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Relationship Between Fertility and the Nonprotein Sulfhydryl Concentration of Seminal Fluid in the Thoroughbred Stallion
Relationship Between Fertility and the Nonprotein Sulfhydryl Concentration of Seminal Fluid in the Thoroughbred Stallion Frederick M. Haag, D.V.M., * and N. T. Werthessen, Ph.D.
IN
AN EARLIER REPORT!
it was shown that an inverse correlation existed
between the nonprotein SH concentration of stallion seminal fluid and the ability of that fluid to maintain motility in spermatozoa. Since motility is one of the properties of spermatozoa which correlates best with fertility, this was some indication that our findings might bear a valid relationship to actual fertility. However, it was recognized that an actual field test would be the only way to demonstrate conclusively a relationship between sulfhydryl concentration and fertility. A unique opportunity to test our hypothesis was given to us by the sponsors of this research, and the following report describes the results of an extensive field trial. Southwest Foundation for Research and Education, San Antonio, Texas, and the Department of Animal Pathology, University of Kentucky, Lexington, Ky. • Guest of the Department of Animal Pathology, University of Kentucky, during the spring of 1955. Supported in part by Greentree Stud, Inc., the Grayson Foundation, and the Keeneland Foundation. The authors wish to express their appreciation to Dr. Hagyard, Dr. W. R. McGee, Dr. F. Saeger, Dr. Horace Davis, and Mr. F. McKenzie, whose kindness and cooperation made it possible to collect adequate specimens for the study, and to Mr. Clarkson Beard, Mr. A. B. Hancock, Jr., Mr. Leslie Combs, and Mr. L. P. Doherty for the trouble these men took in making available the material gathered from the stud farms under their management. To Dr. F. E. Hull of the Department of Animal Pathology, University of Kentucky, their thanks for his making available to Dr. Haag the laboratory facilities of his department. Dr. Goldzieher's assistance in developing the method used in this study was requisite for its success. We wish to record our appreciation for his assistance as well as for his cogent advice as the study progressed. 516
Vol. 7, No.6, 1956
SEMINAL SH AND FERTILITY
517
METHODS This study has taken advantage of the practices and requirements of Thoroughbred horse breeders; since these are quite rigid and specific, and contribute to the study's significance, some detailed description is requisite. One of the objectives in Thoroughbred horse breeding is the delivery of a foal as soon after January 1 as possible, since horses are entered in races according to their age. Irrespective of the date of birth, all Thoroughbred horses become one year of age the first January of their existence. Thus, as far as the rules are concerned, a horse born on January 2 and another born on December 30 in any calendar year are both 1 year old on January 1 of the following calendar year. Such a tremendous age difference hardly ever occurs, but it is clear that under such rules a foal born in June is liable to suffer a serious handicap compared to a future competitor born 4 or 5 months earlier. Other points to be recognized are that (1) artificial insemination is illegal. The stallion must copulate with, or "cover," the mare. (2) Prior to a breeding season (which extends from January to July) each stallion's owner arranges the stallion's breeding activities for the coming season. Approximately 20 to 30 mares are "booked" to a stallion, depending on the stallion's reputation in regard to racing background, fertility, and proved genetic capacity. (3) Stud fees are payable only when the mare is proved pregnant or drops a living foal. (4) Offspring are valueless without a pedigree. (5) Stud fees, in the area and for the stallions on which this work was done, are quite high, reaching $5000 per impregnation. These features have a number of consequences of importance in this study: 1. Chicanery, in the sense of hiding a pregnancy or having a mare copulate with another stallion, is valueless, as the mare's owner either obtains no pedigree and has a completely worthless horse, or pays an enormous sum for a genetic background he could have obtained for far less. 2. Each stallion has so many mares to cover that he is rarely capable of copulating with a mare more than once per heat period. "Double cover" (two matings per cycle) is so rare as to deserve special mention when it occurs, and such data are not included in our statistics. 3. A mare's owner may be paying as much as $5,000 per copulation; this, together with his extreme desire to induce pregnancy early in the season,
518
HAAG & WERTH ESSEN
Fertility & Sterility
has given rise to the practice of placing all available semen into the mare at each copulation. This is accomplished primarily by obtaining and using the "tail end" sample. A stallion's penis when removed from the mare after copulation is not completely flaccid and may still retain from 2 to 15 cc. of semen which did not enter the mare. Breeding practice today calls for the squeezing of this fluid from the penis (the "tail-end sample"), and injecting it into the uterus of the mare. Thus if an aliquot of the tail-end sample could be obtained and if this were representative of the whole ejaculate, measurements could be made on a semen sample actually used for impregnation. Examination of Tail-end Samples
To test this point, a number of ejaculates and the corresponding tail-end samples were obtained from the laboratory's stallions by use of the artificial vagina and subsequent "stripping" of the penis in the usual fashion. Amperometric titration2 demonstrated a sufficiently close and constant correlation between the two fractions to permit us to proceed with a field trial based on the tail-end sample. Further preparations for the field trial involved a study of the effect of freezing in dry ice of an uncentrifuged sample, and the lability in storage of the small samples (1 to 5 cc.) that would be available. No significant alterations were found. The technic of collection as finally developed was as follows: 1. To each breeding farm (or shed) a daily supply of clean plastic centrifuge tubes bearing the stallion's name, laboratory code number, and the serial number of the sample of the particular horse was provided. 2. The dry ice container at each station was replenished daily. 3. Samples were picked up daily from each farm, maintained over dry ice at all times, and brought back to the laboratory for storage over dry ice until assayed. 4. At the time of assay the samples were allowed to melt and were then centrifuged; the supernatant fluid was removed, and two aliquots of 1 cc. each used for determination of the SH concentration whenever the volume permitted. One cubic centimeter of seminal fluid was pipetted directly into 9.0 cc. of the electrolyte used in the titration. Protein precipitation took place and went to completion while the specimen remained in the dry-ice box overnight. The solution was centrifuged and the supernatant used for titration. An additional 20 cc. of electrolyte was added and titration carried out. The amperometric technic of Goldzieher et al. was used. 2
519
SEMINAL SH AND FERTILITY
Vol. 7, No.6, 1956
RESULTS Stallions and mares of all ranges of fertility provided the material for this study. The "raw" data uncorrected for mare sterility are presented in Fig. l. The number of samples in each group is given in brackets and the per cent of pregnacies is plotted as the ordinate. For the sake of simplicity the nonprotein SH concentration is plotted on the abscissa in terms of tenths of mls. of .001N AgN03 used in the determination. This is done in view of the fact that "blank" titrations with p-chloromercuribenzoate usually could not be 60
50
raw
.--
r----
data: 1320 observations
r----
C/)
W 4 0U
Z
« z (!)
W
r----
30
a::
0.. r----
~ Z
20
W U
a::
~
.----
10
o
r----
(441)
(353)
(248)
(133)
(77)
(34)
(15)
(8)
0-0.9
1.0-1.9
2.0-2.9
3.0-3.9
4.0-4.9
5.0-5.9
6.0-6.9
7.0-7.9
SH
Fig. 1.
(II)
8.0
+-
CONCENTRATION
Fertility of single inseminations vs. seminal fluid SH level, raw data.
done; therefore, the observed values are actually slightly higher than the true SH concentration. The values of AgN03 plotted on the abscissa may be converted easily into micrograms of nonprotein sulfhydryl per cubic centimeter of seminal fluid by multiplying by 3.307 (0.1 cc. of O.OOlN AgN03 is equivalent to 3.307 fLg. sulfhydryl). In Fig. 2, matings to mares that returned four times to the stallion as well as those that occurred within their first heat period after foaling have been
520
HAAG & WERTHESSEN
Fertility & Sterility
eliminated. This obviously increases the over-all fertility rate and further sharpens the significance of the correlation to sulfhydryl. It is evident that the fertility of ejaculates with a nonprotein sulfhydryl concentration of about 3 units (10 p.g.jcc.) or higher is greatly impaired; when the level reaches 6 units (20 p.g. / cc. ), fertility is virtually nil. Figure 3 presents information on two selected stallions. Stallion CAB 60
r-r-CJ)
50
r--
L&.J
(3
Z
observations: adjusted for female infertility factor 1052
z
r--
40
(!)
L&.J
a:
a..
30 r-
~
Z L&.J 20 U
a: L&.J
r---
a..
10
( 351)
(284)
r---
(201)
(101)
(64)
(28)
( 10)
2.0-2.9
3.0-3.9
4.0-4.9
5.0-5.9
6.0-6.9
(0 )
(7)
o 0- 0.9
Fig. 2.
LO-I.9
7.0-7.9
8.0+
SH CONCENTRATION Fertility of single inseminations vs. seminal fluid SH level, adjusted for female infertility factor.
maintained a consistently low level of sulfhydryl; his fertility was high, and both the failures in a total of 12 copulations occurred when he was mated with mares who had foaled 9 days previously. Stallion CLH, on the other hand, showed great variability in his sulfhydryl concentration. Eleven fluids showed a level of 3 units or higher (10 p.g.jcc.) out of a total of 30. In the 19 matings where the tail-end sample showed a level lower than 3 units, there were 13 pregnancies; in the eleven samples with a sulfhydryl of 3 or higher, there were only 3 pregnancies. This is statistically significant at the 2 per cent level.
r
521
SEMINAL SH AND FERTILITY
Vol. 7, No.6, 1956
Conclusion. A logical consequence of this observation is that the failure of a particular copulation can be predicted with reasonable certainty, simply by measurement of the sulfhydryl concentration of the tail-end sample of seminal fluid.
IC
Z
0
....
o
4
3
IC
---_ -
-
....
0
-j--
~
= pregnancy
x
=fai lure
8
LLJ U Z
-
KEY:
37%
2
•
•
• •
o
U
IC
:I:
a
en
0 M
0<
0 Fig. 3.
0:
cAB
83%
, o o
60% (difference between 37% 8 60 % significant at the 2 % level)
'" co
.~
CLH
Fertility vs. seminal fluid SH level in consecutive samples from individual stallions.
DISCUSSION These data seem to demonstrate clearly (at least in the Thoroughbred stallion) that the presence of an unknown but measurable factor in the seminal fluid contributes markedly to the fertility of the total ejaculate. When in addition it is recognized that our results are entirely uncorrected for the intrinsic fertility potential of the spermatozoa themselves, as they may vary from sample to sample and stallion to stallion, they appear to gain added significance. Were it possible to measure in addition a fertility index of the spermatozoa, an over-all measure of even greater reliability could result.
522
HAAG & WERTH ESSEN
Fertility & Sterility
The significance of these data as they apply to other species is not known. Few animals provide a suitable quantity of seminal fluid for even the most sensitive technics, nor do the practices concerned with their breeding permit so clear a study as that described. This may well be the reason why observations such as ours have not been made heretofore. Studies in other species, particularly the human, are currently in progress, and the chemical identity of the active factors is under investigative attack. Results of these studies will be reported subsequently. SUMMARY AND CONCLUSIONS It has been demonstrated that a relationship exists in the stallion between fertility and the nonprotein sulfhydryl concentration of seminal fluid. The relationship is inverse; a sharp decline in fertility occurs with concentrations exceeding 10 P,g. sulfhydryl! cc., and infertility of the specimen is virtually certain above 20 P,g.j cc. Fluids containing less than lO P,g.j cc. have a 55 to 60 per cent possibility of causing pregnancy in the group of Thoroughbreds studied. REFERENCES 1.
and WERTHESSEN, N. T. Influence of seminal fluid on sperm motility. Fertil. & Steril. 7:508, 1956. 2. GoLDZIEHER, J. W., RAWLS, W. B., and GOLDZIEHER, M. A. The uptake of sulfhydryl compounds by rat adrenal, liver, and muscle as measured by an improved amperometric technique. ]. Biol. Chern. 203:519, 1953. MARDEN, W.,
IN
AN EARLIER REPORT!
it was shown that an inverse correlation existed
between the nonprotein SH concentration of stallion seminal fluid and the ability of that fluid to maintain motility in spermatozoa. Since motility is one of the properties of spermatozoa which correlates best with fertility, this was some indication that our findings might bear a valid relationship to actual fertility. However, it was recognized that an actual field test would be the only way to demonstrate conclusively a relationship between sulfhydryl concentration and fertility. A unique opportunity to test our hypothesis was given to us by the sponsors of this research, and the following report describes the results of an extensive field trial. Southwest Foundation for Research and Education, San Antonio, Texas, and the Department of Animal Pathology, University of Kentucky, Lexington, Ky. • Guest of the Department of Animal Pathology, University of Kentucky, during the spring of 1955. Supported in part by Greentree Stud, Inc., the Grayson Foundation, and the Keeneland Foundation. The authors wish to express their appreciation to Dr. Hagyard, Dr. W. R. McGee, Dr. F. Saeger, Dr. Horace Davis, and Mr. F. McKenzie, whose kindness and cooperation made it possible to collect adequate specimens for the study, and to Mr. Clarkson Beard, Mr. A. B. Hancock, Jr., Mr. Leslie Combs, and Mr. L. P. Doherty for the trouble these men took in making available the material gathered from the stud farms under their management. To Dr. F. E. Hull of the Department of Animal Pathology, University of Kentucky, their thanks for his making available to Dr. Haag the laboratory facilities of his department. Dr. Goldzieher's assistance in developing the method used in this study was requisite for its success. We wish to record our appreciation for his assistance as well as for his cogent advice as the study progressed. 516
Vol. 7, No.6, 1956
SEMINAL SH AND FERTILITY
517
METHODS This study has taken advantage of the practices and requirements of Thoroughbred horse breeders; since these are quite rigid and specific, and contribute to the study's significance, some detailed description is requisite. One of the objectives in Thoroughbred horse breeding is the delivery of a foal as soon after January 1 as possible, since horses are entered in races according to their age. Irrespective of the date of birth, all Thoroughbred horses become one year of age the first January of their existence. Thus, as far as the rules are concerned, a horse born on January 2 and another born on December 30 in any calendar year are both 1 year old on January 1 of the following calendar year. Such a tremendous age difference hardly ever occurs, but it is clear that under such rules a foal born in June is liable to suffer a serious handicap compared to a future competitor born 4 or 5 months earlier. Other points to be recognized are that (1) artificial insemination is illegal. The stallion must copulate with, or "cover," the mare. (2) Prior to a breeding season (which extends from January to July) each stallion's owner arranges the stallion's breeding activities for the coming season. Approximately 20 to 30 mares are "booked" to a stallion, depending on the stallion's reputation in regard to racing background, fertility, and proved genetic capacity. (3) Stud fees are payable only when the mare is proved pregnant or drops a living foal. (4) Offspring are valueless without a pedigree. (5) Stud fees, in the area and for the stallions on which this work was done, are quite high, reaching $5000 per impregnation. These features have a number of consequences of importance in this study: 1. Chicanery, in the sense of hiding a pregnancy or having a mare copulate with another stallion, is valueless, as the mare's owner either obtains no pedigree and has a completely worthless horse, or pays an enormous sum for a genetic background he could have obtained for far less. 2. Each stallion has so many mares to cover that he is rarely capable of copulating with a mare more than once per heat period. "Double cover" (two matings per cycle) is so rare as to deserve special mention when it occurs, and such data are not included in our statistics. 3. A mare's owner may be paying as much as $5,000 per copulation; this, together with his extreme desire to induce pregnancy early in the season,
518
HAAG & WERTH ESSEN
Fertility & Sterility
has given rise to the practice of placing all available semen into the mare at each copulation. This is accomplished primarily by obtaining and using the "tail end" sample. A stallion's penis when removed from the mare after copulation is not completely flaccid and may still retain from 2 to 15 cc. of semen which did not enter the mare. Breeding practice today calls for the squeezing of this fluid from the penis (the "tail-end sample"), and injecting it into the uterus of the mare. Thus if an aliquot of the tail-end sample could be obtained and if this were representative of the whole ejaculate, measurements could be made on a semen sample actually used for impregnation. Examination of Tail-end Samples
To test this point, a number of ejaculates and the corresponding tail-end samples were obtained from the laboratory's stallions by use of the artificial vagina and subsequent "stripping" of the penis in the usual fashion. Amperometric titration2 demonstrated a sufficiently close and constant correlation between the two fractions to permit us to proceed with a field trial based on the tail-end sample. Further preparations for the field trial involved a study of the effect of freezing in dry ice of an uncentrifuged sample, and the lability in storage of the small samples (1 to 5 cc.) that would be available. No significant alterations were found. The technic of collection as finally developed was as follows: 1. To each breeding farm (or shed) a daily supply of clean plastic centrifuge tubes bearing the stallion's name, laboratory code number, and the serial number of the sample of the particular horse was provided. 2. The dry ice container at each station was replenished daily. 3. Samples were picked up daily from each farm, maintained over dry ice at all times, and brought back to the laboratory for storage over dry ice until assayed. 4. At the time of assay the samples were allowed to melt and were then centrifuged; the supernatant fluid was removed, and two aliquots of 1 cc. each used for determination of the SH concentration whenever the volume permitted. One cubic centimeter of seminal fluid was pipetted directly into 9.0 cc. of the electrolyte used in the titration. Protein precipitation took place and went to completion while the specimen remained in the dry-ice box overnight. The solution was centrifuged and the supernatant used for titration. An additional 20 cc. of electrolyte was added and titration carried out. The amperometric technic of Goldzieher et al. was used. 2
519
SEMINAL SH AND FERTILITY
Vol. 7, No.6, 1956
RESULTS Stallions and mares of all ranges of fertility provided the material for this study. The "raw" data uncorrected for mare sterility are presented in Fig. l. The number of samples in each group is given in brackets and the per cent of pregnacies is plotted as the ordinate. For the sake of simplicity the nonprotein SH concentration is plotted on the abscissa in terms of tenths of mls. of .001N AgN03 used in the determination. This is done in view of the fact that "blank" titrations with p-chloromercuribenzoate usually could not be 60
50
raw
.--
r----
data: 1320 observations
r----
C/)
W 4 0U
Z
« z (!)
W
r----
30
a::
0.. r----
~ Z
20
W U
a::
~
.----
10
o
r----
(441)
(353)
(248)
(133)
(77)
(34)
(15)
(8)
0-0.9
1.0-1.9
2.0-2.9
3.0-3.9
4.0-4.9
5.0-5.9
6.0-6.9
7.0-7.9
SH
Fig. 1.
(II)
8.0
+-
CONCENTRATION
Fertility of single inseminations vs. seminal fluid SH level, raw data.
done; therefore, the observed values are actually slightly higher than the true SH concentration. The values of AgN03 plotted on the abscissa may be converted easily into micrograms of nonprotein sulfhydryl per cubic centimeter of seminal fluid by multiplying by 3.307 (0.1 cc. of O.OOlN AgN03 is equivalent to 3.307 fLg. sulfhydryl). In Fig. 2, matings to mares that returned four times to the stallion as well as those that occurred within their first heat period after foaling have been
520
HAAG & WERTHESSEN
Fertility & Sterility
eliminated. This obviously increases the over-all fertility rate and further sharpens the significance of the correlation to sulfhydryl. It is evident that the fertility of ejaculates with a nonprotein sulfhydryl concentration of about 3 units (10 p.g.jcc.) or higher is greatly impaired; when the level reaches 6 units (20 p.g. / cc. ), fertility is virtually nil. Figure 3 presents information on two selected stallions. Stallion CAB 60
r-r-CJ)
50
r--
L&.J
(3
Z
observations: adjusted for female infertility factor 1052
z
r--
40
(!)
L&.J
a:
a..
30 r-
~
Z L&.J 20 U
a: L&.J
r---
a..
10
( 351)
(284)
r---
(201)
(101)
(64)
(28)
( 10)
2.0-2.9
3.0-3.9
4.0-4.9
5.0-5.9
6.0-6.9
(0 )
(7)
o 0- 0.9
Fig. 2.
LO-I.9
7.0-7.9
8.0+
SH CONCENTRATION Fertility of single inseminations vs. seminal fluid SH level, adjusted for female infertility factor.
maintained a consistently low level of sulfhydryl; his fertility was high, and both the failures in a total of 12 copulations occurred when he was mated with mares who had foaled 9 days previously. Stallion CLH, on the other hand, showed great variability in his sulfhydryl concentration. Eleven fluids showed a level of 3 units or higher (10 p.g.jcc.) out of a total of 30. In the 19 matings where the tail-end sample showed a level lower than 3 units, there were 13 pregnancies; in the eleven samples with a sulfhydryl of 3 or higher, there were only 3 pregnancies. This is statistically significant at the 2 per cent level.
r
521
SEMINAL SH AND FERTILITY
Vol. 7, No.6, 1956
Conclusion. A logical consequence of this observation is that the failure of a particular copulation can be predicted with reasonable certainty, simply by measurement of the sulfhydryl concentration of the tail-end sample of seminal fluid.
IC
Z
0
....
o
4
3
IC
---_ -
-
....
0
-j--
~
= pregnancy
x
=fai lure
8
LLJ U Z
-
KEY:
37%
2
•
•
• •
o
U
IC
:I:
a
en
0 M
0<
0 Fig. 3.
0:
cAB
83%
, o o
60% (difference between 37% 8 60 % significant at the 2 % level)
'" co
.~
CLH
Fertility vs. seminal fluid SH level in consecutive samples from individual stallions.
DISCUSSION These data seem to demonstrate clearly (at least in the Thoroughbred stallion) that the presence of an unknown but measurable factor in the seminal fluid contributes markedly to the fertility of the total ejaculate. When in addition it is recognized that our results are entirely uncorrected for the intrinsic fertility potential of the spermatozoa themselves, as they may vary from sample to sample and stallion to stallion, they appear to gain added significance. Were it possible to measure in addition a fertility index of the spermatozoa, an over-all measure of even greater reliability could result.
522
HAAG & WERTH ESSEN
Fertility & Sterility
The significance of these data as they apply to other species is not known. Few animals provide a suitable quantity of seminal fluid for even the most sensitive technics, nor do the practices concerned with their breeding permit so clear a study as that described. This may well be the reason why observations such as ours have not been made heretofore. Studies in other species, particularly the human, are currently in progress, and the chemical identity of the active factors is under investigative attack. Results of these studies will be reported subsequently. SUMMARY AND CONCLUSIONS It has been demonstrated that a relationship exists in the stallion between fertility and the nonprotein sulfhydryl concentration of seminal fluid. The relationship is inverse; a sharp decline in fertility occurs with concentrations exceeding 10 P,g. sulfhydryl! cc., and infertility of the specimen is virtually certain above 20 P,g.j cc. Fluids containing less than lO P,g.j cc. have a 55 to 60 per cent possibility of causing pregnancy in the group of Thoroughbreds studied. REFERENCES 1.
and WERTHESSEN, N. T. Influence of seminal fluid on sperm motility. Fertil. & Steril. 7:508, 1956. 2. GoLDZIEHER, J. W., RAWLS, W. B., and GOLDZIEHER, M. A. The uptake of sulfhydryl compounds by rat adrenal, liver, and muscle as measured by an improved amperometric technique. ]. Biol. Chern. 203:519, 1953. MARDEN, W.,