Vol. 27, No. 12, December 1976 Printed in U.S.A.
FERTILITY AND STERILITY Copyright = 1976 The American Fertility Society
THE EFFECTS OF ALTERING THE pH OF SEMINAL FLUID ON THE SEX RATIO OF RABBIT OFFSPRING PETER M. MUEHLEIS, M.S.,
AND
SALLY Y. LONG, PH.D.
Department of Anatomy, The Medical College of Wisconsin, Milwaukee, Wisconsin 53233
It has been suggested that the pH ofthe vagina at the time offertilization may have a differential effect on X- or Y-bearing sperm and thereby affect the sex of the offspring. To test this postulate, rabbit semen was collected, diluted 1:10 with a buffer ofpH 5.4,6.9, or 9.6, and after 20 minutes 0.5 ml of semenbuffer mixture was used for insemination in an ovulation-induced female. Newborn pups were examined both externally and internally for gender. The females inseminated with acidic semen had 6 litters, 50 offspring, with 48% males; those with neutral semen had 8 litters, 48 offspring, with 63% males; and those with alkaline semen had 7 litters, 49 offspring, with 49% males. There was no significant difference in these sex ratios from the expected 50% males. Motility ofrabbit sperm at 23° C in buffers ofpH 4.6,5.4,6.9,9.6, and 9.8 was reduced in vitro as the pH deviated from neutrality. Acid conditions were more detrimental than alkaline conditions. Sperm lost their motility more quickly in buffers of3r C than in buffers of 23° C. It was not possible with scanning electron microscopy to distinguish morphologically between X- and Y-bearing sperm. It seems unlikely that a direct effect of pH on sperm can be a single influence on the sex of offspring.
The concept of influencing the sex of one's offspring has been of continuing interest for many centuries. Both physical and chemical properties which might distinguish the X- from the V-bearing sperm have been used to design conditions which would favor the survival of the desired type. There has been little success with in vitro methods such as electrophoresis,1-3 froth flotation, 4 and centrifugation. 5 Also of interest have been the effects of pH on sperm motility and viability, in particular the claim that there is a differential effect on the X or the Y sperm. Unterbergers suggested that a very alkaline seminal fluid would favor males and a very acid vagina would favor females. Accepted June 23, 1976.
Shettles7 is one of the strongest proponents of the theory that, by altering the pH environment of sperm within the human female vagina, one can expect to see a preponderance of male offspring if the cervical fluids are alkaline and a preponderance of female offspring if the cervical fluids are acidic. He also noted that at the time of ovulation the cervical mucus was most alkaline, of lowest viscosity, and most conducive to sperm penetration and survival. On the other hand, the more acidic conditions which prevail during the remainder of the menstrual cycle, except for a day or so before and after ovulation, permit only the more fit sperm to survive. Although both X- and V-bearing sperm survive longer in the slightly alkaline environment found at ovulation, the
1438
Vol. 27, No. 12
EFFECTS OF SEMINAL FLUID pH ON SEX RATIO
smaller, faster, Y sperm is more likely to reach the ovum first and fertilize it. Since the Y sperm is more labile under acidic conditions, the slower X sperm would survive to fertilize the ovum. Consequently, for sex preselection, Shettles recommends carefully timing coitus in relation to ovulation and douching with a vinegar (acid) solution, or baking soda (alkaline) solution to influence the sex of the offspring. Kleegman 8 studied clinical records of cases in which either natural or artificial insemination was used in attempting to select the sex of the offspring. She found that, in those cases in which ovulation could be determined within 1 to 12 hours, insemination that occurred more than 36 hours before ovulation resulted in about 70% females, whereas about 70% males were born when insemination occurred within 2 to 24 hours before ovulation. This is consistent with the theory that Y-bearing sperm might be favored in the alkaline cervical environment of ovulation. Wakim 9 demonstrated an altered sex ratio in the offspring of rabbits in relation to the pH of the vagina at the time of mating. When the cervical pH was 6.5 to 7.3, there was a predominance of females; there was no difference in sex ratio with a pH of 7.3 to 7.5, and at pH 7.5 to 8.3 there was an excess of males. On the other hand, Diasio and Glass 10 prepared capillary tubes filled with Tryode's solution adjusted to pH conditions from 6.5 to 8.4, representing the range of normal human cervical mucus, and immersed them vertically in fresh human semen samples to determine whether there was any difference in the upward rate of migration of Y-bearing sperm. The contents of the tubes were stained with quinacrine dihydrochloride, which reveals a characteristic fluorescent spot on the short arm of the Y chromosome. Under these conditions, no difference in migration rate was observed,
1439
a seeming contradiction to the belief of Shettles and others who maintain that the pH of the cervical mucus affects sperm motility and survival. The controversy persists as to whether pH can affect the sex ratio of offspring. In order to separate the direct effect of pH on the sperm from any other changes that might be occurring in the female's reproductive tract at the time of ovulation, we chose to incubate rabbit sperm in buffers of known pH, to use these to inseminate females in which ovulation was induced, and to examine the sex of the offspring. MATERIALS AND METHODS
Semen was collected from four New Zealand White rabbits, using an artificial vagina warmed to 50° C, as modified from the technique of Bredderman et al. l l The buck readily mounted the artificial vagina when it was beld by the rabbit fur-covered arm of the investigator. Four males were used in the study, and semen from at least two of them was mixed to avoid'the possibility of selecting a rabbit with atypical sperm. The semen was then diluted 1:10 in a range of buffers adjusted to specific pH conditions between 4.6 and 9.8. The buffer solutions consisted of nine parts of Krebs-Ringer solution to one part of buffer. The buffers used were 0.25 M potassium phosphate buffer (pH 7.0), 0.25 M citrate buffer (pH 5.2), and 0.25 M bicarbonate buffer (pH 10.2). Semen (0.1 ml) was added to 1.0 ml of the appropriate buffer and the resulting pH was determined with a Corning model 10 pH meter. However, the addition of semen altered the pH of the original buffer solution somewhat. Throughout this paper, the reference to pH always indicates the pH of the final semen-buffer mixture. The osmolarity of each buffer was determined with a Fiske osmometer, and each was adjusted to within the isotonic range of 300 ± 10 mOsmoles.
December 1976
MUEHLEIS AND LONG
1440
Motility studies were made of sperm fore insemination. This allowed placeat the various pH conditions for up to 30 ment of the semen-buffer mixture as close hours at room temperature (230 C) and to the cervix as possible, rather than in the at 37" C in a water bath. Every hour vaginal canal. after collection, an aliquot was removed, All rabbits were kept in single cages placed on a glass slide under a cover slip, with food and water ad libitum. Newand examined at x 450 magnification. borns were collected the morning after Motility was scored on a scale of 0 to 10 birth, killed with ether, and examined (Table 1), as modified from the method of internally to confirm their sex. All pregChang and Thorsteinsson. 12 The motility nancies were maintained to term rather of a sample was scored within 1 minute than killing the mothers in order to count of being placed under the microscope. implantation sites. This made the female From the results of the motility studies available for subsequent inseminations and pilot attempts at insemination, only and also permitted comparison of birth the pH conditions of 5.4,6.9, and 9.6 were rate rather than conception rate, birth chosen for use in the fertility studies. rate being easier to evaluate with our For artificial insemination, samples present knowledge. Only rarely did a from two males were mixed, diluted 1:10 mother destroy a pup so that it could not with the appropriate buffer, and left in the be sexed. In such a case the pup was not buffer for 20 to 30 minutes at room tem- counted in the total litter size. perature. The sperm solutions were thorIn order to determine the normal moroughly mixed, and 0.5 ml of sperm sus- phology of rabbit sperm, sperm were expension was deposited in the vagina. amined by scanning electron microscopy; Eight does were used for the study. the basic techniques and findings have Ovulation was induced by injecting 351 been described by othersY-15 Semen from U of Gonanome, a commercially available at least two males was collected and human chorionic gonadotropin (Fort mixed. Approximately 0.1 ml of semen Dodge Laboratories, Fort Dodge, Ia.) into was washed and centrifuged three times the marginal vein of the ear 2 hours in normal saline (30 x g) for 10 minutes before insemination. each time. The sperm thus collected was The insemination device was made from fixed in acrolein glutaraldehyde (0.1 M a tuberculin syringe fitted with poly- phosphate buffer at pH 7.4) for 1 hour. ethylene tubing which was inserted One drop of sperm suspension was then through a glass tubing sleeve that was placed on a cover slip and critical pointalso used to stimulate the cervix just be- dried. The cover slips with sperm were then dehydrated in ascending concentrations of alcohol. The cover slips were then TABLE 1. Evaulation of Sperm Motility prepared for viewing with a JSMU -3 scanMotility grade Description of motility ning electron microscope. 10 More than one-half progressively mo9 8 7 6 5 4 3 2 1
o
tile 20-30% progressively motile A few progressively motile More than one-half actively motile (little forward motion) 20-30% actively motile A few actively motile More than one-half sluggishly motile 20-30% sluggishly motile A few sluggishly motile A few showing signs of life Completely immotile
RESULTS
Sperm Motility (in Vitro). The purpose of the motility studies was to determine the limits of pH in both the acidic and basic ranges that would permit sperm motility and to determine whether there were differences in sperm motility at room temperature (23 C) versus 37" C. 0
Vol. 27, No. 12
EFFECTS OF SEMINAL FLUID pH ON SEX RATIO
Sperm which were left in buffers at room temperature showed a sharp decline in motility at a pH below 4.6 (Fig. 1). Likewise, basic pH conditions (above 9.8) also caused reduced sperm motility, although not as severe as those below pH 4.60. Since in our pilot studies there were no pregnancies in rabbits inseminated at either pH 4.6 or 9.8, these extremes were eliminated. The acidic (pH 5.4) and basic (pH 9.6) conditions caused a reduction of sperm motility, but pregnancies were still produced. The control group (pH 6.9) was chosen to be as close to neutrality and normal semen pH as possible. This group exhibited the most sustained motility, reaching zero only after about 30 hours (Fig. 1). Both the degree of motility and length of time the sperm were motile were decreased dramatically by any change in the pH of the buffer, the acidic conditions being the most detrimental. Studies of sperm motility were also conducted at 37' C to determine whether a difference existed from that observed at room temperature (23° C). A comparison of length of time the sperm were motile
at each pH condition at 23° C and 37' C (Fig. 2) shows that the motility and survival time were substantially reduced at 37° C, in some instances to less than one-half that at 23° C. Results of Insemination. Insemination of the does at the three pH conditions (5.4, 6.9, and 9.6) yielded the results shown in Table 2. Importantly, most of the females had successful pregnancies in each pH group. The average litter size in the acidic group was eight; in the control group, six; and in the basic group, seven. These are similar to the normally expected values for rabbit offspring.16 The results of the inseminations in each group are given in Table 3. Each group contained about 50 offspring. The percentages of male offspring were 48% in the acidic group, 63% in the control group, and 49% in the alkaline group. There was no statistical difference (X 2 ) between the results obtained and the 1:1 ratio expected. Sperm morphology was also studied to determine whether there might be two distinct types of sperm which might represent X- and Y-bearing populations. Semen Treated at a pH of
10
4.60 5.40
9
\
.
7
q. \
~
6
Q
C>
...
5
-
4
~ :IE
3
.-.-
2
9.60 9.80
' ,f)
~
."
----••...•...•..•••...•.•.
6.90
8
,
,
,.,. .. , " " " " - • - .'- • - • -
" 0 ..\\ ., , ... \
\
\ \
,
~
•
\
.
.....',., .., . ......... '4r
'. ".
~
"-
"'0
'.'.
0\
1-2 I
,.
., .• ,
.0.•••• 2
3
4
5
6
7
"
8
9
1441
10
II
Time (hrs.)
FIG. 1. Motility of rabbit sperm in buffers of pH 4.6 to 9.8 at 23° C.
1442
MUEHLE IS AND LONG
10
December 1976
Motility Change at pH
5.40 9.60 6. gO
8
23°C 37°C
7
..,.
• 0
6
~ CI
.0
5
...
~
.-0
.
"
-
3
0
~
2
--~ 1
2
3
"
5
6
7
8
9
10
II
12
13
14
15
16
17
18
19 20
Time (hr.'>
FIG. 2. Comparison of rabbit sperm motility in buffers of three pH conditions at 23° C and 31' C.
All sperm were found to be of the oval type (Fig. 3), which is representative of the sperm morphology seen in semen samples from several bucks.
TABLE 2. The Fertility of Female Rabbits Inseminated with Sperm Treated at Different pH Values Female no.
1
2 3 4
Control
Basic
(pH 6.90)
(pH 9.60)
0/0" 0/0 4/6
3/0"
0/2"
110
0/0 4/4 4/3
3/3 0/0 0/0 7/3 617
4/3
Acidic (pH 5.40)
5/4 3/2 0/0 4/3
5
0/0 3/4
6
0/0
0/0 0/0
7
113
6/2 4/4
8
0/0 0/0
" Males/females.
5/2 0/0 0/0 4/4 2/3 4/3 0/0 116
DISCUSSION
There was no statistically significant difference in the sex ratio of offspring of rabbits inseminated with sperm treated with buffers at acidic, neutral, or alkaline pH. This would imply that X and Y sperm are not differentially affected by the pH in the female cervix at ovulation. It was very obvious that pH either below or above 6.9 was deleterious to sperm motility. Although decreased motility does not necessarily reflect viability, we did notice that the fertility rate in the acid and basic groups was lower than that in the controls (42%, 58%, and 72%, respectively). The over-all fertility rate of 57% compares favorably with the 44% success rate of O'Ferrall et a1. 4 and the 15% success of Gordon! for artificial insemination. It was not possible to determine whether the motility of the X and Y sperm were differentially affected by the acidic and basic buffers. The most reliable method of detecting Y sperm is by using quinacrine staining to produce a fluorescent spot on the Y chromosome. Unfortunately, only human and gorilla!O sperm react.
Vol. 27, No. 12
1443
EFFECTS OF SEMINAL FLUID pH ON SEX RATIO
TABLE 3. The Number of Male and Female Offspring Produced with Sperm Buffered at Various pH Conditions PupS/litter
Total"
Buffer group
Total no. of litters
Total no. of offspring
Males
Females
6 3 3 7 4 3
6
50
24
26 b
48
3 1 4 5 3 4 6 4
0 0 3 4 2 3 2 4
8
48
30
18"
63
4 4 5 4 2 4 1
4 3 2 4 3 3 6
7
49
24
25 d
49
Males
Females
pH 5.40
4 3 7 6 3 1
pH 6.90
pH 9.60
% Males
aN one of the male/female ratios are significantly different from that expected. b X2 = 0.08; df 1,0.80; > P > 0.70. e X2 = 3.00; df 1,0.10; > P > 0.05. "X2 = 0.02; df 1, 0.90; > P > 0.80.
FIG. 3. A typical rabbit sperm visualized with scanning electron microscopy (x 11,250).
1444
December 1976
MUEHLEIS AND LONG
A morphologic difference in X and Y ments insemination took place 2 hours sperm head shape, which Shettles l7 ,18 after injection of HCG. In the rabbit, ovudescribed for human sperm by using phase lation occurs about 10 hours after mating microscopy, should be evident with scan- or injection with HCG.19 It takes about ning electron microscopy. After consider- the same time for untreated sperm to ing the great similarity between human reach the site of fertilization. 20 In addiand rabbit sperm, we decided to look tion, capacitation of sperm in the female for such differences. What was assumed genital tract requires at least 4 hours,21 to be normal variation in head size and It thus becomes clear that, regardless of shape was seen (albeit infrequently), and, the time of insemination, sperm must not since sperm are flat, a view from the side only be able to survive for about 10 hours made the sperm appear very oblong, in the female reproductive tract but be whereas a top or bottom view gave a wider able to fertilize an ovum after this period and shorter appearance. No round sperm of time. In this experiment, a moderate were discerned with any regularity suf- success was achieved when insemination ficient to indicate two distinct popula- occurred 2 hours after injection of HCG. tions. In fact, the vast majority of sperm No vaginal douche was used in these were of the oval variety shown in Figure studies. The sperm were suspended di3. These observations closely resemble rectly in a buffer, then placed in the fethose of Hafez and Kanagawa 14 and male's vagina. The conditions in the vaZaneveld et al. 15 for rabbit sperm. gina certainly altered the environment of The question arises, which is the more the sperm somewhat. However, the paraccurate indicator of sperm motility and ticular buffers used were chosen for their survival, those results observed at effective buffering capacity, and the pH 23° C or those observed at 31' C? Sperm should not have been greatly changed. at 23° C had a longer survival time than It still may be possible that the sex of did those at 31' C. One explanation for the offspring could be affected by condithe reduction in motility may be that tions in the female reprod ucti ve tract increased metabolism at higher tempera- beyond the vagina that may affect sperm tures leads to a more rapid depletion of capacitation or zygote viability. However, nutrients in the confined environment it seems unlikely that the pH of the cerof a test tube. In vivo, this decline in vical mucus could affect these processes. motility is probably less marked. It was Perhaps the change in the pH of the cerseen that, in vitro, the survival time of vical mucus indicates more generalized sperm at 31' C at pH 5.40 and 9.60 was changes in the entire female reproductive less than 8 hours, and yet fertilization tract. This might help to explain the data still took place. This would indicate that ofWakim,9 who reported a higher propormeasurements of sperm motility in vitro tion of males to females in rabbits in which at 31' C does not truly indicate their the vaginal pH was 7.55 or greater, and capacity to fertilize. this might also explain the data of 7 The time of insemination after injection Shettles and Kleegman,8 who found a difof human chorionic gonadotropin (HCG) ference in sex ratio in humans when coitus may be crucial. According to Adams,16 was timed in relation to ovulation. j
untreated sperm are equally capable of fertilization anytime during the period from several hours before to 5 hours after injection of the doe with an ovulationinducing agent. In the present experi-
Our data do show that the direct effect of pH on the sperm does not in and of itself alter the sex ratio of offspring. It is intriguing to suppose that one can influence the sex of offspring. However,
Vol. 27, No. 12
EFFECTS OF SEMINAL FLUID pH ON SEX RATIO
the mechanism for accomplishing this probably does not lie in altering exogenous vaginal pH.
10.
11. REFERENCES 1. Gordon MJ: Control of sex ratio in rabbits by electrophoresis of spermatozoa. Proc Natl Acad Sci USA 43:913, 1957 2. Nevo AC, Michaeli I, Schindler H: Electrophoretic properties of bull and of rabbit spermatozoa. Exp Cell Res 23:69, 1961 3. Schorder VN: Physico-chemical analysis of the physiology of spermatozoa. Anim Breed Abstr 3:166,1934 4. O'Ferrall GJM, Meacham TN, Foreman WE: Attempts to separate rabbit spermatozoa by means of froth flotation and the sex ratio of offspring born. J Reprod Fertil16:243, 1968 5. Salamon S: Deep freezing of boar semen. III. Effects of centrifugation, diluent and dilution rate, pellet volume, and method of thawing on survival of spermatozoa. Aust J BioI Sci 26: 239, 1972 6. Unterberger F: Geschlechtsbestimmung und Wasserstoffionenkonzentration. Dtsch Med Wochenschr 58(1):729, 1932 7. Shettles LB: Factors influencing sex ratios. Int J Gynecol Obstet 8:643, 1970 8. Kleegman SJ: Can sex be predetermined by the physician? (abstr). Excerpta Med 109:109, 1966 9. Wakim PE: Determining the sex of baby rabbits by ascertaining the pH of the vagina of the
12.
13.
14.
15.
16. 17. 18. 19.
20.
21.
1445
mother before mating. J Am Osteopath Assoc 72:173, 1972 Diasio RB, Glass RH: Effects of pH on the migration of X and Y sperm. Fertil Steril 22:303, 1971 Bredderman PJ, Foote RH, Yassen AM: An improved artificial vagina for collecting rabbit semen. J Reprod Fertil 7:401,1964 Chang MC, Thorsteinsson T: Effects of osmotic pressure and hydrogen ion concentration on the motility and fertilizing capacity of rabbit spermatozoa. Fertil Steril 9:510, 1958 Bernstein MH, Teichmann RJ: Regional differentiation in the heads of spermatozoa of rabbit, man and bull. Am J Anat 133:165, 1972 Hafez ESE, Kanagawa H: Scanning electron microscopy of human, monkey, and rabbit spermatozoa. Fertil Steril 24:776, 1973 Zaneveld LJD, Gould KG, Humphreys WJ, Williams WL: Scanning electron microscopy of mammalian spermatozoa. J Reprod Med 6(4): 13, 1971 Adams CE: Artificial insemination in the rabbit (abstr). J Reprod Fertil 2:521, 1961 Shettles LB: Head differences in human spermatozoa. J Urol 85:355, 1961 Shettles LB: Sperm morphology and sex ratios. J Urol 86:450, 1961 Harper MJK: The time of ovulation in the rabbit following the injection of luteinizing hormone. J EndocrinoI22:147, 1961 EI-Banna AA, Hafez ESE: Sperm transport and distribution in rabbit and cattle female tract. Fertil Steril 21:534, 1970 Soupart P: Leukocytes and sperm capacitation in the rabbit uterus. Fertil Steril 21:724, 1970
FERTILITY AND STERILITY Copyright = 1976 The American Fertility Society
THE EFFECTS OF ALTERING THE pH OF SEMINAL FLUID ON THE SEX RATIO OF RABBIT OFFSPRING PETER M. MUEHLEIS, M.S.,
AND
SALLY Y. LONG, PH.D.
Department of Anatomy, The Medical College of Wisconsin, Milwaukee, Wisconsin 53233
It has been suggested that the pH ofthe vagina at the time offertilization may have a differential effect on X- or Y-bearing sperm and thereby affect the sex of the offspring. To test this postulate, rabbit semen was collected, diluted 1:10 with a buffer ofpH 5.4,6.9, or 9.6, and after 20 minutes 0.5 ml of semenbuffer mixture was used for insemination in an ovulation-induced female. Newborn pups were examined both externally and internally for gender. The females inseminated with acidic semen had 6 litters, 50 offspring, with 48% males; those with neutral semen had 8 litters, 48 offspring, with 63% males; and those with alkaline semen had 7 litters, 49 offspring, with 49% males. There was no significant difference in these sex ratios from the expected 50% males. Motility ofrabbit sperm at 23° C in buffers ofpH 4.6,5.4,6.9,9.6, and 9.8 was reduced in vitro as the pH deviated from neutrality. Acid conditions were more detrimental than alkaline conditions. Sperm lost their motility more quickly in buffers of3r C than in buffers of 23° C. It was not possible with scanning electron microscopy to distinguish morphologically between X- and Y-bearing sperm. It seems unlikely that a direct effect of pH on sperm can be a single influence on the sex of offspring.
The concept of influencing the sex of one's offspring has been of continuing interest for many centuries. Both physical and chemical properties which might distinguish the X- from the V-bearing sperm have been used to design conditions which would favor the survival of the desired type. There has been little success with in vitro methods such as electrophoresis,1-3 froth flotation, 4 and centrifugation. 5 Also of interest have been the effects of pH on sperm motility and viability, in particular the claim that there is a differential effect on the X or the Y sperm. Unterbergers suggested that a very alkaline seminal fluid would favor males and a very acid vagina would favor females. Accepted June 23, 1976.
Shettles7 is one of the strongest proponents of the theory that, by altering the pH environment of sperm within the human female vagina, one can expect to see a preponderance of male offspring if the cervical fluids are alkaline and a preponderance of female offspring if the cervical fluids are acidic. He also noted that at the time of ovulation the cervical mucus was most alkaline, of lowest viscosity, and most conducive to sperm penetration and survival. On the other hand, the more acidic conditions which prevail during the remainder of the menstrual cycle, except for a day or so before and after ovulation, permit only the more fit sperm to survive. Although both X- and V-bearing sperm survive longer in the slightly alkaline environment found at ovulation, the
1438
Vol. 27, No. 12
EFFECTS OF SEMINAL FLUID pH ON SEX RATIO
smaller, faster, Y sperm is more likely to reach the ovum first and fertilize it. Since the Y sperm is more labile under acidic conditions, the slower X sperm would survive to fertilize the ovum. Consequently, for sex preselection, Shettles recommends carefully timing coitus in relation to ovulation and douching with a vinegar (acid) solution, or baking soda (alkaline) solution to influence the sex of the offspring. Kleegman 8 studied clinical records of cases in which either natural or artificial insemination was used in attempting to select the sex of the offspring. She found that, in those cases in which ovulation could be determined within 1 to 12 hours, insemination that occurred more than 36 hours before ovulation resulted in about 70% females, whereas about 70% males were born when insemination occurred within 2 to 24 hours before ovulation. This is consistent with the theory that Y-bearing sperm might be favored in the alkaline cervical environment of ovulation. Wakim 9 demonstrated an altered sex ratio in the offspring of rabbits in relation to the pH of the vagina at the time of mating. When the cervical pH was 6.5 to 7.3, there was a predominance of females; there was no difference in sex ratio with a pH of 7.3 to 7.5, and at pH 7.5 to 8.3 there was an excess of males. On the other hand, Diasio and Glass 10 prepared capillary tubes filled with Tryode's solution adjusted to pH conditions from 6.5 to 8.4, representing the range of normal human cervical mucus, and immersed them vertically in fresh human semen samples to determine whether there was any difference in the upward rate of migration of Y-bearing sperm. The contents of the tubes were stained with quinacrine dihydrochloride, which reveals a characteristic fluorescent spot on the short arm of the Y chromosome. Under these conditions, no difference in migration rate was observed,
1439
a seeming contradiction to the belief of Shettles and others who maintain that the pH of the cervical mucus affects sperm motility and survival. The controversy persists as to whether pH can affect the sex ratio of offspring. In order to separate the direct effect of pH on the sperm from any other changes that might be occurring in the female's reproductive tract at the time of ovulation, we chose to incubate rabbit sperm in buffers of known pH, to use these to inseminate females in which ovulation was induced, and to examine the sex of the offspring. MATERIALS AND METHODS
Semen was collected from four New Zealand White rabbits, using an artificial vagina warmed to 50° C, as modified from the technique of Bredderman et al. l l The buck readily mounted the artificial vagina when it was beld by the rabbit fur-covered arm of the investigator. Four males were used in the study, and semen from at least two of them was mixed to avoid'the possibility of selecting a rabbit with atypical sperm. The semen was then diluted 1:10 in a range of buffers adjusted to specific pH conditions between 4.6 and 9.8. The buffer solutions consisted of nine parts of Krebs-Ringer solution to one part of buffer. The buffers used were 0.25 M potassium phosphate buffer (pH 7.0), 0.25 M citrate buffer (pH 5.2), and 0.25 M bicarbonate buffer (pH 10.2). Semen (0.1 ml) was added to 1.0 ml of the appropriate buffer and the resulting pH was determined with a Corning model 10 pH meter. However, the addition of semen altered the pH of the original buffer solution somewhat. Throughout this paper, the reference to pH always indicates the pH of the final semen-buffer mixture. The osmolarity of each buffer was determined with a Fiske osmometer, and each was adjusted to within the isotonic range of 300 ± 10 mOsmoles.
December 1976
MUEHLEIS AND LONG
1440
Motility studies were made of sperm fore insemination. This allowed placeat the various pH conditions for up to 30 ment of the semen-buffer mixture as close hours at room temperature (230 C) and to the cervix as possible, rather than in the at 37" C in a water bath. Every hour vaginal canal. after collection, an aliquot was removed, All rabbits were kept in single cages placed on a glass slide under a cover slip, with food and water ad libitum. Newand examined at x 450 magnification. borns were collected the morning after Motility was scored on a scale of 0 to 10 birth, killed with ether, and examined (Table 1), as modified from the method of internally to confirm their sex. All pregChang and Thorsteinsson. 12 The motility nancies were maintained to term rather of a sample was scored within 1 minute than killing the mothers in order to count of being placed under the microscope. implantation sites. This made the female From the results of the motility studies available for subsequent inseminations and pilot attempts at insemination, only and also permitted comparison of birth the pH conditions of 5.4,6.9, and 9.6 were rate rather than conception rate, birth chosen for use in the fertility studies. rate being easier to evaluate with our For artificial insemination, samples present knowledge. Only rarely did a from two males were mixed, diluted 1:10 mother destroy a pup so that it could not with the appropriate buffer, and left in the be sexed. In such a case the pup was not buffer for 20 to 30 minutes at room tem- counted in the total litter size. perature. The sperm solutions were thorIn order to determine the normal moroughly mixed, and 0.5 ml of sperm sus- phology of rabbit sperm, sperm were expension was deposited in the vagina. amined by scanning electron microscopy; Eight does were used for the study. the basic techniques and findings have Ovulation was induced by injecting 351 been described by othersY-15 Semen from U of Gonanome, a commercially available at least two males was collected and human chorionic gonadotropin (Fort mixed. Approximately 0.1 ml of semen Dodge Laboratories, Fort Dodge, Ia.) into was washed and centrifuged three times the marginal vein of the ear 2 hours in normal saline (30 x g) for 10 minutes before insemination. each time. The sperm thus collected was The insemination device was made from fixed in acrolein glutaraldehyde (0.1 M a tuberculin syringe fitted with poly- phosphate buffer at pH 7.4) for 1 hour. ethylene tubing which was inserted One drop of sperm suspension was then through a glass tubing sleeve that was placed on a cover slip and critical pointalso used to stimulate the cervix just be- dried. The cover slips with sperm were then dehydrated in ascending concentrations of alcohol. The cover slips were then TABLE 1. Evaulation of Sperm Motility prepared for viewing with a JSMU -3 scanMotility grade Description of motility ning electron microscope. 10 More than one-half progressively mo9 8 7 6 5 4 3 2 1
o
tile 20-30% progressively motile A few progressively motile More than one-half actively motile (little forward motion) 20-30% actively motile A few actively motile More than one-half sluggishly motile 20-30% sluggishly motile A few sluggishly motile A few showing signs of life Completely immotile
RESULTS
Sperm Motility (in Vitro). The purpose of the motility studies was to determine the limits of pH in both the acidic and basic ranges that would permit sperm motility and to determine whether there were differences in sperm motility at room temperature (23 C) versus 37" C. 0
Vol. 27, No. 12
EFFECTS OF SEMINAL FLUID pH ON SEX RATIO
Sperm which were left in buffers at room temperature showed a sharp decline in motility at a pH below 4.6 (Fig. 1). Likewise, basic pH conditions (above 9.8) also caused reduced sperm motility, although not as severe as those below pH 4.60. Since in our pilot studies there were no pregnancies in rabbits inseminated at either pH 4.6 or 9.8, these extremes were eliminated. The acidic (pH 5.4) and basic (pH 9.6) conditions caused a reduction of sperm motility, but pregnancies were still produced. The control group (pH 6.9) was chosen to be as close to neutrality and normal semen pH as possible. This group exhibited the most sustained motility, reaching zero only after about 30 hours (Fig. 1). Both the degree of motility and length of time the sperm were motile were decreased dramatically by any change in the pH of the buffer, the acidic conditions being the most detrimental. Studies of sperm motility were also conducted at 37' C to determine whether a difference existed from that observed at room temperature (23° C). A comparison of length of time the sperm were motile
at each pH condition at 23° C and 37' C (Fig. 2) shows that the motility and survival time were substantially reduced at 37° C, in some instances to less than one-half that at 23° C. Results of Insemination. Insemination of the does at the three pH conditions (5.4, 6.9, and 9.6) yielded the results shown in Table 2. Importantly, most of the females had successful pregnancies in each pH group. The average litter size in the acidic group was eight; in the control group, six; and in the basic group, seven. These are similar to the normally expected values for rabbit offspring.16 The results of the inseminations in each group are given in Table 3. Each group contained about 50 offspring. The percentages of male offspring were 48% in the acidic group, 63% in the control group, and 49% in the alkaline group. There was no statistical difference (X 2 ) between the results obtained and the 1:1 ratio expected. Sperm morphology was also studied to determine whether there might be two distinct types of sperm which might represent X- and Y-bearing populations. Semen Treated at a pH of
10
4.60 5.40
9
\
.
7
q. \
~
6
Q
C>
...
5
-
4
~ :IE
3
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2
9.60 9.80
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~
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----••...•...•..•••...•.•.
6.90
8
,
,
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\
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0\
1-2 I
,.
., .• ,
.0.•••• 2
3
4
5
6
7
"
8
9
1441
10
II
Time (hrs.)
FIG. 1. Motility of rabbit sperm in buffers of pH 4.6 to 9.8 at 23° C.
1442
MUEHLE IS AND LONG
10
December 1976
Motility Change at pH
5.40 9.60 6. gO
8
23°C 37°C
7
..,.
• 0
6
~ CI
.0
5
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.
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2
3
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5
6
7
8
9
10
II
12
13
14
15
16
17
18
19 20
Time (hr.'>
FIG. 2. Comparison of rabbit sperm motility in buffers of three pH conditions at 23° C and 31' C.
All sperm were found to be of the oval type (Fig. 3), which is representative of the sperm morphology seen in semen samples from several bucks.
TABLE 2. The Fertility of Female Rabbits Inseminated with Sperm Treated at Different pH Values Female no.
1
2 3 4
Control
Basic
(pH 6.90)
(pH 9.60)
0/0" 0/0 4/6
3/0"
0/2"
110
0/0 4/4 4/3
3/3 0/0 0/0 7/3 617
4/3
Acidic (pH 5.40)
5/4 3/2 0/0 4/3
5
0/0 3/4
6
0/0
0/0 0/0
7
113
6/2 4/4
8
0/0 0/0
" Males/females.
5/2 0/0 0/0 4/4 2/3 4/3 0/0 116
DISCUSSION
There was no statistically significant difference in the sex ratio of offspring of rabbits inseminated with sperm treated with buffers at acidic, neutral, or alkaline pH. This would imply that X and Y sperm are not differentially affected by the pH in the female cervix at ovulation. It was very obvious that pH either below or above 6.9 was deleterious to sperm motility. Although decreased motility does not necessarily reflect viability, we did notice that the fertility rate in the acid and basic groups was lower than that in the controls (42%, 58%, and 72%, respectively). The over-all fertility rate of 57% compares favorably with the 44% success rate of O'Ferrall et a1. 4 and the 15% success of Gordon! for artificial insemination. It was not possible to determine whether the motility of the X and Y sperm were differentially affected by the acidic and basic buffers. The most reliable method of detecting Y sperm is by using quinacrine staining to produce a fluorescent spot on the Y chromosome. Unfortunately, only human and gorilla!O sperm react.
Vol. 27, No. 12
1443
EFFECTS OF SEMINAL FLUID pH ON SEX RATIO
TABLE 3. The Number of Male and Female Offspring Produced with Sperm Buffered at Various pH Conditions PupS/litter
Total"
Buffer group
Total no. of litters
Total no. of offspring
Males
Females
6 3 3 7 4 3
6
50
24
26 b
48
3 1 4 5 3 4 6 4
0 0 3 4 2 3 2 4
8
48
30
18"
63
4 4 5 4 2 4 1
4 3 2 4 3 3 6
7
49
24
25 d
49
Males
Females
pH 5.40
4 3 7 6 3 1
pH 6.90
pH 9.60
% Males
aN one of the male/female ratios are significantly different from that expected. b X2 = 0.08; df 1,0.80; > P > 0.70. e X2 = 3.00; df 1,0.10; > P > 0.05. "X2 = 0.02; df 1, 0.90; > P > 0.80.
FIG. 3. A typical rabbit sperm visualized with scanning electron microscopy (x 11,250).
1444
December 1976
MUEHLEIS AND LONG
A morphologic difference in X and Y ments insemination took place 2 hours sperm head shape, which Shettles l7 ,18 after injection of HCG. In the rabbit, ovudescribed for human sperm by using phase lation occurs about 10 hours after mating microscopy, should be evident with scan- or injection with HCG.19 It takes about ning electron microscopy. After consider- the same time for untreated sperm to ing the great similarity between human reach the site of fertilization. 20 In addiand rabbit sperm, we decided to look tion, capacitation of sperm in the female for such differences. What was assumed genital tract requires at least 4 hours,21 to be normal variation in head size and It thus becomes clear that, regardless of shape was seen (albeit infrequently), and, the time of insemination, sperm must not since sperm are flat, a view from the side only be able to survive for about 10 hours made the sperm appear very oblong, in the female reproductive tract but be whereas a top or bottom view gave a wider able to fertilize an ovum after this period and shorter appearance. No round sperm of time. In this experiment, a moderate were discerned with any regularity suf- success was achieved when insemination ficient to indicate two distinct popula- occurred 2 hours after injection of HCG. tions. In fact, the vast majority of sperm No vaginal douche was used in these were of the oval variety shown in Figure studies. The sperm were suspended di3. These observations closely resemble rectly in a buffer, then placed in the fethose of Hafez and Kanagawa 14 and male's vagina. The conditions in the vaZaneveld et al. 15 for rabbit sperm. gina certainly altered the environment of The question arises, which is the more the sperm somewhat. However, the paraccurate indicator of sperm motility and ticular buffers used were chosen for their survival, those results observed at effective buffering capacity, and the pH 23° C or those observed at 31' C? Sperm should not have been greatly changed. at 23° C had a longer survival time than It still may be possible that the sex of did those at 31' C. One explanation for the offspring could be affected by condithe reduction in motility may be that tions in the female reprod ucti ve tract increased metabolism at higher tempera- beyond the vagina that may affect sperm tures leads to a more rapid depletion of capacitation or zygote viability. However, nutrients in the confined environment it seems unlikely that the pH of the cerof a test tube. In vivo, this decline in vical mucus could affect these processes. motility is probably less marked. It was Perhaps the change in the pH of the cerseen that, in vitro, the survival time of vical mucus indicates more generalized sperm at 31' C at pH 5.40 and 9.60 was changes in the entire female reproductive less than 8 hours, and yet fertilization tract. This might help to explain the data still took place. This would indicate that ofWakim,9 who reported a higher propormeasurements of sperm motility in vitro tion of males to females in rabbits in which at 31' C does not truly indicate their the vaginal pH was 7.55 or greater, and capacity to fertilize. this might also explain the data of 7 The time of insemination after injection Shettles and Kleegman,8 who found a difof human chorionic gonadotropin (HCG) ference in sex ratio in humans when coitus may be crucial. According to Adams,16 was timed in relation to ovulation. j
untreated sperm are equally capable of fertilization anytime during the period from several hours before to 5 hours after injection of the doe with an ovulationinducing agent. In the present experi-
Our data do show that the direct effect of pH on the sperm does not in and of itself alter the sex ratio of offspring. It is intriguing to suppose that one can influence the sex of offspring. However,
Vol. 27, No. 12
EFFECTS OF SEMINAL FLUID pH ON SEX RATIO
the mechanism for accomplishing this probably does not lie in altering exogenous vaginal pH.
10.
11. REFERENCES 1. Gordon MJ: Control of sex ratio in rabbits by electrophoresis of spermatozoa. Proc Natl Acad Sci USA 43:913, 1957 2. Nevo AC, Michaeli I, Schindler H: Electrophoretic properties of bull and of rabbit spermatozoa. Exp Cell Res 23:69, 1961 3. Schorder VN: Physico-chemical analysis of the physiology of spermatozoa. Anim Breed Abstr 3:166,1934 4. O'Ferrall GJM, Meacham TN, Foreman WE: Attempts to separate rabbit spermatozoa by means of froth flotation and the sex ratio of offspring born. J Reprod Fertil16:243, 1968 5. Salamon S: Deep freezing of boar semen. III. Effects of centrifugation, diluent and dilution rate, pellet volume, and method of thawing on survival of spermatozoa. Aust J BioI Sci 26: 239, 1972 6. Unterberger F: Geschlechtsbestimmung und Wasserstoffionenkonzentration. Dtsch Med Wochenschr 58(1):729, 1932 7. Shettles LB: Factors influencing sex ratios. Int J Gynecol Obstet 8:643, 1970 8. Kleegman SJ: Can sex be predetermined by the physician? (abstr). Excerpta Med 109:109, 1966 9. Wakim PE: Determining the sex of baby rabbits by ascertaining the pH of the vagina of the
12.
13.
14.
15.
16. 17. 18. 19.
20.
21.
1445
mother before mating. J Am Osteopath Assoc 72:173, 1972 Diasio RB, Glass RH: Effects of pH on the migration of X and Y sperm. Fertil Steril 22:303, 1971 Bredderman PJ, Foote RH, Yassen AM: An improved artificial vagina for collecting rabbit semen. J Reprod Fertil 7:401,1964 Chang MC, Thorsteinsson T: Effects of osmotic pressure and hydrogen ion concentration on the motility and fertilizing capacity of rabbit spermatozoa. Fertil Steril 9:510, 1958 Bernstein MH, Teichmann RJ: Regional differentiation in the heads of spermatozoa of rabbit, man and bull. Am J Anat 133:165, 1972 Hafez ESE, Kanagawa H: Scanning electron microscopy of human, monkey, and rabbit spermatozoa. Fertil Steril 24:776, 1973 Zaneveld LJD, Gould KG, Humphreys WJ, Williams WL: Scanning electron microscopy of mammalian spermatozoa. J Reprod Med 6(4): 13, 1971 Adams CE: Artificial insemination in the rabbit (abstr). J Reprod Fertil 2:521, 1961 Shettles LB: Head differences in human spermatozoa. J Urol 85:355, 1961 Shettles LB: Sperm morphology and sex ratios. J Urol 86:450, 1961 Harper MJK: The time of ovulation in the rabbit following the injection of luteinizing hormone. J EndocrinoI22:147, 1961 EI-Banna AA, Hafez ESE: Sperm transport and distribution in rabbit and cattle female tract. Fertil Steril 21:534, 1970 Soupart P: Leukocytes and sperm capacitation in the rabbit uterus. Fertil Steril 21:724, 1970
