i itre















































F. ,jr. -- :-. I ;.-. .r j..in A deficient rams.






72
















40

ct
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deficient group, suggesting the possibility of a detrimental effect from

treatment with these two hormones. The thyroprotein group maintained an

average sperm production that was above averages found in any of the groups

on the vitamin A deficient diet. Figure 17 illustrates total sperms per

ejaculate from rams on the hormone treated groups throughout phase II.

The rapid decrease in sperm production in testosterone and PMS treated

rams is clearly shown. The beneficial effect from thyroprotein treatment

on total sperm production was apparent until week 20. Irradiation caused

a drop in total sperms per ejaculate of rams in both groups irradiated.

This effect was more severe in the A" irradiated group. Figure 18 illus-

trates total sperms per ejaculate from rams in the control and irradiated

groups. There was a decrease in sperm production in both irradiated groups

up to week 4. Froms weeks 4 to 7 sperm production in both irradiated

groups was comparable to the control, at this time a decreasing trend began

which remained with the A- irradiated group until termination of the ex-

periment, while the normal irradiated group appeared to have recovered in

week 17.

Differences in total sperms per ejaculate between the various treat-

ment groups were not statistically significant. However some biological dif-

ferences were evident. Vitamin A deficiency had a severe effect on total

sperms per ejaculate. This detrimental effect could not be overcome by

treatment with testosterone or PMS. This supports the findings of Lindley

eta. (1949) and is in disagreement with the results of Gunn et al. (1942).

Thyroprotein treatment on vitamin A deficient rams appeared to have dimi-

nished the harmful effects of the vitamin A deficiency on total sperms per

ejaculate. This has not been reported in the literature. The favorable


























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E if ect I:f c. ,r.. r jt : - ', i :p r,, r..u r ,:-, icr,. [ 1 ; y I- I V p d i m-, J : i .-.ce

.i : :ic .-..Cc I n .a.li.i, i o f lI. Ir. li rr... .11 t r I [ .Ti. -, jetllr eClr

r j.-i. :r..c- reductle:r it rtlhe Jert ina epi [tell u* )u (tr'- :eb ,ln. rj

tCab e It It i. p bl I jlar (r .. jpr-:-L. li T t .,-. .J l ,e l IP., hi i 0.3l -

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.-eri.. .n ... II h.:wE r a e Ie i r H C :.I .i .: j d

.a P. DuE C rI:i jc r L, . e.r I ar i Ci, t r- h .I :: I >1 1 1 al Ic : )r.i L ,in L

Occur r. ttE i1 t ,. A -t ii n r -. Tr, et ric t: : r rr r ri .t _t i: :1 :.r ..

prod.J ct ;.:n c 5n Le e l i ir ned i t .r : a :. 5un t l..c ,rr daj 3 i,..,', Ai ecrt d per-

7.E':. c- : ii t. Ir.E arl : t t 3j e t.t :.c ; (r.3 C r ,.. r.e.- :p rma -

C ~c t : re pr. luced *-..'..;al :per pr.:.Jc ht:. .aJ, r : ..i.ed. Tri... -- ld

d* 1i., l I'E rtEC --,. r r, I tr r.T, pr.. u: r i l ,. :... L r e.i ar. ( he t--..r .aI i r r aji e d

gro.,jp 16 ee l, t ie. : r di tat :-r.

I rH. i ; 3u mi t a E l .: I i perTr p r e | aCI. le rilr..e.lu -- u ph e. Ii

.re Ei. Hir. h .d;* t. -p. o 1T 45..t I i .: r r i r-ce' .-r j li cre.ice

t',.Eu :p -rr.. CF r e ic.l te rd r-,.,,, in, TstIe 3'.1 [5ltr...-j. ,) V.. r.'E

rA -.. ii. p .-I

I t ,P .j5p r ri t Iii p;.0 .-'-E t,:, ra,, :-,eral C o.,cd, 1 : E, r. E["

eC rrcti C, th .e a -.Ti. reat-rc *- cr. :. chtr Lcr, i .: 1 t -.-.re doir.j

l : er.phai ed [r.= r ipr.e.. nt aI ruT.Lter: C re 1 i.i t r. I jr e de a r.

E ; t i-. je the .i'Lntie :r .3 r in te e, :.',ler. tr. -. I ri ,-

L- lit, Ir. t ,j e JEpr ,.i the r.l :. C Al i-.J t c c F c .C : t. r r r ..-

t icoal n; i ic r.ct -. t Ia, t r.i c :eiTen *e j-- A .i_ ,n,.; M e. r

ra,,I .f*a. redu ed_ r ir,;' ei fect h. pr. :r .tr [ ut .,re L e.tern-aT H -,,. r..:r-

ji Ioc it ed TI 'i i h ra-.! i .- e -r, cj it r.t C, ...ere *..t e r r- .:j .n ie -id r it[

ut the r .. T. ; c Er C t jn i-t.E-r. ii:l n i ..i pr t, t rt-J rer.-..:-(










with testosterone, PMS or thyroprotein. Sperm motility was severely

affected by a vitamin A deficiency. This effect did not become evident

until the nervous lesions associated with a vitamin A deficiency began

to show in most of the experimental animals. PMS or testosterone treat-

ment had no effect on preventing changes in sperm motility on the vitamin

A deficient rams. Thyroprotein was of some benefit. Irradiation appeared

to have had a detrimental effect on sperm motility values in both groups

Irradiated. An increase in number of abnormal sperm occurred in vitamin

A deficient and irradiated rams. Hormones used had no beneficial effect

in reducing the percentage of abnormal sperms present. Total sperm produc-

tion was severely affected by the vitamin A deficient condition. Manifes-

tation of this effect occurred in association with a decrease in semen

volume. Of the hormones used only thyroprotein appeared to have had any

beneficial effect on sperm production. Histological examination indicated

that the reduced sperm production was due to a reduction in the germinal

epithelium of the seminiferous tubules which caused an impairment in sper-

matogenesis. The ineffectiveness of PMS or testosterone on improving

semen characteristics would suggest that the effects of a vitamin A defi-

ciency on semen traits do not occur through a secondary effect from im-

balances or reduced secretion of hormones normally associated with sperma-

togenesis.


Histoloqical Investigation

Quantitative evaluation of testes of rams from the various treat-

ments is shown in Table 14.




















m






















E
2 8










3 -




















1.-
'5 -
*Qvcu









ro

Yr




CL


-3 0 ON C ( -










c T LA -J rI N -t N
4- [-7- -7 (A C -7 -o ~o




r-^ -37 4-. N .7 4- CC N*





- LA O N^ 0n .7 sO CC
N 0 N 0 0 -











ri liu, cr..:k.-.iss in all rams on the vitamin A deficient diet. The dia-

ic.r *.. r n :eminiferous tubules also appeared reduced. Similar lesions

rha t...r., rtp.rc. d in rats by Mason (1933), Howell et al. (1963), in bulls,

Br:cr.jn j2 (1948) and in rams by Lindley et al. (1949). None of the

hr.:m:ar... ,u.*d nad any effect in preventing testicular damage.

ir. r:critial tissue was not affected by the vitamin A deficient

:j.ji ir., Tt.ere was more interstitial tissue in rams on the deficient

.1ic[ (nr, ir. those on the diet containing vitamin A. The reduced amounts

.:t ;:nrr.riilIl tissue present in the testes of vitamin A supplemented rams

can not be explained. Figures 19 and 20 illustrate the lack of epithelium

thickness in the seminiferous tubules of a vitamin A deficient ram compared

to a control. Note also the sparse interstitial tissue present in the

testes of the control ram.

Thyroid tissues from the control, deficient and A- + Thyroprotein

rams were studied for differences in thickness of the follicular wall cells.

Table 15 shows average epithelium thickness for the 3 treatments. Based

on the small number of thyroid samples studied there appeared to be no

differences in thyroid secretary tissue activity between the 3 treatments.

Histological studies made on the adenohypophisis of rams on the

various treatments revealed no unusual degree of degranulation and vacuo-

lation present. Even though the observations made of the adenohypophisis

were limited in numbers, the lack of abnormal hypophyseal development

suggests no alteration in physiological processes that were dependent upon

the pituitary hormones due to the vitamin A deficiency in these rams.












































Figure 19.--Seminiferous tubules in testes from a control ram.














































Figure 20.--Seminiferous tubules in testes from a vitamin A
deficient ram.












N : (( r :[ r. ,r r "u i-d a iI .* I [ *I ...-. t F .-r r .

I lti i : :r,[ r E.. [rn I r. ... ..r L r,.j I E, I- t I hl I r, v I Ee-.1


S[*C f I ,r. c n r. IT. h I r; .. e.;.Ed i: i t a. i r r

Lie-.[ J i. I E t : r t, 1 ar a .1 I rer.:at c ai e ...,1 3. .= i p-..C.f i

E r. J U: :;.r E r. n L E E i'-- t I. r j... a I [I EJc c t

L,..dic, -i l ( 1 a l c... .I.. .-. L .u I J ,I .: :u t J.. *

r r iC r. F. Mj t r c r. E .: > .-e c r Erb F 1, t e 1 9 ~ I I .ui'd C ;r.L

C I II u t i .. ... l ur j a r 1: .j I I I I ;.,; 1 I 3C .: [

: :r i:r r..ued pC r..' d . i t ..

Ir n .. I .T..e5 *.rre. .r-.[e . I r .e; rad [ ,Ir.l.. [i

.c.-.J.* TALlc .)



TAuLE li

r.Iuji Tar IVE EL.LUAlT I.iN -.,F THE TiivhmCln [ FACar Pi' IN THE
turt7 L, DcIUtCH'. E rt .Ib Li Li rhilAEe-uP i1I dir.



E .. [t.c I ...r TII. L -..
IreaT ..cr. ( Ii:. .:r ....p Ee' ... -


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*.I.,,..uI-,tf C.'.e

T:Lie I A l-: 1ig .:U t .e . .. 7 I ..* .; [r ji.r. a .Jea .:I .

r a.,: r f r. .c .C qr I anfd r .1.h :.c rE [re V rM .-.i are .:... T ac.j




















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Testes were significantly heavier in r --i I.. th. cornticl ir.l re-

turned groups than in all other treatments. Ra..: the "- .rrlral-.J

group had significantly heavier testes than other ra.. : r, Ihe .. i r. ta

deficient diet. This was probably due to the 1fct r-ct -:-t E:.l Ih- rar.:

on this treatment died much earlier in the expel ie.-.n tectaEu: .*1 irra.l.e-

tion exposure thus were subjected to vitamin A cje-.rl. ri ,.. r:r c.-...Or-

able shorter period of time. PMS or testosterone tre.n.li-n. .d.J I'ot preven[

testicular atrophy in vitamin A deficient rams. TI.,r.-: r .. tr tEJ r .,

had slightly heavier testes than rams in the devrcicr.t Jgr.u tut Ihis .ji-

ference was not significant.

Results obtained in this experiment she. it-l.l .i -.-i dotr.c c.

causes a reduction in testicular size. This ei recr d,.,e. ,.:,[ p:,:,r E.. t. r

secondary to a testosterone deficiency since tet:: r :,.i ,.-.i-n : rat. :.,

was not effective in preventing the reduction :- I.:1iCulaI .- c

The reduced testicular weights associate- r ..,r j a- det c..-:c ,

in rams have been reported by Lindley et al. ( .-') ..r..lar re:- r : .-re

reported in rats by Gross (1924), Wolbach (19251 M,,.- anJ Trur.i 1194l

and Howell et a (1963). Mayer and Truant (19'49i .r.recir..-. te:ro .re-

rone were able to obviate testicular atrophy ir. ..ra-r,,i *Jl-t.cenr r ji

Lindley et ai. (1949) were unsuccessful in prec.-,[ ..9 [I:Ciculr cir:.:h;

in rams with injections of testosterone. The re-i c- t r [. : -*.pr,.l-,;

are In agreement with the findings of Lindley Et J. Ii,4,4l.

Epididymal weight comparison between the ar...r.. r-a.T.r,ct 3r:up

is rather inconclusive. Rams in the deficient. A" n: a.-d T-1c, i -

terone groups did not show significantly differe=-r 'e, dd,lii :Ize tr.-r.










the control rams. Rams on the A + Thyroprotein groups showed signifi-

cantly smaller epididymal size than deficient and control rams.

There were no significant differences in seminal vesicle weight

between rams in the different treatments.

The cowpers of rams in the A- + Testosterone were significantly

heavier than those of rams in all other treatments. Rams in both irra-

diated treatments had significantly smaller cowpers than rams in all other

treatments. Vitamin A deficiency appeared to have had no effect on cow-

pers development in rams. The explanation for this irradiation effect is

not known. Testosterone treatment had a stimulating effect on the deve-

lopment of the cowpers. This is to be expected since it has long been

known that the functioning of the cowpers is under testosterone influence

(Turner, 1961).

Statistical differences in adrenal size between the various treat-

ments are not conclusive. However in general rams in the control and re-

turned groups showed smaller adrenals.

A significantly heavier thyroid was present in rams in the A" irra-

diated group. However rams in the control Irradiated group had the smallest

average thyroid size. So that if irradiation per se had an effect on

thyroid development this was only evident in vitamin A deficient rams. No

other effects on thyroid development were observed.

Pituitary weight did not appear to be affected by the vitamin A

status of rams. Treatment with either testosterone or PMS did not appear

to have any definite effect on pituitary development. Had the level of

thyroprotein given been sufficient for normal body functioning, pituitary











Sr..r:.t,i ro r r..:.r r. e :cre[ ld hj.e Let r. reduced CU.-r. uei.C .-[ I a

r Ja .Cl Io .-. t fr.. i i t r r:.- I cL te. rtr .phIc r..r.- r.e : i C .ui t ,,

c.:-ul 3 I I r C ul ted I ir ,i r ere:. irq t rC.IC that Fh ,r.:,rr, I e t r- t -

.-ent did not cause a reduction in thyroid size. However the data are nv:.

conclusive enough to permit a definite statement on this relatior...,-p

Table 25 in the Appendix show individual gland size for .ll ri .:

The reduced number of rams and the variability in gland size bet.-*ee ra-.:

within a treatment suggest caution in making any categoric stateur~c-t: *.

hormonal and nutrrr.onjl interrelationships in these rams.

Analysis of variance for differences in testes, epididyci:. tm--..7a,

vesicles, cowpers, adrenals thyroid and pituitary size are shown i. tie

Appendix Tables 40, 41, 42, 43, 44, 45 and 46 respectively.














SUMMARY


A study was conducted to determine the effects of a vitamin A defi-

ciency on the reproductive capacity of rams. An attempt was made to coun-

teract the harmful effects of the vitamin A deficiency by administering

testosterone, pregnant mare serum (PMS) or thyroprotein. Information was

obtained on the tolerance of vitamin A deprived rams to gamma irradiation.

The experiment was started on February 2, 1963. During phase I of

the experiment 12 yearling rams were assigned as controls to a purified

diet containing 3,000 I.U. of vitamin A per Ib. of feed. Thirty-six year-

ling rams were placed on the vitamin A free diet. During phase II the 12

rams on the control diet with average vitamin A values of 139 mcg./100 ml.

plasma and 156 mcg./g. fresh liver were continued on the same ration with

6 serving as controls and 6 irradiated with 400 r of whole body gamma irra-

diation. During phase II the 36 rams on the vitamin A free diet with

vitamin A values averaging 5 mcg./100 ml. plasma and 5.6 mcg./g.fresh liver

were continued on the vitamin A free diet and were assigned in equal groups

to one of 6 treatments: (1) 400 r of whole body gamma irradiation; (2)

Daily injections of 60 RU of PMS/ 100 Ibs. body weight; (3) Daily injections

of 10 mg. of testosterone/100 Ibs. body weight; (4) Daily capsules of 1 g.

thyroprotein/100 Ibs. body weight; (5) 3000 I.U. of vitamin A/lb. of feed;

(6) No additional treatment. Semen was checked monthly during phase I

and weekly for 21 weeks during phase II.

Night blindness, cloudiness of the cornea, uncoordinated gait and

extreme weakness were symptoms associated with rams on the vitamin A free













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91


reserves. Testicular weight was reduced in all vitamin A deficient rams

Epididymis cowper's, seminal vesicles, adrenals, thyroid and pituitary

weights were not affected by the vitamin A deficiency.




Full Text

PAGE 1

THE EFFECTS OF A VITAMIN A DEFICIENCY ON THE REPRODUCTIVE CAPACITY OF RAMS By ENRIQUE SOSA A DISSERTATION PRESENTED TO THE GRADUATE COUNQL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA August, 1964

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ACKNOWLEDGMENT The writer wishes to express his sincere appreciation to Dr. A. C. Warnick, Chairman of his Graduate Supervisory Cornmittee and Dr. T. J. Cunha and Mr. R. E. Deese under whose guidance and supervision this study was made. Grateful acltnowledgment is extended to Dr. R. L. Shirley, Mr. P. E. Loggins, Dr. H. D. Wallace, Dr. F. E. Neal and Dr. W. P. Callahan for their efforts and counsel extended freely throughout this study. The writer expresses his appreciation to Mr. R. C. Dees, Herdsman, for his assistance in the feeding and handling of the experimental animals. The assistance of his fellow graduate students, Mr. J. Easley, Mr. C. Piedra and other (personnel at the Nutrition Laboratory is also appreciated. The advise of Dr. A. E. Brandt in the statistical analysis is gratefully acknowledged. The writer is indebted to his wife Irene for her patience and encouragement during his graduate study.

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TABLE OF CONTENTS ACKNOWLEDGMENT Methods RESULTS AND DISCUSSION SUMMARY LITERATURE CITED APPENDIX Page ii LIST OF TABLES iv LIST OF FIGURES ^iii INTRODUCTION , LITERATURE REVIEW 3 EXPERIMENTAL PROCEDURE ig Materials )g 22 29 89 92 97

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LIST OF TABLES Tables Page 1 Experimental Design 21 2 Composition of Experimental Ration 23 3 Composition of the Minerals, Mineral Premix, and Vitamin Mixtures Used In the Experimental Rations 2k k Vitamin A Plasma and Liver Values for the Various Groups During Phase i 33 5 Average Vitamin A Plasma and Liver Values for the Various Treatments at Time of Reallotment .... 35 6 Daily Gain and Feed Intake for Rams During Experimental Period 38 7 Differences in Daily Gain for the Various Treatments k\ 8 Average Survival Time for Rams in the Various Treatments 48 9 Per Cent Death Loss for Rams in the Various Treatments hS 10 Differences in Survival Time Between the Various 49 Treatments n Average Semen Volume, Motility, Abnormal Sperm Cells and Total Sperm Cells Ejaculated for all Treatments During Phases 1 and II 52 12 Comparisons Between the Various Treatment Groups » for Differences in Per Cent Motile Sperm Cells — Weeks 8, 9 and 10 60 13 Comparisons Between the Various Treatment Groups for Differences in Total Abnormal Sperm Cells-Weeks 6, 7 and 8 68

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1^ Quantitative Evaluation of Testes from Rams in the Various Treatments 78 15 Quantitative Evaluation of the Thyroid from Rams in the Control, Deficient and A" + Thyroprotein Groups 82 16 Summary of Gland Weights Taken at Time of Slaughter , 83 17 Differences in Gland Weight Between the Various Treatments Qk 18 Initial and Final Vitamin A Plasma and Liver Values for Individual Rams 98 19 Average Daily Gains for Individual Rams During Phase I, Phase II and Phase I and II Combined 101 20 Summary of the l4-Day Periods Weight of Individual Rams and Treatment Groups in Phase I (Pounds) ... ]0k 21 Summary of the 1^-Day Periods Weight of Individual Rams and Treatment Groups in Phase II (Pounds). . . IO7 22 Survival Time for Individual Rams 110 23 Quantitative Evaluation of Testes and Thyroid .... II3 2k Summary of the Semen Volume, Motility, Abnormal Cells and the Total Sperm Cell Production for Individual Rams and Treatment Groups by Weeks ... 116 25 Summary of Individual Ram Endocrine Gland Weight in Grams per 100 Lbs. Body Weight 137 26 Analysis of Variance for the Difference in Average Daily Gain During Phase I ]k] 27 Analysis of Variance for the Difference in Average Daily Gain During Phase II ]k] 28 Analysis of Variance for the Difference in Average Daily Gain During Phases I and II Combined .... l42 29 Analysis of Variance for the Difference in Survival Time Ilf2 30 Analysis of Variance Mean Squares for Semen Volume, Motility, Abnormal Cells and Total Sperm Cell Product ion--Week 1 143

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31 Analysis of Variance Mean Squares for Semen Volume, Motility, Abnormal Cells and Total Sperm Cell Product ion--Week 2 iM» 32 Analysis of Variance Mean Squares for Semen Volume, Motility, Abnormal Cells, and Total Sperm Cell Product ion--Week 3 1^5 33 Analysis of Variance Mean Squares for Semen Volume, Motility, Abnormal Cells, and Total Sperm Cell Product ion--Week k 1^*6 3^* Analysis of Variance Mean Squares for Semen Volume, Motility, Abnormal Cells, and Total Sperm Cell Production— Week 5 I '+7 35 Analysis of Variance Mean Squares for Semen Volume, Motility, Abnormal Cells, and Total Sperm Cell Product ion--Week 6 \k8 36 Analysis of Variance Mean Squares for Semen Volume, Motility, Abnormal Cells, and Total Sperm Cell ^ Product ion--Week 7 1^9 37 Analysis of Variance Mean Squares for Semen Volume, Motility, Abnormal Cells, and Total Sperm Cell Product ion--Week 8 150 38 Analysis of Variance Mean Squares for Semen Volume, Motility, Abnormal Cells, and Total Sperm Cell Product ion--Week 9 151 39 Analysis of Variance Mean Squares for Semen Volume, Motility, Abnormal Cells, and Total Sperm Cell Product ion--Week 10 152 kO Analysis of Variance for the Difference in Mean Size of the Testes 153 k] Analysis of Variance for the Difference in Mean Size of the Epidydlmis 153 ^2 Analysis of Variance for the Difference in Mean Size of the Adrenal Glands 15^ 43 Analysis of Variance for the Difference in Mean Size of the Seminal Vesicles 15*+ kk Analysis of Variance for the Difference in Mean Size of the Cowper's Glands 155 vl

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^5 Analysis of Variance for the Difference in Mean Size of the Thyroid 1 55 ^6 Analysis of Variance for the Difference in Mean Size of the Pituitary Gland ] 56

PAGE 8

LIST OF FIGURES Figures Page 1 Cloudy Condition of the Cornea Present in a Vitamin A Deficient Ram 30 2 A Vitamin A Deficient Ram Showing Extreme Weai
PAGE 9

15 Semen from Vitamin A Deficient Rams 71 16 Effect of Vitamin A Supplementation on Total Sperm Production of Rams 72 17 Effect of Various Hormone Treatments on Total Sperm Production of Vitamin A Deficient Rams . , . Tk 18 Effect of irradiation on Total Sperm Production of Vitamin A Deficient and Normal Rams 75 19 Seminiferous Tubules in Testes from a Control Ram . . 80 20 Seminiferous Tubules in Testes from a Vitamin A Deficient Ram 81

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INTRODUCTION Efficient reproduction in his animals is one of the goals that any producer must achieve in order to successfully compete in todays livestock business. This underlines the importance of research in any aspect of reproductive physiology. In the literature one finds that efforts in this area of research have been directed mostly towards studying various effects of environmental and genetic factors on female reproduction, somewhat underestimating the importance of the male; consequently the basic aspects of the male reproductive physiology have been overlooked to some extent in the past. This study Is part of a broad project being undertaken at the University of Florida to investigate the effects of various nutritional factors on the male reproductive capacity. Other studies in this project have been the work reported by Meacham (1962) and Til ton (1962) in which protein and energy have been investigated. Lack of vitamin A is the only specific dietary deficiency that has been shown with any certainty to affect semen production in farm animals. It is also true however that the studies from which this knowledge was obtained are to some extent outdated in the light of todays improved research methods and techniques. The use of purified diets is indeed a tool of great value in animal research for It facilitates complete knowledge of the nutrients present in the diet, thus allowing a more precise characterization of the role that a specific dietary factor has in the physiology

PAGE 11

of the organism in question. It is with this in mind that the present study was undertai
PAGE 12

LITERATURE REVIEW Vi^arnin A and Reproduction tn the Male As early as 1924 it was found in experiments witli rats by Gross (1924) and Wolbach and Howe (1925) that vitamin A deficiency caused pronounced atrophy of the testes. Since then extensive investigations have been carried out to elucidate the influence of this deficiency on the male germinal epithelium in laboratory and farm animals. In addition to atrophy of the testes this deficiency will also cause impaired development of the epididymis, seminal vesicles, and prostate in young rats. Injection of testosterone can obviate this atrophy in vitamin A deficient rats as shown by Mayer and Truant (1949). Further injections of gonadotrophic hormone will stimulate the development of the accessory sex glands in vitamin A deficient rats according to Mayer and Goddard (1951). This suggests that this deficiency causes a lowered excretion of pitituary gonadotroph! c hormone or a lowered response to this hormone by the interstitial eel Is of the testes. Recent work by Howell et ql . (1963) showed that male rats maintained on a diet in which the vitamin A alcohol had been replaced by vitamin A acid developed lesions in the reproductive tract. The testicular changes were a sloughing of the cells of the germinal epithelium followed by an obliteration of the lumen of the tubule by Sertoli cells. Testicular regeneration was produced by the administration of vitamin A alcohol. The lesions were comparable to those of vitamin A deficiency as described by

PAGE 13

Mason (1933) but were uncomplicated by the secondary manifestations of vitamin A deficiency. This paper is the first to describe lesions in the testes of healthy growing prepubertal rats fed the vitamin A acid diet. It is widely accepted that vitamin A acid can perform all the functions of vitamin A alcohol except for its part in vision. Dowling and Weld (i960) summarized the role of vitamin A acid in a recent symposia on vitamin A. The general tissue functions of vitamin A that support growth and maintenance in the rat are served also by vitamin A acid; but since the rat does not reduce this substance, it forms neither the alcohol, the form in which vitamin A is stored, nor the aldehyde (retinene) needed for the synthesis of visual pigments. For this reason, rats maintained on vitamin A acid, though growing normally and otherwise in good condition, become extremely night blind and eventually blind. The failure to form visual pigments also has specific anatomical consequences; the outer segments of the visual cells deteriorate, followed by the loss of all but a remnant of the cells themselves, in an otherwise normal retina. The findings of Howell et al . (1963) do not support the statements of Dowling and Wald (I960). In bulls a pronounced vitamin A deficiency delays sexual maturity, suppresses sexual interests and causes testicular degeneration and thus a lowering of sperm production and quality. This has been shown by Bratton et al . (19^), Erb et al . (19^7), and Guilbert and Hart (1935). Bratton et al . (19^8) studied the effect of rations low in carotene on the breeding behavior, spermatogenesis and semen production of mature dairy bulls. Levels of 2 to 3 meg. of vitamin A per 100 ml. of blood were recorded before any of the deficiency symptoms appeared.

PAGE 14

Incoordination and loss of the ability to raount without loss of libido were the first signs to appear and they occurred before any marked impairment on semen quality was evident. Percentage of motile spermatozoa was decreased. The per cent of abnormal spermatozoa was increased. Histological examination of the testes showed degeneration of the germinal epithelium of the seminiferous tubules. Hogdson et al . (19^6) also found that vitamin A deficiency apparently does not cause severe disturbance in spermatogenesis until the animals are in advanced stages of deficiency. Erb et al . (1947), Hodgson et al . (19^6), Jones et al . (19^6) and Madsen et ^1 . (19^2) have reported that pituitaries are often cystic In the vitamin A deficient bovine. These findings suggested the possibility that vitamin A deficiency may exert its deleterious effect on reproduction indirectly through the anterior pituitary gland. Gunn et; al . (19^2) improved the semen quality of a ram deficient in vitamin A by injections of pregnant raare serum. Hogdson et al . (19^6) obtained similar results with a vitamin A deficient bull. However Lindley et al . (1949) reported that testosterone propionate injected semiweekly in dosages of 12.5 rag. before and during the deficient condition or of 37.5 mg. after vitamin A deficiency symptoms began to appear resulted in no improvement on semen quality of vitamin A deficient rams. Similar results were obtained from Injections of pregnant mare serum. This work suggests differences between rat and sheep since Mayer and Truant (19^9) were successful In preventing testicular atrophy in the rat resulting from a vitamin A deficiency by administration of testosterone propionate.

PAGE 15

Pathology of Vitamin A Deficiency Moore (i960) described at least three basic lesions occurring in av it aminos i s A: (1) Lacl< of vitamin A, required in the form of its aldehyde for the formation of rhodopsin, causes defective dark adaptation unless complications have ensued this lesion may be regarded as "biochemical" rather than "structural." Thus the response to treatment with vitamin A is rapid and dramatic. (2) Lack of vitamin A causes xerosis or kerat inizat Ion of membranes in many parts of the body, the well-known xerophthalmia belongs to this group of lesions. Epithelia tends to become dry and excessively thick and horny. Those membranes having a columnar structure, often associated with secretions of mucus give place to thick layers of stratified epithelia as found in the outerpart of the epidermis. The most common secondary effects of avitaminosis A arise from the bacterial infection of the abnormal membranes. In some sites such as the urinary bladder, the injury to the membrane may sometimes induce calcification with the formation of stones. (3) During growth, lack of vitamin A can cause defective modeling of the bones. As a result the bones are not compact, strong and well shaped, but cancellous, weak and excessively thick. Lesions of more than a single type are often superimposed, and the possibility of further types cannot always be regarded as secondary effects of the bone lesions. Another abnormality is an increase in the pressure of spinal fluid sometimes associated with hydrocephalus. These lesions might well be regarded as secondary effects of malformation of the skull bones, but a rival theory suggests that deficiency of the vitamin causes the chloroid plexus to secrete unduly large amounts of fluid.

PAGE 16

Vitamin A (qeneran Vitamin A Js an alcohol and forms esters with fatty acJds. Most of the vitamin A in the livers of both mammals and fish is present in the sterified form (White et al . 1959 and Harper 1963). Conversion of provitamin A (carotene) takes place in the intestinal wall in rats, pigs, goats, rabbits, chickens and sheep. According to Barnett and Reid (1961) carotene may be found in the liver of animals suffering from a vitamin A deficiency and does not appear to be capable of utilization for vitamin A synthesis. Dowiing (I960) summarized today's views on vitamin A metabolism as follows: Vitamin A alcohol, the transport form of the vitamin and the storage form as the ester, is oxidized to retinene because of the rapid removal of the aldehyde In its combination with opsin to form rhodopsin. The alcohol is oxidized to the acid in the liver and it is rapidly used in its function of growth and tissue maintenance. Acid is the important form for all functions other than visual, it must be stored as the alcohol and cannot be reconverted to either aldehyde or alcohol. Most of the information on the concentration of vitamin A in the various organs has been gained in studies on rats. Moore (1930 used the antimony trichloride reaction, with the aid of a Lovibond Tintometer, in studies of vitamin A in the tissues of rats which had been given liberal amounts of carotene. By this treatment total stores of up to 50,000 I.U. of vitamin A were accumulated in the liver. Apart from the intestine which contained much yellow pigment the rest of the carcass contributed 100 I.U, of vitamin A. When the various tissues were examined separately the Intraperitoneal fat contained 0.5, 5 and 5 I.U. in four experiments, and the kidneys and lungs each 0,0 and 5 I.U. in three experiments.

PAGE 17

8 No vitamin A could be detected in the brain, heart, pancreas, spleen, thymus or testes. The method of analysis, however, was too crude. Davies and Moore (193^) estimated vitamin A in the organs of rats which had been given either carotene or the preformed vitamin. Rats which had been dosed with carotene until their livers contained 30-90 I.U, vitamin A per gram had 1-3 I.U, of vitamin A per gram in kidney. Similar amounts were found in the lungs of two out of five animals but not in the remaining three animals. By giving vitamin A in large non-toxic doses concentrations of 25,000 I.U. per gram liver, 30 l,U, per gram kidney and 250 I.U. per gram of lungs were obtained. The intraperitoneal fat deposits had 50 I.U. per gram and the adrenal glands 1500 I.U. per gram. By raising the dose in the toxic region the liver contained up to 36,000 I.U. per gram, the kidneys 360 I.U,, lungs 600 I.U. and suprarenal es 30 I.U. Concentrations of up to 15 I.U. per gram were found In the muscles, heart, spleen and brain. The total storage of carotene and vitamin A in the liver and depot fat of cows which had access to a carotene rich rations throughout life was estimated to be 0.6 to 0.7 gram of vitamin A for the younger animals and up to 3.6 grams In aged cows (Maynard and Loosli 1962). Frocn 67 to 93 per cent of the storage was in the liver. In this organ most of the storage was in the form of the vitamin itself but in the fat depots carotene predominated. This work reported was done by Guilbert and Hart (193^ and 1935). Eaton (19510 has shown that In dafry calves approximately four months of age fed a depletion ration devoid of vitamin A activity, the plasma vitamin A level decreases in a linear manner until values of k meg. per 100 ml. are reached. Calves exhibiting

PAGE 18

such low pJasma values for two consecutive weeks were shown to be depleted of liver stores of vitamin A. Unpublished data by Dr. R. L. Shirley and co-workers showed that cattle receiving 30,000 l.U. per day and those receiving no supplemental vitamin A had blood plasma levels of 5^ and 3^ respectively. Another trial conducted during the summer showed levels of kS and 18 meg. of vitamin A per 100 ml. of plasma for groups receiving the supplemental vitamin A and no vitamin A respectively. Vitamin A in liver expressed as racg. per gram of liver dry weight was found to be 119 and 78 for the supplemented group during the first and second trial respectively and 17 and 7 for the second group during the first and second trial respectively. In the second trial vitamin A level in the heart was found to be 0.17 and .05 meg. per gram of heart for the supplemented and non -supplemented groups respectively. In sheep vitamin A storage has not been as extensively studied as It has been in rats. Harm (19^2) reported the average vitamin A content in a gram of liver as 503 l.U. in 19 animals. Moore and F^e (I9't2) reported levels of 460 l.U. per gram of liver in 20 animals. The above data were expressed In terms of grams of dry liver. Recent data in the literature Indicate the levels of vitamin A in the liver of sheep to be within a range from 25 to 5^ meg. per gram of fresh liver, ClJne (1962), Goodrich (1962) and Cllne et al . (1963). Data on the vitamin A content of tissues other than blood and liver are lacking for sheep. The Thvrold and its Role in Reproduction and Vitamin A Metabolism The role of thyroxine in reproduction has been most extensively studied amiong the hormones that may directly or Indirectly influence the

PAGE 19

10 secretion rate of hormones influencing reproductive activity such as the gonadotrophs and the hormooas of the ovary and testes. Berliner and Warbrltton (1937) reported that thyroidectomy of rams produced a decrease in semen volume, spermatozoan concentration and an Increase in the relative numbers of abnormal sperm. Thyroxine administered to the thyroidectomized animal resulted in the production of semen with an increased number of sperm and decreased percentage of abnormal spermatozoa. Bogart and Mayer (19^6) reported that treatment with thyroxine and thyroprotein alleviated symptoms of "summer sterility" resulting from impaired sperraatogenic activity. The feeding of thiouracil during the fall breeding season maintained semen characteristics typical of the sumner months. It was concluded in this study that the thyroid gland is of major importance in the reproductive physiology of the ram. Bhatnagar (1955) studied the seasonal variation in the histology of the thyroid and the testes of Indian buffalo bulls. They found considerable seasonal variations in the histological structures of the two glands, they also observed that during the period when the quality of semen deteriorates in buffalo the secretory activities of the two glands as shown by their histological studies appears to decrease. Goswami (1962) conducted an experiment to study the effect of thyroxine and pregnant mare serum (PMS) hormones on reaction time and semen quality of buffalo bulls. The experiment was conducted in India from June to February. One thousand I.U. of PMS were administered by weekly subcutaneous injections. A daily dosage of 100 mg. of thyroxine was administered orally. Treatment with thyroxine and PMS did not influence the reaction time of the animals.

PAGE 20

Semen volume was reduced significantly in the groups receiving thyroxine and PMS, Initial motility was improved in both groups during treatment. No effect from either hormone was noticed on sperm concentration or total number of spermatozoa during the treatment. The percentage of abnormal spermatozoa was decreased with thyroxine treatment. PMS treatment initially resulted in an increased percentage of abnormal cells but prolonged administration checked this increase. Black et a1 . (1950) studied the effect of testosterone propionate and thyroprotein In rams. Ten mg. of testosterone and 1 gm. thyroprotein were administered dally by subcutaneous injections and orally respectively. One gm, of thyroprotein per hundred lbs. of body weight was given orally to a second group. A third group received both testosterone and thyroprotein at the same level administered to groups one and two. Thyroprotein reduced weight, decreased libido and had no effect on sperm motility, volume, concentration or number of ejaculates. The testosterone treated group showed a significant decrease in semen concentration. They also reported that testosterone could prevent the loss of libido caused by treatment with thyroprotein. Recent studies by Brooks (196I), do not support the observations of Berliner and Warbritton (1937), and Bogart (1946). Brooks (1961) injected thyroxine at levels of 2, 3, or k mg . per hundred lbs. of body weight to rams. Considering the fact that 1 mg. of thyroxine injected equals approximately I gm. of thyroprotein fed (Cole and Cupps 1959) the dosages used in the experiments of Brooks (196!) are comparable to dosagas used in previous work in this field. The results obtained by Brooks (1961) indicated that injections of

PAGE 21

12 thyroxine at high temperatures accentuated the detrimental effect on semen quality or libido. Tapazole was also tested and its use produced a hypothyroid condition but this had no effect on semen. However one must be cautious in making any generalization at this point since the work so far reported was done under a wide range of environmental conditions which would undoubtedly have some effect on the thyroid needs of these animals. Such authors as Turner (1961), Cole and Cupp (1959) and Hafez (1962) agree that the thyroid gland is involved In the reproductive process but in a poorly understood fashion. Another poorly understood function of the thyroid gland is its relationship to vitamin A metabolism. Drill (19^3) reviewed this subject and reached several conclusions: (1) The lack of vitamin A increased thyroid activity and hypervi taminosi s had the opposite effect. (2) Deficiency in vitamin A wi 1 1 produce hypertrophy of the thyroid. (3) Experimental hyperthyroidism increases requirements for vitamin A. {k) In the absence of thyroid carotene could not be converted to vitamin A. Some of these observations have been confirmed by work done since Drill published his review in 19^3Johnson and Bauman (19^7) found that thyroxine increases expenditures of vitamin A deficient rats. However they point out that their results were complicated by accelerated growth observed in the thyroxine fed rats, since this would mean an increase in the animal needs for nutrients including vitamin A. Reddy and Thomas (1962) working with calves, rabbits and bulls reported that in hypothyroidism conversion of carotene to vitamin A was reduced but unaltered in hyperthyroidism. Their experimental numbers of animals were limited.

PAGE 22

13 Clfne Si— aJ.' (1963) found that feeding 150 mg, of tapazole per day or 100 meg. of tri lodo-I -thyronine did not affect liver storages of vitamin A. Any conclusion drawn from work where histological examination of the thyroid glands were not made to confirm thyroidal status should be made with caution. Moore (1957) in his book of vitamin A reviewed the interrelationships of thyroid and vitamin A metabolism. He concluded at the time that the usual effect of thyroxine will be to accelerate any prevailing trend in vitamin A metabolism. If conditions are conductive to expenditures the rate of expenditure Is increased. If dietary conditions are conductive to the storage of vitamin A the amounts stored will be increased by thyroxine, however the why and how of this remains unsolved today. Histology Warbritten and McKenzie (1937) described the pituitary glands of ewes in various phases of reproduction. They found the pituitary body of the ewe to be very complex. They were unable to classify the cells of the pituitary into the conventional acidophils, and chromophobes. They found 9 morphologically different types of cells which they didn't think necessarily represented distinct functional types. In this study the different regions of the pituitary were found to be conspicuously different, that counts made at one level would not be valid for another or for the whole gland. At the time of the studies by Warbritton and McKenzie (1937) present refined histological techniques were not available.

PAGE 23

Based on the chemical nature of the secretory products of the various pituitary cells Pearse (1950) and Wilson and Ezrin (195^) have proposed differential staining of the anterior hypophysis. The periodic acid Schiff technique (PAS) of Pearse (1950) was used by Jubb and McEntee (1955) in the study of the bovine pituitary. They were able to classify granule containing cells stainable by PAS into Beta and Delta types on the basis of positional morphological and functional differences. Beta cells were described as large polyangular finely granulated, and with an ill -defined cytoplasmic outline. They were concentrated in the medulla and sparsely scattered in the cortex of the anterior lobe. The functional relationship of the Beta cells identify them with thyrotropic hormone production. These cells were enlarged, degranulated and vacuolated in thyroid deficiency. The Delta ceils were much smaller, rounded and sharply outlined with coarser granules which were irregular in size and shape. These cells were present in all parts of the adenohypothysi s but concentrated in the medulla of the anterior lobe. These cells were identifiable with gonadotrophtc activity especially LH. They were unable to divide the acidophils. Purves and Griesbach (195^, 1955) described two categories of gonadotrophs or delta cells in the rat using the PAS method. One type they found restricted to peripheral regions and the other to central areas of the glands. In addition to finding those differences in distribution, they have stated that the peripheral gonadotrophs have a coarser granulation and give a more intense reaction with PAS. On the basis of changes occurring in these two types of gonadotrophs after testosterone treatment and after varying periods of gonadectomy these workers suggested that

PAGE 24

15 their "peripheral" gonadotrophs produced FSH and their "central" gonadotrophs produced LH. Wilson and Ezrln (195^) described a modified PAS method in which the Schiff reaction is followed by a methyl blue counterstain by which some but not all of the PAS reactive cells changed from red color to purple color. These workers concluded that the "PAS-red" cell represented the Beta cell or thyrotroph and the "PAS-purple" cell the Delta cell or gonadotroph. Rennels (1957) used a modification of the PAS-methyl blue method which in conjunction with aidehyde fuchsin, enables 'the tinctorial differentiation of two types of gonadotrophs in the pituitary gland of the rat. These two cell types were both gonadotrophic in function as indicated by their development into distinct PAS-red and PAS-purple castration cells following gonadectomy. Thyroidectomy cells were found to be PAS-negative, aldehyde fuchs in-negative and to have an affinity for orange G. Sanders and Rennels (1959) found the predominant chromophilic cell type in grafts producing luteotrophin (LTH) to be an elongated acidophil staining selectively with orange G when the azan stain is used. Pearse (i960), Huroason (1962) and Ham and Leeson (1961) were also used as references for proper histological techniques, in this dissertation. Effects of Irradiation on Reproduction Schubert and Lapp (1958) described the spermatogonia as the most radiosensitive cell in the human body and indicated that 50 roentgens inhibits their development, permanent sterility for the human male requires about 500 to 600 roentgens while 250 roentgens may produce sterility for one to two years. Seminal fluid production was not affected

PAGE 25

16 but no living sperm were present. Tiie germ cell damage proved dose dependent and proportional to dose down to the lowest levels investigated. Craig et , al (I96l) studied the effect of whole-body irradiation on the fertility of the male rat and mouse by systematic weekly matings of the treated animals. In both species irradiation with 300 roentgens produces a short period of sterility associated with oligospermia. This occurred about kS days after treatment in the mouse and 65 days in the rat. Histological examination showed that the main effect is on the spermatogonia! stages but there are obvious differences in the timing and extent of these irradiation effects. The time from treatment to the onset of sterility is a measure of the overall duration of spermatogenesis. The highest dose level used 500 roentgens did not cause a rapid onset of sterility in contrast to the action of some alkylating agents, which rapidly induce infertility. in the cock X-ray doses of up to 10,000 roentgens has been shown by Kosin (19^44) to have no detectable effect on semen motility but the fertilizing capacity of spermatozoa was markedly reduced after the exposure to 200 roentgens, and was destroyed altogether after a dose of 500, supposedly due to damaged chromatin. Mann (195^) found that rams exposed to 100,000 roentgens and examined immediately after exposure had normal motile spermatozoa in their semen, fructolysis and adenosine triphosphate content were also normal. This would indicate that if any damage was done to the ram semen it must be ascertained through other criteria. Murphree and Parish (1956) reported that when mature beef bulls were exposed to a single dose of 100-400 roentgens whole-body gamma irradiation, the first observed semen changes were an increase in per cent abnormal sperm at six weeks after irradiation and a decrease in sperm

PAGE 26

17 concentration at eight weei
PAGE 27

18 were: depression, skin hemorrhaging from eyes and mouth, increased respiration, mild muscular spasms just prior to death, extensive hemorrhage of lymph nodes plus varying amounts of hemorrhage in the kidneys, heart, urinary bladder, intestine and stomach. Shirley et al . (1962) in conjunction with Meacham on the above experiment found that vitamin A supplementation decreased the deposition of copper in the liver while copf>er supplementation increased the level of vitamin A in the liver. Gamma irradiation was shown to increase the deposition of copper in the liver, but had no significant effect on the deposition of vitamin A in the t i ssue.

PAGE 28

EXPERIMENTAL PROCEDURE Material s An i ma 1 s The animals used in this study were k8, 20-months old Florida native rams from Mr. L. A. Maxie's ranch in Frostproof, Florida. These rams were brought as yearlings to the University in June 1962. At this time they were placed in a preliminary experiment in which 12 received 5,000 I.U. of vitamin A per head per day. The remaining 36 received no supplemental vitamin A. The rams were fed rations consisting of 25 per cent cottonseed meal and 75 per cent white corn meal. All rams had access to coastal bermuda hay at all times. This preliminary experiment was continued until January, 1963. At this time two rams were brought in as replacements, from the University Sheep Unit for two that died. From January to February the rams were given a period of adjustment to a new purified diet to be used in the study here reported. On February 2, 1963, the experiment described henceforth was initiated. The average weight of the animals at the start was 87 lbs. with a range from 115 to 7^ lbs. The twelve rams that had previously been receiving vitamin A were put on the control diet consisting of a purified ration shown in Table 2 and 3, containing 3,000 I.U. of vitamin A per lb, of feed. The remaining 36 rams were placed on the vitamin A free diet. Assignment to treatments was accomplished in the following manner, Vitamin A deprived animals were not assigned to a treatment group until

PAGE 29

20 blood analysis showed 15 meg. or less of vitamin A per 100 ml. of plasma. A liver biopsy was also performed when such levels were reached. When 12 animals were accumulated which met the above criteria they were then assigned at random, two to each of 6 treatments. Treatments consisted of an irradiated group (A" Irradiated), a second group received 60 R.U. of PMS/IOO lbs. body weight administered daily by subcutaneous injections (A + PMS). A third group received daily intramuscular injections of testosterone propionate at a rate of 10 mg./lOO lbs. body weight (a" + Testosterone). A fourth group was given daily capsules of thyroprotein , 1 gram/lOO lbs. body weight (A" + Thyroprotein). A fifth group was placed on the vitamin A containing diet (returned) and the sixth group was continued on the deficient diet without further treatment (deficient). At the same time rams that had been receiving vitamin A were also randomly divided, two to each group, a control (control) and a group exposed to irradiation (control irradiated). In this manner eight treatment groups were formed. This procedure was repeated three times so as to allot the experimental animals in their totality. The experimental procedure is outlined in Table I. One of the animals assigned to the deficient group died in the biopsy operation and was excluded from the experimental data. The experiment was conducted at the Physiology Barn at the University of Florida. The area of the barn used in this experiment contained 8 pens, 12 feet wide and k8 feet long, and all pens had concrete floors. A metal roof covered the feed area and 12 feet of th^ pen. The pens were adjacently placed and one water tank served two pens.

PAGE 30

I••-• o + I. o. >~ — UJ < -I H OQ Z o

PAGE 31

22 Scales and a working area were available in which to handle the rams. A small portable chute was used to restrain the rams during the semen collection with the electroejaculator . Rations The composition of the experimental rations is recorded in Table 2. Solka-floc was used to supply fiber in the rations. Corn oil was included in the ration to supply fat, to improve palatability and to insure a uniform blending of the fine texture ingredients with the coarser materials in the rations. Casein was the protein source and corn sugar and starch were included for energy. The mineral, mineral premix and vitamin mixtures used in the rations are shown in Table 3. The mixtures containing 13 essential mineral elements was essentially the same as reported by Meacham (1962) and Tilton (1962). Vitamins A, D, E and choline, in a corn sugar carrier were added to each ration except that the vitamin A was deleted from that of the vitamin A deprived sheep. Methods Management and Feeding The rams were fed daily at 8:00 a.m. all the i'eed they would clean up by the next feeding. Feed refusal was weighed back daily. Fresh water was available at all times. The rams were mantained in the pens at all times except when removed to collect experimental data and for daily hormone treatment. There was one pen for each treatment group and six rams

PAGE 32

23 TABLE 2 COMPOSITION OF EXPERIMENTAL RATION Ingredient Per cent in Ration Cellulose (Solka-floc) 20 Casein (90 per cent protein) 20 Corn Starch 23 Corn Sugar 25 Corn Oil k Trace Mineral Premix 0,5 Vitamin Pre-mix 1 ,0^ Minerals 6.5 100.00 Control group received 3,000 I.U. of vitamin A per b. of feed.

PAGE 33

2k TABLE 3 COMPOSITION OF THE MINERALS. MINERAL PREMIX, AND VITAMIN MIXTURES USED IN EXPERIMENTAL RATIONS A. Minerals

PAGE 34

25 were assigned to each pen. The rams were weighed every two weeks during the experimental period. The hormones were administered daily and all other animals received a daily injection of physiological saline solution to have equal handl ing. I rradiation Irradiation of the rams was performed at the University of Florida Co facility. Rams were irradiated at the time all the other treatments were started. The animals were restrained In wooden crates and subjected to whole body gamma irradiation at the rate of 3.27 roentgens per minute until a total dose of 400 roentgens had been administered. Two hundred roentgens were administered to each half of the body. Semen Evaluation Semen was collected using an electroejaculator . A monthly collection was made on all animals prior to allotment to the eight treatments. A week prior to assignment to a treatment semen was collected eight times, four times in each of two consecutive days. During the first week following initiation of the various treatments, sanen was collected four times in one day. For the remainder of the experiment semen was collected once a week. Volume of semen was determined directly by collecting the semen in graduated centrifuge tubes. Motility was evaluated to the nearest 5 per cent by microscopic examination (X430).of the cells with progressive forward movement. A blood hemocytometer was used to determine the number of sperm per cubic millimeter. From assignment of the various groups to

PAGE 35

26 their respective treatments until slaughter a period of t-venty-one weeks elapsed, during this time semen collected was evaluated for additional data on percentage of abnormal cells present at each collection. Blood Evaluation Blood samples were taken initially and at intervals of 28 days until the biopsy was performed. Blood was evaluated for vitamin A according to the method of Kimble (1939) based on the Carr-Price reaction. Liver Biopsy In order to have a more reliable knowledge of the true reserves of vitamin A in the liver of the animals to be assigned to the various treatments a liver biopsy was performed prior to reallotment. This biopsy was done by Dr. Fred Neal from the Veterinary Science Department of the University of Florida. Essentially the technique consisted of the following. A midlateral incision was made in the area immediately behind the last rib on the right side of the body. A piece of liver was removed by hand of sufficient size as to allow vitamin A determinations on it. The incision was then closed and 10 cc. of penicillin were injected to prevent further infection. Slaughter Data On the twenty-first week from assignment to treatment groups, those rams that had survived were slaughtered. Of the 15 rams allotted in the first group during the last week of April, 10 survived and were slaughtered in the second week of September.

PAGE 36

27 Of the second group 4 rams survived. This group had been allotted in the third vi;eei<. of June and was terminated in the second vveek of November. The third group had been allotted in the second week of July and was slaughtered in the first week of December. Of this last group 6 survived until termination of th« experiment. Weights of the pituitary, thyroid, adrenal, seminal vesicle, cowpers glands, testes and epididymis were taken. At slaughter vitamin A values for blood were determined. Those animals that died before termination of the experiment were examined for gross pathological symptoms as to cause of death. The testes, thyroid and pituitary gland were saved for histological studies from those rams that were seen soon after death. Histological Investigation Tissue samples from the testes, thyroid and pituitary were taken and fixed in 10 per cent formalin solution for histological studies. Thyroid tissue was taken only from animals on the control, thyroprotein and recovery treatment. The procedures of Humason (1962) were followed for f ixi ng, i nbeddi ng , slicing and staining with eos i n-hematoxyl in stain. The procedure outlined by Wilson and Ezrin (195^) was followed to study the different cell types present in the adenohypophi s i s . Microscopic evaluations were made (with a calibrated eye piece) of the size and the development of the secretory tissues in the testes and thyroid. The different cell types in the adenohypophi s i s were studied for evidence of cytoplasmic degranul at ion and vacuolation.

PAGE 37

28 Statii^tical Analysis Tfie daily gain data were summarized into a Pliase I and Phase II and Phases I and II were combined to facilitate statistical analysis. Phase I extended from the date the animals started on the purified diet to the time they were assigned to the various treatments. Phase II extended from the end of Phase I to termination of the experiment after 20 weeks . Data on daily gains, survival time and gland size were analyzed by the analysis of variance method. Semen data was analyzed for weekly differences in semen volume, per cent motile sperm cells, abnormal sperm cells and total sperm cells per ejaculate. Only the first ten weeks of Phase II were analyzed since the reduced numbers present in each group beyond the 10th week did not merit a statistical analysis.? Semen data analysis was programmed by Dr. A. E. Brandt from the University of Florida Statistics Department, and was processed by the University's Computing Center personnel . When a significant effect due to treatment was found Duncans Multiple Range Test was used to seek individual treatments effect on daily gain, survival time and gland weights. Differences in semen characteristics between treatments were determined by the matrix vector product method.

PAGE 38

RESULTS AND DISCUSSION Genera) SvmptOfns of Vitatnin A Deficiency Approximately two and a half months after assignment to the purified diet night blindness was observed in some of the experimental animals on the vitamin A deficient diet. The rams were checked for night blindness by placing a panel across an alley after dark and checking the rams individually for their ability to avoid it when driven through the alley. All animals on the deficient diet eventually showed symptoms of night blindness. There was considerable variation in time of onset and severity of symptoms. Some rams showed a characteristic cocking of the head to one side similar to that described by Lindley et al • (19^9). A number of rams showed a characteristic cloudy cornea. In some, this cloudiness disappeared but in most it persisted. Figure I illustrates this cornea condition in one of the vitamin A deficient rams. Most rams on the deficient diet eventually exhibited an uncoordinated gait and progressive weakness which became so severe in some cases that they were unable to stand as shown in Figure 2. Several rams walked on their knees, apparently as a result of loss of control over the lower part of the leg. This condition was observed in only 3 of the 35 vitamin A deficient rams. Other symptoms exhibited by the deficient rams included loss of apetite and evidence of pain. It is interesting to note that some animals did not shovi any degree of incoordination even when the liver stores of vitamin A had been nearly depleted. 29

PAGE 39

30 Fig. 1 --Cloudy condition of the cornea present in a vitamin A deficient ram. Fig. 2--A vitamin A deficient ram showing extreme

PAGE 40

31 The deficient rams that were returned to a vitamin A containing diet recovered from ail lesions. However even though the vitamin A liver stores of these animals had been drastically reduced no severe symptoms were evident in most of the rams in this group at the time that these began receiving vitamin A. One of the rams receiving PMS treatment exhibited a protruding eye with evident infection. At autopsy the veterinarian diagnosed purulent pleuritis as the immediate cause of death. Another ram on the same treatment developed an abcess in the sheath area. This same ram showed bloody urine before dying. In general the rams on thyroprotein treatment showed less severe symptoms than did other deficient animals. The apetite and growth of these rams was higher than in other deficient rams. Rams in the A" + Testosterone group exhibited symptoms similar in nature to those of other rams on the vitamin A deficient diet. At autopsy no striking gross pathological lesions were evident in rams subjected to the vitamin A deprivation. This author believes that some of the deficiency symptoms observed were in part caused by multiple nutritional deficiencies resulting from the decrease in feed consumption observed in the vitamin A deficient rams. The A + Thyroprotein group had more appetite as well as leSs severity in their symptoms. It should be stated however that the deficient ration would have supplied the required amounts of nutritients had they been consumed at the same level as the control ration. The basic cause of this multiple deficiency is therefore an absence of vitamin A.

PAGE 41

32 General Effects of Ga pfn^ Irradiation Rams exposed to irradiation suffered a gradual loss in appetite until deatli. Those rams surviving the irradiation treatment eventually recovered from the loss of appetite. No signs of external hemorrhaging were evident in any of the rams exposed to irradiation. Several rams exhibited limited anal bleeding prior to death. Most of the rams would lie down, shiver and show signs of pain prior to death. At autopsy multiple hemorrhages were present in the lungs and chest cavity areas. Limited hemorrhagic spots were observed in the cortical region of the kidneys and adrenals and in the urinary bladder of some rams. No characteristic histological lesion was observed in the testes of animals exposed to irradiation. The multiple external and internal hemorrhaging described by Meacham et al . (1962) in pigs exposed to 350 roentgens of whole body gamma irradiation from a Co^° source were not observed in these rams suggesting different mode of action of Irradiation in these two speclds, Plasma and L iver Vitamin A Content Table k shows plasma vitamin A values for rams in the various allotment groups (Group I, 11 and III) and treatments, a summary of all treatment groups is presented in Table 5. It should be noted that the various treatments appearing in Table k were not started until after the biopsy. However the rams appear grouped into the various treatments so that comparisons can be made on the vitamin A reserves of rams assigned to a given treatment. Liver storage of vitamin A determined on biopsy samples is also shown in Table 5-

PAGE 42

33 till 1' I I I o vO r^ tN vO f"^ o <.o r-~. vD MD O O CN cr> fx^ o I I I I La -3" -dosj I I I I I 00 f^ e>l O C^ cr\ raO CNO-d" l (N — 3

PAGE 43

3^ 1-

PAGE 44

35 TABLE 5 AVERAGE VITAMIN A PLASMA AND LIVER VALUES FOR THE VARIOUS TREATMENTS AT TIME OF REALLOTHENT Treatment

PAGE 45

36 The vitaniin A reserves in all rams on the diet containing vitamin A were as expected much higher than in rams on the vitamin A deficient diet. All rams on this latter diet had received similar diets since June 1962; however the drop in plasma vitamin A took place at differing times. The different initial vitamin A reserves in these rams no doubt accounts for most of this variability. Table k shows that the variability in plasma vitamin A occurring from month to month in a given pair of rams was considerably reduced when the plasma vitamin A levels had been drastically diminished, at this latter stage a plateau is reached. It was at this time that the biopsy was performed to ascertain the deficiency condition. In general vitamin A liver reserves were at abnormally lov^i levels at time of assignment of the rams on the deficient diet to the various treatments in phase II. The rams assigned to the returned treatment in group 111 showed higher vitamin A reserves than other deficient rams but considerably lower than those present in the controls or normal irradiated rams. In general one can state that a vitamin A deficiency condition was present at the time of assignment of the rams on the deficient diet to the various treatments in phase II. It should be kept in mind hov-jever that the point at which the liver vitamin A values shown in Table 5 were reached in these rams is unknown, since it is generally agreed that a poor correlation exists between plasma and liver vitamin A values. The only possible way to ascertain when the low liver vitamin A storages found at time of reallotment were reached, would have been to perform a series of biopsies on the same individual rams throughout the experiment. This is not feasible since the stress would

PAGE 46

37 most likely have resulted in heavy death losses. It is also possible to slaughter random samples at different periods and based on these samples infer vitamin A status of the population *i n question. However, in this experiment the latter method could not be followed since only a limited number of rams were available. Table 18 of the Appendix shows individual ram plasma and liver vitamin A reserves at time of reallotment. Also in Table 18 are individual final plasma vitamin A values determined on rams surviving to termination of the experiment. The plasma vitamin A values found present in rams on the vitamin A deficient diet were very low and much lower than those in the control group. Rams in the returned group had final plasma vitamin A values comparable to the controls. Feed lpt.ake Feed intake data for individual ramsiMo^enot possible to record because facilities used in this experiment did not provide for individual allotment. During phase M feed intake data were further confounded because the assignment of rams to one of the 8 treatments was done in three stages so that each pen held increasingly larger numbers of rams until the ky were assigned a treatment. Thus the same number of rams was not present at alt times in a pen chosen for one of the 8 treatments. For these reasons statistical analysis of feed intake data were not possible. However some trends were in evidence so that some conclusions can be drawn from the available data. Table 6 shows feed intake data for the various treatment groups during phase I and It. In phase I rams on the control and vitamin A

PAGE 47

38 Z s i c -a — 01 w. E >0) coo. en X •Z § " (/I — 14. ru *J O (D O C *i J.J — I. w w It) § 2 X. 0)

PAGE 48

39 deficient diet consumed an average of 3-0 and 1.8 lbs. per head daily, respectively. During phase I no widespread occurrence of vitamin A deficiency syraptoms was observed in the rams on the vitamin A deficient diet indicating that the detrimental effect of vitamin A deprivation on feed intake was manifested prior to other external signs of a vitamin A deficient condition. During phase II average daily feed intake per head was 2.1, .85. 1.90, -92, .71, 1.59, .67 and 1.5^ lbs. for the control, deficient, returned; A" + PMS, a" + Testosterone, A' + Thyroprotein, A" + Irradiated and Control Irradiated groups respectively. Rams receiving the vitamin A containing diet during phase II (control, returned and control irradiated groups) consumed more feed than rams on the other treatments, except for the A" + Thyroprotein group. It is interesting to note that the control group consumed less feed during phase II. This resulted from the increased stress to which all rams were subjected during this phase. Thus the decrease in feed consumption evident in rams on the vitamin A deficient diet can be attributed partially to the increased stress. The A" + Thyroprotein group consumed more feed than other rams on the vitamin A deficient diet. PMS or testosterone treatment did not have any beneficial effect on the appetite of vitamin A deficient rams. Irradiation appeared to have had a depressing effect on feed intake on all rams irradiated. Feed consumption in the control group was comparable to that reported by Meacham (1962) but somewhat less than that of Til ton (1962). The latter used corn cobs as a source of fiber in a diet very similar to that used In this experiment. Meacham (1962) used Solka-Floc as the source of fiber which is similar to that used in this experiment. At slaughter,

PAGE 49

ko extensive amount of wool was found in the rumen of rams in all treatments. This was to be expected from the frequent "wool eating" observed in the experimental rams. This suggests that the amount or the source of fiber provided or both were not adequate. The possibility also exists that some other nutritional factor was not adequately provided. Body Weight Changes Table 6 shows average daily gains during phases I, II and I and 11 combined. In phase I rams had not been assigned to the various treatments; however for the purposes of this discussion rams that were assigned to a treatment during phase II appear in this treatment in phase i, so that it is possible to follow growth rate for rams in a group prior to and during treatment. During phase I average daily gains were ,24, .06, .10, .05, .04, .06 and .23 lbs. for the control, deficient, returned, A" + PMS, A" + Testosterone, A" + Thyroprotein, A' + Irradiated and Control Irradiated groups respectively. Table 7 shows the ranked average daily gains for the various treatment groups. Rams on the vitamin A supplemented diet gained significantly more than those on the vitamin A deficient diet. As was to be expected the various groups on the vitamin A deficient diet did not differ significantly between them. Individual daily gains and weights during phase I are shown In Tables 19 and 20. Average daily gains for the various treatment groups during phase II as shown in Table 6 were 0, -.10, II, -.Ok, -.27, -.04, -.23, -.23 lbs. for the control, deficient, returned. A" + PMS, A~ + Testosterone, A + Thyroprotein, A" Irradiated and Control Irradiated groups, respect ively.

PAGE 50

^1 o Q. < O + en < a.

PAGE 51

42 Table 7 shows the ranked average daily gains for the same groups. The returned group gained significantly more than all other treatment groups except for the controls. The controls did not gain significantly more than the A" + PMS , A' + Thyroprotein and deficient groups. The low average daily gains in the control rams is largely due to the drastic weight reduction of ram No. YU3 who lost kS lbs. during phase II. Rams exposed to irradiation had very low average daily gains. The depressing effect of irradiation on average daily gains was equivalent on both control and A" Irradiated groups. Individual weights and average daily gains are shown in Tables 19 and 20 of the Appendix. The combined phase I and II average daily gains are shown in Table 6. Table 7 shows the ranked average daily gain for the various treatment groups during phase I and II combined. All rams on the vitamin A supplemented diet gained significantly more than rams on the deficient diet. Table 19 in the Appendix shows individual average daily gains during phases I and II combined. Figures 3. ^ and 5 illustrate the body weight change in rams on the various treatments. Before discussing these figures it should be noted that the number of rams jjer treatment was small from the start (6 per group) and the heavy death loss inflicted by the vitamin A deficiency drastically reduced the rams per group so that in many instances the values plotted for a given treatment represent the body weight change of a very limited number of rams. It is true however, that rams that died before termination of the experiment were those who had undergone the most drastic weight loss so that the slope of the plotted line would have been steeper had they survived.

PAGE 52

^i -20Fig. 3--Effect of vitamin A supplementation on body weight changes of rams. ^Phase I extended from the start of experiment to assignment to the various treatments. From periods 1 to 6 there were 6 rams per group. From periods 7 to 8 there were k and from periods 9 to 1 1 , 2 rams were present per group. Phase 11 extended from assignment to the various treatments to termination of the experiment. ^Numbers adjacent to plotted values represent animals remaining in each group for a given two week period. Differences in numbers between periods in a given group resulted fron death loss.

PAGE 53

kU PHASE PHASE II CONTROL A"*PMS A-+ THYROPROTEIN A' + TESTOSTERONE 3\ ^ WEEKweight c^I;9ero1':?t1n,irA''d%'J?c?,Jr:f:: '""™"'= °" '^' ;«:= irn£-S^iB-^^S^^SSi^jB.. ..... tion of the IxpeJim^nr"'" ''°"' ="'9™"' '<> ""= "rrous treatments to terminagiven group resulted from death loss, s between periods In a

PAGE 54

kS PHASE I PHASE II WEEK PERIODS Fig. i. — trrect of irradiation on borfy wi?fghr changes or vitamin • A deficient and normal rams. ^Phase I extended from the start of experiment to assignment to the various treatments. From periods 1 to 6 there were 6 rams per group. From periods 7 to 8 tiiere were h and from periods 9 to 1 1 , 2 rams were present per group, "Phase II extended from assignment to the various treatments to termination of the experiment. *^Numbers adjacent to plotted values represent animals remaining in each group for a given two week period. Differences in numbers between periods in a given group resulted from death loss.

PAGE 55

U6 Figure 3 illustrates the body weight changes for the control, deficient, and returned groups. During phase I the body weight change experienced by all rams was similar up to the l^th week (period 8). At this point the control rams continued to gain while rams on the deficient diet began to loose weight. During phase II the rapid recovery experienced by the returned rams is clearly shown. The deficient rams continued to loose weight while the controls increased in weight up to period 6 and then reached a plateau. Figure k shows the body weight change for the control and hormone treated groups. During phase I the significant increase in body weight in the control rams compared to rams on the deficient diet is clearly shown. During phase II rams in the A' + PMS and A" + Testosterone groups suffered a drastic weight reduction. It is interesting to note the beneficial effect thyroprotein had on preventing body weight loss up to period 7. This was partially due to a stimulation in appetite produced by the thyroprotein. Figure 5 illustrates the effects of irradiation on body weight changes. It is clear that all rams exposed to irradiation suffered severe weight losses. This correlates well with the decrease in feed consumption observed In the same rams. Those rams in the control Irradiated group who survived irradiation recovered. The depressing effect on growth normally associated with vitamin A deficiency was observed in this experiment. Raras whose vitamin A reserves had been drastically reduced and were experiencing severe weight losses were able to recover when 3,000 I.U. of vitamin A per lb. of feed were provided. PMS and testosterone treatment did not prevent further weight losses in vitamin A deficient rams. Thyroprotein appeared to have a stimulating effect on appetite resulting partially In less severe weight

PAGE 56

^7 losses, as compared to other rams on the same vitamin A deficient diet without thyroprotein. This finding would appear to contradict the widely accepted theory that an increase in appetite which would normally stimulate growth, if the diet in question is deficient in a nutrient would accelerate needs for the nutrient in question. This would in turn increase severity of deficiency symptoms. Irradiation affected feed intake almost immediately. This was accompanied by a severe weight loss until death. The appetite of rams surviving irradiation returned to normal and did not undergo further weight reduction. Analysis of variance for differences in daily gains between the various treatments in phases I, II and I and II combined are shown In the Appendix Tables 26, 27. and 28 resjiect i vel y , Survival Average survival time expressed in days as shown in Table 8 was 141, 82, 1^1, 85. 97, 125. 46 and 63 for rams in the control, deficient, returned, A" + PMS , A" + Testosterone, A" + Thyroprotein, A' irradiated and control irradiated groups respectively. Survival time for rams in the various allotment groups (l II and III) was also shown in Table 8. Per cent death loss for the same groups as shown in Table 9 was 0, 80, 0, 84, 66, 50, 84, and 66. Table 10 shows the ranked average survival time in days for rams in the various treatment groups. Rams in the control and returned groups suffered no death loss. They survived a significantly larger number of days than rams on the other treatments except for those on thyroprotein. This latter group survived significantly more days than all remaining groups except for rams in the

PAGE 57

k8 — «N i J 1/1 Bo CM — in — O Q. M < U C C O

PAGE 58

^9 TABLE 9 PER CENT DEATH LOSS FOR RAMS IN THE VARIOUS TREATMENTS Treatment

PAGE 59

50 A + Testosterone group. There was no difference in tolerance to gamma irradiation between the control and deficient groups exposed to irradiation as measured by survival time. Differences in survival time between allotment groups (i, II and III) were not significant. Most of the rams whose vitamin A reserves had been depleted eventually died. Treatment with either testosterone or PMS was not effective In preventing death losses resulting from a vitamin A deficiency. Daily capsules of thyroprotefn were effective in reducing death loss of vitamin A deficient rams. Rams whose vitamin A reserves had been drastically reduced but were replenished by feeding 3.000 I.U. of vitamin A per lb. of feed daily suffered no death loss. The vitamin A status of rams exposed to irradiation did not affect significantly the survival time of these umal s Individual survival time for all rams is shown in Appendix Table 21. Analysis of variance for differences in survival time between the various treatment groups is shown in the Appendix Table 28. Semen Character i -,t ;^^ Semen data «re stat i st leal 1 y analyzed during the first ten weeks of phase II. Beyond this point total number of rams per group was too limited for statistical consideration of the data. A series of figures are included which illustrate semen characteristics of rams in the various treatments during all of phase II. It should be noted that in some instances the number of rams per treatment, as indicated by the numbers in parentheses adjacent to the plotted values, is small resulting from heavy death losses in those groups. It is also

PAGE 60

51 true however that death losses in treatment groups on the vitamin A deficient diet occurred when these rams v^ere in advanced stages of avitaminosis A. At this time changes in semen characteristics affected by vitamin A deficiency had taken place. So that in many instances plotted 1 ines. even though based on data from a reduced number of rams, do reflect the trend of the initial population. For example Figure 10 illustrates per cent sperm motility for several treatments. In looking at the A" + Testosterone group one sees that from weeks 11 to 12, 2 rams died. After week !2 a drop in per cent motility was observed. Appendix Table 2^ shows individual semen characteristics for all weeks. One sees that the 2 rams that died were not showing high sperm motility, so that the drop in per cent sperm motility at the twelveth week was not due to death of rams with high sperm motility values but rather to a drop in values in surviving rams. Had the rams survived the change from weeks 12 to 13 the slope of the line would have been more pronounced. This should be kept in mind in drawing conclusions from the experimental data available. A summary of semen data collected monthly during phase I is shown in Table 11, Per cent abnormal sperm cells was not determined during phase I nor was semencbta from phase I analyzed statistically. Volume Average semen volume for rams in all treatment groups during phases I and II as shown in Table 11 were 2.2, 1.4; .8, .9; .6, 1.1; 1.0, 1.0; 8, •5; l.I, -7; 1-9, -9; and 1.0, .8 ml. for the control, deficient returned A + Thyroprotein, A" + PMS , A" + Testosterone, control irradiated and

PAGE 61

52 < — -I < h-i < O _J > rD 2: < LU -5 2: UJ Ic 0) jO Q< — — rr, _ r^ CM vD cr\ — o LA r^ m 00
PAGE 62

53 a' irradiated groups respectively. Rams on the vitamin A containing diet (control and control irradiated groups) showed twice as much semen volume as those on the vitamin A deficient diet during phase 1. From phase I to phase II there was a drop in average semen volume in the control rams. This was probably due to the increased frequency of collections during phase 11. The deficient group did not show a drop in average semen volume during phase II. The returned group increased semen volume during phase II. This change could be attributed to commencement of vitamin A supplementation. Figure 6 illustrates semen volume changes for these 3 groups during phase II. Notice how the control rams always maintained semen volume values above the other 2 groups. The gradual recovery of the returned group is clearly shown. Of the hormone supplemented groups the PMS and testosterone treated rams suffered a further reduction in average semen volume during phase II. Thyroprotein treatment did not have this harmful effect on semen volume. Figure 7 illustrates semen volume changes for the hormone treated groups compared to the controls. Semen volume in the testosterone group was quite variable from weeks 12 to 19 but it remained lower than in the controls. The PMS treated group had low semen volume throughout phase II. The semen volume values for the thyroprotein groups did not drop below their Initial value in any of the 20 weeks. Both groups of irradiated rams showed reduced average semen volume during phase II; this change was more drastic in the control irradiated group. Figure 8 shows this change during phase 11. It should be kept In mind that semen volume differences between all treatments were not of statistical significance when analyzed. Tables 30 through 39 show analysis of variance for differences in semen volume

PAGE 63

54 — 3 (i: o E 1OJ CD 3ivinovr3 a3ci saamniiw al I/) a) 3 .M w J2 *•' E O 3 — C lis

PAGE 64

55 o -o J < u. — o > l 9i OJ o E »I(U Q. 2 4» 3 — W •O u 3ivnnovn3 U3d sasinnim ^%

PAGE 65

56 3 < ro Q. E 3 -

PAGE 66

57 during the first 10 weeks of phase II. However some trends were evident enough to permit certain conclusions. Vitamin A deficiency appeared to have had a depressing effect on average semen volume. This effect could not be overcome with thyroprotein treatment. Treatment with PMS or testosterone resulted in a more pronounced drop in semen volume of the vitamin A deficient rams. Irradiation appeared to have had a harmful effect on average semen volume from normal and vitamin A deficient rams. The reduction in semen volume resulting from a vitamin A deficiency found in this experiment is in agreement with the findings of Lindley et al . (19^9). They also found no beneficial or harmful influence on semen volume of vitamin A deficient rams from treatment with PMS or testosterone, Goswami (1962) reported a reduction in semen volume of bulls treated with thyroprotein during the sunmer months in India. This effect on semen volume was not evident in thyroprotein treated vitamin A deficient rams in the study reported. Black et al . (1950) reported that thyroprotein had no effect on semen volume in rams which is in agreement with our findings. However it is very difficult to compare our data with those reported elsewhere because of dissimilarities In climate, diets, hormones dosage and age of rams present . The harmful effects of irradiation on semen volume apparent in this study have not been reported elsewhere. Individual semen volume values for all rams during phase II are shown in the Appendix Table 2k. >^oXilit;y Average per cent motile sperm cells during phases I and 11 as shown in Table 11 were 71, 72; 29, 22; 66, 68; 8k, k8; 57. 19; 88, 18;

PAGE 67

58 78, kk; and 83, ^+6 for the control, deficient, returned, A" + Thyroprotein , A" + PMS, A" + Testosterone, control irradiated and A" irradiated groups respectively. During phase \ the control group showed motility values higher than rams in the deficient group. Motility values in other groups on the vitamin A deficient diet were comparable to the controls. Average motility values for the control rams were similar in phases I and II. The deficient group suffered further loss in motility values during phase II. Average nratility for the returned group had not been affected during phase I so it was not possible to show whether a drop in motility from a vitamin A deficiency can be overcome by vitamin A supplementation. Figure 9 illustrates changes in f>er cent motile spej^ra during phase II, in the control, deficient and returned groups. Except for week 19 motility values for the deficient group were lower than those of the control and returned groups. Differences in per cent motile sperm between the control and deficient group as shown in Table 12 were significant in weeks 8, 9 and 10 only. Analysis of variance for differences in per cent motile sperm during all of the first 10 weeks of phase II are shown in the Appendix Tables 30 through 39. Average per cent motile sperm values as shown in Table II were lowered during phase II in the hormone treated rams. Thyroprotein treatment overcame somewhat the depressing effects on motility from the vitamin A deficiency. Figure 10 illustrates average per cent motile sperm for the hormone treated and control groups during phase II. From weeks I to 4 a drop in average per cent motile sperm cells was observed in all hormone treated groups. From weeks ^ to 1 1 average motility was variable in the A" + Testosterone group but eventually dropped to from weeks 12 to 21. From weeks k to 16 average per cent motile sperms remained

PAGE 68

59 1Q _i z u o uj z Z Ll 1O UJ UJ o Q Q^ CJ^ — ^ (o ;^ lu re o> o o >^ 3 — 2-g C in ._ « « o e § • (/> 4-. 4) C O

PAGE 69

60 cc

PAGE 70

a. *j 2 '° o V I-a CD E -c O 4)

PAGE 71

I 62 more or less at the same level in the A"+thyroprotein group. The A" + PMS group showed a gradual loss of sperm motility from weeks I to 1^, at this point a value was reached which remained to termination of the experiment, As shown in Table 12 differences in per cent motile sperm betv^en the returned (which had motility values comparable to the control group) and the hormone treated groups were statistically significant at weeks 9 and 10. As shown In Table 11 average per cent motile sperm was lower in phase II for both groups irradiated. In the A" irradiated group sperm motility values were not as low as in other groups on the vitamin A deficient diet. Figure II illustrates changes in motility values in the control and irradiated groups. The tremendous variability in per cent sperm motility in the A" irradiated group can be attributed mostly to the fact that only one ram survived from weeks 7 to 21. In general per cent sperm motility was lower in the irradiated groups than in the control throughout most of phase II. As shown in Table 12 differences in per cent sperm motility between the irradiated groups and their counterparts (control and deficient groups) were not statistically significant in any of the first 10 weeks of phase II. Analysis of variance for differences in per cent sperm motility during phase II are shown in the Appendix, Tables 30 through 39. Results obtained in this experiment indicate that sperm mot i I i ty is severely affected by a vitamin A deficiency. This lowered sperm motility could not be improved by treatment with PMS or testosterone. Thyroprotein alleviated the depressing effect of vitamin A deficiency on sperm motility. This supports the findings of Lindley et al . (1949). They were unsuccessful in preventing or improving low sperm motility in vitamin A deficient rams by treatfoent with testosterone or PMS. Gunn et al . (1942)

PAGE 72

63 t=

PAGE 73

6^4 improved sperm motility of a vitamin A deficient ram by injecting PMS. Hogdson et al . (19^6) obtained similar results in a vitamin A deficient bull. The discrepancy in results between the results of the latter two authors and those of Lindley et al . (19^9) and ours can not be erapharsized since only a very limited number of experimental animals were used by Gunn et al . (19^2) and Hogdson et al . (19^6), The detrimental effects of irradiation on sperm motility was equivalent on both normal and vitamin A deficient rams. However sperm rreDtility was not sufficiently reduced to suppose a serious impairment to the reproductive capacity of these rams. Individual per cent motility for all rams throughout phase II of the experiment is shown in the Appendix, Table 23. Per Cent Abnormal Sperm As shown in Table II per cent abnormal sperm values were 19, 50, 18, 36, 37, 33, ^6, and 37 for the control, deficient, returned, A" + Thyroprotein, A" + PMS, A* + Testosterone, control irradiated and A" irradiated groups respectively. It is apparent from tfiese data that groups on the vitamin A deficient diet showed a higher percentage of abnormal sperms in their semen. Figure 12 illustrates changes in per cent abnormal sperm values for the control, deficient and returned groups during phase II. The control group maintained per cent abnormal sperm at a low level throughout the 21 weeks of phase II. In the fourth week rams in the deficient group began to show an increasingly larger percentage of abnormal sperm while the opposite occurred in the returned group. Differences in percentage of abnormal sperm between these 3 groups were not significant statistically but they appeared to be of biological importance.

PAGE 74

65 O 0) I-o en E x: o

PAGE 75

66 As shown in Table II it appeared that treatment with testosterone, PMS or thyroprotetn was not effective In overcoming the harmful effect of vitamin A deficiency on sperm abnormality in rams, rather it appeared that hormone treatment had a detrimental effect. Figure 13 illustrates changes in percentage of abnormal sperm during phase II in the hormone treated and control groups. The A' + PMS group had a higher percentage of abnormal sperm than the control throughout phase II. The thyroprotein treated group showed a temporary improvement in sperm abnormality percentages from weeks 3 to 10, from then on an increase in sperm abnormality was evidentTestosterone treatment produced a gradual decline in sperm abnormality from weeks k to 7From weeks 7 to 1 2 values were variable. After week 12 no further determination of per cent abnormal sperm was possible because of the reduced sperm concentration present in the semen of these rams. These phenomena occurred in the A~ + PMS group from weeks l8 to 21. Differences in per cent abnormal sperm between the returned group (with comparable values to the control group) and the hormone treated groups were not of statistical significance. Irradiation appeared to have increased sperm abnormality. As shown in Table 13 this effect was of statistical significance during weeks 6, 7 and 8 of phase II. However only 2 rams were present in each of the Irradiated groups at this time. Table 13 also shows that the effects of irradiation on sperm abnormality were more drastic during weeks 6 and 8 on the control irradiated rams. Figure 1^ illustrates changes in per cent abnormal sperm values for the control and both irradiated groups throughout phase II. Analyses of variance for differences between treatments in per cent abnormal sperm during the first 10 weeks of phase ii are shown in the Appendix Tables 30 through 37.

PAGE 76

67 UJ Z 2 Q. cn O O -I c/, tr Io ^ > t/5 or „ X UJ . QI(E E I0) O 1^ 3 (D 4^ it:

PAGE 77

68 • O O I U.CO o o z. a: < o to < -I (> UJ UJ — oc o 00 q: Z3 UJ O QUJ a: jC o I/) -D 1QJ (U C +J 0) 14-J l_ C 0) o > V 3 >/> OJ C O u ,2! ° .!! I(U -DO + C — I" "O I QJ O ^ < — Q n I-D I-D lU 3 C *J c O

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69 r*^--^__ §5 X N c^! w I U)(£ (0 I (U

PAGE 79

70 Results obtained in this experiment indicate that vitamin A deficiency increased the presence of abnormal sperm in the semen of rams on a vitamin A deficient diet. This effect could not be overcome with treatment of testosterone, PMS or thyroprote i n . Figure 15 shows a broken tail condition characteristic of sperm from vitamin A deficient rams. Results obtained in this study are in agreement with those reported by Lindiey SJL^. (19^9) in rams, and Bratton et al . (19^8), Erb et al . (19^7) and Guilbert and Hart (1935) in bulls. Total Sp erm per Ejaculate As shown in Table 11 total sperm per ejaculate (xlO^) during phases I and II were 29^5, 1723; 637, ^61; 605, 118^; 1627, 1163; 1025, 234; I365, 313; 3036, 1346; and 1421, 67O for the control, deficient, returned. A" + Thyroprotein. A" + PMS, A" + Testosterone, control irradiated and A" irradiated groups respectively. During phase I total sperm production was somewhat less in rams on the vitamin A deficient diet. The deficient group had a further drop in sperm production during phase II. A similar trend was observed in the controls. However the returned group experienced a recovery during phase II. Figure 16 illustrates changes in total sperm production for the control, deficient and returned groups. The almost inmediate increase in total sperm per ejaculate In the returned group occurred from the third week on. The deficient group maintained a low sperm production throughout the 21 weeks of phase II. As shown in Table II the PMS and Testosterone treated rams underwent a further drop in sperm production during phase II. The total sperm per ejaculate of rams in these two groups was slightly lower than in the

PAGE 80

^: WS^M: .ssx Figure 15. --Semen from vitamin A deficient rams.

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72 3 O — -o 3iv-inovn3 y3d waads ivioi

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73 deficient group, suggesting the possibility of a detrimental effect from treatment with these two hormones. The thyroprotein group maintained an average sperm production that was above averages found in any of the groups on the vitamin A deficient diet. Figure 17 illustrates total si^erms per ejaculate from rams on the hormone treated groups throughout phase II. The rapid decrease in sperm production in testosterone and PMS treated rams is clearly shown. The beneficial effect from thyroprotein treatment on total sperm production was apparent until week 20. Irradiation caused a drop in total sperms per ejaculate of rams in both groups irradiated. This effect was more severe in the A" irradiated group. Figure 18 illustrates total sperms per ejaculate from rams in the control and irradiated groups. There was a decrease in sperm production in both irradiated groups up to week k. Froms weeks ^ to 7 sperm production in both irradiated groups was comparable to the control, at this time a decreasing trend began which remained with the A" irradiated group until termination of the experiment, while the normal irradiated group appeared to have recovered in week 17. Differences in total sperms per ejaculate between the various treatment groups were not statistically significant. However some biological differences were evident. Vitamin A deficiency had a severe effect on total sperms per ejaculate. This detrimental effect could not be overcome by treatment with testosterone or PMS. This supports the findings of Lindley et al . (19^9) and Is in disagreement with the results of Gunn et a1 . (19^2). Thyroprotein treatment on vitamin A deficient rams appeared to have diminished the harmful effects of the vitamin A deficiency on total sperms per ejaculate. This has not been reported in the literature. The favorable

PAGE 83

7^ — O 1CO.: i o< ->-a u ox OJ o II 'c en (0 10 (U U > (U a§ C in « (U 3ivnnDvr3 y3d swy3ds nvioi

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75 gOI X 3l\nnOVn3 U3d SWd3dS IVIOI

PAGE 85

76 effect of thyroprotein on sperm production is not easily explained since histological examination of the testes from all the vitamin A deficient rams showed a reduction in the germinal epithelium of the seminiferous tubules. It is possible that thyroprotein might have delayed the histological changes thus allowing continuation of spermatogenesis for a longer period. To confirm this however, a series of testicular biopsies would have been necessary to ascertain when the histological lesions began to occur in the vitamin A deficient rams. The effects of irradiation on sperm production can be explained if one assumes that irradiation affected spermatogenesis in the early stages of the process, so that when new spermatocytes were produced normal sperm production was resumed. This would explain the recovery in sperm production observed in the normal irradiated group 16 weeks after irradiation. Individual data on total sperm per ejaculate throughout phase II are shown in the Appendix, Table 2k. Analysis of variance' for differences in total sperm per ejaculate are shown in Tables 30 through 39 in the Appendix. It is appropriate at this point to draw several conclusions on the effects of the various treatments on semen characteristics. Before doing so It Is emphasized that experimental numbers were limited and large death losses made the number of observations even smaller. This and the variability In the data deprived the biological differences observed of statistical significance in most Instances. Seoien volume of vitamin A deficient rams was reduced. This effect was present before the external signs normally associated with a vitamin A deficiency were observed on the majority of the rams. This effect on semen volume was not prevented by treatment

PAGE 86

77 with testosterone, PMS or thyroprotein. Spjerm motility was severely affected by a vitamin A deficiency. This effect did not become evident until the nervous lesions associated with a vitamin A deficiency began to show in most of the experimental animals. PMS or testosterone treatment had no effect on preventing changes in sperm motility on the vitamin A deficient rams. Thyroprotein was of some benefit. Irradiation appeared to have had a detrimental effect on sperm motility values in both groups irradiated. An increase in number of abnormal sperm occurred in vitamin A deficient and irradiated rams. Hormones used had no beneficial effect in reducing the percentage of abnormal sperms present. Total sperm production was severely affected by the vitamin A deficient condition. Manifestation of this effect occurred in association with a decrease in semen volume. Of the hormones used only thyroprotein appeared to have had any beneficial effect on sperm production. Histological examination indicated that the reduced sperm production was due to a reduction in the germinal epithelium of the seminiferous tubules which caused an impairment in spermatogenesis. The ineffectiveness of PMS or testosterone on improving semen characteristics would suggest that the effects of a vitamin A deficiency on semen traits do not occur through a secondary effect from imbalances or reduced secretion of hormones normally associated with spermatogenes i s. Histological Investigation (luant itative evaluation of testes of rams from the various treatments is shown in Table 1^.

PAGE 87

78 < cc :=> < -J > < LA UPi vO — 0) (U o ~ c ». u ». 4J — 3

PAGE 88

79 Vitamin A deficiency resulted in a reduction in the germinal epithelium thickness in all rams on the vitamin A deficient diet. The diameter of the seminiferous tubules also appeared reduced. Similar lesions have been reported in rats by Mason (1933), Howell et al . (1963), in bulls, Bratton et al . (19^8) and in rams by Lindley et al . (19^9). None of the hormones used had any effect in preventing testicular damage. Interstitial tissue was not affected by the vitamin A deficient condition. There was nnore interstitial tissue in rams on the deficient diet than in those on the diet containing vitamin A. The reduced amounts of interstitial tissue present in the testes of vitamin A supplemented rams can not be explained. Figures 19 and 20 illustrate the lack of epithelium thickness in the seminiferous tubules of a vitamin A deficient ram compared to a control. Note also the sparse interstitial tissue present in the testes of the control ram. Thyroid tissues from the control, deficient and A" + Thyroprotein rams were studied for differences in thickness of the follicular wall cells. Table 15 shows average epithelium thickness for the 3 treatments. Based on the small number of thyroid samples studied there appeared to be no differences in thyroid secretory tissue activity between the 3 treatments. Histological studies made on the adenohypophisi s of rams on the various treatments revealed no unusual degree of degranulat ion and vacuolation present. Even though the observations made of the adenohypophi si s were limited in numbers, the lack of abnormal hypophyseal development suggests no alteration in physiological processes that were dependent upon the pituitary hormones due to the vitamin A deficiency in these rams.

PAGE 89

80 *V V'.^ w^ ...,.«» V .-^I Figure 19. -Seminiferous tubules in testes from a control n

PAGE 90

T :^^ Figure 20. --Semi n i f erous tubules in testes from a vitamin A deficient ram.

PAGE 91

82 No cystic pituitaries were found in any of the vitamin A deficient rams. This is contrary to the findings of Lindley et al . (19^9); they reported cystic pituitaries in 3 to 4 month old rams exposed to a vitamin A deficient diet. it is possible that the difference in age and development at the time of vitamin A deprivation between the rams in the studies of Lindley et al . (19^9) and those used in this study could account for differences in pituitary condition. However Erb et al . (l 9^7) found cystic pituitaries in mature dairy bulls exposed to a vitamin A deficient diet over a prolongued period of time. Individual measurements on testes and thyroid are shown in the Appendix Table 23TABLE 15 QUANTITATIVE EVALUATION OF THE THYROID FROM RAMS IN THE CONTROL. DEFICIENT, AND A" + THYROPROTEIN GROUPS Epithelium Thickness Treatment No. of Samples (mm) Control 5 .038 Deficient 3 .05^ A' + Thyroprotein 5 .Ok\ Slaughter Data Table 16 shows weights of the various glands taken at death of rams. Differences in gland weights due to treatment are shown in Table 17.

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83 il 3 o Lr\ oo — CM — a -D •u — (0 O 1-

PAGE 93

8k C -D o to O 1+ V < O o Q. < O "O — — C (D (DOS O

PAGE 94

85

PAGE 95

86 Testes were significantly heavier in rams in the control and returned groups than in all other treatments. Rams in the A" irradiated group had significantly heavier testes than other rams in the vitamin A deficient diet. This was probably due to the fact that most of the rams on this treatment died much earlier in the experiment because of irradiation exposure thus were subjected to vitamin A deprivation for a considerable shorter period of time. PMS or testosterone treatment did not prevent testicular atrophy in vitamin A deficient rams. Thyroprotein treated rams had slightly heavier testes than rams in the deficient group but this difference was not significant. Results obtained in this experiment show that vitamin A deficiency causes a reduction in testicular size. This effect does not appear to be secondary to a testosterone deficiency since testosterone administration was not effective in preventing the reduction in testicular size. The reduced testicular weights associated with vitamin A deficiency in rams have been reported by Lindley et al . (19^9). Similar results were reported in rats by Gross (1924), Wolbach (1925), Mayer and Truant (19^9) and Howell et al . (1963). Mayer and Truant (19^9) by injecting testosterone were able to obviate testicular atrophy in vitamin A deficient rats, Lindley et al . (19^9) were unsuccessful in preventing testicular atrophy in rams with injections of testosterone. The results of this experiment are in agreement with the findings of Lindley et al . (19^9). Epididymal weight comparison between the various treatment groups is rather inconclusive. Rams in the deficient, A" + PMS and A' + Testosterone groups did not show significantly different epididymal size from

PAGE 96

87 the control rams. Rams on the A' + Thyroprotein groups showed significantly smaller epididymal size than deficient and control rams. There were no significant differences in seminal vesicle weight between rams in the different treatments. The cowpers of rams in the A" + Testosterone were significantly heavier than those of rams in all other treatments. Rams in both irradiated treatments had significantly smaller cowpers than rams in all other treatments. Vitamin A deficiency appeared to have had no effect on cowpers development in rams. The explanation for this irradiation effect is not known. Testosterone treatment had a stinujlating effect on the developoient of the cowpers. This is to be expected since it has long been known that the functioning of the cowpers is under testosterone influence (Turner, 1961). Statistical differences in adrenal size between the various treatments are not conclusive. However in general rams in the control and returned groups showed smaller adrenals. A significantly heavier thyroid was present In rams in the A" irradiated group. However rams in the control irradiated group had the smallest average thyroid size. So that if irradiation per se had an effect on thyroid development this was only evident in vitamin A deficient rams. No other effects on thyroid development were observed. Pituitary weight did not appear to be affected by the vitamin A status of raras. Treatment with either testosterone or PMS did not appear to have any definite effect on pituitary development. Had the level of thyroprotein given been sufficient for normal body functioning, pituitary

PAGE 97

88 thyrotrophic hornxjne secretion would have been reduced. Consequently a reduction in thyroid size from lack of thyrotrophic hormone stimulation could have resulted. It is interesting to note that thyroprotein treatment did not cause a reduction in thyroid size. However the data are not conclusive enough to permit a definite statement on this relationship. Table 25 in the Appendix show individual gland size for all raras. The reduced number of rams and the variability in gland size between rams within a treatment suggest caution in making any categoric statements on hormonal and nutritional interrelationships in these rams. Analysis of variance for differences in testes, epididymis, seminal vesicles, cowpers, adrenals thyroid and pituitary size are shown in the Appendix Tables kO , k] , k2, ^3 , V*, ^+5 and kS respectively.

PAGE 98

SUMMARY A study was conducted to determine the effects of a vitamin A deficiency on the reproductive capacity of rams. An attempt was made to counteract the harmful effects of the vitamin A deficiency by administering testosterone, pregnant mare serum (PMS) or thyroprotei n. Information was obtained on the tolerance of vitamin A deprived rams to gamma irradiation. The experiment was started on February 2, 1963During phase I of the experiment 12 yearling rams were assigned as controls to a purified diet containing 3.000 I.U. of vitamin A per lb. of feed. Thirty-six yearling rams were placed on the vitamin A free diet. During phase II the 12 rams on the control diet with average vitamin A values of 139 mcg./lOG ml. plasma and 156 mcg./g. fresh liver were continued on the same ration with 6 serving as controls and 6 irradiated with 400 r of whole body gamma irradiation. During phase II the 36 rams on the vitamin A free diet with vitamin A values averaging 5 mcg./lOO ml. plasma and 5-6 mcg./g. fresh liver were continued on the vitamin A free diet and were assigned in equal groups to one of 6 treatments: (1) 400 r of whole body gamtna irradiation; (2) Daily injections of 60 RU of PMS/ 100 lbs. body weight; (3) Daily injections of 10 mg. of testosterone/100 lbs. body weight; (4) Daily capsules of 1 g. thyroprotein/lOO lbs. body weight; (5) 3000 I.U. of vitamin A/lb. of feed; (6) No additional treatment. Semen was checked monthly during phase I and weekly for 21 weeks during phase II. Night blindness, cloudiness of the cornea, uncoordinated gait and extreme weakness were symptoms associated with rams on the vitamin A free 89

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90 diet. Limited Internal hemorrhaging in the chest cavity was characteristic of rams exposed to irradiation. Rams on the vitamin A deficient diet consumed less feed and lost more weight than ccntrols. During phase II PMS or testosterone treatment did not have any beneficial effect on appetite or growth of vitamin A deficient rams. The thyroprotein treated rams consumed more feed and lost less weight than other rams on the vitami n A deficient diet. Irradiation appeared to have had a depressing effect on feed intake and growth of all animals irradiated. Rams whose vitamin A reserves had bean drast i cal 1 y reduced and were experiencing severe weight losses were able to recover and suffered no death losses when 3,000 l,U. of vitamin A per lb. of feed were provided. Most of the rams whose vitamin A reserves had been depleted eventually died. Treatment with either testosterone or PMS was not effective in preventing death losses in deficient rams. Thyroprotein capsules did reduce death loss of deficient rams. The vitamin A status of rams exposed to irradiation did not affect their survival time. Vitamin A deficiency caused a reduction of germinal epithelium thickness and in the diameter of the seminiferous tubules of testes. Histological studies of the thyroid and adenohypophi si s revealed no changes that could be attributed to vitamin A status of rams. Semen volume, sperm motility, per cent abnormal sperm and total sperm production were severely affected by vitamin A deficiency. Sperm motility and total sperm production changes were ameliorated with thyroprotein treatment. Irreparable damage of semen traits studied did not result from a drastic reduction of vitamin A reserves. Irradiation appeared to increase per cent of abnormal sperm cells and reduced motility values in rams with normal vitamin A

PAGE 100

91 reserves. Testicular weight was reduced in all vitamin A deficient rams Epididymis cowper's, seminal vesicles, adrenals, thyroid and pituitary weights were not affected by the vitamin A deficiency.

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LITERATURE CITED Barnett, A. J. G. , and R. L. Reld. 1961. Reactions In the rumen. Edward Arnold Ltd. , London. Berliner, \l . , «nd \l . Warbritton. 1937. The pituitary and thyroid in relation to sperm production in rams. Proc . Am. Soc. of Anim. Prod. 30th Ann. Meeting. Bhatnagar, D. S., D. P. Mukherjee and P. Bhattacharya. 1955. Ind. Vet. J., 25:293Black, J. W. , A. L. Schick, A. L. Pope, and L. E. Casida. 1950. Some effects of testosterone propionate and thyroprotein in rams. J. Anim. Sci., 9:l86. Bogart, R. . and D. Y. Mayer. 19^6. Missouri Agr. Expt . Sta. Res. Bull. No. 402. Bratton, R. W. , G. W. Salisbury, T. Tanabe, C. Branton, E. Mercier and J. K. Loosll. 1948. Breeding behavior, spermatogenesis and semen production of mature dairy bulls fed rations low in carotene. J. Dairy Science, 31 :779. Brooks, J. B. , and C. W. Ross, 1961. Role of the thyroid gland in the fertility of rams. J. Anim. Sci., 20:967, Cljne, T. R., E. E. Hatfield and U. S. Garrigus, 1962. Effects of potassium nitrate, alpha-tocopherol , thyroid treatments and vitamin A on weight gain and liver storage of vitamin A in lambs. J. Anim. Sci. , 21:1010. 1963. Effect of potassium nitrate, alpha-tocopherol, thyroid treatments and vitamin A on weight gain and liver storage of vitamin A in fattening lambs. J. Anim. Sci. 22:911. Cole, H, H. and P. T. Cupps. 1959. Reproduction in domestic animals. Academic Press, N. Y. and London. Craig, A. W. , B. W. Fox and H, Jackson. I96I. Effect of radiation on male mouse and rat fertility. J. of Reprod. and Fertil. 2:466. Davles, A. W. and T. Moore. 1934. J. Blochem. 28(288). Dowling. J. E. and G. Wald. I960. The role of vitamin A acid. Vitamins and Hormones 18:515. 92

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93 Drill, v. A. 19^3. Interrelations between thyroid function and vitamin metabolism. Physiol. Rev. 23:335. Eaton, H. D. 1951. Effect of vitaoiin A depletion on live weight, plasma and liver vitamin A and microanatomy in young calves. J. Dairy Sci. 3^:386. Erb, R. E., F. N. Andrews, S. M. Hauge and W. A. King. 19^7Observations on vitamin A deficiency in young dairy bulls. J. Dairy Sci. 30:687. Freund, M, and R. L, Murphree. I960. Effect of whole-body gamma irradiation on the characteristics and metabolism of bull semen during the early post-irradiation period, J. Dairy Sci. it3:ll30. Goodrich, R. D., R. J. Emerick and L. B. Embry. 1962. Effects of sodium nitrate on vitamin A status of sheep. J. Anim. Sci. 21:997. Goswami , S. B. 1962. The effect of administration of thyroxine and PMS hormones on reaction time and semen quality of buffalo bulls. Ind. Vet. J. , 39:12. Gross, L. 1924. The effects of vitamin deficient diets on rats. J. Pathol Bacteriol. 27:27. Guilbert, H. R. and G, H, Hart. 193^. Storage of vitamin A in cattle. J. Nutri., 8:25. . 1935. Minimum vitamin A requirements with particular reference to cattle. J. Nutri., 10:^+09. Gunn. R. M. , R. N, Sanders and W. Granger, 19^2. Studies in fertility in sheep. Commonwealth of Aust. Council for Sci. and Indust. Res. Bull. 148:140. Hafez, E, S, E. 1962. Reproduction in farm animals. Lea and Febiger, Philadelphia. Halmi , N. S. 1950. Two types of basophils in the anterior pituitary of the rat and their respective cytophysiological significance. Endocrinol. 47:289. Ham, A. W. and T. S. Leeson, I96I, Histology. J. Lippincott Co., Philadelphia and Montreal. Harm, F. 1942. Vitamin A and animal health. Thesis, Hannover Hoch schule 32; Niedersachsi sche Buchdruckerei , Uelsen. Harper, H. A. J963. Review of physiological chemistry. Lange Medical Publ i cations, Los Altos, California.

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9k Hogdson, R. E. , S. R. Hall, W. J. Sweetman, H. G. Wiseman and H. T. Converse. 19^6. The effect of vitamin A deficiency on reproduction in dairy bulls. J. Dairy Sci. 29:669. Howell, J., J. N. Thompson and G. A. Pi It. 1963Histology of the lesions produced in the reproductive tract of animals fed a diet deficient in vitam n A alcohol but containing vitamin A acid. J. Reprod. and Pert. 5:159. Humason, G, L. 1962. Animal tissue techniques. W, H. Freeman and Co., San Francisco and London. Johnson, R. M. and C. A. Bauman. 19^7The effect of thyroid in the conversion of Carotene Into vitamin A. J. Biol. Chem. 171:513Jones, I. R., J. 0. Schautz and J. R. Hagg. 19^6. Relation of carotene levels to fertility in dairy bulls, J. Dairy Sci. 29:522. Jubb, K. v. and K. McEntee, 1955. Observations on the bovine pituitary gland. II. Architecture and cytology with special reference to basophil cell function. The Cornell Vet. ^45:593. Kimble, M. S. 1939. The photoelectric determination of vitamin A and carotene in human plasma. J. Lab. Clin. Med. 24:1055Kosin, I. L. I9M*. Some aspects of the biological action of X rays on cock spermatozoa. Physiol. Zoology, 17:289. Lindley, C. E. , H. H. Brugman, T. J. Cunha and E. J. Warwick. 19^9. The effect of vitamin A deficiency on semen quality and the effect of testosterone and pregnant mare serum on vitamin A deficient rams. J. Anim. Sci. 8:590. Madsen, L. L. , S. R. Hall and H. T. Converse. 19^*2. Cystic pi tituaries in young cattle with vitamin A deficiency. J. Nutri. 24:15. Mann, T. 195'+. The biochemistry of semen. John Wiley and Sons, New York, N. Y. Mason, K, E. 1933. Differences in testes injury and repair after vitamin A deficiency, vitamin E deficiency and inhition. Amer. J. Anat . 52:153. Mayer, J. and A. P. Truant. 19^9. Effects of administration of testosterone on vitamin A deficiency. Proc. Soc. Exptl. Biol. Med. 72:^36. and J. W, Goddard. 1951. Effects of administration of gonadotrophlc hormone on vitamin A deficient rats. Proc, Soc. Exptl. BIpl. Med. 76:1^+9. Maynard, L. A, and J. K. Loosil. 19^2. Animal nutrition. McGraw-Hill Book and Company, Inc., New York, Toronto and London.

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95 Meacham, T. N. . T. J. Cunha, G. E. Combs, H. 0. Wallace, A. C. Warnick, R. L. Shirley and C. F. Simpson. 1962. Effect of gamma irradiation on pigs fed low vitamin A rations. Proc. Soc. Exptl . Biol. Med. 11:30. . 1962. Effect of level of nitrogen on growth and reproductive physiology of young bulls and rams. Doctoral Dissertation, University of Florida, Gainesville, Florida, Moore, T. 1931. The distribution of vitamin A and carotene in the body of the rat. Biochem. J. 25:275. and J. E. Payne. 19^2. Sheep level of vitamin A storage. Biochem. J. 36:3^. __. 1957. Vitamin A. Elsevier Publishing Co. i960. The pathology of vitamin A deficiency. Vitamins and Hormones 18:^99Murphree, R. L. and N. R. Parish, 1956. Effects of whole body gamma radiation on semen characteristics in bulls. J. Anim. Sci . 15:1300. Pace, H. B. , E. W. Hupp and R, L. Murphree. I962. Changes in semen and blood of boars following total body gamma irradiation. J. Anim. Sci. 21:615. Pearse, A. G. E. 1950. Differential stain for human and animal anterior hypophisis. Stain. Technol . , 25:95. i960. Histochemistry. Little, Brown and Co., Boston. Purves, H. D, and W. E. Griesbach. 195^. The site of follicle stimulating and lutenislng hormone production in the rat pituitary. Endocrinol, 55:785. . 1955. Changes In the gonadotrophs of the rat pituitary after gonadectomy. Endocrino. 56:37^. Reddy, B. S. and J. W. Thomas. 1962. Interrelationships between thyroid status and nitrate on carotene conversion. J. Anim. Sci. 21:1010. Rennels, E. G. 1957. Two tinctorial types of gonadotrophic cells in the rat hypophisis. Zeitschrift fur Zel Iforschung , Bd. 45, S. kSk. Sanders, A. E. and E. G. Rennels, 1959Evidence on the cellular source of luteotrophin derived from a stud/ of rat pituitary autografts. Zeitschrift fur Zell forschung , Bd., S. 263Schubert, J. and R. E. Lapp. 1958. Radiation, what it is and how it affects you. Viking Press, New York, New York.

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96 Shirley, R. L. , T, N. Meacham, A, C. Warnick, H. D, Wallace, J. F, Easley, G. K, Davis and T. J. Cunha. 1962. Gamma irradiation and interrelation of dietary vitamin A and copper on their deposition in the liver of swine. J, Nutri. 78:^54. Tilton, W. A. 1962. Effect of energy and protein deficiencies on growth and reproductive performance of young rams. Masters Thesis, University of Florida, Gainesville, Florida. Turner, C. D. I96I. General endocrinology. W. B. Saunders, Philadelphia and London. Wald, G. i960. The visual function of the vitamins A. Vitamin and Hormones 18:515. Warbritton, V., and F. F. McKenzie. 1937The pituitary glands of ews in various phases of reproduction. Missouri Agr. Expt . Sta. , Res. Bull 257. White, A. D., P. H. Handler, E. L. Smith and D. S. Stetten. 1959. Principles of biochemistry. McGraw-Hill Book Co., Inc., New York, Toronto, and London. Wilson, W. D. and C. Ezrin. 195^. Three types of chromophll cells of the adenohypophisi s. Araer. J. Pathol. 30:891. Wolbach, S. B. and P. R. Howe. 1925. Tissue changes following deprivation of fat soluble vitamin A. J. Exp. Med. ^2:753.

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APPENDIX

PAGE 107

98 TABLE 18 UNA PLAS INDIVIDUAL RAMS Initial F' nal Treatment Group meg. /I 00 ml. meg . /g . fresh and Animal No. plasma liver mcg./lOO ml. plasma Control 13 21 130 5^ 15 ^5 162 21 Y97 54 228 60 22 33 15^ 34 G9 54 127 27 Y'43 30 23 15 Deficient Y19 .6 9 B9 9.0 ' '' -^ B32 1.5 B83 1.2 Y44 6.0 Returned G99 .6 42 G94 3.0 1.5 30 Y47 7.5 1.4 ko B2 1.5 35 Y22 12 69.6 24 Y99 27 34.6 30 130

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99 TABLE 18 (Continued) LuLLLaJ Final Ireatment Group meg ./ I 00 ml. racg./g. fresh and Animal Nc._. P'asma liver meg /I 00 ml. plasma A" + Thyroprotein Yi6 3.0 .6 Y24 6.0 1.5 Y83 1 . t il9 1.2 Y95 1.5 2.0 G87 1.2 4.5 Y'^^ 3.0 4.6 9.0 3.0 Y98 k.5 -c G26 6.0 4.3 _c Y75 3.0 3.8 -<= A" + PMS B2 1 .9 3 4.2 Y8I 9 2.9 -c Y20 18 1,7 .c Y84 1,9 ,9 _c Y50 A" + Testosterone Y80 6.0 ,6 6.0 YlOO 4,5 14 _c c

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100 Treatment and Animal

PAGE 110

101 TABLE 19 Treatment Group

PAGE 111

102 TABLE 19 (Continued) Treatment

PAGE 112

103 TABLE 19 (Continued) Treatment

PAGE 113

\ok TABLE 20 SUMMARY OF THE li+-DAY PERIODS WEIGHT OF INDIVIDUAL RAMS AND TREATMENT GROUPS IN PHASE I (POUNDS) Treatment Group

PAGE 114

105 TABLE 20 (Continued) Treatment Group Per iods and Animal No. 1 2 3 '+ 5 6 A" + Thyroprotein Y76 97 88 93 93 95 99 Y24 90 89 96 97 10» 105 Y83 87 8k 92 9^ 90 91 91 95 Y98 81 79 78 83 90 91 92 9^ G26 79 78 88 96 10^* 107 105 1 06 105 108 108 Y75 98 96 102 102 10^ 105 105 105 105 105 108 a" + PMS B21 81 81 77 72 7^ 71 3 lOi* 100 106 104 106 112 Y8I 95 95 100 103 107 nit 118 117 Y20 94 85 86 86 91 95 95 98 Y8U 74 77 84 84 92 92 89 96 98 99 99 Y50 91 88 93 85 86 91 91 96 99 103 105 A" + Testosterone Y80 78 79 79 75 74 76 B19 84 84 90 88 95 95 YlOO 78 78 84 73 76 87 85 83 Y95 83 83 84 78 88 91 81 87 G87 78 78 78 81 91 95 102 107 106 107 110 Y46 112 112 114 114 118 116 120 127 118 118 118

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106 TABLE 20 (Continued) Treatment Group Periods and Animal No. 12 3^5 Control Irradiated G90

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107 TABLE 2] SUMMARY OF THE J4-DAY PERIODS WEIGHT OF INDIVIDUAL RAMS AND TREATMENT GROUPS IN PHASE II (POUNDS) Treatment Group

PAGE 117

108 TABLE 21 (Continued) Treatment

PAGE 118

TABLE 21 (Continued) 109 Treatment

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110 TABLE 22 SURVIVAL TIME^ FOR INDIVIDUAL RAMS Treatment Group and Animal No. Days Survived Control 13 14! 15 1*1 Y97 1^1 22 ]k\ G9 -li+l \k3 ]l4] Av. --\k] Def icient Y19 — 141 B9 136 B32 kk B83 1 YM+ 88 Av. kk Returned G99 141 G94 141 Y47 141 B2 -— 141 Y2^ 141 Y99 141 Av. ___ 14I

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I II TABLE 22 (Continued) Treatment Group and Animal No. Days Survived A + Thyroprotein Y76 141 724 I4l Y83 I4l Y98 133 G26 95 Y75 98 Av. 96 a" + PMS B21 -66 3 38 Y8l 120 Y20 83 Y84 --64 Y50 I4l Av. 102 A" + Testosterone Y80 141 819 124 YlOO — 81 Y95 — 21 G87 -72 Y46 I4i Av. -106

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112 TABLE 22 (Continued) Treatment Group and Animal No. Days Survived Control Irradiated G90 \k] G97 28 6 25 21 2k Y79 141 16 20 Av. 80 A" Irradiated Gl ---25 G92 \k] Y74 21 Y85 2k B52 25 37 — 37 Av. --36 From reassignment to death resu ting from treatment or slaughtering after l4l days.

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113 TABLE 23 QUANTITATIVE EVALUATION OF TESTES AND THYROID

PAGE 123

TABLE 23 (Continued) Treatment and Animal

PAGE 124

115 TABLE 23 (Continued) Testes Thvruid Epithelium Tubule Interstitial Epithelium Treatment Group Thickness Diameter Tissue^ Thickness and Animal No. (mm. ) (mm.) (mm.) (mm.) .^53 3 .h%e 3 _b _b _b b .452 3.8 • 783 2 .681 5 .5^0 2 -d _d .729 _d _d .683 2.1 G87

PAGE 125

116

PAGE 126

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124 JLTV I 00 vO LA ir\ tvj o^ cr> r^ — fM LTS lA vO ^ r> o o 00 LA o^ 00 OO OO I LA CA -dCO OO I CM LA

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135 < ^g r^ — 00 1^ (V^ CM vO CM CO — urv CTl <\l U> 00 ir» — 00 r-v v£> o -at^ -dLA LA <\l r^ — — LA rr\ O U\ ». — — m — CO LA O 00 LA LA O^ O vD r^ 00 — CVJ r^ LA PO LA LA — OA 00 o r--.

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139 CM — m 00 tN 00 OO O vD o cr\ — >>-

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140 r-o CM
PAGE 150

]k\ TABLE 26 ANALYSIS OF VARIANCE FOR THE DIFFERENCE IN AVERAGE DAILY GAIN DURING PHASE I Source

PAGE 151

142 TABLE 28 ANALYSIS OF VARIANCE FOR THE DIFFERENCE IN AVERAGE DAILY GAIN DURING PHASES I AND II COMBINED Source of Var i at ion

PAGE 152

143 o

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151 o uj Z UJ

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152 I5^ O LU CC LU < O00 _j ^^ < o E c

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153 TABLE kO ANALYSIS OF VARIANCE FOR THE DIFFERENCE IN MEAN SIZE OF THE TESTES Source

PAGE 163

\5h TABLE k2 ANALYSIS OF VARIANCE FOR THE DIFFERENCE IN MEAN SIZE OF THE ADRENAL GLANDS Source

PAGE 164

155 TABLE kk ANALYSIS OF VARIANCE FOR THE DIFFERENCE IN MEAN SIZE OF THE COWPER'S GLANDS Source

PAGE 165

156 TABLE 46 ANALYSIS OF VARIANCE FOR THE DIFFERENCE IN MEAN SIZE OF THE PITUITARY GLAND Source

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BIOGRAPHICAL SKETCH Enrique Sosa was born March 17, '9^0 at Camaguey, Cuba. In June 1957 he was graduated from Colegio de Belen in Habana, Cuba. He received the degree of Bachelor of Science in Agriculture in January 1961 at the University of Florida. He enrolled in the Graduate School of the University of Florida in February I96I and obtained a degree of Master of Science in Agriculture in June I962. Membership is maintained in the American Society of Animal Science, Phi Sigma and Sigma Xi. He is married to the former Irene Tremols and is the father of two daughters. The author is now a candidate for the degree Doctor of Philosophy.

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This dissertation was prepared under the direction of the chairman of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August, 1964 ^2^ ^^ ^^ean, College of Agriculture SuD^rvi sory yCommi ttee Chai rman Dean, Graduate School

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