Effect of Sexual Rest and Frequency of Ejaculation on Sperm Acrosomal Morphology 1

M. E. WELLS, O. A. AWA, L. G. JAY, and S. S. FANCY Department of Animal Sciences and Industry, Oklahoma State University

Sti]lwater 74074

Abstract

Effects of sexual rest and frequency of ejaculation on the acrosome were investi- gated. I n the first phase, two groups of three bulls each were used to determine the possible effect of sexual rest and collection frequency on acrosome morphology. All bulls had been ejaculated once weekly for several months. Aged acrosomes in Group 1 bulls, switched from once weekly ejacu- lation to two ejaculations, two days per week, with no sexual rest, decreased from 17.0 to 5.0%. Conversely, aged acrosomes in Group 2 bulls, sexually rested for 6 weeks, increased from a prerest 18% to a postrest 42.2%. Resumption of four-time weekly ejaculation reduced the incidence similar to Group 1 within 2 weeks.

In the second phase of the study, four of the bulls were given 4 weeks sexual rest and then assigned to groups ejaculated either once or four times weekly for 1 month. The groups then switched frequen- cies and were collected for 1 month. Four times weekly ejaculation consistently re- sulted in fewer aged or aging sperm cells and greatly increased the total yield of sperm cells with no morphological evidence of aging.

Introduction

Considerable research has been conducted during recent years to determine effects of fre- quency of semen collection upon semen quality of the bull. In general, studies (1, 7, 9) have shown greater sperm cell yield by bulls associ- ated with increased frequencies of collection. Several studies have suggested that per cent live cells or per cent normal cells per ejaculate are not materially affected by frequency of ejaculation (3, 9). Few reports exist wherein the relationship between frequency of ejacula- tion and acrosomal characteristics is defined. l=lafez and Darwish (8) suggested that short

Received for publication August 11, 1970. Journal Paper 2071 of the Oklahoma Agricul-

tural Experiment Station, Stillwater 74074.

intervals between collections led to detached acrosome caps and enlarged midpieces. Wells et al. (15) found in dairy bulls that once weekly ejaculation after sexual rest reduced aged acro- somes from 29.3% initially to 12.5% by the sixth week. Saacke et al. (12) and Wells and Awa (14) reported significant weekly varia- tions in per cent of altered acrosomes among bulls and among collection periods on ejacula- tion frequencies of twice weekly and once weekly. No reports exist detailing the possible effect of ejaculation frequency on acrosome characteristics. A par t of our study was de- signed to yield information on this problem.

The effect of sexual rest on reproductive per- formance is controversial. Bonnadonna (6) sug- gested that periods of sexual rest exceeding 2 to 3 weeks may cause a gradual deterioration of semen quantity and quality. Schmidt et al. (13), using sexual rest periods up to 13 days, concluded that optimum semen quantity was obtained with a 4-day rest period. )Sartig et al. (11) reported, however, that a 44-week sexual rest period has no observable effects of the freezability of bull semen. Bedford (4) ob- served that older sperm cells lost the acrosome more readily in the uterus of either estrous or pseudopregnant rabbits. Blom (5) and Wells et al. (15) and Wondafrash (16) suggested that periods of sexual rest of 2 to 3 weeks or more will lead to high percentages (18 to 21.2%) of altered acrosomes. They also found that weekly semen collection significantly decreased percent- ages of acrosomal anomalies. The second facet of this report is to present fur ther information concerning effects of sexual rest on acrosome characteristics.

Experimental Procedures

Six bulls, 3 to 4 years of age and managed similarly, were utilized for the first par t of this study. These bulls had been ejaculated once weekly for 13 months and the state of the acro- some as well as the variation encountered was established. To secure preliminary indications of the possible effect of frequency of ejaculation and sexual rest on the acrosome state, three of these bulls (Group 1) were changed, with no

526

S P E R ~ ACROSOMAL ~ O R P H O L O G Y 527

sexual rest period, to a frequency of four times weekly (2 ejaculates per day on Mondays and Thursdays) for 5 weeks.

The other three bulls (Group 2) were placed on sexual rest for 6 weeks and then ejaculated four times weekly until their acrosome state was similar to that of the bulls in Group 1 that received no sexual rest.

The results of our prel iminary trials prompted a tr ial designed to shed more light on the effects of sexual rest and frequency of ejaculation on acrosomal aging. Two bulls had to be elimi- nated from the previously described groups. Af te r completion of the prel iminary trials, the remaining 4 bulls were placed on sexual rest for 4 weeks and then randomly assigned to pairs subjected to either a weekly ejaculation or the four-time weekly ejaculation as in the prel iminary trial. Af ter i month at these fre- quencies, the groups were switched, with no rest period, with the weekly bulls changing to four times weekly and the four-time bulls chang- ing to the weekly rate. Bulls were subjected to these frequencies for 1 month.

Semen was collected with the artificial vagina with a 5 to 8 min stimulation period, including 1 false mount, being typical for first ejaculates. Second ejaculates, where appropriate, were typ- ically collected within 30 min of the first ejacu- lates with the teaser animal being changed and a similar stimulation period.

Semen samples were maintained at 30 to 35 C while data were being collected. Ejaculate vol- ume was recorded directly from graduated col- lection tubes. Sperm cell concentration was de- termined colorimetrically. The percentage live sperm was determined from 200-cell-counts on slides prepared by using the nigrosin-eosin dif- ferential stain described by Hancock (10). The percentage of morphological abnormalities was determined from the live-dead stains, disregard- ing the live-dead differentiation. Ejaculate p H was measured, shortly after collection, with a p H meter. Acrosomal characteristics were de- termined by the differential staining procedure of Awa (2). Two hundred sperm were counted per stained preparat ion and were recorded as possessing a nonaged acrosome, i.e., a smooth, closely adherent acrosome, or showing evidence of aging of the acrosome. Aging acrosomes in- cluded cells with swollen, irregular outline, ele- vated, and disintegrating acrosomes as well as those with missing aerosomes. Knobbed acro- somes, a genetic abnormality~ were observed on the sperm cells of 1 bull throughout the study. These were recorded separately from the aged category and are not included in any of the data in this report.

Appropr ia te analyses of variance were used in all statistical comparisons.

Results and Discussion

The results of the preliminary trial are in Figure 1. Group 1 bulls, which were changed from 1 ejaculate to 4 ejaculates per week with no sexual rest, showed a steady decline and less variation in the percentage of aged aerosomes per ejaculate. The average acrosome state for the 5 weeks at the high frequency was 11.8% sperm with aged acrosomes, or, 88.2% of the cells showed no morphological evidence of ag- ing. The average of the same 3 bulls for the 10 weeks pr ior to increasing the frequency of ejaculation was 23.7% aged sperm. These dif- ferences were highly significant (P < .005) and indicate that the state of the acrosome can be greatly influenced by frequency of ejaculation.

F igure 1 indicates that the 6 weeks of sexual rest resulted in deterioration of acrosome qual- i ty for Group 2 bulls. There was no significant difference between the groups at the initiation of sexual rest. However, aged aerosomes rose from 18.0 to 42.2% in the group rested for 6 weeks. On resumption of ejaculation four times weekly, the level dropped precipitously in 2 weeks to levels similar to the group that had not been rested. I t is of interest that the per- centage of aged acrosomes for both groups on four times weekly ejaculation was reduced to about half that apparent ly typical on once weekly ejaculation. Both sexual rest and fre- quency of ejaculation can have a rather drastic influence on apparent age of the acrosome.

The results of the second phase of the study are in Figure 2. The 4 weeks of sexual rest again had detrimental effects on the acrosomal state. Percentage of sperm cells with no up-

• • GroupGr°up 21 lx/wk 4x/wk

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~ ... ~ - ~

% o I I [ I 1 ] 1 I I

1 2 3 5 7 8 9 10 11 TIME IN WEEKS

FzG. 1. Effects of sexual rest and changing ejaculation frequency on acrosomal state, a

a Each line is the average of 3 bulls, with either one ejaculate or two ejaculates per collection day.

JOURNAL O~ DAIRY SCIENCE VOL, 5~, NO. 4

528 WELLS ET AL

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90

80 o=

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- - • Group 1 Group 1: 4x/wk • Group 2 Group 2: l x / w k

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so I I I Levels prior to 1 2. 3 4 5 6 7 8 sexual re~t TIME IN WEEKS

~IG. 2. Effects of sexual rest and ejaculation frequency on levels of nonagecl acrosomes. ~

Each line is the average of 2 bulls with either one ejaculate or two ejaculates per collection day.

parent aging of the acrosome for Groups 1 and 2 decreased from 94.2 and 94.8% in the average of the last two ejaculates prior to sexual rest to an average of 63.2% and 67.8% in the first two ejaculates following that period. Conversely, the proportions of aged acrosomes increased from 5.8 and 5.2% to 36.8 and 32.2%. This illustrates a deterioration in acrosomal quality from this 4-week period of sexual rest.

The effects of ejaculation frequency on this deteriorated state is shown in F ig . 2. Nonaged sperm cells in both groups were within 5% of each other in the first ejaculates following sex- ual rest. However, the percentage of nonaged sperm cells in Group 1 bulls, ejaculated four times weekly, rose sharply from 63.2 to 91.5% in the 4 weeks~ and averaged significantly higher {P < .05) than Group 2 which rose gradually from 67.8 to 81.5% in the same 4 weeks. At this point, ejaculation frequency changed. Group 2 bulls, switched from one to four times weekly ejaculation, showed a continued rise in the per- centage of nonaged sperm cells and finished 4 weeks later at 93.2% nonaged cells. Group 1 bulls switched from four to one-time ejaculation showed a reversal in their trend and finished signifieantly lower (P < .05) than Group 2 with 77.1% nonaged sperm cells. The incidence of aged acrosomes at these two frequencies of collection indicates that the acrosome state while the sperm cell is in the epididymis is subject to change. ]%at-time weekly ejaculation frequency apparently will maintain 90 to 95% nonaged acrosomes whereas the one-time weekly fre- quency will apparently maintain 75 to 80%.

Figure 3 presents a comparison of percentage of aged acrosomes in first and second ejacu- lates on increased frequency of collection. Ob- viously the degree of aging was typically lower

30 - - ~ 1x/wk14x/wk • 1st Ejaculate • 2rid Ejaculate

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COLLECTION DAYS

Fro. 3. Aged acrosomes in first and second ejaculates, a

Each line is the average of 3 bulls.

in second ejaculates. The total difference was 6.5% in the initial ejaculates on the increased frequency and decreased to 1.0% by the 5th week on the increased frequency. Additionally these data support the contention that aging does occur in the epididymis and that the de- gree of aging can be influenced by ejaculation frequency.

Table 1 gives an indication of how the acro- somal changes compare with changes in other ejaculate characteristics. Ejaculate character- istics for 3 bulls are compared for 5 weeks at one ejaculate we@ly and 5 weeks at four ejac- ulates weekly. There were expected significant decreases in volume, sperm concentration per milliliter, and the number of live sperm per ejaculate, and an increase (P < .005) in total yield of sperm cells with more frequent collec- tion. Percentages of llve sperm cells and ab- normal sperm cells did not change measurably. However, acrosome state is sensitive to manip- ulation in that large and significant decreases occurred in aged acrosomes upon increased fre- quency of collection.

The sequence of aging begins with the nero- some showing a wrinkled or irregular outline and some slight swelling of the aerosome. The swelling progresses and is usually accompanied by progressive elevation as the acrosome be- comes loosened from the sperm cell nucleus. In the latter stage the acrosome may become com- pletely detached, or, may fragment with por- tions still being attached to the nucleus. Vari- ous stages of aging were easily discernible. Ejaculates after sexual rest contained all stages of aging with free acrosomes being read- ily apparent. Regular ejaculation typically re- sulted in fewer cells showing the latter stages of aging. The predominant aerosome state seen in ejaeulates of bulls ejaculated four times

JOURI~AL O F D A I R Y S O I E N C E WC~OL. 5 4 , N O . 4

SPER~¢[ ACROSOMAL MORPHOLOGY

TABLE 1. Comparison of ejaculate characteristics at two ejaculation frequencies, a

529

Average of Per cent in- four ejaeulates crease ( + )

Average of five per week for or decrease weekly ejacu- five weeks fol- ( -- ) after lares prior to lowing the in- increasing

Ejaculate increasing ejacu- crease in ejacu- ejaculation characteristic lation frequency lation frequency frequency

Ejaculate volume (ml) 5.0 3.9 --22.3 .05 Sperm concentration

per ml ( × 106) 1,153.8 979.3 --15.4 .05 Live sperm (%) 68.3 65.7 --3.8 N.S. Number live sperm per

ejaculate ( × 106) 4,116.6 2,920.9 --29.0 .005 Nmnber sperm per

ejaculate ( × 106) 5,787.6 3,756.2 --35.1 .005 Number sperm per

week ( × 106) 5,787.6 15,024.8 +159.5 .005 Abnormal sperm (%) 16.3 14.8 --8.9 N.S. Semen p H 6.3 6.5 +3.1 .05 Sperm with nonaged

acrosomes (%) 75.2 87.9 +16.9 .005

a Each value is the average of three bulls.

weekly was a wrinkled, roughened, and slightly swollen condition.

Conclusions

A period of sexual rest of four weeks or more results in large increases in aged acro- somes. An ejaculation frequency of four times weekly reduces aged acrosomes and maintains significantly fewer than will a frequency of once weekly. The acrosome is labile and sus- ceptible to internal or external environmental circumstances or both. Thus, at higher frequen- cies of ejaculation, sperm cells will spend less time in the epididymis, and there will be less opportunity for acrosomal aging to occur. I f aging of the acrosome is related to fertility of an animal, then judicious use of frequent e~aeu- lation will result in a greater harvest of useful sperm cells.

References

(1) Almquist, J. O., and D. C. Cunnlngham. 1967. Reproductive capacity of beef bulls. I. Postpuberal changes in semen production at different ejaculation frequencies. J. Animal Sci., 26: 174.

(2) Awa, O. A. 1968. The development and efficacy of a new stain for assessing acro- somal characteristics of spermatozoa. Mas- ter's thesis. Oklahoma State University, Stillwater.

(3) Baker, F. N., N. C. VanDemark, and G. W. .~'OUR~AL OF DAIRY SCIE!"~CE VOL. 54, NO. 4

Salisbury. 1955. The effect of frequency of ejaculation on the semen production, seminal characteristics, and libido of bulls during the first post-puberal year. J. Dairy Sci., 38 : 1000.

(4) Bedford, J. M. 1963. Morphological reac- tion of spermatozoa in the female reproduc- tive tract of the rabbit. J. Reprod. Fertil., 6 : 245.

(5) Blom, E. 1946. Spontaneous detachment of the galen capitis in spermia of bull and stallion. Animal Breed. Abstr., 14: 84.

(6) Bonnadonna, T. 1956. On some bioIogical and non-biological factors that may affect the collection and quality of the semen. Proe. 3rd. Int. Congr. Animal Reprod, p. 105.

(7) Bratton, R. W., and /~. It. Foote. 1954. Semen production and fertility of dairy bulls ejaculated either once or twice at intervals of either four or eight days. J. Dairy Sci., 37: 1439.

(8) ttafez, E. S. E., and Y. H. Darwish. 1956. Effect of successive ejaculations on semen characteristics in the buffalo. J. Agric. Sci., 47: 191.

(9) Itafs, H. D., R. S. Hoyt, and R. W. Bratton. 1959. Libido, sperm characteristics, sperm output, and fertility of mature dairy bulls ejaculated daily or weekly for thirty-two weeks. J. Dairy Sci., 42:626.

(10) Hancock, J. L. 1952. The morphology of bull spermatozoa. J. Exptl. Biol., 29: 445.

(11) Martig, /~. C., J. O. Almquist, and R. P. Amaun. 1966. Bull sperm freezability after

5 3 0 W~LLS EW AL

varying ejaculation frequencies. Abstr. J . Animal Sei., 25: 927.

(12) Saacke, R. G., R. P. Amann, and C. E. Marshall. 1968. Acrosomal cap abnormali- ties of sperm from subferti le bulls, ft. A~imal Sci., 27:1391.

(13) Sclunidt, K., H. Strassburg, and G. Korriath. 1957. Effect of sexual rest on various sperm characters and fert i l i ty in breeding bulls. Animal Breed. Abstr., 25: 161.

(14) Wells, M. E., and O. A. Awa. 1970. l~ow technique for assessing acrosomal char-

acteristies of spermatozoa. J. Dairy Sei., 53 : 227.

(15) Wells, M. E., T. Wondafrash, O. A. Awa, and D. i% Stephens. 1970. Effect of sexual rest and subsequent regular collection on acrosome characteristics of bull spermatozoa. ft. Animal Sci., 31: 67.

(16) Wondafrash, T. 1968. The effect of ab- stinence on aerosome characteristics of spermatozoa of Hereford and Angus bulls. Master's Thesis. Oklahoma State University, Stillwater.

ff0IY~lfAI, OF DAIRy S01~NO ~. V0X,. 54, NO. 4