E F F E C T OF E X H A U S T I V E E J A C U L A T I O N ON C O M P O S I T I O N

OF B O V I N E S E M E N 1

M. L. HOPWOOD, L. C. FAULKNER, ,xxo F. X, GASSNER

Endocrine and Animal Reproduction Sections, Colorado State University, Ft. Collins

SUMMARY

To obtain information on the nature of sperm and of the glandular fluids produced through exhaustive ejaculation, normal, infertile, or bilaterally vasoli- gated bulls of various breeds were subjected to exhaustive semen collection by artificial vagina (AV) service, electroejaculation, or a combination of these. Electric stimulation was applied at approximately 3-rain intervals. Collections were n-rode during periods of tess than 3 hr, and quantities of seminal fluid between 85 and 225 ml were obtained. Fructose, nitrogen, and sperm concen- tration in the ejaculates were correlated, whereas pH was inversely related to these components. Free amino acid and protein patterns were normal in intact and infertile bulls during early ejaculates, but became distinctly abnormal as exhaustion was approached. In an infertile bull, abnormal sperm of the sec- ondary type decreased as primary abnormalities increased. The percentage of sperm showing no affinity for nigrosin-eosin vital stain (presumably live sperm) increased during exhaustive ejaculation.

Use of exhaustive ejaculation was reported by Walton and Edwards in 1938 (17). Ten ejaculates were collected from a bull by means of an artificial vagina (AV) over a 12-hr pe- riod. Almquist and co-workers (1, 2, 7, 8) later reported use of the technique for deple- tion of epididymal sperm reserves. Swanson and Boyd (16), in partial exhaustion tests, ex- amined semen collected by the AV for morpho- logical changes in the spermatozoa.

Ejaculation by electric stimulation has been used in the bull to obtain semen from aninlals not trained to serve the AV or from bulls in- capable of natural service. By electric stimu- lation of bulls, Lutwak-Mann and Rowson (13) were able to obtain two distinct fractions from the ejaculate. Differences in pH, volume, fruc- tose concentration, and globulin components have been shown to exist between senlen sam- ples collected by the AV and by electroejacu- lation in preliminary studies (10). The fer- tility of semen collected by electric stimulation appears to be unaffected (9).

In man, the boar, and the stallion the process of ejaculation is a protracted one during which

Received for publication May 14, 1963.

This investigation was supported by a PHS Research Grant, GM 07120, from the Division of General Medical Sciences, Public Health Service, as well as through cooperation in Regional Project W-49, Physiological Causes of Breeding Failure in Cattle. Colorado Agricultural Experiment Station, Scientific Series No. 864.

the secretions from the accessory sex glands are delivered in a definite sequence (15). Ejacula- tion in the bull under physiological conditions is an ahnost instantaneous, explosive process in which, with exception of the prespenn por- tion, the ejaculate is delivered with sinmlta- neous mixing of the seminal vesicular fluid with the spermatozoa. Interest in the chemical com- position of these secretions, their influence on sperm metabolism, and in the mechanism of ejaculation led us to collect semen of the bull by exhaustive ejaculation. This process was considered by us to be an ideal procedure by which the morphology of spermatozoa derived from various parts of the bull's reproductive tract could be evaluated, especially because a supply of epididymal ceils would be readily produced, as indicated by the studies of Bialy and Smith (3).

EXPERIh[ENTAL PROCEDURE

Normal, infertile, or bilaterally vasoligated bulls were subjeted to exhaustive semen collec- tion by means of AV service, electric stinm]a- tion with a Marden Eleetroejaeulator, or by a combination of these. During electric ejacula- tion all seminal fractions were included in the ejaculate. The pH, fructose concentration, ni- trogen, and free amino acid levels were deter- mined. Fructose was determined by the Seli- wanoff reaction adapted by Mann (14). Free amino acids were measured according to Hop- wood and Gassner (11). Nitrogen was deter- mined by Kjeldahl's method. Sperm morphol- ogy was classified according to Biota (4).

1409

1410 5i . 1.. H t I P \ \ ' O [ . ) I . ) . L ( ' , I " A U I A - : N F I I . A N D I-'. X. ( ; A S S N I ' ; R

Elec t rophore t i e p a t t e r n s of seminal p ro te ins were obta ined according to Grassman and I I an - nig (6) , us ing amido black s ta in and measur- ing opt ical dens i ty in a Welch Densiehron. Areas u n d e r the curves were measured by a p lanimeter , to es t imate the concent ra t ion of the p ro te in components .

R E S U L T S

Table 1 shows the effect of" exhaust ive ejacu- la t ion on p H , volume, and seminal cons t i tuents of a ma tu re S h o r t h o r n bull. Over a per iod of 2 h r the bull was subjected to eight A V collec- tions, followed by eight collections b.v electro- e jaculat ion. The p i t rose s teadi ly unt i l the fifth ejaculate , increased a b r u p t l y at the sixth, and remained elevated fo r the balance of the collections, a p p a r e n t l y as a resul t of deplet ion. Yolume was grea t ly increased as a resul t of e leetroejaeulat ion. Ni t rogen levels and num- bers of sperm p e r e jacula te reached a peak a t the fou r th e jacula te ( A V ) , d iminished as the volume remained s teady in the next fore ' A V collections, and were increased as the w)lume of semen inereased. Both cons t i tuents reaehed peak values a t the t en th ejaculate. Del ivery of amino acids (a lanine, glycine, serine, aspar t ie . and g lu tamie acids) was correla ted with the delivery of nit~'ogen. F ree glutamie acid. con- sidered separa te ly , a p p e a r e d more near ly cor- re lated wi th the del ivery of sperm and was less dependen t on the volume of the ejaculate . The f ructose content pe r e jacula te rose to a maxi- mum in the f o u r t h ejaculate , and ita ou tpu t was independen t of the volume of seminal fluid.

The seminal p ro te in pa t te rn~ appea red nor- real t h rough the first four collections; there- af ter , the components cor responding to a-glob- ulins (12) d iminished (F igu re 1). No a lbumin was detected in any of the f ract ions . The pa t - t e rns f rom the e leet roejaeula tes were dis t inct ly

S H - - 5 AV C O L L E C T I O N

2 0 0 I

180 i 16C

I4C FRACT. FRACT. FRACT. . F R4ACT. ~

S.I Ioc

8 0

go

g 20

120

I 0 0 FRACT. FRACT. 7 8 8 0

6 0

4 0

2 0 %

0 rare rata mm mm

PI(;. 1. Electrophoretie pat terns from semen of a Shorthorn bull (SH-5) collected b v artificial vagilm. The major peaks correspond to a-globulins (a:,, a~, and a~, respectively) of bovine sermn.

TABLE 1

Constituents in semen of a normal Shorthorn bull (SH-5) collected by AV (eight ejaculates) followed by eleeiroej/culation (eight ej ~¢ulates)

Constituents per ejaculate

Total Free free glu-

Frac- Nitro- ]"rue- amino ramie tion pH Volume gen S p e r ~ l l r o s e acids acid

(*lO.) (ml) (mq) (X 10 ~) (rag). 1 6.(; 1.(; 7.4 1,40~ 11.7 0.51 0.31 2 6.8 1.0 3.7 59o 5.2 0.22 0.18 3 7.0 2.8 10.5 560 20.2 0.40 0.23 4 7.1 4.2 18.9 2,058 29.8 0.49 0.36 5 7.2 2.0 (1.2 1,100 13.2 0.34 0.16 6 7.8 2.2 5.5 836 13.3 0.21 0.06 7 7.8 2.3 6.2 575 13.7 0.20 0.06 8 7.!) 2.3 7'.0 460 9.2 0.11 0.05 9 7.8 11.2 13.4 10J 9.5 0.82 0.22

10 7.5 11.3 27.3 2,260 ]I).~ 0.73 0.3. ~) 11 7.7 12.'2 l(}.q 1,46"4 1.2 0.83 o.36 12 7.6 11.0 8.7 1,650 5.5 0.71 0.29 13 7.2 10.9 5.4 43t; 5.S 0.40 ( .1 ) 14 8.0 9.9 6.9 99 4.0 0.51 0.0!~ 15 7.5 11.0 6.6 4(,)5 5.5 0.69 0.2() 16 7.(i 10.0 5.o ] 00 :l .0 0.52 0.12

('()MPOSI'PII)X ()1." B(IVINI,; N1-]MI,:N 1411

abnormal (]J~igure 2) and the eoneel~tration of protein was drastically diminished.

F igure 3 shows the pat tern obtained when fructose concentration (rag per 100 ml), sperm eount (eells per m~d), and pH were eompared for each eolleetion from a normal Holstein bull sul)jeeted to eleetroejaeulation. In 3 hr the bull

SH-5 ELECTRO. COLLECTION

,O,o,o~i E.C,. ~E"'C~ ,]~'"'I~" ~ERyT mm mm mm mm

FRAC'[ FRAC'[ FRACT. FRACT. 13 t4 15 16

a mm ram mm mm

Pro. '2. Abnormal eleetrophoretie, la t terns ob- tained from semen of .q Shorthorn bull (SH-5i eolleeted by e e(troejaeulation following eight A V colleetions.

delivered 34 ejaculates, totMling 156 ml of fluid. The fruetose content and sperm count were posit ively era'related, whereas p H fluctu- ated inversely with the fructose and sperm eoneentrations.

A Guernsey bull was subjected to eleetro- ejaeulation and 195 ml of fluid (25 ejaculates) obtained in 3 hr. F igure 4: shows the correla- tion between fructose mM .~l)ernl eoneentration obserw,d earlier (F igure 3), as well as the rela- tionship of these faetors to the seminal nitro- gen which ire'teased and declined in a fashion similar to fru(.tose and sperm.

In tta. HI)splice Of sl)er[ l iatoz()~l , s e m i n a l l l i -

trogen fluetuates with the t'ruetose eoneentra- tion. A bi lateral l ) wtsoligated Holstein bull (F igure 5) was subjeeted to exhaustive elee- troejacuhlt ion and 34 ejaculates, totall ing 190 ml of fluid, were obtained. As observed in other subjects, the p H and fruetose concentrations were inversely related, whereas seminal nitro- gen resembled fruetose in its delivery. I t is obvious that the fluetuation in ni trogen is not d u e to ~l}erln IlllHl})l,rs, I Io r {'10 t h e ~ lmino a c i d s

emltribute great ly to the seminal nitrogen. Free amino acids in semen ot2 the normal

bull (Table 1) showed an appreciable concen- t ra t ion oi glutamie acid. lit the vasoligated bull, however. (Fig'ure (i). it is eh, ar that ala-

S P E R M C O U N T ml X 1 0 0 0

. IOOO

F R U C T O S E r a g / l O O m / X E B - - 13

2 0 0 " I I

X / ¢ ~ - - - - - - 0 F R U C T 0 S E C O N C E N T R A T I O N /

~ , I , , / X - - - - - - X S P E R M C O U N T 7 5 0

, o / / t x

5 0 O ~ 0 0

/\ ,o I l l / - ; ' , . o

I X b . I iX / I , : ' , f I', ,x, x , I / ^ ~'x,X,.x//V. / / " "~x.X--X-_ . / ~ ~" ~,,

X-- A - X - - ~ x - - . 0 o ML-O I0 20 ~Jo 4 0 50 60 70 80 90 i00 I [0 t20 130 140 150 160

9 . 0 -

pH

8 . 0

I 3 5 7 9 II 13 15 17 19 21 23 25 27 29 31 35

F R A C T I O N N U M B E R

];IG. ;~, Vfll']fltJOll i l l Sl)el'lll eOUllt. /;rttcto~e ~'OllcetLtl';Irioli. ~llH] p l l ill ,~(,lllell of ,¢t ] l o [ s t e in bull (la~B-]3) subjected to exhaustive (,levtroe.jacul:ltim~.

1412 ~ . L. HOP~VOOD, L, C. I zAULKNER, AND F. X. G A S S N E R

F R U C T O S E S P E R M C O U N T rag/-- I O O m l F R U C T O S E • • m! X I O 0 0

3 0 0 - S P E R M C O U N T x - - - x - 6 0 0 X - - - - - - x

4 I 0 0 - I i 200

/x.,.... ,x. ~x'04,

rag, / I O O m l N I T R O G E N 50~"

40~

301

201

! 0 ,

i

pH

S'Oo ' io " ,io ' 6o " io ' ~oo ~io ~io r6o " " ~ o " zoo V O L U M E IN ml

Fro. 4. Fructose and nitrogen levels and pH of semen of a Guernsey bull (G-18) sub- jected to exhaustive eleetroejaculation.

F R U C T O S E t o O / I O O m l

500 '

4 0 0 "

300"

200-

,oo x , l I \ !

o Xx~( -~ ~( mg N I T R O G E N p e r moo m l

5 0 0 '

X

I Ii

I I

1

E B - - 19 0 0 pH X - - - - - X F R U C T O S E

/i

; ~ x .x- - , I " x \ x / \ / \ / ,, / )~._xj.. x \ , ~1 \

x - x - - X

pH "8.8

8 . 4

8 .0

'7.6

"7.2

6.11

4 0 0

3 0 0 '

2 0 0

lO0

0 0 20 40 60 80 I00 IZU I q u ~ov ,~v

T O T A L m l F L U I O

FIG. 5. Fructose and nitrogen levels and pH of seminal fluid of a bilaterally vasoligated Holstein bull (EB-19) subjected to exhaustive electroejaeulatiom

( ~ O M P O S I T I O N OF B O V I N E S E M E N 1413

M 9 j l O O m l E B - 1 9

':[ _ GLUTAMIC ACID

A L A N I N E

,o[ 0

':L ':[

m m

m m m

GLYCINE

S E R I N E

B.5 p H

i FRACTION NO,--I 3 5 T 9 II 13 IS IT 19 21 23 25 ?.7 29 31 ~ ~}S

FIG. 6. Free amino acids and pH of seminal fluid of a bilaterally vasoligated ttolstein bull (EB-19) subjected to exhaustive electroejaculation.

nine was the predomilmnt amino acid. Further- more, the alanine was present in the later col- lections in concentrations as high as in the normal bull and became increased in these ejaculates from which the other acids were ab- sent. It is interesting that the alanine concen- tration fluctuated inversely with the pH.

It is again to be noted (Figure 7) that glu- tamie acid is more highly correlated to the de- livery of spermatozoa than the other free amino acids in semen of a Holstein bull from which 21 ejaculates (80 ml of fluid) were ob- tained. As previously shown (11), glutamie acid has been implicated as being delivered by the epididymides.

An infertile Hereford bull having a high quantity of primary and secondary sperm ab- normalities was subjected to exhaustive ejacu- lation. This collection process took 9 hr. The bull delivered 16 AV ejaculates followed by eight eleetroejaculates. Fluids from this bull resembled those obtained from the other ani- mals, in that sperm numbers decreased in cor- relation with fructose concentration. Initially, the bull delivered sperm 45% of which were classified as secondary abnormalities and 22% as primary. As successive ejaculates were ob- tained, the nmnber of primary abnormalities (head deformities) increased markedly as the secondary abnormalities (mainly separation of heads and tails) declined (Figure 8).

Figure 9 shows the sperm count and per- centage of live sperm delivered in successive

m g / l O O m t

3 0

20

tO

o

'° t IO

0

:1

E B - - 2 7

TAMIC ;CID

A L A N I N E

B I B m _ _ ~ L

GLYCINE

SERINE

I O | ~ ~ m ~ mB B m ~ 0 J

A S P A R T I C

I01 ACID

o . . . . . - : - , , - - . . . . - 7 " , . - - ' , , ~ ,

F R A C T I O N N U M B E R

0.5 ~0 75 4,= $0 3/'$ 4S io~$ 3z 57

S P E R M C O U N T p e r ml X I 0 0 0

FIG. 7. Free amino acids and spernl numbers in semen of a Holstein bull (EB-27) subjected to ex- haustive electroejaculation.

ejaculates from the same bull• Most interesting is the change in percentage of live sperm. These values were low in the first four collec- tions, became elevated in the next six collec- tions, and were again lowered as the AV col- lections were carried to exhaustion. Subsequent electric stimulation gave ejaculates that showed

1414 ~xl. L . H O t ' W O O D . L . t ' . I "AUI , I ,~NEIC A N D F. X, ( L \ S S N I ' : R

NO. ?a?

ASNOR~IAL SPERN

IOC

a ¢ i

7e

50 ib---~ pmuA~y I X ~ ~CONDARy i

40 \ Y

I

' ° V "\ . . . . 1 / \ , / \ " \ x / - -X~ . x / "NX/I X~X.~x~X

. . . . . . . , , , , , , , , . I . . . . ~OLL( CTION 2 4 6 e tO ~ 14 t ~ IQ 20" ~2 ' ~4

NO" ARTIFICIAl" VA6tNA I (LECTROEJACULATION

FiG. 8. Effect of exhaustive ejaculation on sperm morphology" of an infertile Hereford hull (no. 727).

no increase in spenn numbers, lint the per- centage of' live sperm in these fraeti<ms in- creased considerably. For several weeks fol- lowing this treatment the bull yMded semen of better quality, in that morphological ah- normalities attd dead spermatozoa were les- sened on a pereentagc hasis.

D I S C U S S I O N

Sperm coneentrati(m is highest during earlier ejaculates and declines steadily in subsequent ejaculates with but few exceptions. On a basis of total sperm delivered, however, this de(.line is not observed. Bialy and ~mith (3) have de-

duced, from slaughterhouse speeimens, that the aml)ulht and ductus deferens of the hull do not uontain sufficient cells to form a normal ejacu- late, whereas the eauda epididymis, containing about nine billion spermatozoa, should furnish suttieient cells for a nmuber of ejaculates. By the techniques employed here, between 6 and 11 billion sl)ermatozoa were flushed from the reproduetive tract which distinctly should he delivered by the epididymis.

The fructose concentration, correlated in- versely with the pH, indi<.ates that the fruc- tose-containing fluid (from the seminal vesicles) of the ejaculate is responsible for the acidic nature of freshly ejaculated semen and that the fluid with the higher pH is a product of the bulbo-urethral glands, since it is also low in nitrogen (13). Even in the vasoligated bull this relationship between nitrogen, fructose, and 1)It holds. It would, therefore, appear that the sperm are delivered with seminal vesicular fluid, and are not released concurrently with hulbo-urethral, or possibly, with pr<>statie fluid which contribute little t'ruetose or seminal ni- trogen.

The relationship of the sen,inal g'lutamie acid to sl)ermat<~zoa as earlier observed hy u,~ (11) is further established in these exl>eriments. sinee the delivery of this eomp<ment fluctuates with the sperm concentration of the ejaculate and is uegligihle if the testieula,' l)V<)¢lu<.ts are

N O . 7 2 7

% L I V E SPERM

IOO

8O

TO

60

° ! 4 0

3 0

2 0

/ X

COUNT

~, ~, L IVE SPERM

X

X /

X-.... / " t

SPERM COUNT rnm 3 I000

I000

9 0 0

8 0 0

7 0 0

6 0 0

5 0 0

4 0 0

300

2 0 0

I00

0 0 I I Y , . , . , . v v v X I I _

COLLECT ION 2 4 ' 6 6 IO - - ~ 14' 16 ~ 16 20 22 24

NO. ART IF IC IAL VAGINA I ELECTROEJACULATION

~ . 9. Effecf of exhaustive ejaculation on sperm numbers and percentage of live sperm in the seminal fluid of an infertile Hereford bull (no. 727).

COMPOSITI()N (H." B()VINI,: SEMEN 1415

not delivered to the seminal fluid ,)r if the sperm eoneent ra t ion is low.

Of in teres t is the morphology of the sl>er- matozoa. Swanson and Boyd (16) repor ted tha t morphologica l abnormal i t i e s decreased du r ing successive collections, whereas moti l i ty improved in three Je r sey bull~ with coiled-tail deformit ies . I n our exper i lnents wi th the in- fer t i le bull, successive e jacula t ions resul ted in decreased number s of secondary abnormal i t ies , accord ing to Biota (4) of ep id idymal origin, and an increased n m n b e r of p r i m a r y a b n o f realities of' tes t icular origin. The decrease in secondary abnormal i t i e s may be i n t e rp re t ed to mean t ha t cer ta in ep id idymal sperm were ini- t ia l ly flushed out to be replaced wi th sperm whose existence in the o rgan was of shor te r dura t ion . The increase in live spermatozoa on repea ted e jacula t ion is especially in teres t ing , since it has been de termined t ha t sperm f rom the p rox imal por t ions of the epid idymis show a g rea te r stainino' affinity to eosin than those f rom the eauda (5) . The increase in the per- centage of live sperm is indicat ive of an epi- d idymal dysfunc t ion in t ha t the s torage of sperm in this o rgan a p p e a r e d to be at fau l t in the reproduct ive problem of the bull.

ACKNOWLEDGMENTS

We are grateful to Dr. E. J. Carroll and Sir. J. J. Prosser for help in eolleetiolt and evalua- tion of semen and to Mr. J. F. Masken for assistance in the analyses.

REFERENCES

( l ) AL~IQUIST, J. O., A.~LXN~', R. P., AXD O'I)ELL, W. T. Spenn Reserves of Dairy Bulls as Deternfined by Depletion Trials and Post- Slaughter Sperm Counts. J . DMry Sei., 41: 733. 1958.

(2) AMANN, R. P., AND ALMQUIST, J. O. Repro- ductive Capacity of Dairy Bulls. I I . Gonadal and Extra-Gonadal Sperm Reserves as De- termined by Duct Counts and Depletion Trials; Dimensions and Weight of Genitalia. J . Dairy Sci., 44: 1668. 1961.

(3) BIALY, O, AND S~[I'I'H, V. R. Number of Spermatozoa in Different Par ts of the Re- productive Tract of the Bull. J. Dairy Sci., 41: 1781. 195,%

(4) B~.t>_~r, E. Interpretat ion of Spcrmatie Cytol- ogy ill Bulls. Fertil. and Steril., 1: 223. 1950.

(5) BRO('HA~'~', 3[. Origin of Half-Stained Sper- matozoa Obtained from Live-Dead Differ- ent iat ing Stains. Proc. Soe. Stud. Yertil., 5: 82. 1!!53.

((;) GaASS~AX, W., axD H~xxm, K. Ein ei~- faehes Verfahren zur koutinvierlichen Tren- mmg yon Stoffgemisehen auf Fil terpapier durch Elektrophorese. Natul.wisseuschaften, 37: 397. 1950.

(7) I-IALE, E. B., xxI) AL-~QU*Sa', J. O. Relation of Sexual Behavior to Germ Cell Output in Farm Animals. J. Dairy Sci., 43 (suppl.) : 145. 1960.

(8) "[{ALE, E. B., ALMQUIST, J. O., AND THACKLR, D . L . Observations on the Sexual Behavior and Semen Production of Dairy Bulls. J. Dairy Sci., 36: 573. 1953.

(!)) ]ilLL, H. J., SCOTT, F. S., HOMAN, -~., AND GASSXEg, F. X. Electroejaeulation in the Bull. J . Am. Vet. Med. Assoc., 128: 375. 1956.

(10) HOWWOOD, M. L., AND GASSNER, F. X. Ex- haustive Eleetroejaeulation in tile Bull. Fed- eration :Proe., 19: 370. 1960.

( ] ] ) "HOP'iVOOD~ M. L., AND GASSNER, ~. X. The Tree Amino Acids of Bovine Semen. Fertil . and Steril., 13:290. 1962.

(72) L_x~sox, B. L., A.Xl) SALISmmY, G. W. The Proteins of Bovine Seminal Plasma. I. Pre- liminary and Electrophoretie Studies. 5. Biol. Chem., 206: 74]. 1954.

(13) IA'TWAK-3]I.kNN, C., AND ROWSON, L. E. A. The Chenfical Composition on the Pre-sperm ]Praetion of Bull Ejaculate Obtained by Electrical Stimulation. J. Agr. Sci., 43: 131. 1953.

(14) 3I_~xx, T. Fructose ( 'ontent and Fruetolysis in Semen. Practical Application in the Eval- uation of Semen Quality. J. Agr. Sei., 38: 323. 1948.

(15) MAXX, T. The Biochemistry of Semen. p. 35. John Wih'y and Sons. New York. 1954.

(16) SWAXSOX, E. W., .xxD BoYI), L. J. Factors Affecting Coiled-tail Spermatozoa in the Bull. Am. J. Vet. ]~eseareh, 23: 300. 1962.

(17! \Vx[,'l,oy, A., aXD Emvam)s, G. Criteria of l"ertility in the Bull. 1. The Exhaus¢ion Test. Proe. Am. Soc. Animal Production, ;~1 : 254. 1938.