14, 483-491
CRYOBIOLOGY
(1977)
The Low Temperature Preservation of Boar Spermatozoa. 3. The Fertilizing Capacity of Frozen and Thawed Boar Semen I. WILMUT A.R.C.
1
Unit of Reproductive Physiology and Biochemistry, 307 Huntingdon Road, Cambridge, U.K.
It is now well established that boar spermatozoa are peculiarly sensitive to glycerol ‘at ambient temperatures (11, 18) and furthermore that fertilizing capacity after freezing and thawing is greatly influenced by the concentration of glycerol (5, 17). When boar semen frozen and thawed in the presence of 3-570 glycerol was deposited in the oviduct, full fertilization was achieved, whereas such semen achieved almost no fertilization ( 2.870 ) even when large numbers of spermatozoa were deposited at the tip of the uterine horn (13). By contrast, conception following insemination through the cervix has been achieved with semen frozen and thawed in the presence of lower concentratios of glycerol (O-270 ) than are required to achieve maximum postthawing motility (4-80/o glycerol) (5, 14, 15, 17). A comparison has been made previously of the effect on motility and on morphology of boar spermatozoa of freezing and thawing in the presence of one of a number of cryoprotective agents all of which were presumed to permeate the cells (19). Only three, glycerol, acetamide, and erythritol, increased the proportion of motile spermatozoa, whereas all of the compounds caused Received
August
G. POLGE
AND
a decrease in the number of normal acrosomes as the concentration of agent increased. By contrast, when spermatozoa were frozen and thawed in diluents which contained only sugar and egg yolk, a relatively large proportion of the acrosomes were normal and in the presence of fructose and lactose postthawing motility was equal to that in the presence of lower concentrations of glycerol (20). The objective of the present experiment was to examine in detail the effect of perand nonpermeating protective meating agents on the fertilizing capacity of frozen and thawed boar semen and to contrast the effect on fertilizing ability with the previously defined effects on motility and morphology. Use of surgical techniques of insemination has made possible a direct comparison of two treatments in each gilt and facilitated a study of the transport and survival of frozen and thawed spermatozoa. MATERIALS
AND
METHODS
Freezing and Thawing Unless otherwise stated, the techniques em’ployed were those described previously (19, 20). The ‘diluents which were used for dilution of the semen contained either 315 mM glucose ‘or 180 mM inositol with 22.5% egg yolk. Small volumes of semen were thawed by dropping two or three
16, 1976.
1 Present address: Animal Breeding Research Organisation, West Mains Road, Edinburgh, EH9 SJQ, Scotland. 483 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any Iorm reserved.
ISSK
0011-2240
484
WILMUT
AND
pellets into a sterile dry test tube which was shaken in ,a water bath at 50°C. As the last ice disappeared, the tube was transferred to a bath at 20°C. Larger volumes were thawed by scattering the pellets upon a Teflon coated baking tray as it floated on a water bath at 50°C. Just before the last ice disappeared, the semen was poured into a bottle at room temperature (ca. 20°C). Insemination
Techniques
Insemination through the cervix was carried out with a spiral rubber catheter (10) with a collapsible bottle from which the semen could be delivered by gentle pressure. Care was taken to minimize the pressure in order to reduce any backflow through the vagina. In order to carry out insemination into the oviduct or the tip of the uterine horn, anesthesia was induced by intravenous injection of sodium pentobarbitone (Nembutal; Abbott Labs., Ltd.) and maintained by use of Fluothane (ICI, Ltd.) in a closed circuit system. The reproductive tract was exposed by a midventral laparotomy, and a blunt-ended 18-gauge needle was pushed into the uterine lumen about 2 cm from the tip of the horn. Semen was deposited either in the region of the utero-tubal junction, or, after pushing the needle through the junction, directly into the isthmus. Experimental
Animals
The animals used for these inseminations were Large White or Landrace x Essex gilts weighing 120-150 kg. After they had shown at least one heat period, their estrous cycles ,and time ‘of ovulation were synchronized by the use of Methallibure ( 12). The gilts were induced to superovulate by injection of 1000 IU of pregnant mares serum ( Gestyl; Organon, Ltd. ) followed 84-96 hr later by an injection of 500 IU of human chorionic gonadotrophin (Pregnyl; Organon, Ltd.). Ovulation was expected to occur
POLCE
approximately (3, 9).
40 hr after the Iast injection
Recovery and Examination
of Eggs
The gilts were slaughtered at a local abattoir and the reproductive tract was removed after the animal had been stunned and bled, but before the carcass was scalded. Tracts were transported to the laboratory in a vacuum flask, where the corpora lutea and follicles were counted. The oviducts were dissected free of the supporting ligaments and each oviduct was flushed from the ovarian end with 20 ml of warm saline. The eggs were mounted, fixed, cleared in acetic-alcohol ( 1:3; glacial acid : 1000/o ethanol), and stained with orcein (13). Experimental
Design
In those experiments in which semen was deposited at the tip of the uterus or in the oviduct, it has been possible to compare two treatments within each female. In experiments 2 and 4, each treatment has been compared with every other treatment in .a randomized block. The fertilization results were analyzed by weighted mean analysis of variance after transforming the percentages to angles by the arcsine transformation. EXPERIMENTAL
Experiment 1: Estimation of the Proportion of Motile Frozen and Thawed Spermatozoa Which Are Capable of Fertilization when deposited in the Oviduct A comparison was made of the minimum number of freshly collected or frozen and thawed spermatozoa required for full fertilization when the semen was deposited in the oviduct. From such a titration it was possible to estimate the proportion of the motile frozen and thawed spermatozoa that had retained a normal fertilizing ability. Collections were taken from two
LOW TEMPERATURE
PRESERVATION TABLE
OF BOAR SPERMATOZOA
485
1
The Effect of the Number of Motile Spermatozoa Deposited in the Oviduct on t,he Percentage Fertilized after Insemination of Freshly Collected Semen Which Had Been Diluted Eit.her with Plasma or Glucose-Yolk Diluent Type
Number of Number of Number of Number of Percentage
motile spermatozoa (X 103) sites ovulations eggs recovered of normal eggs fertilized
10 7 84 56 36.1
boars and 4 ml of a sperm-rich fraction from each boar was mixed and diluted to contain 2.4 x lo8 spermatozoa/ml, with diluent which contained 315 mM glucose and egg yolk. The concentration of egg yolk in the diluted semen was 15% (v/v). The semen was then diluted 1: 1 with diluent containing 315 mM glucose, 15% egg yolk, and 6% glycerol. When frozen and thawed, such semen was estimated to contain 2.0 x lo6 motile spermatozoa in the volume of 0.05 ml which was used for all inseminations. Lower concentrations of spermatozoa were obtained by dilution after thawing with medium which contained 315 mM glucose, 15% egg yolk, and 3% glycerol. Freshly collected semen was diluted either with sperm-free seminal plasma from the same ejaculate ‘or with glucoseyolk-glycerol ‘diluent. Sperm-free seminal plasma was obtained by centrifuging a portion of the same sperm-rich fraction at 20,OOOgfor 20 min. As the dilution ratio of the freshly collected semen approached 1 in 2000, the addition was made only, 1 or 2 min before the semen was deposited in the oviduct. The gilts were inseminated 4 hr before the expected time of ovulation and slaughtered 26 hr after the operation. The proportion of eggs fertilized by the freshly collected semen was influenced by the diluting medium (Table 1) . When 10,000 or 50,000 motile spermatozoa were inseminated, the proportion of eggs fertilized was significantly greater (p < 0.05)
Seminal
plasma
50 7 83 52 40.0
100 4 73 58 100.0
of Eggs Seminal
of dilution Diluent
250 5 63 32 71.9
10
50
3 26
4 42
19
30
250 3 28 22
.52.6
82.1
100.0
after dilution with glucose diluent (70.2% ) than after dilution with seminal plasma (38.2% ). The difference may have been exaggerated by the fact that only 40.0% of the eggs were fertilized after insemination of 50,000 spermatozoa diluted in seminal plasma. Nonetheless, further evidence for a difference is presented in Table 3 in which the stage of development of the fertilized eggs is shown. A significantly greater jproportion of the fertilized eggs reached the two-celled stage if the semen had been diluted with glucose-yolk diluent than after dilution with seminal plasma ( p < 0.001) . There was no difference between the proportion of eggs which were fertilized after insemination of 250, 1000, or 2000 X lo3 motile frozen and thawed spermatozoa, but the proportion was significantly lower after insemination of 100,000 motile spermatozoa (Table 2). After insemination of frozen and thawed semen, the stage of development of the fertilized eggs was TABLE
2
The Effect of the Number of Motile Spermatozoa Deposited in the Oviduct on the Percentage of Eggs Fertilized after Insemination with Frozen and Thawed Semen Number of motile spermatozoa (X 109 Number of sites Number of ovulations Number of eggs recovered Percentage of normal eggs fertilized
100 7 69 66 20.3
250 5 42 38 70.3
1,000
2,000
3 28 19
4 67 52
88.2
68.6
486
WILMUT
AND POLGE
TABLE 3 The Effect of the Type of Semen Inseminated on the Stage of Development of Fertilized Eggs Type
of semen
Stage
of developnxmt
One cell
Two or more cells
-
Fresh in diluent Fresh in plasma Frozen and thawed
2 56 34
52 23 55
midway between that of the two samples obtained from gilts that had been inseminated with freshly collected semen (Table 3) and statistically different from them both (p < 0.001). Experiment 2: Effect of Number of Spermuto,zoa, Glycerol Concentration, and Time of Insemination on the Proportion of Eggs Fertilized when Frozen and Thawed Semen was Deposited in the O&duct The experiment was a 2 x 2 x 2 X 2 factorial in an incomplete block design. It was not practicable to compare the effect of different times of insemination within animals, but all other treatments were contrasted within animals. The concentrations of glycerol were 3 and 5% in the diluted semen, the times of insemination were 6 hr before and 4 hr after the expected time of ovulation, and either 1 x lo6 or 5 X lo6 motile spermatozoa were deposited in the standard volume of 0.05 ml. Every comparison was made in two gilts and so each treatment was used six times in all.
Table 4 shows the effects of the treatments on the number of gilts in which some eggs were fertilized and on the percentage of fertilized eggs. There were no significant differences in the proportion of eggs fertilized because of the great variation within treatments (a range of 0 to 100% fertilization in five of the eight treatment groups), but there were some interesting differences. A greater proportion of the eggs were fertilized after insemination of semen frozen and thawed in the presence of 3% glycerol than 5% (64.2 vs 43.2% ). The proportion fertilized was higher when insemination was carried out after, rather than before, ovulation (57.2 vs 50.2% ), and when 5 x lo6 motile spermatozoa were inseminated when compared to 1 x 10” ( 56.1 vs 50.570 ). There were no interactions between the factors and maximum fertilization was obtained by insemination 4 hr after the expected time of ovulation of 5 X 10fi motile spermatozoa which had been frozen and thawed in the presence of 3% glycerol (71.9% ). Experiment 3: A Study of the Eflect of Six Concentrations of Glycerol on the Fertilizing Ability of Frozen and Thawed Boar Semen Which was Deposited Near the Utero-Tubal Junction The pooled sperm-rich fraction was diluted 1: 2 ( semen: diluent ) with glucoseyolk diluent which contained glycerol at 1.5 times the desired final concentration.
TABLE 4 Effect, of t,he Number of Spermatozoa, Concentration of Glycerol, and Time of Insemination on the Proport,ion of Eggs Fertilized when Frozen and Thawed Semen Was Deposited in the Oviduct Time
(3.1)~
Number of gilts with fertilized eggs Number of ovulations Number of eggs recovered Normal eggs fertilized (%) Fertilized in gilts with fert.ilixation (70)
5 62 -50 47.9 51.1
of insemination After
ovulation
(3x4
(5.1)
(5,s)
(3.1)
(3,5)
(5,l)
(5,s)
4 40 34 40.6 65.0
2 49 41 20.0 43.8
4 48 38 33.3 40.7
5 68 44 51.2 68.8
6 57 46 52.3 71.9
3 43 37 22.9 38.1
4 48 34 33.3 50.0
Before
ovulation
P Figures in paren theses represent [glycerol concentration (%), number of mot.ile spermatozoa (X 106)].
LOW
TEMPERATURE
PRESERVATION TABLE
Effect
OF BOAR
SPERMATOZOA
487
5
of Glycerol Concentration on the Fertilizing Ability of Frozen and Thawed Boar Semen Deposited at t.he Utero-Tubal Junction Glycerol 0
Number of ovulations Number of eggs recovered Normal eggs fertilized (“/c) Motile sperm (%) Normal acrosomes (%) * Means with different
subscripts
1
65 58
78 64
l&b*
27ab
5 17.9,
8 24.0,
differ at the .5r7Gprobability
Although the experiment was not a randomized block design, two treatments were always compared in a gilt. The insemination volume of 1 ml contained 5.0 x lo* or 4.0 x lo* spermatozoa in the two batches of semen which were studied. Each treatment was used six times, three times with each batch of semen. The semen was asSessed in two ways: first by an estimate of the postthawing percentage of motile spermatozoa, and second, by a study of nigrosin-eosin smears. The smears were coded and randomized, and the spermatozoa were assessed as stained or unstained and as having normal or abnormal acrosomes. One hundred spermatozoa were analyzed on each of four slides for every treatment. Maximum fertilization was obtained in the presence of 2% glycerol (Table 5). The effect of glycerol concentration did not achieve significance in the analysis of variance ( p = 0.1)) although a comparison by Duncan’s Multiple Range Test showed the fertilization in the 2% group to be significantly greater than that with 3 or 4% glycerol (p < 0.05). The rather high figure for the 5% glycerol group was a result of good fertilization in one gilt which contributed a large number of eggs, and only 10% of the eggs were fertilized in the other sites. The treatments had no effect on the proportion of spermatozoa which were not stained by eosin, and the proportion (50.570 ) was a great deal larger
than the estimates of the percentage of
concentration 2
64 61 50, 16 17.4,
(%I 3
‘1 t
4
5
hl 40
1.h
33 29
21&t’
8b
23 1.5.1cd
25 8.8d
22 14.0,d
level.
motile spermatozoa (5-25s ). There were more normal acrosomes in the presence of 0, 1, or 2% glycerol than in the presence of 4% glycerol (p < 0.05, Duncan’s Multiple Range Test). Estimates of the percentage of motile spermatozoa were not compared statistically because of the difficulty of obtaining unbiased estimates on a small number of treatments. In parallel with this experiment, inseminations were carried out with semen that had been frozen and thawed after dilution with 420 mM fructose or 210 mM lactose diluents which contained 22.5% egg yolk. Very few eggs were fertilized after insemination of semen frozen and thawed after dilution with these sugar-yolk diluents (Table 6). Experiment 4: The Fertilizing Ability of Semen Frozen and Thawed in Three Diluents and Deposited in the Oviduct A sperm-rich fraction from two boars was pooled and diluted 1: 2 (semen: diluTABLE
6
Number of Eggs Fertilized when Semen Frozen in Fructose-Yolk or Lactose-Yolk Diluent Was Deposited at the Utero-Tubal Junction Type
Number Number Number
of inseminat,ions of eggs fertilized
of diluent
Fructwe
Lactose
4 37 2
4 40 1
488
WILMUT TABLE
7
The Effect of Erythritol and Acetamide on the Moti1it.y and Morphology of Frozen and Thawed Spermatozoa and on the Proportion of Eggs Fertilized when Semen Was Deposited in the Oviduct Protective xone Number of sites Number of ovulations Number of eggs recovered Normal eggs fertilized (%) hlotile spermatozoa Normal aerosomes
(%)
(%) .-
agent
Erythritol
Acetamide
4 38
5 42
4 .55
20
32
27
70.0 5 lti.5
s-i.4 17 12.2
67.7 27 0.4 -
ent) with diluents containing 315 mM glucose, 22.5% egg yolk, and either 0.375 M erythritol, 1.125 M acetamide, or no cryoprotective agent. Hence, the spermatozoa were frozen and thawed in the presence of 0.25 M erythritol, 0.75 M acetamide, or in the absence of any permeating cryoprotective agent. Insemination of 3.5 X lo7 spermatozoa in 0.1 ml was carried out 4 hr after ovulation. The numbers were too small for an analysis of variance because of the great variability in the proportion of eggs fertilized (0-1000/o ). A majority of eggs in all treatments were fertilized, although the proportions of motile and morphologically normal spermatozoa varied greatly (Table 7). Experiment 5: A Study of the Effect of Eythritol, Acetamide, and Glycerol on the Fertilizing Capacity of Frozen and Thawed Boar Semen Deposited at the Utero-Tubal Junction Glucose-yolk diluents which contained two concentrations of acetamide or erythritol were compared with diluent without cryoprotective agent and with diluent which contained glycerol (Table 8). Semen from two boars was pooled and diluted with glucose-yolk diluents ( 1:2, semen: diluent ) . There were 2.91 x lo* spermatozoa in the l-ml insemination volume. Each treatment was compared with all
AND
POLGE
other treatments. Insemination was carried out 39 hr after injection of HCG and the gilts slaughtered 24 hr later. There were no significant differences between the treatments and only a small proportion of the eggs was fertilized. Maximum fertilization (33.3% ) was obtained in the 2% glycerol group although 26.2% were fertilized in the 0.125 M erythritol group. Experiment 6: A Study of the Fertilizing Ability of Semen Frozen and Thawed in the Presence of Glycerol and Inseminated through the Cervix Glycerol concentrations were 0, 1, 2, or 3% in the diluted semen, and glucose or inositol was used as the osmotic basis of two diluents. Gilts were inseminated with 85 to 100 ml of semen diluted with glucose diluent or 50 ml of semen diluted with inositol diluent. These volumes contained 3.2 to 3.4 x lOlo spermatozoa and 1.15 to 1.6 x lOlo spermatozoa, respectively. Inseminations were carried out 8 to 10 hr before the expected time of ovulation, and the gilts were slaughtered 24 to 30 hr later. Some fertilization was obtained with each treatment except when there was no glycerol in the inositol diluent where no spermatozoa were motile after being frozen and thawed (Table 9). Maximum fertilization was obtained with semen frozen and thawed in the presence of 2% glycerol in the inositol diluent and fertilization was clearly less in the presence of 3% glycerol (42.5 vs 4.6%). Results were rather variable with semen diluted with glucose diluent. DISCUSSION
The present experiments demonstrated clearly that the maximum fertilizing ability of frozen and thawed boar spermatozoa is obtained in the presence of a lower concentration of glycerol than is required for maximum postthawing motility, and that
LOW
TEMPERATURE
PRESERVATION
OF BOAR
TABLE The Effect
of Erythritol, and Thawed
Acetamide, Spermatozoa
489
SPERMATOZOA
8
and Glycerol on the Fertilizing Deposited at. the Utero-Tubal
Capacity Junction
of Frozen
Diluent
2%
Acetamide
Erythritol
Glycerol
0.125
Number of sites with fertilized eggs Number of ovulat,ions Number of eggs recovered Normal eggs fertilized (%)
2 36 27 33.3
2 52 41 9.8
poor fertilizing ability of semen frozen and thawed in the presence of the higher concentrations of glycerol is associated with damage to the acrosome region of the spermatozoa. Furthermore, confirmation has been obtained of the previous observation that the fertilizing ability of frozen and thawed boar spermatozoa depends very greatly upon the site of insemination (13). In a comparison of the fertilizing capacities of different numbers of freshly collected or frozen and thawed spermatozoa, it was found that at least five times as many motile frozen and thawed spermatozoa were required for full fertilization (Table 1 and 2). In the presence of 3% glycerol less than 5% of the spermatozoa had morphologically normal acrosomes, although between 20 and 25% were motile.
0.5 M
3 52 49 8.2
0.125
2 38 32 6.3
4 47 42 26.2
Inositol Glycerol
was through
the cervix.
2 47 40 15.0
of Semen Frozen
GlUCOSe
a Insemination
0.5 M
9
of Glycerol Concentration on the Fertilizing Capacity and Thawed in Glucose or Inositol Diluenta
Number of pigs Number with fertilization Number of ovulations Number of eggs Normal eggs fertilized (%) Motile spermatozoa (%) Normal acrosomes (‘%)
M
The requirement for at least five times as many motile frozen and thawed spermatozoa can be explained by the assumptions that a spermatozoan must be motile and have a “normal” acrosome in order to achieve fertilization, and that the retention of motility and a normal acrosome are independent events. If the retention of motility and a normal acrosome are independent events, the probability of a cell having both characteristics is the product of the individual probabilities, and for glycerol concentrations of 0, 1, 2, 3, 4, and 5% the probabilities can be estimated to be 0.0065, 0.0104, 0.0160, 0.0069, 0.0025, and 0.0022, respectively. These probabilities have the same rank as that of the percentage of eggs fertilized when frozen and thawed semen was deposited in the uterus, except that
TABLE The Effect
Y
concentration
0
1
2
0
1
2
3
7 3 105 89 18.0 5 14
7 2 118 94 2.1 8 13
8 4 131 99 11.1 17 11
6 0 91 69 0 0 14
6 4 93 83 19.3 10 12
6 4 114 87 42.5 18 6
5 2 104 87 4.6 25 3
490
WILMUT
AND POLGE
rather more fertilization was achieved by semen frozen and thawed in the presence of 5% glycerol. Similarly, the fertilization achieved by semen deposited in the oviduct after being frozen and thawed in glucose-yolk diluent or in the presence of erythritol or acetamide, rank in the same order as the combined estimate of the probability of motility and a normal acrosome (experiment 4). Although these estimates of the probability of a spermatozoan being motile and having a normal acrosome provide a useful indication of the fertilizing ability of semen frozen and thawed in the presence of glycerol, acetamide or erythritol, and deposited in either the oviduct or the utero-tubal junction, they did not provide any indication of the fertilizing ability of semen frozen in lactose-yolk or fructose-yolk diluents. These probabilities can be estimated to be 0.0195 and 0.224, respectively (20), which are the highest in these experiments, and yet only 2.5 and 5.4% of the eggs were fertilized when frozen and thawed semen was deposited at the utero-tubal junction (Table 6). Although assessment of the proportion of normal acrosomes provided useful information, it apparently does not, even in conjunction with an estimate of motility, provide an indication of fertilizing ability. Similarly, estimates of enzyme release (2, 6) have provided understanding of the effects of a number of stresses including freezing and thawing, but have not provided indications of fertilizing ability, which can apparently only be determined directly. The comparison of glycerol with other permeating agents and with diluents which contained only sugar and egg yolk has failed to reveal a more satisfactory cryoprotective agent than glycerol. Furthermore, as has been noted, the methods which have recently been reported to give good fertility after insemination through the cervix of frozen and thawed semen all
employed low concentrations of glycerol at the time of freezing. Confirmation is provided in these experiments of the earlier observation that the fertilization achieved by frozen and thawed boar semen depends greatly upon the site of deposition of the semen ( 13). If deposited in the oviduct, semen frozen by a great variety of treatments was capable of fertilization, whereas only with a very few treatments was semen able to achieve fertilization from the uterus. This difference probably reflects the important function of the utero-tubal junction and isthmus region of the oviduct in limiting the number of spermatozoa which enter the ampullar region of the oviduct (7, 8). Whereas several thousand million spermatozoa in 100 to 500 ml are ejaculated more or less directly into the uterus during coitus, only a few thousand spermatozoa ever reach the oviduct (16). At estrus the opening to the oviduct at the utero-tubal junction is effectively closed by polypoid processes and these prevent the passage of large volumes of fluid into the oviduct (7, 8). There is agreement that dead spermatozoa enter the lower region of the oviduct, but not about the relative ability of “dead” and live spermatozoa to do SO (1, 4). It may be that the utero-tubal junction or the oviduct selects against frozen and thawed spermatozoa with damaged plasma membranes, whether or not the cells are motile. A model of this kind could be tested by determining the minimum numbers of freshly collected and frozen and thawed spermatozoa required for full fertilization when deposited in the uterus and oviduct. If selection is taking place against some damaged frozen and thawed spermatozoa, the ratio of the numbers of fresh and frozen and thawed spermatozoa would be different at the two sites of insemination. ACKNOWLEDGMENTS We wish to thank L. Smith and J. Doggett for care of the animals used in this series of experi-
LOW TEMPERATURE
PRESERVATION
ments and D. E. Walters for carrying out most of the statistical analyses. One of us (I.W.) gratefully acknowledges receipt of a postgraduate scholarship from the Meat and Livestock Commission.
REFERENCES 1. Baker, R. D., and Degen, A. Transport of live versus dead boar spermatozoa within gilts. J. Reprod. Feti. 23, 542 ( 1970). 2. Crabo, B. G., Bower, R. E., and Graham, E. F. The effect of glycerol on bull and boar spermatozoa in vitro measured as loss of intracellular glutamicoxaloacetic transaminase. Proc. 11th Now?. Vet. CongT., Bergen, 242 ( 1970). 3. Dziuk, P. J., and Baker, R. D. Induction and control of ovulation in swine. J. Anim. Sci. 21, 698-699 ( 1962). 4. First, N. L., Short, R. E., Peters, J. B., and Stratman, F. W. Transport and loss of boar spermatozoa in the reproductive tract of the sow. 1. Anim. Sci. 27, 1037-1040 (1968). 5. Graham, E. F., Rajamannan, A. H. J., Schmeh!, M. K. L., Maki-Laurila, M., and Bower, R. E. Fertility studies with frozen boar spermatozoa. A.I. Digest 19(6), 6, (1971). 6. Graham, E. F., and Pace, M. M. Some biochemical changes in spermatozoa due to freezing. Cryobiology 4, 75-84 ( 1967). 7. Hunter, R. H. F. Physiological aspects of sperm transport in the domestic pig, SUS scrofu. I) Semen deposition and cell transport. Brit. Vet. J. 131, 565-573 (1975a). 8. Hunter, R. H. F. Physiological aspects of sperm transport in the domestic pig, SUS scrofu. 2) Regulation, survival and fate of cells. Ibit. Vet. J. 131, 681-690 ( 1975b). 9. Hunter, R. H. F., and Polge, C. Maturation of follicular oocytes in the pig after injection of human chorionic gonadotrophin. J. Reprod. Fe& 12, 525-531 (1966). 10. Melrose, D. R., and O’Hagan, C. Investiga-
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
OF BOAR SPERMATOZOA
491
tions into the technique of insemination in the pig. 4th Int. Congr. Anim. Reprod. Al., The Hague 4, 855-859 ( 1961). Polge, C. Some experiments on the preservation of boar semen. Ann. Zootech. (Paris), Series D, Suppl. 8, 113-120 ( 1959). Polge, C., Day, B. N., and Groves, T. W. Synchronisation of ovulation and artificial insemination in pigs. Vet. Rec. 83, 136142 ( 1968). Polge, C., Salamon, S., and Wilmut, I. Fertilizing capacity of frozen boar semen following surgical insemination. Vet. Rec. 87, 424-428 ( 1970). Purse], V. G., and Johnson, L. A. Fertility of gilts intracervically inseminated with frozen boar spermatozoa. Proc. VlIth Int. Congr. Anim. Reprod., Munich II, 1653 ( 1972). Purse], V. G., and Johnson, L. A. Freezing of boar spermatozoa: Fertilizing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 40 (l), 99-102 (1975). Rigby, J. P. The persistence of spermatozoa at the uterotubal junction of the sow. J. Reprod. Pert. 11, 153-155 (1966). Wilmut, I., and Polge, C. The freezing of boar spermatozoa. PTOC. VlIth Int. Congr. Anim. Reprod., Munich II, 1611 ( 1972). Wilmut, I., and Po!ge, C. The fertilizing capacity of boar semen stored in the presence of glycerol at 20, 5 and -79°C. J. Reprod. Fert. 38, 105-113 ( 1974). Wi!mut, I., and Po!ge, C. The low temperature preservation of boar spermatozoa. 1. The motility and morphology of boar spermatozoa frozen and thawed in the presence of permeating protective agents. Cryobiology 14, 471-478 ( 1977). Wilmut, I., and Polge, C. The low temperature preservation of boar spermatozoa. 2. The motility and morphology of boar spermatozoa frozen and thawed in diluent which contained only sugar and egg yolk. Cryobiology 14, 479-482 ( 1977).
CRYOBIOLOGY
(1977)
The Low Temperature Preservation of Boar Spermatozoa. 3. The Fertilizing Capacity of Frozen and Thawed Boar Semen I. WILMUT A.R.C.
1
Unit of Reproductive Physiology and Biochemistry, 307 Huntingdon Road, Cambridge, U.K.
It is now well established that boar spermatozoa are peculiarly sensitive to glycerol ‘at ambient temperatures (11, 18) and furthermore that fertilizing capacity after freezing and thawing is greatly influenced by the concentration of glycerol (5, 17). When boar semen frozen and thawed in the presence of 3-570 glycerol was deposited in the oviduct, full fertilization was achieved, whereas such semen achieved almost no fertilization ( 2.870 ) even when large numbers of spermatozoa were deposited at the tip of the uterine horn (13). By contrast, conception following insemination through the cervix has been achieved with semen frozen and thawed in the presence of lower concentratios of glycerol (O-270 ) than are required to achieve maximum postthawing motility (4-80/o glycerol) (5, 14, 15, 17). A comparison has been made previously of the effect on motility and on morphology of boar spermatozoa of freezing and thawing in the presence of one of a number of cryoprotective agents all of which were presumed to permeate the cells (19). Only three, glycerol, acetamide, and erythritol, increased the proportion of motile spermatozoa, whereas all of the compounds caused Received
August
G. POLGE
AND
a decrease in the number of normal acrosomes as the concentration of agent increased. By contrast, when spermatozoa were frozen and thawed in diluents which contained only sugar and egg yolk, a relatively large proportion of the acrosomes were normal and in the presence of fructose and lactose postthawing motility was equal to that in the presence of lower concentrations of glycerol (20). The objective of the present experiment was to examine in detail the effect of perand nonpermeating protective meating agents on the fertilizing capacity of frozen and thawed boar semen and to contrast the effect on fertilizing ability with the previously defined effects on motility and morphology. Use of surgical techniques of insemination has made possible a direct comparison of two treatments in each gilt and facilitated a study of the transport and survival of frozen and thawed spermatozoa. MATERIALS
AND
METHODS
Freezing and Thawing Unless otherwise stated, the techniques em’ployed were those described previously (19, 20). The ‘diluents which were used for dilution of the semen contained either 315 mM glucose ‘or 180 mM inositol with 22.5% egg yolk. Small volumes of semen were thawed by dropping two or three
16, 1976.
1 Present address: Animal Breeding Research Organisation, West Mains Road, Edinburgh, EH9 SJQ, Scotland. 483 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any Iorm reserved.
ISSK
0011-2240
484
WILMUT
AND
pellets into a sterile dry test tube which was shaken in ,a water bath at 50°C. As the last ice disappeared, the tube was transferred to a bath at 20°C. Larger volumes were thawed by scattering the pellets upon a Teflon coated baking tray as it floated on a water bath at 50°C. Just before the last ice disappeared, the semen was poured into a bottle at room temperature (ca. 20°C). Insemination
Techniques
Insemination through the cervix was carried out with a spiral rubber catheter (10) with a collapsible bottle from which the semen could be delivered by gentle pressure. Care was taken to minimize the pressure in order to reduce any backflow through the vagina. In order to carry out insemination into the oviduct or the tip of the uterine horn, anesthesia was induced by intravenous injection of sodium pentobarbitone (Nembutal; Abbott Labs., Ltd.) and maintained by use of Fluothane (ICI, Ltd.) in a closed circuit system. The reproductive tract was exposed by a midventral laparotomy, and a blunt-ended 18-gauge needle was pushed into the uterine lumen about 2 cm from the tip of the horn. Semen was deposited either in the region of the utero-tubal junction, or, after pushing the needle through the junction, directly into the isthmus. Experimental
Animals
The animals used for these inseminations were Large White or Landrace x Essex gilts weighing 120-150 kg. After they had shown at least one heat period, their estrous cycles ,and time ‘of ovulation were synchronized by the use of Methallibure ( 12). The gilts were induced to superovulate by injection of 1000 IU of pregnant mares serum ( Gestyl; Organon, Ltd. ) followed 84-96 hr later by an injection of 500 IU of human chorionic gonadotrophin (Pregnyl; Organon, Ltd.). Ovulation was expected to occur
POLCE
approximately (3, 9).
40 hr after the Iast injection
Recovery and Examination
of Eggs
The gilts were slaughtered at a local abattoir and the reproductive tract was removed after the animal had been stunned and bled, but before the carcass was scalded. Tracts were transported to the laboratory in a vacuum flask, where the corpora lutea and follicles were counted. The oviducts were dissected free of the supporting ligaments and each oviduct was flushed from the ovarian end with 20 ml of warm saline. The eggs were mounted, fixed, cleared in acetic-alcohol ( 1:3; glacial acid : 1000/o ethanol), and stained with orcein (13). Experimental
Design
In those experiments in which semen was deposited at the tip of the uterus or in the oviduct, it has been possible to compare two treatments within each female. In experiments 2 and 4, each treatment has been compared with every other treatment in .a randomized block. The fertilization results were analyzed by weighted mean analysis of variance after transforming the percentages to angles by the arcsine transformation. EXPERIMENTAL
Experiment 1: Estimation of the Proportion of Motile Frozen and Thawed Spermatozoa Which Are Capable of Fertilization when deposited in the Oviduct A comparison was made of the minimum number of freshly collected or frozen and thawed spermatozoa required for full fertilization when the semen was deposited in the oviduct. From such a titration it was possible to estimate the proportion of the motile frozen and thawed spermatozoa that had retained a normal fertilizing ability. Collections were taken from two
LOW TEMPERATURE
PRESERVATION TABLE
OF BOAR SPERMATOZOA
485
1
The Effect of the Number of Motile Spermatozoa Deposited in the Oviduct on t,he Percentage Fertilized after Insemination of Freshly Collected Semen Which Had Been Diluted Eit.her with Plasma or Glucose-Yolk Diluent Type
Number of Number of Number of Number of Percentage
motile spermatozoa (X 103) sites ovulations eggs recovered of normal eggs fertilized
10 7 84 56 36.1
boars and 4 ml of a sperm-rich fraction from each boar was mixed and diluted to contain 2.4 x lo8 spermatozoa/ml, with diluent which contained 315 mM glucose and egg yolk. The concentration of egg yolk in the diluted semen was 15% (v/v). The semen was then diluted 1: 1 with diluent containing 315 mM glucose, 15% egg yolk, and 6% glycerol. When frozen and thawed, such semen was estimated to contain 2.0 x lo6 motile spermatozoa in the volume of 0.05 ml which was used for all inseminations. Lower concentrations of spermatozoa were obtained by dilution after thawing with medium which contained 315 mM glucose, 15% egg yolk, and 3% glycerol. Freshly collected semen was diluted either with sperm-free seminal plasma from the same ejaculate ‘or with glucoseyolk-glycerol ‘diluent. Sperm-free seminal plasma was obtained by centrifuging a portion of the same sperm-rich fraction at 20,OOOgfor 20 min. As the dilution ratio of the freshly collected semen approached 1 in 2000, the addition was made only, 1 or 2 min before the semen was deposited in the oviduct. The gilts were inseminated 4 hr before the expected time of ovulation and slaughtered 26 hr after the operation. The proportion of eggs fertilized by the freshly collected semen was influenced by the diluting medium (Table 1) . When 10,000 or 50,000 motile spermatozoa were inseminated, the proportion of eggs fertilized was significantly greater (p < 0.05)
Seminal
plasma
50 7 83 52 40.0
100 4 73 58 100.0
of Eggs Seminal
of dilution Diluent
250 5 63 32 71.9
10
50
3 26
4 42
19
30
250 3 28 22
.52.6
82.1
100.0
after dilution with glucose diluent (70.2% ) than after dilution with seminal plasma (38.2% ). The difference may have been exaggerated by the fact that only 40.0% of the eggs were fertilized after insemination of 50,000 spermatozoa diluted in seminal plasma. Nonetheless, further evidence for a difference is presented in Table 3 in which the stage of development of the fertilized eggs is shown. A significantly greater jproportion of the fertilized eggs reached the two-celled stage if the semen had been diluted with glucose-yolk diluent than after dilution with seminal plasma ( p < 0.001) . There was no difference between the proportion of eggs which were fertilized after insemination of 250, 1000, or 2000 X lo3 motile frozen and thawed spermatozoa, but the proportion was significantly lower after insemination of 100,000 motile spermatozoa (Table 2). After insemination of frozen and thawed semen, the stage of development of the fertilized eggs was TABLE
2
The Effect of the Number of Motile Spermatozoa Deposited in the Oviduct on the Percentage of Eggs Fertilized after Insemination with Frozen and Thawed Semen Number of motile spermatozoa (X 109 Number of sites Number of ovulations Number of eggs recovered Percentage of normal eggs fertilized
100 7 69 66 20.3
250 5 42 38 70.3
1,000
2,000
3 28 19
4 67 52
88.2
68.6
486
WILMUT
AND POLGE
TABLE 3 The Effect of the Type of Semen Inseminated on the Stage of Development of Fertilized Eggs Type
of semen
Stage
of developnxmt
One cell
Two or more cells
-
Fresh in diluent Fresh in plasma Frozen and thawed
2 56 34
52 23 55
midway between that of the two samples obtained from gilts that had been inseminated with freshly collected semen (Table 3) and statistically different from them both (p < 0.001). Experiment 2: Effect of Number of Spermuto,zoa, Glycerol Concentration, and Time of Insemination on the Proportion of Eggs Fertilized when Frozen and Thawed Semen was Deposited in the O&duct The experiment was a 2 x 2 x 2 X 2 factorial in an incomplete block design. It was not practicable to compare the effect of different times of insemination within animals, but all other treatments were contrasted within animals. The concentrations of glycerol were 3 and 5% in the diluted semen, the times of insemination were 6 hr before and 4 hr after the expected time of ovulation, and either 1 x lo6 or 5 X lo6 motile spermatozoa were deposited in the standard volume of 0.05 ml. Every comparison was made in two gilts and so each treatment was used six times in all.
Table 4 shows the effects of the treatments on the number of gilts in which some eggs were fertilized and on the percentage of fertilized eggs. There were no significant differences in the proportion of eggs fertilized because of the great variation within treatments (a range of 0 to 100% fertilization in five of the eight treatment groups), but there were some interesting differences. A greater proportion of the eggs were fertilized after insemination of semen frozen and thawed in the presence of 3% glycerol than 5% (64.2 vs 43.2% ). The proportion fertilized was higher when insemination was carried out after, rather than before, ovulation (57.2 vs 50.2% ), and when 5 x lo6 motile spermatozoa were inseminated when compared to 1 x 10” ( 56.1 vs 50.570 ). There were no interactions between the factors and maximum fertilization was obtained by insemination 4 hr after the expected time of ovulation of 5 X 10fi motile spermatozoa which had been frozen and thawed in the presence of 3% glycerol (71.9% ). Experiment 3: A Study of the Eflect of Six Concentrations of Glycerol on the Fertilizing Ability of Frozen and Thawed Boar Semen Which was Deposited Near the Utero-Tubal Junction The pooled sperm-rich fraction was diluted 1: 2 ( semen: diluent ) with glucoseyolk diluent which contained glycerol at 1.5 times the desired final concentration.
TABLE 4 Effect, of t,he Number of Spermatozoa, Concentration of Glycerol, and Time of Insemination on the Proport,ion of Eggs Fertilized when Frozen and Thawed Semen Was Deposited in the Oviduct Time
(3.1)~
Number of gilts with fertilized eggs Number of ovulations Number of eggs recovered Normal eggs fertilized (%) Fertilized in gilts with fert.ilixation (70)
5 62 -50 47.9 51.1
of insemination After
ovulation
(3x4
(5.1)
(5,s)
(3.1)
(3,5)
(5,l)
(5,s)
4 40 34 40.6 65.0
2 49 41 20.0 43.8
4 48 38 33.3 40.7
5 68 44 51.2 68.8
6 57 46 52.3 71.9
3 43 37 22.9 38.1
4 48 34 33.3 50.0
Before
ovulation
P Figures in paren theses represent [glycerol concentration (%), number of mot.ile spermatozoa (X 106)].
LOW
TEMPERATURE
PRESERVATION TABLE
Effect
OF BOAR
SPERMATOZOA
487
5
of Glycerol Concentration on the Fertilizing Ability of Frozen and Thawed Boar Semen Deposited at t.he Utero-Tubal Junction Glycerol 0
Number of ovulations Number of eggs recovered Normal eggs fertilized (“/c) Motile sperm (%) Normal acrosomes (%) * Means with different
subscripts
1
65 58
78 64
l&b*
27ab
5 17.9,
8 24.0,
differ at the .5r7Gprobability
Although the experiment was not a randomized block design, two treatments were always compared in a gilt. The insemination volume of 1 ml contained 5.0 x lo* or 4.0 x lo* spermatozoa in the two batches of semen which were studied. Each treatment was used six times, three times with each batch of semen. The semen was asSessed in two ways: first by an estimate of the postthawing percentage of motile spermatozoa, and second, by a study of nigrosin-eosin smears. The smears were coded and randomized, and the spermatozoa were assessed as stained or unstained and as having normal or abnormal acrosomes. One hundred spermatozoa were analyzed on each of four slides for every treatment. Maximum fertilization was obtained in the presence of 2% glycerol (Table 5). The effect of glycerol concentration did not achieve significance in the analysis of variance ( p = 0.1)) although a comparison by Duncan’s Multiple Range Test showed the fertilization in the 2% group to be significantly greater than that with 3 or 4% glycerol (p < 0.05). The rather high figure for the 5% glycerol group was a result of good fertilization in one gilt which contributed a large number of eggs, and only 10% of the eggs were fertilized in the other sites. The treatments had no effect on the proportion of spermatozoa which were not stained by eosin, and the proportion (50.570 ) was a great deal larger
than the estimates of the percentage of
concentration 2
64 61 50, 16 17.4,
(%I 3
‘1 t
4
5
hl 40
1.h
33 29
21&t’
8b
23 1.5.1cd
25 8.8d
22 14.0,d
level.
motile spermatozoa (5-25s ). There were more normal acrosomes in the presence of 0, 1, or 2% glycerol than in the presence of 4% glycerol (p < 0.05, Duncan’s Multiple Range Test). Estimates of the percentage of motile spermatozoa were not compared statistically because of the difficulty of obtaining unbiased estimates on a small number of treatments. In parallel with this experiment, inseminations were carried out with semen that had been frozen and thawed after dilution with 420 mM fructose or 210 mM lactose diluents which contained 22.5% egg yolk. Very few eggs were fertilized after insemination of semen frozen and thawed after dilution with these sugar-yolk diluents (Table 6). Experiment 4: The Fertilizing Ability of Semen Frozen and Thawed in Three Diluents and Deposited in the Oviduct A sperm-rich fraction from two boars was pooled and diluted 1: 2 (semen: diluTABLE
6
Number of Eggs Fertilized when Semen Frozen in Fructose-Yolk or Lactose-Yolk Diluent Was Deposited at the Utero-Tubal Junction Type
Number Number Number
of inseminat,ions of eggs fertilized
of diluent
Fructwe
Lactose
4 37 2
4 40 1
488
WILMUT TABLE
7
The Effect of Erythritol and Acetamide on the Moti1it.y and Morphology of Frozen and Thawed Spermatozoa and on the Proportion of Eggs Fertilized when Semen Was Deposited in the Oviduct Protective xone Number of sites Number of ovulations Number of eggs recovered Normal eggs fertilized (%) hlotile spermatozoa Normal aerosomes
(%)
(%) .-
agent
Erythritol
Acetamide
4 38
5 42
4 .55
20
32
27
70.0 5 lti.5
s-i.4 17 12.2
67.7 27 0.4 -
ent) with diluents containing 315 mM glucose, 22.5% egg yolk, and either 0.375 M erythritol, 1.125 M acetamide, or no cryoprotective agent. Hence, the spermatozoa were frozen and thawed in the presence of 0.25 M erythritol, 0.75 M acetamide, or in the absence of any permeating cryoprotective agent. Insemination of 3.5 X lo7 spermatozoa in 0.1 ml was carried out 4 hr after ovulation. The numbers were too small for an analysis of variance because of the great variability in the proportion of eggs fertilized (0-1000/o ). A majority of eggs in all treatments were fertilized, although the proportions of motile and morphologically normal spermatozoa varied greatly (Table 7). Experiment 5: A Study of the Effect of Eythritol, Acetamide, and Glycerol on the Fertilizing Capacity of Frozen and Thawed Boar Semen Deposited at the Utero-Tubal Junction Glucose-yolk diluents which contained two concentrations of acetamide or erythritol were compared with diluent without cryoprotective agent and with diluent which contained glycerol (Table 8). Semen from two boars was pooled and diluted with glucose-yolk diluents ( 1:2, semen: diluent ) . There were 2.91 x lo* spermatozoa in the l-ml insemination volume. Each treatment was compared with all
AND
POLGE
other treatments. Insemination was carried out 39 hr after injection of HCG and the gilts slaughtered 24 hr later. There were no significant differences between the treatments and only a small proportion of the eggs was fertilized. Maximum fertilization (33.3% ) was obtained in the 2% glycerol group although 26.2% were fertilized in the 0.125 M erythritol group. Experiment 6: A Study of the Fertilizing Ability of Semen Frozen and Thawed in the Presence of Glycerol and Inseminated through the Cervix Glycerol concentrations were 0, 1, 2, or 3% in the diluted semen, and glucose or inositol was used as the osmotic basis of two diluents. Gilts were inseminated with 85 to 100 ml of semen diluted with glucose diluent or 50 ml of semen diluted with inositol diluent. These volumes contained 3.2 to 3.4 x lOlo spermatozoa and 1.15 to 1.6 x lOlo spermatozoa, respectively. Inseminations were carried out 8 to 10 hr before the expected time of ovulation, and the gilts were slaughtered 24 to 30 hr later. Some fertilization was obtained with each treatment except when there was no glycerol in the inositol diluent where no spermatozoa were motile after being frozen and thawed (Table 9). Maximum fertilization was obtained with semen frozen and thawed in the presence of 2% glycerol in the inositol diluent and fertilization was clearly less in the presence of 3% glycerol (42.5 vs 4.6%). Results were rather variable with semen diluted with glucose diluent. DISCUSSION
The present experiments demonstrated clearly that the maximum fertilizing ability of frozen and thawed boar spermatozoa is obtained in the presence of a lower concentration of glycerol than is required for maximum postthawing motility, and that
LOW
TEMPERATURE
PRESERVATION
OF BOAR
TABLE The Effect
of Erythritol, and Thawed
Acetamide, Spermatozoa
489
SPERMATOZOA
8
and Glycerol on the Fertilizing Deposited at. the Utero-Tubal
Capacity Junction
of Frozen
Diluent
2%
Acetamide
Erythritol
Glycerol
0.125
Number of sites with fertilized eggs Number of ovulat,ions Number of eggs recovered Normal eggs fertilized (%)
2 36 27 33.3
2 52 41 9.8
poor fertilizing ability of semen frozen and thawed in the presence of the higher concentrations of glycerol is associated with damage to the acrosome region of the spermatozoa. Furthermore, confirmation has been obtained of the previous observation that the fertilizing ability of frozen and thawed boar spermatozoa depends very greatly upon the site of insemination (13). In a comparison of the fertilizing capacities of different numbers of freshly collected or frozen and thawed spermatozoa, it was found that at least five times as many motile frozen and thawed spermatozoa were required for full fertilization (Table 1 and 2). In the presence of 3% glycerol less than 5% of the spermatozoa had morphologically normal acrosomes, although between 20 and 25% were motile.
0.5 M
3 52 49 8.2
0.125
2 38 32 6.3
4 47 42 26.2
Inositol Glycerol
was through
the cervix.
2 47 40 15.0
of Semen Frozen
GlUCOSe
a Insemination
0.5 M
9
of Glycerol Concentration on the Fertilizing Capacity and Thawed in Glucose or Inositol Diluenta
Number of pigs Number with fertilization Number of ovulations Number of eggs Normal eggs fertilized (%) Motile spermatozoa (%) Normal acrosomes (‘%)
M
The requirement for at least five times as many motile frozen and thawed spermatozoa can be explained by the assumptions that a spermatozoan must be motile and have a “normal” acrosome in order to achieve fertilization, and that the retention of motility and a normal acrosome are independent events. If the retention of motility and a normal acrosome are independent events, the probability of a cell having both characteristics is the product of the individual probabilities, and for glycerol concentrations of 0, 1, 2, 3, 4, and 5% the probabilities can be estimated to be 0.0065, 0.0104, 0.0160, 0.0069, 0.0025, and 0.0022, respectively. These probabilities have the same rank as that of the percentage of eggs fertilized when frozen and thawed semen was deposited in the uterus, except that
TABLE The Effect
Y
concentration
0
1
2
0
1
2
3
7 3 105 89 18.0 5 14
7 2 118 94 2.1 8 13
8 4 131 99 11.1 17 11
6 0 91 69 0 0 14
6 4 93 83 19.3 10 12
6 4 114 87 42.5 18 6
5 2 104 87 4.6 25 3
490
WILMUT
AND POLGE
rather more fertilization was achieved by semen frozen and thawed in the presence of 5% glycerol. Similarly, the fertilization achieved by semen deposited in the oviduct after being frozen and thawed in glucose-yolk diluent or in the presence of erythritol or acetamide, rank in the same order as the combined estimate of the probability of motility and a normal acrosome (experiment 4). Although these estimates of the probability of a spermatozoan being motile and having a normal acrosome provide a useful indication of the fertilizing ability of semen frozen and thawed in the presence of glycerol, acetamide or erythritol, and deposited in either the oviduct or the utero-tubal junction, they did not provide any indication of the fertilizing ability of semen frozen in lactose-yolk or fructose-yolk diluents. These probabilities can be estimated to be 0.0195 and 0.224, respectively (20), which are the highest in these experiments, and yet only 2.5 and 5.4% of the eggs were fertilized when frozen and thawed semen was deposited at the utero-tubal junction (Table 6). Although assessment of the proportion of normal acrosomes provided useful information, it apparently does not, even in conjunction with an estimate of motility, provide an indication of fertilizing ability. Similarly, estimates of enzyme release (2, 6) have provided understanding of the effects of a number of stresses including freezing and thawing, but have not provided indications of fertilizing ability, which can apparently only be determined directly. The comparison of glycerol with other permeating agents and with diluents which contained only sugar and egg yolk has failed to reveal a more satisfactory cryoprotective agent than glycerol. Furthermore, as has been noted, the methods which have recently been reported to give good fertility after insemination through the cervix of frozen and thawed semen all
employed low concentrations of glycerol at the time of freezing. Confirmation is provided in these experiments of the earlier observation that the fertilization achieved by frozen and thawed boar semen depends greatly upon the site of deposition of the semen ( 13). If deposited in the oviduct, semen frozen by a great variety of treatments was capable of fertilization, whereas only with a very few treatments was semen able to achieve fertilization from the uterus. This difference probably reflects the important function of the utero-tubal junction and isthmus region of the oviduct in limiting the number of spermatozoa which enter the ampullar region of the oviduct (7, 8). Whereas several thousand million spermatozoa in 100 to 500 ml are ejaculated more or less directly into the uterus during coitus, only a few thousand spermatozoa ever reach the oviduct (16). At estrus the opening to the oviduct at the utero-tubal junction is effectively closed by polypoid processes and these prevent the passage of large volumes of fluid into the oviduct (7, 8). There is agreement that dead spermatozoa enter the lower region of the oviduct, but not about the relative ability of “dead” and live spermatozoa to do SO (1, 4). It may be that the utero-tubal junction or the oviduct selects against frozen and thawed spermatozoa with damaged plasma membranes, whether or not the cells are motile. A model of this kind could be tested by determining the minimum numbers of freshly collected and frozen and thawed spermatozoa required for full fertilization when deposited in the uterus and oviduct. If selection is taking place against some damaged frozen and thawed spermatozoa, the ratio of the numbers of fresh and frozen and thawed spermatozoa would be different at the two sites of insemination. ACKNOWLEDGMENTS We wish to thank L. Smith and J. Doggett for care of the animals used in this series of experi-
LOW TEMPERATURE
PRESERVATION
ments and D. E. Walters for carrying out most of the statistical analyses. One of us (I.W.) gratefully acknowledges receipt of a postgraduate scholarship from the Meat and Livestock Commission.
REFERENCES 1. Baker, R. D., and Degen, A. Transport of live versus dead boar spermatozoa within gilts. J. Reprod. Feti. 23, 542 ( 1970). 2. Crabo, B. G., Bower, R. E., and Graham, E. F. The effect of glycerol on bull and boar spermatozoa in vitro measured as loss of intracellular glutamicoxaloacetic transaminase. Proc. 11th Now?. Vet. CongT., Bergen, 242 ( 1970). 3. Dziuk, P. J., and Baker, R. D. Induction and control of ovulation in swine. J. Anim. Sci. 21, 698-699 ( 1962). 4. First, N. L., Short, R. E., Peters, J. B., and Stratman, F. W. Transport and loss of boar spermatozoa in the reproductive tract of the sow. 1. Anim. Sci. 27, 1037-1040 (1968). 5. Graham, E. F., Rajamannan, A. H. J., Schmeh!, M. K. L., Maki-Laurila, M., and Bower, R. E. Fertility studies with frozen boar spermatozoa. A.I. Digest 19(6), 6, (1971). 6. Graham, E. F., and Pace, M. M. Some biochemical changes in spermatozoa due to freezing. Cryobiology 4, 75-84 ( 1967). 7. Hunter, R. H. F. Physiological aspects of sperm transport in the domestic pig, SUS scrofu. I) Semen deposition and cell transport. Brit. Vet. J. 131, 565-573 (1975a). 8. Hunter, R. H. F. Physiological aspects of sperm transport in the domestic pig, SUS scrofu. 2) Regulation, survival and fate of cells. Ibit. Vet. J. 131, 681-690 ( 1975b). 9. Hunter, R. H. F., and Polge, C. Maturation of follicular oocytes in the pig after injection of human chorionic gonadotrophin. J. Reprod. Fe& 12, 525-531 (1966). 10. Melrose, D. R., and O’Hagan, C. Investiga-
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
OF BOAR SPERMATOZOA
491
tions into the technique of insemination in the pig. 4th Int. Congr. Anim. Reprod. Al., The Hague 4, 855-859 ( 1961). Polge, C. Some experiments on the preservation of boar semen. Ann. Zootech. (Paris), Series D, Suppl. 8, 113-120 ( 1959). Polge, C., Day, B. N., and Groves, T. W. Synchronisation of ovulation and artificial insemination in pigs. Vet. Rec. 83, 136142 ( 1968). Polge, C., Salamon, S., and Wilmut, I. Fertilizing capacity of frozen boar semen following surgical insemination. Vet. Rec. 87, 424-428 ( 1970). Purse], V. G., and Johnson, L. A. Fertility of gilts intracervically inseminated with frozen boar spermatozoa. Proc. VlIth Int. Congr. Anim. Reprod., Munich II, 1653 ( 1972). Purse], V. G., and Johnson, L. A. Freezing of boar spermatozoa: Fertilizing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 40 (l), 99-102 (1975). Rigby, J. P. The persistence of spermatozoa at the uterotubal junction of the sow. J. Reprod. Pert. 11, 153-155 (1966). Wilmut, I., and Polge, C. The freezing of boar spermatozoa. PTOC. VlIth Int. Congr. Anim. Reprod., Munich II, 1611 ( 1972). Wilmut, I., and Po!ge, C. The fertilizing capacity of boar semen stored in the presence of glycerol at 20, 5 and -79°C. J. Reprod. Fert. 38, 105-113 ( 1974). Wi!mut, I., and Po!ge, C. The low temperature preservation of boar spermatozoa. 1. The motility and morphology of boar spermatozoa frozen and thawed in the presence of permeating protective agents. Cryobiology 14, 471-478 ( 1977). Wilmut, I., and Polge, C. The low temperature preservation of boar spermatozoa. 2. The motility and morphology of boar spermatozoa frozen and thawed in diluent which contained only sugar and egg yolk. Cryobiology 14, 479-482 ( 1977).
