Artificial Insemination of Cattle

2314

JOURNAL OF DAIRY SCIENCE

(]3) MBLAMPY, 1%. M., EMMFACSON,M. A., fAKIrS, J. M., HANKA, L. J., AND EN~SS, P. G. The Effect of Progesterone on the Estrous Response of Estrogen-Conditioned Ovarleetomized Cows. J. Animal Sei., 16: 967. ]957. (14) NF~LOI~, Y. E., AHI~IVHOLI),J. E., AND N]~Lso~¢, 1%. H. Influence of Oral Administration of 6-Methyl-17-acetoxy-progesterone on Follicular Growth and Estrous Behavior in Beef Heifers. 5. Animal Sei., 19: 1331. 1960. (Abstract) (]5) NF~,LOrb J. E., Awl) COL~, H. H. The Hormonal Control of Estrns and Ovulation in the Beef Heifer. J. Animal Sci., 15: 6,50. 1956. (16) RAY, D. E., EI~MERSON, M. A., AN]) M~L~MPY, R. M. Effect of Exogenous Progesterone on Reproductive Activity in the Beef Heifer. J. Animal Sci., 20: 373. 1961. (17) ROBEI~TS, S. J. Veterinary Obstetrics and Genital Diseases. Edwards Bros., Ann Arbor, Mich. 1956. (18) SI~ZPsoN, M. E. 1%oleof the Anterior Pituitary Gonadotrophins in 1%eproduetive Processes. Reproduction in Domestic Animals. Chap. 3, Vol. 1. Ed., 1[. 1[. Cole and P. T. Cupps. Academic Press, New York, N. Y. 1959. (19) S~ITH, V. R., McS~tA~¢, W. H., AND CASmA,

ARTIFICIAL

L. E. On Maintenance of the Corpora Lutea of the Bovine with Lactogen. J. Dairy Sci., ~¢0':443. 1957. (20) Som~Ns~¢, A. M., HANS~'~, W., Am'~SVRON~, D. T., McE~TE~, K., AN]) Bm~TOI¢, 1%. W. Causes and Prevention of Reproductive Failures in Dairy Cattle. Cornell Univ. Agr. Expt. Sta., Bull. 936. 1959. (21) STAPLES, 1~. E., ANI) H2~.Q'S]~) W. Luteal Function and Embryo Survival in the Bovine. 5. Dairy Sol., 4~: 2040. 1961. (22) TRI~BF.aGE]~, G. W., ~ FINC]~En, M. G. 1%egularity of Estrus, Ovarian Function and Conception 1%ares in Dairy Cattle. Cornell Univ. Agr. Expt. Sta., Bull. 911. 1956. (23) ULBE~, L. C. Synchronization of Estrous Cycles. Proe. Centennial (II) Sympos. Reproduction and Infertility, p. 10~. Michigan State Univ. 1955. (24) U L B ~ , L. C., AND LI~¢])~, C. E. Use of Progesterone and Estrogen in the Control of Reproduction Activities in Beef Cattle. J. Animal Sci., 19: 1132. 1960. (25) WrS~BA~K, g. N., AN]) CASmA, L. E. Alteration of Ovarian Activity by Hysterectomy. J. Animal Sci., 15: 13.4~. 1956. (26) ZI~B]~LI~, 1%. G. The Control of Estrus and Ovulation in Heifers by Orally Administered 6-Methyl-17-acetoxy-progesterone. J. Dairy Sci., 44: 1195. 1961.

INSEMINATION

OF

CATTLE

•. L. VANDEYfAlCK Department of Dairy Science, University of Illinois, Urbana Artificial insenlination offers one of the most effective means of improving reproductive efficiency. Although making improvements in reproductive efficiency in the cow population through artificial insemination in most respects is not much different than in natural mating, the potential with the male is enormous. Maximmu reproductive efficiency is interpreted to m e a n - - g e t t i n g the maximum nmnber of calves from the parent stock per unit of time. To be sure, artificial insemination may be useful in insuring protection from disease for the cow, thus permitting a longer useful life and more frequent calving. However, earlier calving and subsequent shorter calving intervals through shorter gestation periods and optimal post-partum breeding intervals are usually as readily obtainable by natural mating as with artificial insemination (34). W i t h the male, on the other hand, artificial insemination brings with it the possibilities of increasing the offspring of a desirable sire several hundred-fold. Consider expressing reproductive efficiency by an equation proposed by the Cornell workers (14) some time ago:

Calves per bull

No. live sperm produced

per year No. live sperm per insemination X % semen used X % cows calving F r o m this, we can readily see the role played by artificial insemination in increasing reproductive efficiency is primarily through increasing the utilization of sperm produced by the bull. Maximum sperm production by bulls is still not fully realized, but great strides have been made in recent years in describing and defining many of the factors affecting sperm production. Likewise, great strides have been made following the early Cornell studies on limits of extending semen (25, 27, 28, 31). The next item in the equation, per cent of semen used, was for many years, and under certain conditions still is, a great deterrent to the full realization of a bull's potential, because of the loss of semen through aging. The last item of the equation has been left to the other speakers of this symposium, except as that factor is dependent upon the other factors of the equation for optimal conception rates. In 1957, Foote and Bratton (t4) integrated the above-mentioned factors and presented a table, partly reproduced in Table 1, to show

SYMPOSIUM

TABLE

2315 1

Interrelationship of factors affecting the possible number of progeny produced a Motile sperm per bull per year

No. of motile sperm used per cow

Per cent of sperm used

Per cent conceiving (one insem.)

50 50 10 10 10 10 10 10 5

25 25 25 25 50 50 50 50 50

50 70 50 70 50 70 50 70 65

No. of progeny

---(million) 250,000 250,000 250,000 250,000 250,000 250,000 750,000 750,000 750,000

625 875 3,125 4,375 6~250 8,750 18,750 26,250 48,750

Modified from Foote and Bratton (14). the potential offspring of a bull under conditions resulting in changes in efficiency in the several items of the equation. This table does not show the even lower number of offspring-perhaps 25 to 5 0 - - t h a t would result from natural mating. Sperm production is considered from 250 billion motile sperm per year, a reasonable estimate for a mature bull producing one ejaculate per week, to 750 billion, a conservative estimate in view of later evidence. Sperm used per insemination covers the range from early use, 50 million motile sperm per insemination or about a 1:20 extenmon rate, to five million motile sperm per insemination, a level established in the early experiments that appears to be near the minimum even today. The percentage of semen utilized is considered from 25%, a realistic picture in some operations even today, to 50%, a level still f a r from maximal. Finally, the conception rates shown are realistic of the changes that occur in many A.I. orgaaizations as they go through the trials of early growth. Although units frequently start with conception rates as low or lower than 50% (60- to 90-day nonreturns), conception rates of 70% and above have been obtained consistently by some units performing thousands of inseminations. Still, in the United States in 1960, the average A.I. sire was used to insenfinate only about 3,000 cows. Additional evidence in several areas suggests that changes are possible that will push the potential offspring of a bull many times beyond that now being obtained and even beyond the projections shown in Table 1. No attempt has been made here to cite all the references pertaining to the items discussed. However, the reviews and papers mentioned do refer to many other pertinent references. INCREASING SPER/~ PRODUCTION One of the obvious limitations to the number of calves that can be sired by a particular bull is the number of sperm produced by the

bull. Extending the useful life of a bull is one way of increasing his potential of siring offspring. I n recent years, it has been shown also that sperm production can be increased by more intense preparation and stimulation before collection and by more frequent collections of" semen. Extending the useful life of bulls. In 1954, Becker et al. (6) showed that the average tenure of a bull in artificial insemination service was only 2.72 yr. Yet they showed that the anticipated f u r t h er useful life of a 5-yr.-old bull in natural service was over 5 yr. This suggests that bulls in A.I. service were not lasting as long as they should. As shown in Table 2, few bulls went out of A.I. service TABLE 2 Reasons why bulls go out of A.I. service " Reason

Lost

(%) Low fertility, sterility, poor semen, or refusal to work Poor physical condition (including bloat) Infectious causes Accidents and injuries Old age Other unidentified or unstated Total

61.1 11.0 8.3 7.1 4.0 8.5 100.0

aCompiIed from Becker et al. (6). because of old age, but many went out f o r reasons such as accidents, injuries, and infections, which careful management should help to prevent. The greatest losses came from low fertility, sterility, poor semen production, and refusal to work. Some of the research reviewed below has probably helped to avoid some of the losses in this category, but f a r too little emphasis in research today is directed at avoiding or correcting low f er t i l i t y and sterility.

2316

JOURNAT, OF D A I R Y S C I E N C E

Becker (5) recently pointed out that, since 1954, bulls have remained in A.I. service for an average of over 4 yr. This a p p a r e n t l y means t h a t g r e a t e r care has been used in selecting and maintaining bulls in A.I. usage. Perhaps more young bulls have been taken into service and fewer old bulls that will meet the rigors and standards of A.I. have been pressed into A.I. usage. There are some suggestions that the useful life of bulls may be extended by getting bulls started at an earlier age. This is indicated by work from a number of quarters that shows r a p i d early growth brings on early semen production (see reviews in References 23 and 34). However, such studies need to be continued throughout the lifetime of bulls, to be certain that r a p i d growth and early m a t u r i t y do not shorten the useful life. Rapid early grovzth and semen production. During the growing period there is a high correlation between body size and testes size [r : .90 for 65 bulls (37)] and between testes size and semen production [r = . 8 0 f o r 11 bulls (37), and r----.92 for nine bulls (46)]. This high degree of relationship, however, disappears in mature bulls (46), probably as a result of the ravages of disease and other factors t h a t tend to inhibit testes function.

One of the first noticeable effects of retarded growth on reproductive function is shown by the delay in puberty. This is shown in F i g u r e 1, where the effect of varying levels of total digestibIe nutrient intake during early growth in trials at three stations have been brought together and plotted against age at first semen production (8, 12, 42). Retarding ~rowth apparently results not only in delayed puberty and low sperm production earIy in life, but from limited investigations it appears that irreparable damage may be done if the retardation is continued through the normal growing period. Illinois workers found no recovery of semen-producing ability of Holstein bulls in a full year with normal feeding following a 4-yr. period when the bulls were kept on a ration containing only 60% of the recommended TDN requirements (41). Thus, it appears t h a t further investigations are needed to determine whether r a p i d early growth does increase a bull's lifetime sperm-producing potential. Adequate preparation for collection. A number of studies in recent years, following earlier suggestive evidence (reviewed i n - R e f e r e n c e s 17, 34, and 37), have shown that semen volume and sperm numbers can be increased considerably by adequate p r e p a r a t i o n for collection.

170

160

X

x

x

CORNELL

DATA

o PENN. DATA

Lcl

A

,4

I LLI N ~ A T A

140

l-Z Z C3

120

1--

14. 0

I00

.-I I.=.I

>

x " ~ ~

l.iJ

o

80

60 i

35

I

40 AGE

I

I

45

50

AT

PUBERTY

I

55

I

60

I

65

(WEEKS)

FIG. 1. Effect of level of TDN intake during growth on the age at first semen production of Holstein bulls (regression equation Y----65.22--0.1878X).

s~POSlV~ Both r e s t r a i n t n e a r the t e a s e r cow and false m o u n t i n g p r i o r to collection b r i n g on sexual excitement r e s u l t i n g in increased yields of sperm. A n u m b e r of the studies in this a r e a have been compiled in Table 3. I n c r e a s e s in sperm o u t p u t in these studies have r a n g e d f r o m 14 to as much as 250% o v e r the n u m b e r of sperm p r o d u c e d in ejaculates w h e n the same bulls were allowed to w o r k at will. Studies at P e n n s y l v a n i a h a v e done much to show t h a t changing teasers and changing the .position of the teaser g r e a t l y aid in stimulating a bull to w o r k (17). T h e i r studies h a v e shown t h a t bulls a p p a r e n t l y depleted of sperm in exhaustion trials are f r e q u e n t l y s t i m u l a t e d to produce sizable quantities of semen w h e n a new stimulus (teaser) animal is supplied. 1YIaintaining sex drive seems to be more of a l i m i t i n g f a c t o r to the p r o d u c t i o n of large numbers of sperm t h a n a shortage of sperm p r o d u c t i o n by the testes. M o r e f r e q u e n t collection. I n c r e a s i n g the frequency of collection was one of the first approaches to o b t a i n i n g l a r g e r numbers of sperm f r o m bulls. F o r m a n y years it was common p r a c t i c e to limit collections to one or two ejaculates e v e r y week or two. I t was believed t h a t more f r e q u e n t collection would r e s u l t in lowered f e r t i l i t y or sterility. This b e l i e f no doubt stems f r o m p a s t u r e breeding, where ten to 20 services p e r cow in h e a t are not unusual f o r young, vigorous bulls (45). Thus, w i t h a f a i r l y large h e r d the e j a c u l a t i o n f r e quency would soon deplete the sperm reserves and sex drive would likely be diminished. Controlled i n v e s t i g a t i o n s f r o m a n u m b e r

2317

of places have changed the old view w i t h reg a r d to collection-frequency effects on sperm output. Collection frequencies of two and three times p e r week g r e a t l y increase the sperm o u t p u t o f old and y o u n g bulls alike. E v e n w i t h exhaustive collections (see Table 4), the depleted sperm supply is largely replenished 1 wk. a f t e r depletion. R a t h e r f r e q u e n t collections are possible w i t h o u t l o w e r i n g f e r tility. As seen in Table 5, t a k e n f r o m H a l e and A]mquist (17), collections daily or six days a week f o r 26 to as long as 76 wk., h a v e yielded large numbers of sperm (enough to ~)reed more t h a n 500 cows a d a y ) , w i t h f e r tility levels in the ideal range of 70 to 81% 60- to 90-day nonreturns. O t h e r work at P e n n s y l v a n i a , in which six (twice on Monday, W e d n e s d a y , and F r i d a y ) , seven ( d a i l y ) , 14 (twice d a i l y ) , or 70 (ten daily) ejaculates p e r week were collected, showed t h a t six or seven ejaculates p e r week w i t h d r e w n e a r l y as m a n y sperm as the testes produced. M o r e f r e q u e n t collections increased sperm o u t p u t slightly. Thus, sperm production should be considered on the basis of the bull's p o t e n t i a l output, r a t h e r t h a n on a prescribed n u m b e r of collections p e r unit of time. On such a basis, sperm collections can be made w i t h the artificial v a g i n a or w i t h e l e c t r o e j a c u l a t o r equipment, even though some m a y consider the l a t t e r an undesirable method, w i t h o u t stopping f a r short of a bull's p o t e n t i a l or exceeding it. NUIV~BER OF SPERI~ TO I N S E M I N A T E

The question of the n u m b e r of sperm required p e r i n s e m i n a t i o n was first considered

TABLE 3 Effect of sexual preparation on total sperm output Per cent increase

l~O,

ej acu

Investigator (s)

Type of preparation

No. of bulls

lutes per week

First ejaculate:

Second ejaculate

Collins et al. (9)

None vs. 2, to 3"R ~ + 1 F M

22

:1

36

....

Branton et aL (7)

None vs. 1 FM b None vs. 9 FM

6 6

1 1

4.2 41

.... ....

Branton et al. (7)

None vs. 1 FM None vs. 2 FIVI

9 9

2 2

50 6.7

Baker (4')

None vs. 1 FM None vs. 3'R

9 9

1-3 1-3

22 14

.... ....

Crombaeh (10)

None vs. 1 FIVI None vs. 10'R Nolle vs. 5/R + 1 FlY[ None vs. 5'R + 1 FlY[

6 2 4 10

4 4 4 4

129 147 251 112

.... .... 109

Almquist et al. (2)

None vs. 1 FM 1 FM vs. 2'R + 3 YlYI None vs. 2'R + 3 F!V[ 1 F)£ vs. 2'R + 3 F M

6 6 3 3

2 2 6 6

72 64 .... 44

29 27 45 ....

"i~ = restraint. b F ~ = false m o u n t .

. . . .

....

J O U I ~ N A L OF DAIP~Y S C I E N C E

2318

TABLE 4 Recovery of semen characteristics 1 wk. after depletion ~ Per cent recovery of

No. of bulls

Reference Hale and Almquist Hale and Almquist Frederick Boyd and VanDemark

26 13 6 6

Av. No. Av. No. Sperm No. of ejac. in ejac. in Semen Sperm concentrials depletion recovery volume motility tration 46 17 6 6

24 22 24 10

2 5 21 10

84 84 77 74

99 100 92 96

:No. of sperm per ejac.

103 112 105 145

86 94 85 100

From Hale and Almquist (17). TABLE 5 Fertility of bulls with high sperm output over extended periods "

Bull

Weeks

Frequency of ejaeulation

Hafs et al. (Cornell) D 32 B 32 S 3'2 H 32 C 32 Almquist et al. (Pennsylvania) G1 76 Hu 32 H "~ 50 b 26

Daily Daily Daily Daily Daily 6/Week 6/Week 6/Week 6/Week

Daily sperm output

60- to 90-day nonreturns

First services

(billio¢ts) 2.7 5.6 5.7 5.9 4.3

(%) 81 71 72 70 72

531 1,795 3,038 982 762

4.5 4.8 6.0 7.4 b

70 74 78 79

544 286 881 1,709

F r o m Hale and Almquist ( 1 7 ) .

Prepared with three false mounts before each ejaculate. by Salisbury and associates at Cornetl in the early and mid-forties, with a series of experiments in which they attempted to establish maximum extension rates for bull semen (25, 27, 28, 31). As a result of those studies, Salisbury and Bratton (28) wrote that "the minimum number of spermatozoa consistent with optimum f ert i l i t y rests at 5 to 10 millions from bulls of known fertility." Subsequent investigations by a number of workers have not altered these original estimates of minimal sperm numbers for fertility in artificially inseminating the cow. Figure 2 shows the results of several of these investigations (34). From these it is apparent that a sharp drop in fertility oceurs if the number of motile sperm per insemination is reduced much below five million, but increasing the number of live sperm above five milhon does not result in corresponding increases in fertility. PERCENTAGE OF SEMEN ?STILIZED

To take advantage of the large sperm-producing capacity of a bull, and to breed many cows with a single ejaculate by extending the semen so that each cow gets only a limited number of sperm, good methods of semen preservation must be available. This is an-

other area in which great improvements in efficiency have been made in the last 20 yr. The main problem in getting a high percentage of semen utilization is in maintaining viability and fertility over an extended period of time, to permit distribution of the semen and hold it in readiness f o r the presentation of estrous cows f o r service. Two main approaches taken to preserve semen have been that of reducing temperature to slow metabolism and motility and that involving reversible chemical inhibition, or combinations of the two. A third method, lyophilization or freeze-drying, also appears possible and suggests some promise. Supplying diluents that help to maintain optimal conditions by furnishing nutrients and removing waste products also greatly prolongs the storage life of spermatozoa. Salisbury (26) reviewed the status of bovine semen diluents in Animal Breeding Abstracts in 1957. A later and more detailed consideration of the merits of various diluents can be found in the recent book of Salisbury and VanDemark (34). Storage at refrigerator (above-freezing) temperatures. The wide-spread use of cooling to refrigerator temperatures to prolong sperm life in this country dates back to about 1940. J1 fact, the protection from cold shock af-

SYMPOSIUM 75

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i

I

I

I

I

|

|

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l

2319 I

I

f

I

I

i

I

I

t• ~

70

u~

65

,

~

~

~ / ~ " ~

j~

-

~--D- ~ - a ~ "

-

~ 6o !

g :"

o

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45 I ~11~. I 0 2

I

I 4

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20

MILLIONS OF SPERM INSEMINATED )[~IG. o,. :Effect o~ n u m b e r of sperm i n s e m i n a t e d on fertility of dairy Cows. ( F r o m P h y s i o l o g y of Reproduction and Artificial I n s e m i n a t i o n of Cattle, b y G. W . S a l i s b u r y and N. L. V a n D e m a r k . W. H. F r e e m a n and Company, S a n Francisco. 1961.)

forded sperm by the yolk diluents [Phillips' yolk-phosphate (22) and the Salisbury et at. yolk-citrate (32)] permitted cooling and greatly lengthened the useful life of semen. Even with a storage life of three or four days on a routine basis, some A.I. operations poured down the drain over 80% of the semen collected and processed. Addition of antibacterial agents improved storage life by eliminating much of the competition of bacteria and improved fertility, too (3, 19). A good example of the effects of antibacterial agents is seen in the results presented by the Cornell workers, comparing f e r t i l i t y results before and after addition of antibacterial agents (13). During a 6-mo. period before antibiotics, f e r t i l i t y averaged 64%; in the 6 mo. a f t e r antibiotics were added, f e r t i l i t y rose to 73% (60- to 90-day nonreturns). The presence of antibiotics and sulfanilamide in extended semen results not only in slightly better conception rates, but fewer cows return to estrus at irregular intervals after unsuccessful artificial insemination (21). Additions of penicillin to semen have been shown to increase the f e r t i l i t y of some lowfertility bulls (1). Little is known of the actual effect of semen processing and handling procedures on fertility. As semen ages in storage at r e f r i g e r a t o r temperatures, and other

temperatures, too, there is a decline in the f e r t i l i t y (34, 35). I t has been shown t h a t DNA (the chemical substance associated with the genetic potentials of the sperm) is lost during storage at r e f r i g e r a t o r temperatures (30). This loss may be responsible for the gradual increase in apparent embryonic death that occurs following the insemination of cows with semen stored over longer and longer periods (29). I t is tempting to postulate that storage conditions may be responsible for such phenomena as decreased f e r t i l i t y and increased embryonic death, but the proof is not yet at hand. However, such items as the oxygen damage that occurs in sperm in p a r t i a l l y filled tubes (43), and when semen is mixed excessively and aerated (a damage that can be prevented by the use of catalase), indicate a possible oxidative damage which could involve the DNA. Many improvements in semen diluents have been made which enhance the effectiveness of reduced temperatures in slowing metabolism and prolonging sperm survival (34). Storage at room temperatures utilizing chemical inhibition. One new approach to semen

preservation, made in the last 10 yr., was the attempt to simulate the conditions of the epididymis and keep sperm in a constant-flow

2320

JOURNAL

OF DAIRY

dialysis s y s t e m (40). A l t h o u g h u n e x t e n d e d s e m e n l i v e d f o r o v e r a w e e k u n d e r s u c h conditions, c o m p a r e d to a d a y or less in t h e t e s t t u b e a t room t e m p e r a t u r e , t h e r e a l beneficial d i s c o v e r y f r o m t h o s e s t u d i e s was t h e effect o f c a r b o n dioxide on sperm. C a r b o n dioxide i n c o n c e n t r a t i o n s above t h o s e n o r m a l l y f o u n d in extended semen reduces metabolic activity of s p e r m a t o z o a a n d i n h i b i t s m o t i l i t y (33). O u t of such s t u d i e s was d e v e l o p e d t h e carbonated Illini variable temperature (IVT) d i l u e n t (44). U n d e r c o n d i t i o n s in w h i c h t h e a m b i e n t t e m p e r a t u r e does n o t rise f o r too l o n g above 80 ° F., t h i s t y p e of d i l u e n t , s a t u r a t e d w i t h c a r b o n dioxide, h a s m a i n t a i n e d quite good f e r t i l i t y f o r two or t h r e e days. H o w e v e r , u n d e r c o n d i t i o n s w h e r e t h e t e m p e r a t u r e rem a i n s long a b o v e 80 ° F., a n d w h e r e care is n o t t a k e n to k e e p h i g h levels of CO~ in t h e d i l u e n t , d i s a p p o i n t i n g r e s u l t s h a v e b e e n obtained.

Combining chemical inhibition and reduced

SCIENCE

temperatures. A d v a n t a g e h a s been t a k e n of reversible chemical inhibition while eliminati n g t h e difficulties e n c o u n t e r e d a t a m b i e n t temperatures by refrigerating semen extended w i t h the c a r b o n a t e d diluents. A n u m b e r of such diluents, all of r a t h e r s i m i l a r composition, a r e s h o w n i n T a b l e 6. All e x c e p t t h e New Y o r k C U E are g a s s e d w i t h COs. T h e C U E is s e l f - c a r b o n a t i n g , g e n e r a t i n g C02 f r o m t h e m i x t u r e of s o d i u m b i c a r b o n a t e a n d c i t r i c acid. F e r t i l i t y levels p u b l i s h e d f r o m t h e use of some o f t h e s e d i l u e n t s a r e s h o w n in T a b l e 7. All a t t e m p t s to use t h i s m e t h o d of s e m e n p r e s e r v a t i o n h a v e n o t b e e n as successful as those shown, b u t t h e p o o r e r r e s u l t s a r e k n o w n to h a v e b e e n due, in some cases a t least, to i m p r o p e r p r o c e d u r e a n d care i n k e e p i n g t h e CO~ i n t h e s t o r a g e a m p u l e o r tube. Preservation by freezing. F r e e z i n g exceeds all o t h e r d e v e l o p m e n t s to d a t e f o r t h e p r e s e r v a t i o n of b o v i n e semen. The B r i t i s h h a v e

TABLE 6 Some carbonated diluents for bovine semen a Orig. IVT

Ingredients

Mod. IVT

Ark. No. 2

Sodium bicarbonate 0.21 0.42 Sodium citrate 2.00 1.00 Potassium chloride 0.04 0.04 Glucose 0.30 1.20 Sulfanilamide 0.30 0.3.0 Egg yolk (%) 10 15 Penicillin (I.U/mt) 1,000 1,000 Streptomycin (~g/ml) 1,000 1,000 Catalase ...... 0.01 Glycine . . . . . . . . . . . . Glutathione (reduced) ............ Citric acid Gassed with C:O2 CO~ Cited from References .................... (44) (39)

Tenn. YCCG

0.21 1.00 ...... 0.30 0.30 10 1,000 1,000

Germany EIBL

0.21 1.60 0.04 1.00 0.30 2'0 5.00 500 ............

0.18 1.67 0.03 0.25 0.25 30' 500 500

1.00

. . . . . . . . . . . .

0.15~

............

N.Y. CUE 0.21 1.4.5 0.04 0.30 0'.30 20 1,000 1,000 0.9@ 0.087

CO~ (18)

CO~ (36)

CO2 (11)

(15) 16)

Grams per 100 ml. unless otherwise indicated. TABLE 7 Per cent 60- to 90-day nonreturns from inseminations with carbonated diluents compared with control diluents Investigation and diluents used Age of semen

Arkansas CO2 No. 2

Tennessee ¥C

YCCG

New York

MYGLy

CUE

YC

(days) 0-1

78

74

. . . . . . . . . . . .

1-2

76

68

75

2-3 72 3-4 66 4-5 63 5-6 63 6-7 67 Over-all 70.9 No. cows bred 8,657 Cited from References .......... (18)

78

78

62 78 72 75 60 75 70 73 .... 71 69 ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66.6 74.4 72.6 76.6 1,481 2,015 1,974 6,280 (36) (15.)

~

72 67

73.4 6,06.9

2321

sY~POSiUM

gotten pregnancies from semen stored over 8 yr. (24) and we must be near or exceeding that in the United States by now. Although the period of storage can be long, thus making possible high percentage utilization, use of the freezing technique is not without wastage (see Review References 34, 38). In the first place, semen of some bulls just will not come through the freezing process without a high loss of motile sperm. Secondly, even with the semen that does freeze fairly well, there is a sizable loss in motile sperm resulting from freezing. And, lastly, there is a loss of motile sperm during storage in the frozen state. To date, no special treatment is known to improve the freezability of semen from hard-to-freeze bulls or to eliminate the 20 to 30% kill commonly encountered in freezing. However, holding the storage temperatures much below the danger point by means of liquid air or liquid nitrogen has helped to reduce loss of motile sperm during storage in the frozen state. F r o m the practices being followed by those using frozen semen, it appears t h a t a liquid semen program that utilized 50% of the semen would be as efficient at a lower cost than an all-frozen semen program. However, a number of other itenls in addition to utilization of bull power must be considered, as well, in deciding between a liquid and frozen semen program. Freeze-drying for semen preservation. The dream of researchers, and practical men as well, that semen might be preserved indefinitely in a dry, unrefrigerated state after lyophilization or freeze-drying, has recently appeared hopeful. Some of the early reports of success with this technique with laboratory and farm animals in Russia were recently reviewed (38). The report of Meryman and Kafig (20), of the successful impregnation of a cow in Maryland with freeze-dried sperm, appears to be the first such report in cattle. Unfortunately, with this procedure, as with many new and revolutionary techniques, the original investigators and others have not yet succeeded in repeating this feat. So, once again, perfection of the procedure may take months or even years before it becomes practical. However, now it would appear to be the ideal method of semen preservation and utilization. THE CHALLEN-GE Even without using freezing, or awaiting the perfection of freeze-drying as a semen preservation technique, the potential of most bulls would appear to be f a r greater than many artificial organizations are utilizing at the present. I t appears, from the investigations cited above and hoping' for nearer 100% utilization of the semen produced, that the following values inserted into the formula for

reproductive efficiency (given earlier) may be reachable at least for some bulls: 5 billion live sp er m / d ay )< 300/yr 5 million live sperm/insem × 100% usage × 75% calving ~ 225,000 calves/yr. Semen production and an operation at this level of efficiency is a real challenge to the cattle artificial insemination industry. An even greater challenge is that of selecting bulls of genetic worth that should be used as extensively as such an operating efficiency would permit. I%EFERENCES

(1) AL~QUIS% J. O. The Effect of Penicillin upon the ~'ertility of Semen from Relatively Infertile Bulls. J. Dairy Sci., 32 : 950. 1949. (2) AL:MQUIS~ J. O., HAL]~, E. B., Am) AMANN, R.. P. Sperm Production and Fertility of Dairy Bulls at High-Collection Frequencies with Varying Degrees of Sexual Preparation. J. Dairy Sci., 41:73~3'. 1958. (3) AL~IQUIST, J. O., TItOI~P, W. T. S., AND KNOD~, C. B. The Effect of Penicillin upon the Livability, Fertility and Bacterial Cvntent of Bull Semen. J. Animal Sci., 5: 400. ]946. (4) BAKe, F. ~. The Effect of Frequency of ]~jaclflation and Epinephrine Injections on the Semen Production and Libido of Yonng Bulls. Ph.D. thesis, University of Illinois. 1954. (5) B]~Cs:E~, R. B. Personal communications. 1901. (6) BECKEa, R. B,, DIX ARNOLD, P. T., AND S P ~ o c K , A. H. Productive Life Span of Dairy Cattle. Florida Agr. Expt. Sta., Bull. 540. ]954. (7) BFoANTON,C., D 'Ar~NsBouI~, G., AN~ JOHNS~N, J. E. Semen Production, Fructose Content of Semen, and Fertility of Dairy Bulls as Related to Sexual Excitement. J. Dairy Sci., 35: 801. 1952. (8) BtCATTON-, R. W., MUSGRAVE~,S. D., DUNN, H. 0., AN]) F00Tm, i~. If. Causes and Prevention ef Reproductive Failures in Dairy

Cattle. II. Influence of Underfeeding and Overfeeding from Birth to 80 Weeks of Age on Growth, Sexual Development and Semen Production of Holstein l~ulls. Cornell University Agr. E~pt. Stu., Buff. 9~0. 1959. (9) COLLINS, W. J., BP.ATTON, I~. W., AN]) I~FSN])]~USON, C. R. The Relationship of Semen Production te Sexual Excitement of Dairy Bulls. J. Dairy Sci., 34: 224. 1951. (]0) Cr~o~B~0~r, J. J. M. L. De Invloed van Voerbereidlng veer hot Dekken op de Spermaproduktie en de Bevruchtingsresultaten van K. I. Stieren. (The Effect of the Preparation of A. I. Bulls Before Service on Semen Production and Conception Rate.) Tijdsehr. Dierffeneesk., 83: 137. 195,8. (11) EmL, K., ZOI)F~, It. F., ANn HAH~r, R. Preservation of Semen by Means of Carbon Dioxide. (Translated Title.) Zuchtwahl u. Besamung, Special ~umber, Neustadt Aisch. June, 1960.

2322

JOURNAL OF DAIRY SCIENCE

(12) FLIPSE, R. J., AND ALI~QUIST, J. O. Unpublished data. Pennsylvania Agr. Expt. Sta. 1959. (13) Foo,z~, R. H., ~,N]) BRA~U~ON, R. W. The Influence of Antibiotics on Delayed Returns in Artificial Breeding. J. Dairy Sci., 35: 261. 1952. (14) F ~ , R. H., AN]) Bm~T~ON, R. W. Fertility as I t Relates to Genetic Improvement. Intern. J. Fertility, 2: 231. 1957. (15) FooT~, R. H., GR~Y, L. C., YouNg, D. C., A~]) DUNN, H. O. Fertility of Bull Semen Stored up to Four Days at 5 ° C.. in 20% Egg Yolk Extenders. J. Dairy SEA., &3: 1330. 1960. (16) FOO~E, R. H., YouNg, D. C., ANI) DUN~, H. O. Fertility of Bull Semen Stored for One and Two Days at 5 ° C. in 20% Yolk-Citrate-Glycine-Glucose Extenders. J. Dairy Sei., 41: 732. 1958. (17) HA/E, E. B., AND ALMQUIST~ J. O. Relation of Sexual Behavior to Germ Cell Output in F a r m Animals. I n The Effect of Germ Cell Damage on Animal Reproduction. J. Dairy Sci., Suppl. 43:145. 1960. (18) HAY])F~, J. S., AN]) ST~,LLCUP, O. T. Storing Bovine Semen at Room Temperature. Arkansas F a r m Research, 7: 6. 1958. (19) KNOD~, C. B., AND SAMSBU~¥, G. W. The Effect of Sulfanilamide upon the Livability and Metabolism of Bovine Spermatozoa. J. Dairy Sci., 29:285. 1946. (20) MF~YI~AN, N. T., ~ K-~Fm, E. Survival of Spermatozoa Following Drying. ]Nature (London), 184: 470. 1959. (21) Olms, D., OLZV~, L., AN]) SF~TI~, D. M. The Effect of Antibiotics on the Fertility of Bull Semen and Their Relationship to the Estrous Cycle Length of Dairy Cattle Following Artificial Insemination. J. Dairy Sci., 3.4: 966. 1951. (22) PHrULn~S, P. H. The Preservation of Bull Semen. J. Biol. Chem., 130: 415. 1939. (23) R~-~m, J. T. Effect of Energy Intake upon Reproduction in F a r m Animals. I n The Effect of Germ Cell Damage on Animal Reproduction. J. Dairy Sci., Suppl. 43:103. 1960. (24) Rowso~, L. E. A. Personal communication, Cambridge, England. January, 1961. (2~) SA~ISBU'aY, G. W. Fertility of Bull Semen Diluted at 1:100. J. Dairy SEA., 29: 695. 1946. (26) SA/mBU~Y, G. W. Recent Developments with Bull Semen Diluents. Animal Breeding Abstr., 25: 111. 1957.

(27) SALISBURY, G. W., BECK, G. H., CUPPS, P. T., AND ELLI(nn~, I. The Effect of Dilution Rate on the Livability and the Fertility of Bull Spermatozoa Used for Artificial Insemination. J. Dairy Sci., 2~: 1057. 1943. (28) SALmnu~v, G. W., A~r~ B ~ r o ~ , R. W. Fertility Level of Bull Semen Diluted at 1:400 with and Without Sulfanilamide. J. Dairy SEA., 31: 817. 19¢8. (29) SALISnin~Y, G. W., B ~ r O N , R. W., AN]) FOOT~, R. H. The Effect of Time and Other Factors on the Nonreturn to Service Estimate of Fertility Level in Artificial Insemination of Cattle. J. Dairy Sci., 35: 256. 1952. (30) SALISBLrlcy, G. W., DE LA TOEPJS, L., BIRG]~, W. J., AND L~D~, ~. R. Effect of 5" C.

(31)

(32)

(33)

(3~)

(35)

(36) (37)

(38)

(39)

(4~0)

Storage in Yolk-Citrate on Feulgen-Positive Material (DNA) of Sperm Heads. J. Dairy Sci., ~3: 882. 1960. SALISBURL G. W., Er.~IO~, I., AN]) VA~DE}~A~K, N. L. Further Studies of the Effect of Dilution on the FertilitF of Bull Semen Used for Artificial Insemination. J. Dairy Sei., 28: 233. 1945. SAMSBU~¥, G. W., FUImE~, H. K., AN]) WILLF/~, E. L. Preservation of Bovine Spernmtozoa in Yolk-Citrate Diluent and Field Results from Its Use. J. Dairy ScL, 24: 905. 1941. SALISBURY, G. W., AN]) VANDE:MARK, N. L. Carbon Dioxide as a Reversible Inhibitor of Spermatozoan Metabolism. Nature (London), 180: 989. 1957. SALISBURY, G. W., AN]) VANDI~iAKK, N. L. Physiology of Reproduction and Artificial Insemination of Cattle. W. H. Freeman and Company, San Francisco. 1961. SOHuL'rz]:, A. B., DAWS, H. P., B L U R , C. T., AN]) OLOUrA, M. M. The Influence of Length of Storage of Bovine Semen on Conception Rate Under Field Conditions. Nebraska Agr. Expt. Sta., Research Bull. 154. 1948. SWANSON,E. W., AN]) MCFF~E, A. F. P u t t i n g Bull Power to Work. Tennessee F a r m and Home Science. p. 6. June, 1959. VANDi~MARK, N. L. Quantitative Aspects of Semen Production in Bulls. 3rd Intern. Oongr. Animal Reproduction, Plenary Section. pp. 80-88. 1956. VAxDE~AaK, N. L. Preservation of Animal Breeding Stocks Through Storage of Germ Plasm. In Germ Plasm Resources. pp. 337354. Am. Assoc. Adv. Sci., Publ. No. 66, Washington, D. C. 1961. VANDEMARK,N. L., ANI) BARTLETT~ F. D., JB.. Prolonged Survival of Bovine Sperm in the Illini Variable Temperature Diluent. J. Dairy Sci., 41: 732. 1958. VAND]~I~ARK, N. L., ANO COUT~RIE~, L. R. Flow Dialysis as a Means of Preserving Bovine Semen at Room Temperature. J. Dairy ScI., 41: 530. 1958.

(41) VANDEI~[AEK, N. L., FI~ITZ, G. R., EWING, L. L., AND MAUGE5~, R. E. Limiting Effects

(42) (43)

(44)

(45)

(46)

of Underfeeding During Growth on Sperm Production of Mature Bulls. J. Animal SEA., 19: 1338. 1960. V A N D E ~ K , N. L., AN]) MAU(~F~, R. E. Unpublished data. Illinois Agr. Expt. Sta. 1959. VANDE~ARK, N. L., SALISBURY, G. W., AND Bma~ON, R. W. Oxygen Damage to Bull Spermatozoa. and Its Prevention by Catalase. J. Dairy Sci., 32: 353. 1949. YANDEI~ARK,N. L., AND SHAI~i~A, U. D. Preliminary Fertility Results from the Preservation of Bovine Semen at Room Temperatures. J. Dairy Sci., 40: 438. 1957. Vm~VKI~A, T. P., ANn S~m~ov-U~Rzu~ov, D. V. The Activity of Bulls in Natural Mating. (Translated Title.) Probh Zivotn., 3: 178. 1938. Animal Breeding Abstr., 8: 35. 1940. WILLETT, E. L., AND O ~ S , 5. I. Measurement of Testicular Size and I t s Relation to Production of Spermatozoa by Bulls. J. Dairy Sci., 40: 1559. 1957.