Glycolytic Activity in Mixed Semen Samples1, 2

G L Y C O L Y T I C A C T I V I T Y I N M I X E D S E M E N S A M P L E S 1." ~I. H. E H L E R S , N. S. K U S I { W A t l A , _xx~) R. E. ERB

Departme~t of Dairy Seiel,'~,, ,State Colb, ge of W(isl~i~lgto~t. Pidlma~t

SUMMARY Glyeolytic activity in mixed seme~_ was compared by mea~urino" fru~.tose and lactic acid changes during 1 hr. of iueubation at 37 ° C. A m~x was made of the semen from two bulls and ultimately semea from each bull wa,~ mixed with semen from each of the other six bulls. M.ixing of semen from two bulls ~/t a time was replicated four times for a total of 84 mixes with accompanying unmixed controls. On an over-all basis, fructose utilization and lactic acid accumulation were stinmluted by nfixing; lactic acid ehauges were somewhat less pronounced. Mean values showed mixed sample fructose util.ization to be 8 ~ higher. A part of this increase appeared to be due to a more favorable fructose ~upply in some samples owing to nfixing, but other stimulation related to mixing of semen from two sourees is probably involved. Although semen from the diff'eretat bulls varied substalltially in quality, there was no strong individual bull influence contributing t~) the stimuhttion. Hess et al. (9) r e p o r t e d t h a t m i x i n g semen of three or f o u r b u l l s gave a h i g h e r n o n r e t u r n rate t h a n m n u i x e d c o n t r o l semen. The m i x e d semen had a g r e a t e r p e r c e n t a g e of live s p e r m a n d b e t t e r m o t i l i t y d u r i n g seven days of r e f r i g erated storage. C a m p b e l l a n d Jaffe (3), however, observed no i m p r o v e m e n t i n m o t i l i t y w i t h m i x i n g . G a s s n e r ct al. (7) f o u n d t h a t n f i x i n g of b u l l semen h a d n o d e t r i m e n t a l effects on the a b i l i t y of s p e r m to use fructose. M i x i n g of s e m e n obviously can have little direct a p p l i c a t i o n i n cattle b r e e d i n g . However, it m a y be possible to use this i n f o r m a t i o n , if the n a t u r e of a n y beneficial effects can be established. E n h a n c e m e n t of f e r t i l i t y would a p p e a r to be d u e to h a v i n g s p e r m f r o m more t h a n one source, to h a v i n g s e m i n a l p l a s m a from more t h a n one source, or to both. The p u r p o s e of this e x p e r i m e n t was to d e t e r m i n e w h e t h e r m i x i n g affected glyeolytic a c t i v i t y of semem T h r e e p r i n c i p a l a l t e r n a t i v e r e s u l t s were v i s u a l i z e d : (a) no change i n g l y c o l y s i s ; (b) s t i m u l a t i o n ; (c) i n h i b i t i o n . None of these exeluded the others, because semen samples m i g h t v a r y in response to m i x i n g . Beneficial effects m i g h t not n e c e s s a r i l y be those associated with s t i m u l a t i o n : it is conceivable t h a t i n h i b i t i o n of glyeolysis is desirable. EXPERIMENTAL PLANS AND PROCEDURES S e v e n b u l l s p r o d u c i n g semen w i t h s u b s t a n t i a l v a r i a t i o n i n a c t i v i t y were selected on the basis of pre-experimel~tal i n f o r m a t i o n . W i t h such semen, a n y effects due to m i x i n g should be more detectable. These bulls were f r o m the A n g u s , G u e r n s e y , Holstein, a n d J e r s e y breeds, a n d r a n g e d f r o m 2 to 7 yr. i n age. Received for publication April 2, 1958. 1This investigation was supported in part by funds provided for biological and medical research by the State of W~lshington Initiative 3[easure No. 171. 2Scientific Paper No. 1709, Washington Agricultural Experiment Stations, Pullman. Project 1193. 1395

1396

M. I-I. EI-II, E R S , N. S. K I S H W A H A ,

A N D R. E. E R B

Semen f r o m each bull was mixed with semen from each of the other six bulls. A given mixing was made with semen from two bulls. Thus, there were 21 combinations of mixed semen from the seven animals. Combinations were replicated four times for a total of 84 mixings. Each mix had as its controls the unmixed semen from the two contributing samples. About 15 rain. elapsed between collection and the beginning of controlled incubation. The mixed and the unmixed portions were incubated under essentially anaerobic conditions with four p a r t s of citrate buffer for 60 rain. at 37 ° C. The general procedure has been previously described (6). Fructose measurement was by the method of Roe (13) as modified by this laboratory (6). Lactic acid deternfination was as outlined by B a r k e r and Summerson (1), except that 0.5 g. of calcium hydroxide was used. F o r the initial and 60-rain. lactic acid measurements, filtrate equivalent was 0.05 and 0.01 ml. of semen, respectively. Fructose and lactic acid changes during 60 rain. of incubation were expressed on a " p e r milliliter of s e m e n " and " 1 0 "~ s p e r m " basis. Estimated fructose changes in I0 rain. were derived using first-order reaction formulae (6. t0). " E x p e c t e d " fructose and lactic acid chanzes per 1 ml. mixed semen = actual change in 1/z ml. of semen from one contributing bull (first control) + actual change in 1/2 ml. of semen f r o m the other contributor (second control). Expected changes by 109 sperm were calculated from these values. Differences between the expected glycolytie changes and those actually observed in mixed semen were examined statistically by the " t " test (14). Motility estimates were on the basis of percentage progressively motile at the beginning of incubation in the citrate-diluted semen. 5[otility was not rated in mixed semen because of the subjective nature of such evaluation. S p e r m concentration was determined by hemocytometer. RESULTS

Table 1 shows glyco]ytic data for the umnixed controls to serve as a comparison with other studies. The desired variability of semen was achieved as indL cated in the fructose utilization by 109 sperm in 60 rain., where bull means ranged f r o m ].24 to 2.31 rag. Motility ranged from 45 to' 6 9 ~ , and other values in Table 1 show sizable differences between bulls. Note, however, that none of the bulls produced semen having extremely high levels of fructose (6). The differences between actual and expected glyeolytic changes in mixed semen are shown (Table 2). Fructose utilization per milliliter of mixed semen averaged 2.00 mg. in 60 rain. (Table 2), which was 0.16 rag. higher than expected (P < 0.01). Utilization by 109 sperm was 2.02 mg., which was higher than expected by 0.18 rag. ( P < 0.01). Fruetolytie rates depend upon sperm numbers, the amount of fructose, and the length of iueubation (10, 12). Therefore, mixing of semen should, with some samples, result in a more favorable sperm-to-fructose ratio, p a r t i c u l a r l y with longer incubations. This would simply be a matter of nutrient availability. F r u e tolytie rates are generally underestimated in semen that has high numbers of

TABLE

1

Glyeolytic data from independently incubated seme~ (12 ejaculates

F;ull

G-9 G-I o ,J-] 8 ] 1-32 H-34 A-60 A-64 Menn

Initial motility

Colleentration per ml. o f semen

(%)

(10' sperm)

62 52 {19 69 69 45 57 6(}

1.34 1 .()s 1.10 1.16 o.97 (/.83 0.75 1.03

" Esfimafed

Initial fructose p e r ml. of semen

Fructose

-

per bull) utilization .

P e r nil. of semen in 60 rain.

B y 109 s p e r m in (i(I r a i n .

P e r m l. of Sellle]l

in 10 r a i n . "

.

.

.

By l 0" S])ornl ill 1 l) rain."

Initial lactic a('id p e r nil. of semen

l , a c t i e "reid a c c u m u l a t i o n P e r nil. of seln¢ll in 60 r a i n .

Sl)erlll ill

B y 10 o

1.69 1.1.5 1.57 1.40 1.46 0.78 1.09 1.30

1.21 1.07 1.51 1.25 1.54 0.95 1.40 1.28

60 r a i n .

(m:/.) 5.90 3.90 4.60 5.65 5.44 :L36 3.40 4.61

o n }raMs o f f i r s t - o r d e r r e a c t i o n

2.18 1.34 2.(15 2.54 2.02 1.22 .1.53 1.84 (see

R e f e r e n c e 6').

1.62

1.24 ] .90 2.31 2.09 1.93

2.119 1.88

0.50 0.;~3 0.4,~ 0.60

0.44 0.27 (I.34 0.42

0.36 O.31 0,43 ().5:1 0.44 (L39 0.4(~ 0.41

().5". 0.5:~ 0.5a (}.48 0.5~ 0.28 0.32 0.4(;

M. H. E H L E R S , N. S. K I S H W A H A . AND R. E. ERB

1398

TABLE 2 Differences between actual and e x p e c t e d y l y e o l y t i e vaMes f o r :nixed semen

(21 bull combinations; 84 mixes l Lactic acid accumulation

Fructose utilization Senlen from Measurement 6 other bulls in mixed mixed with: semen

Per ml. semen in 60 rain.

By 10 ~' s p e r m ill

61) rain.

Per nil. senleu in 10 lniu. ~

By 10' sperm in 10 lnin. a

(rag,) -

G-9 G-11) J-] 8 H-32 H-34

Actual Difference b Actual Difference Actua[ Difference Actual Difference Actual

A-60 A-64 All combinations

Difference Actual Difference Actual Difference Actual Difference

2.11 +0.15 1.7(J +0.09 1.92 +1).16 2.47 +g.18

1.84 ~-0.18 1.51 +IU)7 2.0(1 +9.14 2.32

±0.19

0.47 +(~,(11 0.:',9 0.(m 11.43 =0.1.)2 O.56 --fUJi

Per ml. semen in 60 nliu.

By 10 ~' sperm in 6{Prain.

1.59 +0.18 1.27 --(1.05 1.40 +o.04 1.36 +0.14

1.:45 +t~.15 1.14 --0.06 1.46 +t/.o:I 1.22 +0.11 1.42

-

0.4(* +0.1)3 tl.35 ~0.(11 0.43 +1).~)1 0.52 ~1.02

2.q74

2.14

q~.44

1~.45

1.41)

+0.18 1.79 +0.24 1.95 +0.11

m11.24

=-11..:~ 0.4{~ ~¢~.tJ{i 0.4.5 ±0.02 II.45 +0.(}:i

-11.04 0.46

--0.00 1.09

+0.07

~-1).02

0.51 +0.04 0.44 +0.(1:~

1.40 +0.06 1.36 +0.1)6

2.11 +O..~1 2.22

+0.17

2.00

2.02

+0.16

+0.18

--(I.02

1.24 +/).04 1.53 +0.06 1.34 +11.04

" Estimated on basis of first-order reaction (see Reference 6). b Difference from expected value based on independent incubation of contributing samples. s p e r m or low levels of f r u c to s e , or both. U n d e r e s t i m a t i o n of f r u e t o l y s i s is accent u a t e d as the i n c u b a t i o n t i m e increases. E s t i m a t i o n of f r u c t o s e u t i l i z e d in 10 rain., u s i n g f i r s t - o r d e r r e a c t i o n f o r m u l a e , p r o v i d e s a u s e f u l t e c h n i q u e in m i n i m i z i n g v a r i a t i o n a t t r i b u t a b l e to s p e r m n u m b e r s a n d a m o u n t s of f r u c t o s e (6'). A p p l i c a t i o n of this t e c h n i q u e s h o u l d i n d i c a t e w h e t h e r t h e r e is s t i m u l a t i o n ( o r p e r h a p s i n h i b i t i o n ) of f r u e t o l y s i s a p a r t f r o m t h a t causeft by a l t e r i n g s p e r m and fructose relationships. E s t i m a t e s f o r 10 rain. show m i x e d semen u t i l i z a t i o n of 0.45 a n d 0.44 rag. p e r m i l l i l i t e r semen a n d by 109 sperm, r e s p e c t i v e l y . These e x c e e d e d e x p e c t e d v a l u e s f o r 10 rain. by 0.02 a n d 0.03 rag. ( P < 0.025). M i x e d sem en f r u c t o s e u t i l i z a t i o n p e r m i l l i t e r s e m e n in 60 rain. was 8.0% h i g h e r t h a n ex p ect ed , w h e r e a s e s t i m a t e d u t i l i z a t i o n in 10 rain. was 5.1c/, h i g h e r t h a n expected. C o m p a r a b l e u t i l i z a t i o n s b y 109 s p e r m w e r e 9.2 a n d 7.0qYc h i g h e r t h a n expected. Thus, p a r t of the h i g h e r f r u c t o s e u t i l i z a t i o n in 60 rain. in m i x e d semen is d u e to m o r e a v a i l a b l e s u b s t r a t e . b u t t h e r e also a p p e a r s to be a f r u e t o l y t i e s t i m u l u s f r o m a n o t h e r f a c t o r or factors. On an over-all basis, lactic acid a c c u m u l a t i o n p er m i l l i l i t e r semen in 60 min. a n d by 10 "~ s p e r m ( T a b l e 2) was o n l y s l i g h t l y h i g h e r t h a n e x p e c t e d ( P < 0.20). T h e r e is no p r e s e n t basis f o r e s t i m a t i n g lactic acid c h a n g e s in a s h o r t e r t i m e i n t e r v a l , such as was a p p l i e d to th e f r u c t o s e values. Th e d a t a w e r e also a n a l y z e d f o r i n d i v i d u a l bull influences on g l y e o l y t i c c h a n g e s in m i x e d semen. I n d i v i d u a l l y , 59 mixes showed m o r e an d 25 showed less t h a n expected, or no difference in f r u c t o s e u t i l i z a t i o n d u r i n g 60 rain. 0 f the 84 mixes, however, o n l y 46 showed m o r e lactic acid a c c u m u l a t i o n t h a n expected.

G L Y C O L Y T I C A C T I V I T Y I N SEMEN

1399

This f u r t h e r indicates that lactic acid production is less influenced by mixing. Some bull combinations (four mixes to a two-1)ull combination) had less fructose utilization than the unmixed controls, but when the six combinations to which a bull contributed were considered, a(.tual fructose utilization in mixed samples averaged higher t h a n expected for all bulls (Table 2). With regard to lactic acid acemnulation, two of the seven bulls, considering all of their combinations, had less than expected lactic acid aeemnulation in mixed semen. The difference between actual and expected fructose utilization per milliliter of semen in 60 rain. ranged from + 0.09 rag. for combinations including semen of G-10 to + 0.24 rag. for combinations including A-60 (Table 2). Fiducial limits at the 959} confidence level were calculated for differences between actual and expected fructolytic activity in mixed samples of the individual bulls (as shown in Table 2). At least one of the ficlucial limits for the combinations of a particular bull fell within the rauge established for each of the other bulls. This overlapping of fiducial limits for differences in activity between actual mixed and expected value was true for all of the other measurements of fructose and lactic acid changes. These lmlls produced semen having substantial variation in metabolic activity (Table 1), but the differences attributable to mixing did not have a strong individual bull influence. D[SCUSSION

The report of Hess ct al. (9) indicates that mixing of semen m a y improve fertility. The present work shows that mixing m a y increase fructose utilization and to a lesser extent lactic acid accumulation during 60 rain. of incubation at 37 ° C. I n s o f a r as fructolytie aspects are concerned, some of this increase is a p p a r e n t l y a result of more available fructose attributable to mixing and particularly evident with longer incubation. However, p a r t of this increase is probably due to other stimulation. Chang (4) found that dead spernl f r o m a bull improved the fertilizing capacity of rabbit semen. Thus. there is the possibility that improved fertility and higher glyeolytic activity of mixed bull semen are a result of having sperm f r o m more than one source. The other principal alternative explanation for a ~timulus is the presence of a foreign seminal plasma. L a r d y and Ghosh (11) noted no fertility difference between epididymal and ejaculated sperm f r o m several bulls which, in effect, minimizes the contribution of the fluid portion of semen. Change ( l ) , however, found that seminal plasma from the rabbit improved the fertilizing capacity of ejaculated rabbit sperm. Ghosh and L a r d y (8) reported that seminal fluid of the bull a p p a r e n t l y activated sperm through a regulator associated with the epididymal sperm. Bishop and Salisbury (2) suggest that some constituent of semen depresses oxygen uptake of whole semen. None of these observations, however, provides a direct answer to the nature of any stimulus attributable to mixing of semen. REFERENCES (1) BARKER,S. B., AN]) SUM~ZRSOX,W. H. The Colorimetric Determination of Lactic Acid in Biological Material. J. Biol. Clt,.m., 138: 535. 1941.

1400

M. H. EHLERS, N. S. KISI[WAHA, AND !R. E. ERB

(2) BISHOP, M. W. H., AND S.~.LISBURY, (~. ~V. Effect of S p e r m C o n c e n t r a t i o n on the O x y g e n U p t a k e of Bull Semen. Am. J. Physiol., 180: 107. 1955. (3) CAMPBELL, 1~. C., A-','D JA~'FE, W. P. The Motility of Mixed Semen. J. :tgr. Sci., 50: 65. 1958. (4) Cm~-~G, M. C. Effect of Testis ]Hya]uroni~blse a n d S e m i n a l F h f i d s on the F e r t i l i z i n g C a p a c i t y of l~abbit Spermatozoa. Proc. Soc. E.,'ptl. Biol. Mrd., 66: 51. 1947. (5) CHANO, M. C. E f f e c t s of H e t e r o l o g o u s S e m i n a l P l a s m a a n d S p e r m Cells on F e r t i l i z i n g C a p a c i t y of R a b b i t Spermatozoa. P,'oc. Soc. E.rptl. Biol. lied., 70: 32. 19:~9. (6) ERB, ]:~. E., PLEICCHINGER, F. H., EHLERS, ~][. H., AND GASSNER, F. X. M e t a b o l i s m of Bull Semen. I I . F r u c t o ] y s i s Rebltionshit)s with S p e r m ConcentratimL a n d F e r t i l i t y . J. Dairy Sei., 39: 326. 1956. (7) GASSNEg, F. X., RUTI-IERY0RD, E. ~., HOPWOOD, ~[. L., AND ]=IILL, I]. J. Effect of C a s t r a tion a n d Steroid T h e r a p y on Senlinal P l a s m a with Respect to F r u c t o s e U t i l i z a t i o n by N o r m a l Bull Sperm. Proc. Soc. IZ.rptl. Biol. M~d., 81: 43. 1952. (8) GH0SH, D., AND LAR~Y, H. A. The N a t u r e of a Y e a s t - S t i m u l a t i n g A g e n t in M a m m a l i a n Spermatozoa. J. ,4~iimal Sei., 12: 543. ]952. (9) HESS, E. A., LUDWICK, T., RI('KAF,D, H. C., AND ELY, F. Some of the Influences of Mixed E j a c u l a t e s u p o n Bovine Fertility. J. Dairy Sei., 37: 649. 1954. (10) HOPWOOD, M. L., RUTHERFORD. E. R., .XXD GASSXER, P. X. T h e Concept of F r u c t o s e Utilization by Bull S p e r m a n d I t s Relation to Fertility. J. Dairy Sei., 39: 51. 1956. (11) LARDY, H. A., AND GHOSH, ~). C o m p a r a t i v e Metabolic B e h a v i o r of E p i d i d y m a l a n d E j a c u l a t e d M a n m m l i a n S p e r m a t o z o a . . 4 n ~ . X. Y. Acad. Sci., 55: 594. 1952. (12) MIXNEP~,J. P., MATHER, R. E., A.',-DPREUND, hi. Bovine Semen Metabolism. I. M e t h o d s for E x p r e s s i n g F r u c t o l y t i e Activity. J. Dairy Sei., 40: 142. 1957. (13) ROE, J. H. A Colorimetrie M e t h o d for the D e t e r m i n a t i o n of F r u c t o s e in Blood a n d Urine. J. Biol. Chem., 107: 15. 1934. ( t 4 ) SNE[mCOR, G. W. Statistical Methods. 5th ed. T h e Iowa S t a t e College Press, Ames. 1956.