ON EQUINE FERTILITY 1
B. W. Pickett I , L. D. Burwash 1,3, j. L. Voss 2 and D. G. Back 2,4 Colorado State University, Fort Collins 80523
Summary Pregnancy rate, spermatozoan motility and spermatozoan agglutination were significant.ly lower in an extender containing .349% Tris than in an extender utilizing 2.4% Tris. First-cycle pregnancy rates of 22.2 and 11.I% were obtained with 500 and 100 X 106 motile spermatozoa in. a 2.4% Tris extender, and a rate of 61.1% (P < .05) was obtained with 500 • 106 motile spermatozoa in raw semen. After four cycles there was no significant difference in fertility obtained with 2.4% Tris and the raw semen when 500 X 106 spermatozoa were utilized (72.2 vs 88.9%). First cycle pregnancy rates of 37.5, 75.0 and 75.0% were obtained in 1973 when mares were inseminated with 500 • 106 motile spermatozoa extended in 2.4% Tris, and cream-gel extenders and with raw semen, respectively. After three cycles, mares on the Tris-seminal treatment had a pregnancy rate of 75.0%, which was lower (P < .05) than the 95.8 and 91.7% for the cream-gel and raw-seminal treatments, respectively. The presence of glycerol in the Tris extenders may have been at least partially responsible for the depression of fertility. Pregnancy rates of 75.0 and 91.7% were obtained after one and three cycles, respectively, with a mean insemination volume of 1.5 ml of raw semen. Thus, a large insemination volume may not be necessary for maximum fertility in the mare.
Introduction
Loy, 1970); evaporated milk-glucose-glycerol (McCall and Sorensen, 1971); egg yolk-sugarglycerol (Krause and Grove, 1967; Oshida et al., 1968); mare's milk (Kamenev, 1955); egg yolk-glucose-glycerol (Nishikawa et al., 1968); skim milk-glucose (Rajamannan et al., 1968); and Tris-glucose-citric acid-egg yolk (Cranwell, 1970; Anderson, 1971). A stallion seminal extender is needed to prolong spermatozoan survival, enhance fertility, provide a media for antibiotic treatment of semen and permit a more convenient volume to be utilized, particularly when small numbers of spermatozoa are inseminated.
Materials and Methods General. Three groups ( 1 9 7 1 , 1972 and 1973) of nonlactating, clinically normal mares between 3 and 20 years of age were obtained for these studies between February 1 and April 1. All mares were maintained at the Equine
t Animal Reproduction Laboratory, Department of Physiology and Biophysics. Supported in part by grants-in-aid from American Breeders Service; National Association of Animal Breeders; and the Arabian Horse Registry of America, Inc., through Morris Animal Foundation. Portions of these data were presented at the Fifth N.A.A.B. Tech. Conf. A. I. Reprod., Chicago, IL, 1974, and the "First International Symposium Upon Equine Reproduction," Cambridge, England, 1974. 2 Department of Clinical Sciences. apresent address: Alberta Department of Agriculture, Animal Industry Division, Edmonton, Alberta, Canada. 4present address: Gleannloch Farms, SpringCypress Rd., Spring, TX 77393.
Many seminal extenders have been utilized to extend and store stallion semen. The types of extenders utilized include whole milk (Ebertus, 1963); cream-gelatin (Hughes and 1136
J O U R N A L O F A N I M A L S C I E N C E , vol. 4 0 , n o . 6, 1 9 7 5
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EFFECT OF SEMINAL EXTENDERS
1137
SEMINAL EXTENDERS ON EQUINE FERTILITY TABLE 1. EXTENDER COMPOSITION Tr~
Tris ( w / v ) Glucose (anhydrous) (w/v) Citric acid (monohydrate) (w/v) Egg y o l k ( v / v ) G l y c e r o l (v/v)
.349 b
2.4 b
.............
(%)
.349
2.4
3.655 .151 22.8
1.163 1.326 22.8
2.4 c
Cream-gelatin e
...............
2.4 .45 1.255 22.8
m m
5.41
5.41
5.25
Half & half cream d ( v / v )
--
--
--
88.7
K n o x gelatin e ( w / v )
--
--
--
1.3
67.8
69.5
67.9
10.0
Water (v/v) Osmolality (mOs) pH
350
350
298
7.00
7.00
6.99
348
6.65
a C o n t a l n s 1 , 0 0 0 ~ g s t r e p t o m y c i n sulfate and 1,00O units s o d i u m p e n c i l l i n G / m l . b1971 and 1972. c1973. d C o n t a i n e d a p p r o x i m a t e l y 1 1 % b u t t e r f a t , 2 . 8 % c a s e i n , .9% a l b u m i n a n d 7 8 . 9 % w a t e r . eKnox Gelatin, Inc., Johnstown, New York.
inseminated on days 3, 5, 7, etc, of estrus with semen collected daily from a single stallion. Mares were inseminated with 500 X 10 6 motile spermatozoa extended in either .349 or 2.4% Tris extender (table 1) with a final volume of t> 10 ml but .10) between the 26.7 and 12.5% pregnancy rates obtained in cycle 2 with semen extended in the 2.4 and .349% Tris, respectively. The lower pregnancy rate during
the second cycle could have resulted if the more fertile mares conceived during the first cycle. There is also the pgssibility that the mares had developed an immunological response to the extender, because it contained 22.8% egg yolk. The 40.6% pregnancy rate over two cycles for mares inseminated with semen extended in 2.4% Tris extender was higher (P < .10) than the 15.4% pregnancy rate in mares inseminated with semen extended in .349% Tris extender. The first-cycle pregnancy rate (52.9%) for semen extended in 2.4% Tris is in agreement with that reported,, for Thoroughbred and Quarter Horses (Pickett and Voss, 1972). There were no differences between number of inseminations per cycle or volume inseminated for the .349 or 2.4% Tris-seminal treatments. The influence of .349 and 2.4% Tris extenders on spermatozoal motility, agglutination score and clumping score at 0, 1 and 2 hr after seminal collection is presented in table 3. There was no difference (P > .05) in spermatozoal motility, between the two extenders at 0 hr, but motility was depressed after 1- and 2-hr incubation in the .349% Tris extender (P < .05). The spermatozoan-agglutination score was higher (P < .05) in the 2.4% Tds extender than the .349% Tris extender regardless of incubation period. Also, spermatozoan agglutination increased (P < .05) after 1 hr incubation in the 2.4% Tris extender. The clumping score was higher (P < .05) at 0-hr incubation in the 2.4% Tris extender than in the .349% Tris extender, but the clumping did not differ after incubation.
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Cycle
No. of mares inseminated
SEMINAL EXTENDERS ON EQUINE FERTILITY
membrane over the acrosomal and post-acrosomal regions (Bedford, 1965, 1970). Most of the agglutination that occurred involved the "sticking together" of the sperm at the acrosomal region, but tail-to-tail agglutination has been reported (Bedford, 1965). He has also shown that the concentration of electrolytes in the medium influenced the head-to-head agglutination of rabbit spermatozoa, which may account for the differences in degree of clumping observed in the present study. It has been generally assumed that agglutination renders the sperm infertile. Although, Bedford (1970) has reported that spermatozoa from one rabbit that showed agglutination were fertile, and agglutination in this study was associated with increased fertility. 1972. In this experiment, pregnancy rates obtained with seminal treatments utilizing 100 • 106 and 500 • 1 0 6 motile spermatozoa in 10 ml of 2.4% Tris extender were compared to 500 X 1 0 6 motile spermatozoa in raw semen In cycle 1, the 61.1% pregnancy rate was higher (P < .05) for mares inseminated with 500 • 1 0 6 motile spermatozoa in raw semen than the 22.2 and 11.1% pregnancy rate for mares inseminated with 500 X 1 0 6 o r 100 • 1 0 6 motile spermatozoa extended in 10 ml of Tris extender (table 4). There were no differences (P > .05) in pregnancy rates obtained during cycles 2, 3 or 4. The 27.8% pregnancy rate over four cycles for mares inseminated with 100 X 1 0 6 motile spermatozoa in Tris extender was lower (P < .05) than the 72.2 or 88.9%, respectively, for the mares inseminated with
T A B L E 3. M O T I L I T Y , A G G L U T I N A T I O N S C O R E A N D C L U M P I N G S C O R E F O R S P E R M A T O Z O A 1N . 3 4 9 A N D 2 . 4 % T R I S E X T E N D E R S ( 1 9 7 1 )
Agglutination
M o t i l i t y (%) Incubation time (hr)
.349
Clumping
score a 2.4
.349
score b 2.4
.349
2.4
0
65.0 c
65.0 c
.0 d
1.1 c
.0 d
1
53.3 d
61.8 c
.3 d
1.9 e
1.1 c
1 . 6 c'e
1.1 c
2
36.3 e
55.2 d
.4 d
2.1 e
1.4 c,e
2.3 e
a 0 = n o a g g l u t i n a t i o n , 1 = 2 0 % , 2 = 4 0 % , 3 = 6 0 % , 4 - 80% and 5 = 1 0 0 % agglutination. b 0 = n o s p e r m a t o z o a per clump, 1 = 2-4, 2 = 5 - 1 0 , 3 = 1 1 - 1 5 , 4 = 1 6 - 2 0 a n d 5 = ~ 2 0 s p e r m a t o z o a per clump. c,d,eMeans under each major heading followed b y different superscripts are significantly different at the 5% level of probability (Analysis of variance and Studentized range test).
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Senger and Saacke (1973) reported that agglutinated bovine spermatozoa maintained both motility and intact acrosomes longer than nonagglutinated spermatozoa. It is tempting to suggest that conditions which permit agglutination are beneficial to spermatozoal motility and perhaps fertility, since 40.6% of the mares became pregnant when spermatozoa were extended in the 2.4% Tris extender and only 15.4% when mares were inseminated with semen extended in .349% Tris extender. The clumping of stallion spermatozoa is highly seasonal (Pickett et al. 1970), probably due to changes in the chemical composition of the seminal plasma. Motility of stallion spermatozoa also appeared to be affected by season. However, when semen was placed in an extender that prevented clumping, motility was relatively constant throughout the year. Thus, it was concluded that clumping interfered with motility, and for accurate motility estimations stallion semen must be extended in a diluent that prevents clumping (Pickett and Voss, 1972). Clumping, or agglutination of spermatozoa, appears to be a normal phenomenon in the semen of several species including rabbit, guinea pig, boar, and stallion (Bedford, 1965, 1970; Pickett et al., 1970). This occurrence probably has little or no relationship to the antigenic properties of spermatozoa following isoimmunizations with semen or testicular tissue (Henle et al., 1938; Chang, 1947; Smith, 1949). In all probability, "normal" agglutination is due to different surface properties of the plasma
1139
1140
PICKETT ET AL.
TABLE 4. THE E F F E C T OF SEMINAL TREATMENTS ON PREGNANCY R A TE OF MARES (1972)
Extendeda I00 b
Pregnancy rate
No. of mares inseminated
100 b
Cycle
No. of mares inseminated
Pregnancy rate
1
18 (4) c
22.2 d
18 (2)
11,1 d
18 (11)
61.1 e
2 3 4 Total
14 (3) 11 (4) 7 (2) (13)
21,4 36.4 28.6 72.2 d
16 (2) 13 (0) 12 (1) (5)
12.5 .0 8.3 27.8 e
7 (4) 2 (1) 1 (0) (16)
57.1 50.0 .0 88.9 d
(%)
No. of mares inseminated
(%)
Pregnancy rate
(%)
a E x t e n d e d in 10 ml of 2.4% Trls e x t e n d e r . b(10 6 ) motile spermatozoa. CNumbers in parentheses axe t h e n u m b e r o f maxes t h a t b e c a m e pregnant. d , e p e z c e n t a g e s within rows followed by a d i f f e r e n t superscript are significantly different a t the 5% level of p r o b a b i l i t y (chi-squaxe analysis).
500 X 106 motile spermatozoa in Tris extender or in raw semen. There was no statistically significant difference (P :> .05) in pregnancy rate over four cycles between the two seminal treatments utilizing 500 X 106 motile spermatozoa, although there was an observed difference of 16.7 percentage points in favor of the raw-seminal treatment. The 2.4% Tris extender depressed fertility, and the low pregnancy rate with 100 X 106 m o t i l e spermatozoa was due to an interaction between spermatozoal numbers and extenders. From these data, it is suspected that to properly evaluate a seminal extender the number of motile spermatozoa per insemination should not exceed 100 X l 0 6 when mares are inseminated every other day. Early pregnancies are important to the equine industry. After two cycles, 83.3% of the mares inseminated with raw semen became pregnant, while only 38.9% o f the mares inseminated with an equal number of spermatozoa in 10 ml o f 2.4% Tris extender became pregnant. Inseminating with raw semen containing 500 • 106 motile spermatozoa must be recommended. In the event pathogenic organisms are present, an extender containing the appropriate antibiotic is recommended. The excellent pregnancy rate obtained with raw semen was higher than normally anticipated and may have occurred due to the small number of mares, or because properly managed
mares are not as inherently infertile as previously suspected. Pregnancy rates on a per cycle basis were different (P < .05) among treatments, averaging
TABLE 5. THE EFFECT OF SEMINAL TREATMENTS ON REPRODUCTIVE EFFICIENCY IN MARES (1972)
Variable No. of mares Pregnancy rate/ cycle (%) No, o f cycles/ mare No. of inseminations]cycle Vol./insemination (ml) Efficiency o f pregnancy e
Spermatozoa (106 ) Extendeda Raw 500 100 100 18
18
18
26.0 b
8.5 c
57.1d
2.8 b
3.3b
1.6 c
2.6
2.9
2.7
13.4b
10.6 c
4.2d
5.3b
2.2 c
8.2 b
a E x t e n d e d in 10 mi of 2.4% Tris extender. b,c,dNumbers w i t hi n rows followed b y a d i f f e r e n t superscript axe significantly different a t t he 5% level of proba bi l i t y (S t ude nt i z e d range test a nd chi-square analy sis). e E a e h m a ~ t h a t became p r e g n a n t o n cycle 1 was awarded 10 points, eight p o i n t s for cycle 2, six p o i n t s for cycle a and four p o i n t s for cycle 4.
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500 b
Raw
1141
SEMINAL EXTENDERS ON EQUINE FERTILITY
mares inseminated with semen in Tris became pregnant, compared to 75.0% for both the cream-gel and raw-seminal treatments. These differences were not statistically significant (P > .05). However, after three cycles, mares inseminated with the Tris-seminal treatment had a 75.0% pregnancy rate, which was lower (P < .01) than the 95.8 and 91.7% for the cream-gel and raw-seminal treatments, respectively. Although direct comparisons among the 1971, 1972 and 1973 experiments could not be made, there appeared to be little or no influence of day of first insemination on pregnancy rate. In 1971 and 1972, semen was collected daily and mares inseminated on days 3, 5, 7, etc, or days 2, 4, 6, etc, of estrus, respectively; while in 1973 semen was collected every other day, and all mares in estrus for two days or longer were inseminated. This resulted in some mares being bred on days 2, 4, 6, etc, and others on 3, 5, 7, etc. Since this procedure resulted in less labor, more spermatozoa per ejaculate (Pickett et al., 1975), fewer spermatozoa lost per collection (Pickett et al., 1974) and acceptable pregnancy rates (table 6), it must be recommended. The influence of extenders on other measures of reproductive performance is presented in table 7. A 40.9% pregnancy rate per cycle was obtained for mares in the
TABLE 6. THE EFFECT OF SEMINAL TREATMENTS ON PREGNANCY RATE OF MARES (1973)
Extended a Tris No. of mares
Cycle
inseminated
Cream-gel
Pregnancy rate
No. o f mares inseminated
(%)
Raw b
Pregnancy rate
No. o f mares Pregnancy inseminated rate
(%)
(%)
1
24 (9) c
37.5
24 (18)
75.0
24 (18)
75.0
2
13 (5)
38.5
5 (4)
80.0
6 (4)
66.7
3
7 (4)
57.1
I00.0
1 (0)
Total
(18)
75.0 d
I (I)
(23)
95.8 e
(22)
.0 91.7 e
a s o 0 X 10 6 m o t i l e s p e r m a t o z o a in 10 ml e x t e n d e r . b s o 0 X 10 6 m o t i l e s p e r m a t o z o a . CNumbets in parentheses are the n u m b e r o f mares that b e c a m e pregnant. d,epercentages w i t h i n r o w s f o l l o w e d b y a different superscript are significantly different at t h e 1% level o f probability (Analysis of variance).
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26.0, 8.5 and 57.1% for the 500 and 100 • 106 extended- and raw-seminal treatments, respectively (table 5). The average of 1.6 cycles per mare in the raw-seminal treatment was less (P < .05) than the 2.8 and 3.3 cycles per mare for the two extended-seminal treatments. There was no difference (P > .05) in the number of inseminations per cycle among the treatments. There was a difference (P < .05) in the volumes inseminated, 13.4 and 10.6 ml for the 500 and 100 X 10 6 motile spermatozoa in the extended treatments and 4.2 ml for the raw-seminal treatment. Mares inseminated with 500 or 100 X 106 motile spermatozoa in the extended-seminal treatments and 500 X 106 motile spermatozoa in raw semen had mean efficiency scores of 5.3, 2.2 and 8.2 points, respectively. There was no difference in efficiency scores between the two 500 X 10 6 treatments, but they were both statistically larger (P < .05) than the 2.2 points for the 100 • 10 6 treatment (table 5). 1973. From the previous results (tables 2 and 4) it was obvious another extender was required. In 1973, a cream-gel extender recommended by Hughes and Loy (1970) was utilized. Mares were inseminated with 500 X 10 6 motile spermatozoa, in 10 ml of 2.4% Tris or cream-gel extender, or in raw semen. Presented in table 6 are the fertility results over three cycles. In cycle 1, only 37.5% of the
PICKETT ET AL.
1142
No. cycles/no. pregnant
2.4
1.3
1.4
a s 0 0 X 106 motile spermatozoa. b l 0 ml of extender. C,dNumbers within rows followed by a different superscript are significantly different at the 1% level of probability (chi-square and analysis of variance).
Literature Cited Anderson, E. W. 1971. Equine .semen extenders. M.S. Thesis. Colo. State Univ., Fort Collins, 80523. Bedford, J. M. 1965. Non-specific tail-tail agglutination of mammalian spermatozoa. Exptl. Cell Res. 38: 654.
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1972) and the pH was much lower (6.65) than stallion semen (7.47) (Pickett e t al., 1970). A mean volume of 1.5 ml was used to inseminate the mares in the raw-seminal-treatment group, which was significantly less (P < .01) than the 11.8 or 11.6 ml used with the extended:seminal treatments (table 7). Since highly acceptable pregnancy rates were obtained with the raw semen, it is suspected that insemination volume is relatively unimportant in the mare. The factor(s) in Tris extenders responsible for the reduction in fertility of stallion spermatozoa is unknown. When the concentration of Tris was increased from .349 to 2.4% fertility also increased. Concomitantly the concentration of citric acid increased and glucose decreased. Further, the Tris extenders contained glycerol, and the presence of glycerol in porcine seminal extenders reduced fertility (King and Macpherson, 1966). Thus, there may be an interaction between glycerol and the concentration of other ingredients in the Tris extenders affecting fertility; particularly since the Tris extenders used in 1971 contained the same quantity of glycerol, yet fertility was lower when the concentration of Tris was only .349%. It is suspected that the addition of glycerol to the cream-gel extender would depress fertility. Further, the removal of TABLE 7. INFLUENCE OF SEMINAL TREATMENTS ON REPROglycerol from the 2.4% Tris extender might DUCTIVE PERFORMANCE enhance fertility. OF MARES (1973) Fertility was excellent when mares were inseminated with semen in the cream-gel Seminal treatmentsa extender (table 6). However, this extender has CreamRaw the disadvantage that motility and agglutination Variable Trisb gelb of the spermatozoa are obscured by the fat No. of mares 24 24 24 globules. Further, when the cream-gel extender Pregnancy rate/ 71.0d was cooled to approximately 5C, solidification cycle (%) 40.9 c 76.7 d occurred. No. of cycles 44 30 31 The relationship between motility and No. cycles/mare 1.8 c 1.2 d 1.3d fertility appears to be poor (table 3). Therefore, No. inseminaimprovements through laboratory studies will tion/cycle 3.1 2.6 3.2 be difficult until an extender is developed that Voi./inseminawill provide excellent fertility and in which the tion (ml) 11.8 c 11.6 c 1.5d spermatozoa can be viewed microscopically.
Tris-treatment group, which was lower (P < .01) than the 76.7 and 71.0% for mares in cream-gel and raw-seminal treatments, respectively. Mares inseminated with the Tris-treatment had more (P < .01) cycles per mare than mares in the other two treatments (1.8 vs 1.2 and 1.3). There was no difference ~ > .05) in the number of inseminations per cycle. The number of cycles per pregnancy was in favor of the cream-gel and raw-seminal treatments (1.3 and 1.4) when compared to the Tris-seminal treatment (2.4). Pregnancy rates of 75% during a single cycle were much higher than normally reported for nonlactating mares and would be considered excellent for other classes of livestock, including cattle. It has been assumed that fertility is lower in the equine than other species of livestock. Obviously, this is not true if proper management conditions are established and followed. It was somewhat surprising that the cream-gel extender provided such a satisfactory pregnancy rate, since the osmolality (348 mOs) was considerably higher than for stallion seminal plasma (302 mOs) (Pickett and Voss,
SEMINAL E X T E N D E R S ON EQUINE F E R T I L I T Y
Oshida, H., S. Horiuchi, H. Takahashi, T. Tomizuka and H. Nagase. 1968. Fertility of frozen stallion semen and some factors affecting to it. Prec. VI. lnternat. Congr. Anim Reprod. A.I. 2: 1597. Pickett, B. W. and D. G. Back. 1973. Procedures for preparation, collection, evaluation, and insemination of stallion semen. Colo. Sta. Univ. Exp. Sta. Anim. Reprod. Lab. Gen. Series 935. Pickett, B. W., M. R. Gebauer, G. E. Seidel, Jr. and J. L. Voss. 1974. Reproductive physiology'of the stallion: Spermatozoal losses in the collection equipment and gel. J.A.V.M.A. 165:708. Pickett, B. W., L. C. Faulkner and T. M. Sutherland. 1970. Effect of month and stallion on seminal characteristics and sexual behavior. J. Anim. Sci. 31: 713. Pickett, B. W., J. J. Sullivan and G. E. Seidel, Jr. 1975. Reproductive physiology of the stallion. V. Effect of frequency of ejaculation on seminal characteristics and spermatozoal output. J. Anita. Sci. 40:917. Pickett, B. W. and J. L. Voss. t972. Reproductive management of the stallion. Prec. 18th Ann. Cony. A.A.E.P., p. 501. Rajamannan, A. H. J., R . Zemjanis and J. Ellery. 1968. Freezing and fertility studies with stallion semen. Prec. VI. Internat. Congr. Anim. Reprod. A.I. 2: 1601. Senger, P. L. and R. G. Saacke. 1973. Effects of blood serum on bovine spermatozoa. J. Anim. Sci. 37: 328. Smith, A. U. 1949. Some antigenic properties of mammalian spermatozoa. Prec. Royal See. B. 136: 46.
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Bedford, J. M. 1970. Observations on some properties of a potent sperm-head agglutinin in the semen of a fertile rabbit. J. Reprod. Fertil. 22:193. Chang, M. C. 1947. The effects of serum on spermatozoa. J. Gen. Physiol. 30: 321. Cranwell, J. E. 1970. Factors affecting survival of frozen equine spermatozoa. M.S. Thesis. Colo. State Univ., Fort Collins, 80523. Ebertus, R. 1963. The dilution of stallion semen with whole cow milk. Animal Breed. Abstr. 31: 313. Henle, W., G. Henle and L. A. Chambers. 1938. Studies on the antigenic structure of some mammalian spermatozoa. J. Exp. Med. 68: 335. Hughes, J. P. and R. G. Loy. 1970. Artificial insemination in the equine. A comparison of natural breeding and artificial insemination of mares using semen from six stallions. Comell Vet. 60: 463. Kamenev, N. 1955. The use of milk diluent. Animal Breed. Abstr. 23:255. King, G. J. and J. W. Macpherson. 1966. The effects of glycerol on fertility of liquid boar semen. A.I. Digest 14 (12): 6. Krause, D. and D. Grove. 1967. Deep-freezing of jackass and stallion semen in concentrated pellet form. J. Reprod. Fertil. 14: 139. McCall, 1. P., Jr. and A. M. Sorensen, Jr. 1971. Evaporated milk as an extender for stallion semen. A.I. Digest 19 (12): 8. Nishikawa, Y., Y. Waide and S. Shinomiya. 1968. Studies on deep freezing of horse spermatozoa. Prec. V1. lnternat. Congr. Anim. Reprod. A.I. 2: 1589.
1143
B. W. Pickett I , L. D. Burwash 1,3, j. L. Voss 2 and D. G. Back 2,4 Colorado State University, Fort Collins 80523
Summary Pregnancy rate, spermatozoan motility and spermatozoan agglutination were significant.ly lower in an extender containing .349% Tris than in an extender utilizing 2.4% Tris. First-cycle pregnancy rates of 22.2 and 11.I% were obtained with 500 and 100 X 106 motile spermatozoa in. a 2.4% Tris extender, and a rate of 61.1% (P < .05) was obtained with 500 • 106 motile spermatozoa in raw semen. After four cycles there was no significant difference in fertility obtained with 2.4% Tris and the raw semen when 500 X 106 spermatozoa were utilized (72.2 vs 88.9%). First cycle pregnancy rates of 37.5, 75.0 and 75.0% were obtained in 1973 when mares were inseminated with 500 • 106 motile spermatozoa extended in 2.4% Tris, and cream-gel extenders and with raw semen, respectively. After three cycles, mares on the Tris-seminal treatment had a pregnancy rate of 75.0%, which was lower (P < .05) than the 95.8 and 91.7% for the cream-gel and raw-seminal treatments, respectively. The presence of glycerol in the Tris extenders may have been at least partially responsible for the depression of fertility. Pregnancy rates of 75.0 and 91.7% were obtained after one and three cycles, respectively, with a mean insemination volume of 1.5 ml of raw semen. Thus, a large insemination volume may not be necessary for maximum fertility in the mare.
Introduction
Loy, 1970); evaporated milk-glucose-glycerol (McCall and Sorensen, 1971); egg yolk-sugarglycerol (Krause and Grove, 1967; Oshida et al., 1968); mare's milk (Kamenev, 1955); egg yolk-glucose-glycerol (Nishikawa et al., 1968); skim milk-glucose (Rajamannan et al., 1968); and Tris-glucose-citric acid-egg yolk (Cranwell, 1970; Anderson, 1971). A stallion seminal extender is needed to prolong spermatozoan survival, enhance fertility, provide a media for antibiotic treatment of semen and permit a more convenient volume to be utilized, particularly when small numbers of spermatozoa are inseminated.
Materials and Methods General. Three groups ( 1 9 7 1 , 1972 and 1973) of nonlactating, clinically normal mares between 3 and 20 years of age were obtained for these studies between February 1 and April 1. All mares were maintained at the Equine
t Animal Reproduction Laboratory, Department of Physiology and Biophysics. Supported in part by grants-in-aid from American Breeders Service; National Association of Animal Breeders; and the Arabian Horse Registry of America, Inc., through Morris Animal Foundation. Portions of these data were presented at the Fifth N.A.A.B. Tech. Conf. A. I. Reprod., Chicago, IL, 1974, and the "First International Symposium Upon Equine Reproduction," Cambridge, England, 1974. 2 Department of Clinical Sciences. apresent address: Alberta Department of Agriculture, Animal Industry Division, Edmonton, Alberta, Canada. 4present address: Gleannloch Farms, SpringCypress Rd., Spring, TX 77393.
Many seminal extenders have been utilized to extend and store stallion semen. The types of extenders utilized include whole milk (Ebertus, 1963); cream-gelatin (Hughes and 1136
J O U R N A L O F A N I M A L S C I E N C E , vol. 4 0 , n o . 6, 1 9 7 5
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EFFECT OF SEMINAL EXTENDERS
1137
SEMINAL EXTENDERS ON EQUINE FERTILITY TABLE 1. EXTENDER COMPOSITION Tr~
Tris ( w / v ) Glucose (anhydrous) (w/v) Citric acid (monohydrate) (w/v) Egg y o l k ( v / v ) G l y c e r o l (v/v)
.349 b
2.4 b
.............
(%)
.349
2.4
3.655 .151 22.8
1.163 1.326 22.8
2.4 c
Cream-gelatin e
...............
2.4 .45 1.255 22.8
m m
5.41
5.41
5.25
Half & half cream d ( v / v )
--
--
--
88.7
K n o x gelatin e ( w / v )
--
--
--
1.3
67.8
69.5
67.9
10.0
Water (v/v) Osmolality (mOs) pH
350
350
298
7.00
7.00
6.99
348
6.65
a C o n t a l n s 1 , 0 0 0 ~ g s t r e p t o m y c i n sulfate and 1,00O units s o d i u m p e n c i l l i n G / m l . b1971 and 1972. c1973. d C o n t a i n e d a p p r o x i m a t e l y 1 1 % b u t t e r f a t , 2 . 8 % c a s e i n , .9% a l b u m i n a n d 7 8 . 9 % w a t e r . eKnox Gelatin, Inc., Johnstown, New York.
inseminated on days 3, 5, 7, etc, of estrus with semen collected daily from a single stallion. Mares were inseminated with 500 X 10 6 motile spermatozoa extended in either .349 or 2.4% Tris extender (table 1) with a final volume of t> 10 ml but .10) between the 26.7 and 12.5% pregnancy rates obtained in cycle 2 with semen extended in the 2.4 and .349% Tris, respectively. The lower pregnancy rate during
the second cycle could have resulted if the more fertile mares conceived during the first cycle. There is also the pgssibility that the mares had developed an immunological response to the extender, because it contained 22.8% egg yolk. The 40.6% pregnancy rate over two cycles for mares inseminated with semen extended in 2.4% Tris extender was higher (P < .10) than the 15.4% pregnancy rate in mares inseminated with semen extended in .349% Tris extender. The first-cycle pregnancy rate (52.9%) for semen extended in 2.4% Tris is in agreement with that reported,, for Thoroughbred and Quarter Horses (Pickett and Voss, 1972). There were no differences between number of inseminations per cycle or volume inseminated for the .349 or 2.4% Tris-seminal treatments. The influence of .349 and 2.4% Tris extenders on spermatozoal motility, agglutination score and clumping score at 0, 1 and 2 hr after seminal collection is presented in table 3. There was no difference (P > .05) in spermatozoal motility, between the two extenders at 0 hr, but motility was depressed after 1- and 2-hr incubation in the .349% Tris extender (P < .05). The spermatozoan-agglutination score was higher (P < .05) in the 2.4% Tds extender than the .349% Tris extender regardless of incubation period. Also, spermatozoan agglutination increased (P < .05) after 1 hr incubation in the 2.4% Tris extender. The clumping score was higher (P < .05) at 0-hr incubation in the 2.4% Tris extender than in the .349% Tris extender, but the clumping did not differ after incubation.
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Cycle
No. of mares inseminated
SEMINAL EXTENDERS ON EQUINE FERTILITY
membrane over the acrosomal and post-acrosomal regions (Bedford, 1965, 1970). Most of the agglutination that occurred involved the "sticking together" of the sperm at the acrosomal region, but tail-to-tail agglutination has been reported (Bedford, 1965). He has also shown that the concentration of electrolytes in the medium influenced the head-to-head agglutination of rabbit spermatozoa, which may account for the differences in degree of clumping observed in the present study. It has been generally assumed that agglutination renders the sperm infertile. Although, Bedford (1970) has reported that spermatozoa from one rabbit that showed agglutination were fertile, and agglutination in this study was associated with increased fertility. 1972. In this experiment, pregnancy rates obtained with seminal treatments utilizing 100 • 106 and 500 • 1 0 6 motile spermatozoa in 10 ml of 2.4% Tris extender were compared to 500 X 1 0 6 motile spermatozoa in raw semen In cycle 1, the 61.1% pregnancy rate was higher (P < .05) for mares inseminated with 500 • 1 0 6 motile spermatozoa in raw semen than the 22.2 and 11.1% pregnancy rate for mares inseminated with 500 X 1 0 6 o r 100 • 1 0 6 motile spermatozoa extended in 10 ml of Tris extender (table 4). There were no differences (P > .05) in pregnancy rates obtained during cycles 2, 3 or 4. The 27.8% pregnancy rate over four cycles for mares inseminated with 100 X 1 0 6 motile spermatozoa in Tris extender was lower (P < .05) than the 72.2 or 88.9%, respectively, for the mares inseminated with
T A B L E 3. M O T I L I T Y , A G G L U T I N A T I O N S C O R E A N D C L U M P I N G S C O R E F O R S P E R M A T O Z O A 1N . 3 4 9 A N D 2 . 4 % T R I S E X T E N D E R S ( 1 9 7 1 )
Agglutination
M o t i l i t y (%) Incubation time (hr)
.349
Clumping
score a 2.4
.349
score b 2.4
.349
2.4
0
65.0 c
65.0 c
.0 d
1.1 c
.0 d
1
53.3 d
61.8 c
.3 d
1.9 e
1.1 c
1 . 6 c'e
1.1 c
2
36.3 e
55.2 d
.4 d
2.1 e
1.4 c,e
2.3 e
a 0 = n o a g g l u t i n a t i o n , 1 = 2 0 % , 2 = 4 0 % , 3 = 6 0 % , 4 - 80% and 5 = 1 0 0 % agglutination. b 0 = n o s p e r m a t o z o a per clump, 1 = 2-4, 2 = 5 - 1 0 , 3 = 1 1 - 1 5 , 4 = 1 6 - 2 0 a n d 5 = ~ 2 0 s p e r m a t o z o a per clump. c,d,eMeans under each major heading followed b y different superscripts are significantly different at the 5% level of probability (Analysis of variance and Studentized range test).
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Senger and Saacke (1973) reported that agglutinated bovine spermatozoa maintained both motility and intact acrosomes longer than nonagglutinated spermatozoa. It is tempting to suggest that conditions which permit agglutination are beneficial to spermatozoal motility and perhaps fertility, since 40.6% of the mares became pregnant when spermatozoa were extended in the 2.4% Tris extender and only 15.4% when mares were inseminated with semen extended in .349% Tris extender. The clumping of stallion spermatozoa is highly seasonal (Pickett et al. 1970), probably due to changes in the chemical composition of the seminal plasma. Motility of stallion spermatozoa also appeared to be affected by season. However, when semen was placed in an extender that prevented clumping, motility was relatively constant throughout the year. Thus, it was concluded that clumping interfered with motility, and for accurate motility estimations stallion semen must be extended in a diluent that prevents clumping (Pickett and Voss, 1972). Clumping, or agglutination of spermatozoa, appears to be a normal phenomenon in the semen of several species including rabbit, guinea pig, boar, and stallion (Bedford, 1965, 1970; Pickett et al., 1970). This occurrence probably has little or no relationship to the antigenic properties of spermatozoa following isoimmunizations with semen or testicular tissue (Henle et al., 1938; Chang, 1947; Smith, 1949). In all probability, "normal" agglutination is due to different surface properties of the plasma
1139
1140
PICKETT ET AL.
TABLE 4. THE E F F E C T OF SEMINAL TREATMENTS ON PREGNANCY R A TE OF MARES (1972)
Extendeda I00 b
Pregnancy rate
No. of mares inseminated
100 b
Cycle
No. of mares inseminated
Pregnancy rate
1
18 (4) c
22.2 d
18 (2)
11,1 d
18 (11)
61.1 e
2 3 4 Total
14 (3) 11 (4) 7 (2) (13)
21,4 36.4 28.6 72.2 d
16 (2) 13 (0) 12 (1) (5)
12.5 .0 8.3 27.8 e
7 (4) 2 (1) 1 (0) (16)
57.1 50.0 .0 88.9 d
(%)
No. of mares inseminated
(%)
Pregnancy rate
(%)
a E x t e n d e d in 10 ml of 2.4% Trls e x t e n d e r . b(10 6 ) motile spermatozoa. CNumbers in parentheses axe t h e n u m b e r o f maxes t h a t b e c a m e pregnant. d , e p e z c e n t a g e s within rows followed by a d i f f e r e n t superscript are significantly different a t the 5% level of p r o b a b i l i t y (chi-squaxe analysis).
500 X 106 motile spermatozoa in Tris extender or in raw semen. There was no statistically significant difference (P :> .05) in pregnancy rate over four cycles between the two seminal treatments utilizing 500 X 106 motile spermatozoa, although there was an observed difference of 16.7 percentage points in favor of the raw-seminal treatment. The 2.4% Tris extender depressed fertility, and the low pregnancy rate with 100 X 106 m o t i l e spermatozoa was due to an interaction between spermatozoal numbers and extenders. From these data, it is suspected that to properly evaluate a seminal extender the number of motile spermatozoa per insemination should not exceed 100 X l 0 6 when mares are inseminated every other day. Early pregnancies are important to the equine industry. After two cycles, 83.3% of the mares inseminated with raw semen became pregnant, while only 38.9% o f the mares inseminated with an equal number of spermatozoa in 10 ml o f 2.4% Tris extender became pregnant. Inseminating with raw semen containing 500 • 106 motile spermatozoa must be recommended. In the event pathogenic organisms are present, an extender containing the appropriate antibiotic is recommended. The excellent pregnancy rate obtained with raw semen was higher than normally anticipated and may have occurred due to the small number of mares, or because properly managed
mares are not as inherently infertile as previously suspected. Pregnancy rates on a per cycle basis were different (P < .05) among treatments, averaging
TABLE 5. THE EFFECT OF SEMINAL TREATMENTS ON REPRODUCTIVE EFFICIENCY IN MARES (1972)
Variable No. of mares Pregnancy rate/ cycle (%) No, o f cycles/ mare No. of inseminations]cycle Vol./insemination (ml) Efficiency o f pregnancy e
Spermatozoa (106 ) Extendeda Raw 500 100 100 18
18
18
26.0 b
8.5 c
57.1d
2.8 b
3.3b
1.6 c
2.6
2.9
2.7
13.4b
10.6 c
4.2d
5.3b
2.2 c
8.2 b
a E x t e n d e d in 10 mi of 2.4% Tris extender. b,c,dNumbers w i t hi n rows followed b y a d i f f e r e n t superscript axe significantly different a t t he 5% level of proba bi l i t y (S t ude nt i z e d range test a nd chi-square analy sis). e E a e h m a ~ t h a t became p r e g n a n t o n cycle 1 was awarded 10 points, eight p o i n t s for cycle 2, six p o i n t s for cycle a and four p o i n t s for cycle 4.
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500 b
Raw
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SEMINAL EXTENDERS ON EQUINE FERTILITY
mares inseminated with semen in Tris became pregnant, compared to 75.0% for both the cream-gel and raw-seminal treatments. These differences were not statistically significant (P > .05). However, after three cycles, mares inseminated with the Tris-seminal treatment had a 75.0% pregnancy rate, which was lower (P < .01) than the 95.8 and 91.7% for the cream-gel and raw-seminal treatments, respectively. Although direct comparisons among the 1971, 1972 and 1973 experiments could not be made, there appeared to be little or no influence of day of first insemination on pregnancy rate. In 1971 and 1972, semen was collected daily and mares inseminated on days 3, 5, 7, etc, or days 2, 4, 6, etc, of estrus, respectively; while in 1973 semen was collected every other day, and all mares in estrus for two days or longer were inseminated. This resulted in some mares being bred on days 2, 4, 6, etc, and others on 3, 5, 7, etc. Since this procedure resulted in less labor, more spermatozoa per ejaculate (Pickett et al., 1975), fewer spermatozoa lost per collection (Pickett et al., 1974) and acceptable pregnancy rates (table 6), it must be recommended. The influence of extenders on other measures of reproductive performance is presented in table 7. A 40.9% pregnancy rate per cycle was obtained for mares in the
TABLE 6. THE EFFECT OF SEMINAL TREATMENTS ON PREGNANCY RATE OF MARES (1973)
Extended a Tris No. of mares
Cycle
inseminated
Cream-gel
Pregnancy rate
No. o f mares inseminated
(%)
Raw b
Pregnancy rate
No. o f mares Pregnancy inseminated rate
(%)
(%)
1
24 (9) c
37.5
24 (18)
75.0
24 (18)
75.0
2
13 (5)
38.5
5 (4)
80.0
6 (4)
66.7
3
7 (4)
57.1
I00.0
1 (0)
Total
(18)
75.0 d
I (I)
(23)
95.8 e
(22)
.0 91.7 e
a s o 0 X 10 6 m o t i l e s p e r m a t o z o a in 10 ml e x t e n d e r . b s o 0 X 10 6 m o t i l e s p e r m a t o z o a . CNumbets in parentheses are the n u m b e r o f mares that b e c a m e pregnant. d,epercentages w i t h i n r o w s f o l l o w e d b y a different superscript are significantly different at t h e 1% level o f probability (Analysis of variance).
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26.0, 8.5 and 57.1% for the 500 and 100 • 106 extended- and raw-seminal treatments, respectively (table 5). The average of 1.6 cycles per mare in the raw-seminal treatment was less (P < .05) than the 2.8 and 3.3 cycles per mare for the two extended-seminal treatments. There was no difference (P > .05) in the number of inseminations per cycle among the treatments. There was a difference (P < .05) in the volumes inseminated, 13.4 and 10.6 ml for the 500 and 100 X 10 6 motile spermatozoa in the extended treatments and 4.2 ml for the raw-seminal treatment. Mares inseminated with 500 or 100 X 106 motile spermatozoa in the extended-seminal treatments and 500 X 106 motile spermatozoa in raw semen had mean efficiency scores of 5.3, 2.2 and 8.2 points, respectively. There was no difference in efficiency scores between the two 500 X 10 6 treatments, but they were both statistically larger (P < .05) than the 2.2 points for the 100 • 10 6 treatment (table 5). 1973. From the previous results (tables 2 and 4) it was obvious another extender was required. In 1973, a cream-gel extender recommended by Hughes and Loy (1970) was utilized. Mares were inseminated with 500 X 10 6 motile spermatozoa, in 10 ml of 2.4% Tris or cream-gel extender, or in raw semen. Presented in table 6 are the fertility results over three cycles. In cycle 1, only 37.5% of the
PICKETT ET AL.
1142
No. cycles/no. pregnant
2.4
1.3
1.4
a s 0 0 X 106 motile spermatozoa. b l 0 ml of extender. C,dNumbers within rows followed by a different superscript are significantly different at the 1% level of probability (chi-square and analysis of variance).
Literature Cited Anderson, E. W. 1971. Equine .semen extenders. M.S. Thesis. Colo. State Univ., Fort Collins, 80523. Bedford, J. M. 1965. Non-specific tail-tail agglutination of mammalian spermatozoa. Exptl. Cell Res. 38: 654.
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1972) and the pH was much lower (6.65) than stallion semen (7.47) (Pickett e t al., 1970). A mean volume of 1.5 ml was used to inseminate the mares in the raw-seminal-treatment group, which was significantly less (P < .01) than the 11.8 or 11.6 ml used with the extended:seminal treatments (table 7). Since highly acceptable pregnancy rates were obtained with the raw semen, it is suspected that insemination volume is relatively unimportant in the mare. The factor(s) in Tris extenders responsible for the reduction in fertility of stallion spermatozoa is unknown. When the concentration of Tris was increased from .349 to 2.4% fertility also increased. Concomitantly the concentration of citric acid increased and glucose decreased. Further, the Tris extenders contained glycerol, and the presence of glycerol in porcine seminal extenders reduced fertility (King and Macpherson, 1966). Thus, there may be an interaction between glycerol and the concentration of other ingredients in the Tris extenders affecting fertility; particularly since the Tris extenders used in 1971 contained the same quantity of glycerol, yet fertility was lower when the concentration of Tris was only .349%. It is suspected that the addition of glycerol to the cream-gel extender would depress fertility. Further, the removal of TABLE 7. INFLUENCE OF SEMINAL TREATMENTS ON REPROglycerol from the 2.4% Tris extender might DUCTIVE PERFORMANCE enhance fertility. OF MARES (1973) Fertility was excellent when mares were inseminated with semen in the cream-gel Seminal treatmentsa extender (table 6). However, this extender has CreamRaw the disadvantage that motility and agglutination Variable Trisb gelb of the spermatozoa are obscured by the fat No. of mares 24 24 24 globules. Further, when the cream-gel extender Pregnancy rate/ 71.0d was cooled to approximately 5C, solidification cycle (%) 40.9 c 76.7 d occurred. No. of cycles 44 30 31 The relationship between motility and No. cycles/mare 1.8 c 1.2 d 1.3d fertility appears to be poor (table 3). Therefore, No. inseminaimprovements through laboratory studies will tion/cycle 3.1 2.6 3.2 be difficult until an extender is developed that Voi./inseminawill provide excellent fertility and in which the tion (ml) 11.8 c 11.6 c 1.5d spermatozoa can be viewed microscopically.
Tris-treatment group, which was lower (P < .01) than the 76.7 and 71.0% for mares in cream-gel and raw-seminal treatments, respectively. Mares inseminated with the Tris-treatment had more (P < .01) cycles per mare than mares in the other two treatments (1.8 vs 1.2 and 1.3). There was no difference ~ > .05) in the number of inseminations per cycle. The number of cycles per pregnancy was in favor of the cream-gel and raw-seminal treatments (1.3 and 1.4) when compared to the Tris-seminal treatment (2.4). Pregnancy rates of 75% during a single cycle were much higher than normally reported for nonlactating mares and would be considered excellent for other classes of livestock, including cattle. It has been assumed that fertility is lower in the equine than other species of livestock. Obviously, this is not true if proper management conditions are established and followed. It was somewhat surprising that the cream-gel extender provided such a satisfactory pregnancy rate, since the osmolality (348 mOs) was considerably higher than for stallion seminal plasma (302 mOs) (Pickett and Voss,
SEMINAL E X T E N D E R S ON EQUINE F E R T I L I T Y
Oshida, H., S. Horiuchi, H. Takahashi, T. Tomizuka and H. Nagase. 1968. Fertility of frozen stallion semen and some factors affecting to it. Prec. VI. lnternat. Congr. Anim Reprod. A.I. 2: 1597. Pickett, B. W. and D. G. Back. 1973. Procedures for preparation, collection, evaluation, and insemination of stallion semen. Colo. Sta. Univ. Exp. Sta. Anim. Reprod. Lab. Gen. Series 935. Pickett, B. W., M. R. Gebauer, G. E. Seidel, Jr. and J. L. Voss. 1974. Reproductive physiology'of the stallion: Spermatozoal losses in the collection equipment and gel. J.A.V.M.A. 165:708. Pickett, B. W., L. C. Faulkner and T. M. Sutherland. 1970. Effect of month and stallion on seminal characteristics and sexual behavior. J. Anim. Sci. 31: 713. Pickett, B. W., J. J. Sullivan and G. E. Seidel, Jr. 1975. Reproductive physiology of the stallion. V. Effect of frequency of ejaculation on seminal characteristics and spermatozoal output. J. Anita. Sci. 40:917. Pickett, B. W. and J. L. Voss. t972. Reproductive management of the stallion. Prec. 18th Ann. Cony. A.A.E.P., p. 501. Rajamannan, A. H. J., R . Zemjanis and J. Ellery. 1968. Freezing and fertility studies with stallion semen. Prec. VI. Internat. Congr. Anim. Reprod. A.I. 2: 1601. Senger, P. L. and R. G. Saacke. 1973. Effects of blood serum on bovine spermatozoa. J. Anim. Sci. 37: 328. Smith, A. U. 1949. Some antigenic properties of mammalian spermatozoa. Prec. Royal See. B. 136: 46.
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Bedford, J. M. 1970. Observations on some properties of a potent sperm-head agglutinin in the semen of a fertile rabbit. J. Reprod. Fertil. 22:193. Chang, M. C. 1947. The effects of serum on spermatozoa. J. Gen. Physiol. 30: 321. Cranwell, J. E. 1970. Factors affecting survival of frozen equine spermatozoa. M.S. Thesis. Colo. State Univ., Fort Collins, 80523. Ebertus, R. 1963. The dilution of stallion semen with whole cow milk. Animal Breed. Abstr. 31: 313. Henle, W., G. Henle and L. A. Chambers. 1938. Studies on the antigenic structure of some mammalian spermatozoa. J. Exp. Med. 68: 335. Hughes, J. P. and R. G. Loy. 1970. Artificial insemination in the equine. A comparison of natural breeding and artificial insemination of mares using semen from six stallions. Comell Vet. 60: 463. Kamenev, N. 1955. The use of milk diluent. Animal Breed. Abstr. 23:255. King, G. J. and J. W. Macpherson. 1966. The effects of glycerol on fertility of liquid boar semen. A.I. Digest 14 (12): 6. Krause, D. and D. Grove. 1967. Deep-freezing of jackass and stallion semen in concentrated pellet form. J. Reprod. Fertil. 14: 139. McCall, 1. P., Jr. and A. M. Sorensen, Jr. 1971. Evaporated milk as an extender for stallion semen. A.I. Digest 19 (12): 8. Nishikawa, Y., Y. Waide and S. Shinomiya. 1968. Studies on deep freezing of horse spermatozoa. Prec. V1. lnternat. Congr. Anim. Reprod. A.I. 2: 1589.
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