3°7

NOTES ON OVULATION AND FERTILISATIONIN THE FERRET

BY J. HAMMOND AND A. WALTON.(School of Agriculture, Cambridge.)

(Received 10th January, 1934.)

CONTENTS.PAGE

In t roduct ion . . . . . . . . . 307Experimental results

T h e t ime of ovulation 307Appearance of the vulva after ovulation . . . . 309T h e ascent of the spermatozoa . . . . . . 3 1 1At tempts at artificial inseminat ion 312T h e durat ion of coitus . . . . . . . - 3 1 3T h e t ime that the ova remain capable of fertilisation . . 315

S u m m a r y . . . . . . . . 3 1 8References . . . 3 1 8

INTRODUCTION.SINCE the ferret ovulates only after coitus, it is a most suitable species in which toobtain accurate data concerning the timing of the processes of reproduction involvedin fertility. It is useful also to compare the results obtained with the ferret with thosealready obtained with the rabbit (Hammond, 1934), the only other species (with thedoubtful exception of the cat) known to ovulate only after coitus.

While much of the physiology of reproduction in the ferret is known through thework of Marshall (1904, 1933), Robinson (1918), Hammond and Marshall (1930),Hill and Parkes(i93i, 1933), Parkes, Rowlands and Brambell (1932), Murr(i932fland b), Bissonnette (1932) and others, the problems connected with the control offertility have received but little attention. The solution of these problems was theobject of the present investigation, and the results are given below in sections dealingwith various aspects of the problem. While under several of these sections the amountof data obtained is too small to sustain any but very tentative conclusions, the obser-vations have been included in the paper, as owing to pressure of other work thereseem3 little likelihood of time and space being available in the immediate future tomake a more extensive series.

EXPERIMENTAL RESULTS.

The time of ovulation.

The first step was to find out whether the ferret ovulates at a definite time aftercopulation as in the rabbit, or whether, as Robinson (1918) was inclined to believe,there is no definite fixed period but that ovulation may occur at any time from 30 to75 hours after coitus. Since in the rabbit ovulation occurs at a fixed time after

308 J. HAMMOND and A. WALTON

coitus (10 hours) as a result of stimulation of the anterior pituitary by the orgasm(Fee and Parkes, 1929), we were not prepared to accept Robinson's conclusionswithout verification. His data (his Tables VIII and IX) were therefore analysed tofind out if another interpretation could be put upon his facts. The stage of develop-ment of the ova found in the different animals killed was plotted against the timeafter coitus in 6-hour intervals. The results are shown in Table I.

Table I. Stages of development of ferret ova at different intervals of time after coitus.Numbers of animals {from Robinson's data—his Tables VIII and IX).

Day after coitus

Hours after coitus

Had not ovulatedIn tubes:

PronuclearJust fertilised1-2 cells4-8 cells8-16 cells16-32 cellsMorulaBias tula

In uterus:MorulaBiastula

19-

2425- 3i- 37- 43-30 36 42 48

49- 55- 61- 67-54 60 66 72

73- 79- 85- 91-78 84 go 96

Day after coitus

Hours after coitus

Had not ovulatedIn tubes:

PronuclearJust fertilisedi-2 cells4-8 cells8-16 cells16-32 cellsMorulaBias tula

In uterus:MorulaBias tula

97- 103- 109- 115-102 108 114 120

•

t

t22

6

121- 127- 133- 139-126 132 138 144

111

11

62

As we see it these results point to a fairly definite time of ovulation in the regionof 30-36 hours after coitus, for at no one interval of time after coitus is there anygreat variation in the stage of development of the embryos found, at any rate notmuch greater than might be expected to occur in the normal course of developmentof any set of ova shed at the same time. The apparent exceptions to this statementare capable of alternative explanations. They fall into two categories: (1) Thoseanimals which apparently failed to ovulate within periods of 48, 72 and 96 hours.

Ovulation and Fertilisation in the Ferret 309

Failure to ovulate, even when coitus is known definitely to have taken place, some-times occurs in the rabbit and may well occur in the ferret. In addition, failure toovulate in the ferret may be due to incomplete coitus, since in this animal coitustakes a considerable time (see below p. 313), and it is frequently difficult to determinewhether penetration of the penis has actually taken place or not. (2) The presence ofova in the pronuclear stage at 78, 96 and 120 hours after coitus. These facts do notprovide conclusive evidence of delayed ovulation, since the ova might have beenshed earlier and not fertilised owing to lack of sufficient spermatozoa in the semen ofthe male used. We have failed to obtain young on several occasions after matingswith males deficient in sperm, not only in the ferret but also in the rabbit, and wewould therefore attribute these cases to sperm deficiency of the males rather than todelayed ovulation.

In order to test these conclusions a few ferrets were killed at different times aftercoitus and the ovaries examined fresh under a binocular microscope to see if ovula-tion had occurred. The ovaries were then fixed in 10 per cent, formalin and theobservations confirmed by cutting serial sections. Owing to the long and variabletime taken in coitus (see below p. 313) it is not possible to fix the time of ovulation asaccurately as in the rabbit, and the ferrets were therefore killed at intervals of 6hours, from 24 to 48 hours, after the beginning of coitus.

Both the ferrets killed 24 hours after the beginning of coitus had not yet ovu-lated, while those killed at 30, 36, 42 and 48 hours had all ovulated with the excep-tion of one—No. 21, killed at 42 hours. This animal was mated late in the season(August 25th), and it seems quite probable that it would never have ovulated at all,owing to insufficient anterior pituitary substance or to some other cause associatedwith the time of year at which the mating was made.

Our conclusion is therefore that the ferret ovulates normally about 30 hoursafter coitus.

Appearance of tlie vulva after ovulation.In anoestrum the vulva is so small that, even if desire were present, it would be

impossible for coitus to occur. During oestrus on the other hand not only do thelips swell up to large size (see Marshall, 1933) but the lumen becomes much largeralso. In pregnancy and pseudo-pregnancy the vulva rapidly atrophies and goesback to the anoestrous condition. When planning the experiments of matings with afertile male at different intervals of time after a sterile coitus (see p. 315 below) itwas important to know precisely when the turgidity of the vulval lips began todisappear and for how long after the first sterile coitus (and subsequent ovulation)it would be possible to get a second coitus.

Fortunately for our experiment sterility (as a result of the second mating at 66hours after the first sterile mating) was obtained before the time when the vulvabecame too small to admit of coitus, or before the female ceased to have desire forcoitus. Up to a period of 72 hours after the first coitus, or a period of 42 hours afterovulation, no difficulty was experienced in getting a second mating with a fertilemale, and there was no case of failure to mate through lack of desire by the female, orthrough the decrease in size of the vulva.

310 J. HAMMOND and A. WALTON

The first change which occurs in the vulva at the beginning of pseudo-preg-nancy is the loss of turgidity of the lips, which become flaccid; this occurs beforeactual decrease in size of the lumen takes place. While making the fertile matings(see p. 315 below) at different intervals of time after a first sterile mating (andtherefore at different intervals of time in relation to ovulation) we made a few inci-dental observations on the state of the vulva, in order to ascertain if possible whatthe time relation was between ovulation and the loss of turgidity of the vulval lips.It was hoped by this means to be able to detect quickly those animals which had notovulated. A summary of these observations is as follows:

24 hours after the beginning of coitus (6 hours before ovulation):

No. 38 (30. vii. 30)—Still quite turgid and swollen.No. 33 (12. viii. 30)—Still quite turgid and swollen.

30 hours after the beginning of coitus (at time of ovulation):

No. 34 (20. vii. 30)—Still quite large and turgid.

36 hours after the beginning of coitus (6 hours after ovulation):

No. 37 (17. vii. 30)—Quite swollen and showing no signs of flabbiness.

54 hours after the beginning of coitus (24 hours after ovulation):

No. 37 (5. v. 30)—Very slightly flabby.No. 41 (5. v. 30)—Very slightly flabby.

60 hours after the beginning of coitus (30 hours after ovulation):No. 39 (22. iv. 30)—Still fairly turgid.No. 32 (22. iv. 30)—Becoming flabby.No. 32 (20. vi. 30)—Slightly flaccid.

66 hours after the beginning of coitus (36 hours after ovulation):

No. 40 (27. vi. 30)—Becoming flaccid.No. 40 (29. iv. 30)—Becoming flaccid.No. 42 (29. iv. 30)—Moderately turgid.

72 hours after the beginning of coitus (42 hours after ovulation):

No. 36 (6. v. 30)—Still fairly turgid but becoming smaller.No. 38 (17. v. 30)—Slightly flabby: going down.No. 34 (17. v. 30)—Flabby: going down.No. 39 (27. vi. 30)—Becoming flaccid.

The data are scanty but tentatively it can be said that no change in the turgidityof the vulva occurs up to 36 hours after coitus, but that the lips begin to becomeflaccid by about the 6o-66th hour, although there is some variation in individuals inthe rate at which it occurs. From this we conclude that by about 30-36 hours afterovulation the action of oestrous hormone on the vulval swelling ceases.

Ovulation and Fertilisation in the Ferret 31 1

The ascent of the spermatozoa.

Since the ovary of the ferret is contained within a closed capsule, in which it isprobable that the ova are fertilised, it is possible to obtain an estimate of the numbersof sperm which reach this position at different intervals of time after coitus. A fewincidental observations were made on this point when animals were killed for otherpurposes. The method used for estimating the numbers in the capsule was to dissectout the reproductive tract and to open the capsule carefully on a glass slide, collect-ing the whole of the fluid contained within it. If the amount of fluid was small thesperms in it were counted directly, but if the amount was large, it was measured in acapillary tube and the number of sperm in an aliquot portion of it counted by meansof a haemocytometer. A drop of fluid from the upper ends of the uterine horns wasalso examined in the same way; in this case no actual count was made, but theapproximate number in a drop of the fluid was noted. The results obtained areshown in Table II.

Table II. Spermatozoa in female tract after coitus.

Hoursafter

beginningof coitus

3

6

24

24

24

3°

36

10 days

10 days

No.of

female

36

3 i

38

21

33

34

37

40

39

No.of

male

S

O

s

JJ

s

0

s0

Dat

4 vn

3- v-

29. vii

28. vni

I I . Vlll

19. vn

15. vii

27. vi.27. vi.

c

3 0

30

3 0

• 29

3 0

3 0

30

3030

Durationof coitushrs rrun

1

2

1

1

1

1

1

00

55

2 0

5O

45

3°

55

45

4530

Spermatozoa in

Ovarian capsules

0

75 on one side0 on other side

10 on one side8 on other side

16000

17 on one side10 on other side

2—dead. not muchfluid in capsule

00

Drop of fluid fromupper end ofuterine horn

200—motile

Not examined

500-50 % quitemotile

Not examined

0—not much fluidin uterus

200—many motile

500

00

Drop of fluidfrom upper

vagina

Not examined

Not examined

100-50 % quitemotile

Not examined

0

Not examined

Not examined

Not examined

Not examined

While the number of cases examined is small and the results very variable, itwould seem that the spermatozoa do not reach the capsule in 3 hours after the begin-ning of coitus, although plenty were found in the upper part of the uterus at thistime (No. 36). They reach the capsule, however, within 6 hours of coitus. In all theanimals except one (No. 33) in which no sperm were found in the tract at death,although they were obtained from the vulva after mating, spermatozoa were presentin the capsule from 6 to 36 hours after mating, and many sperms were present in theupper part of the uterus during this time. By 10 days after mating however nosperms at all could be found in any part of the tract.

When making matings, the fact that normal coitus had occurred was always21-3

312 J. HAMMOND and A. WALTON

checked by examining a drop from the vulva of the female immediately after themale had withdrawn. It was found that the number of sperms contained in such asample varied considerably in different males at any one time and in one male atdifferent times. Variable fertility in the male may have had an effect on the numbersof sperm found in the female tract after coitus. One other factor which may haveaffected these results is the duration of coitus (see p. 313 below), but no definitecorrelation is shown in the data available.

Attempts at artificial insemination.

An experiment was planned to find out how long the spermatozoa would remaincapable of fertilisation in the female tract, by artificially inseminating females atdifferent intervals of time before coitus with a sterile (vasectomised) male to induceovulation. A similar experiment has already been done with the rabbit (Hammondand Asdell, 1926).

Both epididymes of a male were minced in Tyrode solution, and, after ascer-taining that large numbers of motile spermatozoa were present, five females wereinseminated. One female of each set was left as a control to determine whether theact of artificial insemination would cause ovulation or not: in four such femalesinseminated during April and May ovulation was not caused by this means, for thevulva remained turgid and no pregnant or pseudo-pregnant changes took place. Theresults of this set of experiments were as follows:

Exp. I. Both epididymes of <$R minced in 2-5 c.c. Tyrode and 0-4 c.c. injectedinto each female. 37-5 millions of sperm in epididymes; 15,000 sperm in 1 c.mm.of fluid injected. Six millions of sperm injected into each female. Control femaleinseminated only: did not become either pregnant or pseudo-pregnant. Threefemales inseminated and mated with vasectomised male at 24 hours, 72 hours and5 days respectively; all became pseudo-pregnant but not pregnant.

Exp. II. Both epididymes of <$Q minced in 1-5 c.c. Tyrode and 0-2 c.c. injectedinto each female. 96 millions of sperm in epididymes; 64,000 sperm in 1 c.mm. offluid injected. 12-8 millions of sperm injected into each female. Control female in-seminated only; did not become either pregnant or pseudo-pregnant. Four femalesinseminated and mated with vasectomised male at 24 hours, 4 days, 5 days and 7 daysafter insemination respectively; all became pseudo-pregnant but not pregnant.

Exp. III. Both epididymes of c?O minced in 0-5 c.c. Tyrode and o-i c.c. injectedinto each female. 36 millions of sperm in epididymes; 72,000 sperm in 1 c.mm. offluid injected. 7-2 millions of sperm injected into each female. Control female in-seminated only; did not become either pregnant or pseudo-pregnant. Four femalesinseminated and mated with vasectomised male at 48 hours, 72 hours, 4 days and6 days after insemination respectively: all became pseudo-pregnant but not pregnant.

Exp. IV. Both epididymes of $V minced in 0-25 c.c. Tyrode and 0-04 c.c.injected into each female. 100 millions of sperm in epididymes; 400,000 sperm in1 c.mm. of fluid injected. 16 millions of sperm injected into each female. Controlfemale inseminated only; did not become either pregnant or pseudo-pregnant. Four

Ovulation and Fertilisation in the Ferret 313

females inseminated and mated with vasectomised male at 48 hours, 72 hours, 4 daysand 6 days after insemination respectively; all became pseudo-pregnant but notpregnant.

Exp. V. Both epididymes of <JC2 minced in a few drops of Tyrode. Fluidcontained a lot of motile sperm. Four females inseminated; two mated with vasecto-mised male immediately after insemination and two mated with vasectomised maleat 24 hours and 48 hours respectively after insemination; all became pseudo-pregnant but not pregnant.

Four sets of females were inseminated and were mated with a vasectomised maleat different intervals afterwards—in all three at 1 day (24 hours), three at 2 days,three at 3 days, three at 4 days, two at 5 days, two at 6 days, and one at 7 days—but no young at all were produced, although all these animals became pseudo-pregnant, as shown by the atrophy of the vulva and the growth of the nipples. If weallow 30 hours after coitus for ovulation to occur, these results might suggest thatthe spermatozoa are not capable of fertilising ova after 54 hours (30 and 24 hours) inthe female tract. We were, however, by no means certain that spermatozoa gainedentrance to the uterus, for, contrary to experience with the rabbit, we found greatdifficulty in introducing fluid in such a way as to be retained in the vagina. In thefirst set 0-4 c.c. of fluid was injected into the vagina, but much of it was extruded; inthe second set the volume of fluid was reduced to 0-2 c.c, using a more concentratedsperm suspension; in the third set o-1 c.c. of fluid was used; and in the fourth set itwas reduced to only 004 c.c. But even with this small quantity of fluid (in whichthere was approximately 400,000 sperms per c.mm.) some of the fluid was stillextruded from the vagina after insemination. One test (Exp. V) was made to seewhether this lack of fertility was actually due to the extrusion of the injected fluid orto the short life of the sperm in the female tract. In this experiment two controlfemales were mated with a vasectomised male immediately after they were in-seminated ; neither of these became pregnant, although they ovulated and becamepseudo-pregnant. We therefore concluded that the lack of fertility was due to theextrusion of the inseminated fluid. It may be that the function of the prolongedcopulation which occurs in the ferret is that of ensuring that the semen remains inthe vagina for a sufficient length of time for the spermatozoa to penetrate the cervix,the penis of the ferret acting as does the vaginal plug in rats and mice in preventingthe extrusion of the semen.

The duration of coitus.

A number of observations were made on the time occupied in coitus by a numberof different males, and the results are shown in Table III. The average duration isabout 2 hours, while the range of variation is from \ to 3 hours. As might beexpected vasectomy in no way affects the duration of coitus. The shortest coitusfrom which young were produced was one hour, but there is not sufficient evidenceto say that short copulations are less effective.

Ovulation and Fertilisation in the Ferret 315

The time that the ova remain capable of fertilisation.

The next problem was to determine how long the ova remained capable offertilisation after ovulation. It has previously been found in the rabbit (Hammond,1928, 1934) that the ova can only be fertilised for a period of about 6 hours afterovulation. It is important to know whether this is exceptional or whether it is trueof all species. Similar methods to those used with the rabbit were tried. Females onheat were mated first with a sterile (vasectomised) male, and then at different in-tervals of time afterwards mated with a fertile male; that is, the sperm were put inlater than normal in relation to the time of ovulation. The first (sterile) matingwould induce ovulation at a definite time, and then the second mating could be madeat any time desired in relation to this time of ovulation.

The results of these experiments are given in Table IV; it will be seen that as thesperms are put in later than normal (which is 30 hours before ovulation—-see abovep. 308)—from 12 hours before to 42 hours after ovulation—so the percentage ofmatings producing young dropped from 100 per cent, to o. The average litter size inthose matings which proved fertile also decreased from 67 at 6-12 hours before ovu-lation, to 4-2 at 12-18 hours after ovulation, and to i-o at 24-30 hours after ovulation,after which time no young at all were produced. Thus the ova only remain capable offertilisation for a period of about 30 hours after ovulation, while from matings duringa period of 18-30 hours after ovulation litters of small size (1-3 young) only areproduced. This is just what occurs in the rabbit (see Hammond, 1928, 1934),although in that species the ova remain capable of fertilisation for a much shorterlength of time (6 hours). As a possible explanation of this difference in time betweenthe two species, the fact that the ova pass down the tubes into the uterus moreslowly in the ferret than in the rabbit may be relevant. In the latter species the ovatake 4 days to reach the uterus, while according to Robinson's (1918) data on theferret (see Table I) the uterus is not reached until the sixth day. Pincus (1930) hasfound in the rabbit that the ova remain held up for several hours after ovulation in amass of debris consisting of cumulus cells and liquor folliculi within the upper endof the Fallopian tube, and that it is here that they are fertilised. As soon as theybegin to pass down the tube they are no longer capable of fertilisation, and hencewhen the sperms are put in late the first sperms do not reach the tops of the tubesuntil all or most of the ova have started on their way down, and are therefore in-capable of fertilisation, with the result that small litters or none at all are produced.We suggest therefore that in the ferret the passage of ova down the Fallopian tubesis considerably delayed, and hence that there is an increased period during whichthey remain fertilisable in the ovarian capsule.

That failure to produce young (when the fertile copulation was made at intervalsof 60 hours or more after the first sterile coitus) was due to unfertilisability of the ovarather than to disturbance of gestation is shown by the fact that all the nine animalsmated at 66 and 72 hours after the first sterile coitus underwent the typical pseudo-pregnant changes, as shown by the atrophy of the vulval swelling, the enlargementof the nipples and the falling out of the hair. To make quite certain that this was so,

316 J. HAMMOND and A. WALTON

Table IV. Fertility of matings made at time intervals before and after ovulation.

Coitus with fertile maleAfter ist (sterile) coitus—

hours

Before ( - ) or after ( + )ovulation—hours

Number of female •2 1

252728252 02 33 02 42 1

2622

3537274 1

2439322 0

2 2

3 -4 0

4 2

3°2340163«3439

Number of matings% FertileAverage litter sizeNumber of young per mating

18

— 12

24

- 6

94

.

—

11

3

41 0 0

6 7

6-7

3 0

0 + 6

42

+ 12

48

+ 18

54

+ 24

60

+ 3°

Number of young in litter

50

.

-

80

•

4

5°6-5

3' 2

94

31

0

5

80

4 2

3 4

0

0

1

0

0

1

0

1

1

9

441 0

0 4

66

+ 36 + i

.

0

0

0

0

0

0

0

0

0

90

0

0

Table V. Post-mortem results on ferrets which had been mated after

ovulation had occurred.

Female No.

324039

Time of fertile mating—hours

After sterilemating

606672

Afterovulation

3°3642

Time aftercoitus that

killed—weeks

4i

No. of corporalutea in

8 + 54 + 44 + 4

No. of foetusesin uterus

1+00 + 00 + 0

Ovulation and Fertilisation in the Ferret 3:7we killed three such animals mated at the 6oth, 66th and 72nd hour after the firststerile coitus (or at 30, 36 and 42 hours after ovulation), with the results shown inTable V.

These results show that the failure to conceive at 36 and 42 hours was not due tothe failure to ovulate or of the corpora lutea to develop. It also shows that, when themating was made at a time (30 hours after ovulation) during which small litters wereproduced, the cause of these was failure of fertilization, for although there werethirteen large corpora lutea in the ovaries only one embryo was present in the uterusand there were no traces of any other attachments having been made on the uterinemucosa, such as occurs in foetal atrophy. These results are similar to those alreadyobtained in the rabbit.

It is thus possible not only to define the length of time the ova remain capable offertilisation (30 hours after ovulation), but also to state the time (18 30 hours afterovulation) during which a mating may produce small litters from females whichunder normal conditions would have produced large litters, i.e. in which only aproportion of the ova shed will become fertilised.

The time during which the ova remain capable of fertilisation after ovulation iscomparatively short. In the ferret it is at most 30 hours, while during a period offrom 18 hours to 30 hours after ovulation only a small proportion of the ova shed arecapable of fertilisation. In the rabbit the time is shorter still—at the most some 6hours only (see Hammond, 1928, 1934). In both these species ovulation occursnormally only as a result of coitus, but there is also strong presumptive evidence thatthe fertilisability of the ovum is also very short in species which ovulate spontane-ously. In the mare we have found that a mating on the last day of heat (which lastson the average about 7 days) gives a lower percentage fertility than one made 3 daysbefore the end of the heat (ovulation occurring about 1 day before the end of heat).In the sheep, Quinlan, Mare and Roux (1932) found that lowered fertility followsservices after the 30th hour following the onset of oestrus, whereas fertilitywas high in sheep served at the onset of oestrus. Lewis (1911) mated thirty-four sows after the true period of heat was over (and consequently late in relation toovulation, which occurs during the latter part of heat); seven mated on the first dayafter heat produced only two litters, while in the remaining thirty-two cases nolitters were obtained. He concluded that the ovum does not live for more than a fewhours after being liberated from the follicle.

In the monkey ovulation occurs about the 14th day of the cycle, and the fer-tility of matings made after this time falls rapidly, as the following figures taken fromHartman's (1932) results show:

Day of cycle atwhich mated

Number of matinpsmade

Number ofmatings fertile

% fertile

3-8

12

0

0

9-10

42

2

5

n-14

269

42

16

15-18

80

10

,2

19-23

17

O

24-28

4

0

0

318 J. HAMMOND and A. WALTON

Asdell (1927), who collected the statistics of various workers concerning humanfertility, found that the percentage conception rate, after being at its highest justbefore the time of ovulation (14th day) fell off rapidly after the time of ovulation tothe end of the cycle.

Thus it would seem that, although the exact time during which the ova remaincapable of fertilisation is slightly variable in different species, it is quite short inall the species for which there are any adequate data.

The reason for this short life of the ovum after ovulation needs further investiga-tion. In the rabbit, as soon as the ovum begins to pass down the tube, a thick layerof albumin is put on, but this albumin layer is absent or slight in other species. Inthe rabbit fertilisation occurs while the ova are in, or as they pass out of, the plug atthe top of the tubes, and if it does not occur here it cannot take place at all. We wouldsuggest that the short time during which the ova are capable of fertilisation is due tosome environmental condition of this sort rather than to the viability of the ovumitself, since Pincus (1930) found that unfertilised rabbit ova taken from the tubes upto 63 hours after ovulation could be made to undergo parthenogenetic divisions intissue culture medium, showing that they were reactive at this time although nolonger fertilisable.

SUMMARY.

1. The ferret ovulates normally about 30 hours after coitus.2. The average duration of coitus is about 2 hours but varies from \ hour to

3 hours. There was no evidence that short copulations were less fertile than longones.

3. For 36 hours after coitus the vulva remains turgid, but becomes flaccid byabout 60 hours after coitus, presumably from absence of oestrin.

4. Spermatozoa reach the upper part of the uterus within about 3 hours aftercoitus, but during this time do not reach the ovarian capsule. Spermatozoa werefound in the ovarian capsule 6 hours after coitus. No spermatozoa were found in theovarian capsule or uterus 10 days after coitus.

5. An attempt to determine the possible duration of fertilising capacity of thespermatozoa in the female tract was made, but no conclusive results were obtainedowing to the difficulty of performing artificial insemination in the ferret.

6. In the ferret the ovum remains capable of fertilisation for not more than 30hours after ovulation. From 18 to 30 hours after ovulation only a small proportionof the ova remain capable of fertilisation, and small litters are produced from matingsmade at this time.

REFERENCES.ASDELL, S. A (1927). Journ. Amer Med Ass 89, 509BISSONNETTE, T. H. (1932). Proc. Roy. Soc. B, 110, 322.FEE, A. R. and PARKES, A. S. (1929). Journ. Phys. 67, 383.HAMMOND, J. (1928). Zilchtungskunde, 3, 523.

(1934). Journ. Exp. Biol. 11, 140.HAMMOND, J. and ASDELL, S. A. (1926). Brit. Journ. Exp Biol 4, 155.HAMMOND, J. and MARSHALL, F. H. A. (1930). Proc. Roy. Snc. B, 105, 607.

Ovulation and Fertilisation in the Ferret 319HARTMAN, C. G. (1932). Contributions to Embryology, No. 134.HILL, M. and PARKES, A. S. (1931). Proc. Roy. Soc. B, 107, 39.

(i933). Proc Roy. Soc. B, 113, 537 and 541.LEWIS, L. L. (191 I ) . Oklahoma Agric. Exp. Sta. Bull. No. 96.MARSHALL, F. H. A. (1904). Quart. Journ. Micros. Sci. 48, 323.

(i933) Quart. Journ. Exp. Phys. 23, 131.MURR, E. (1932,0). Zeit f. vergleich Phys. C, 17, 591.

(1932,6). Der Zool Gartens, Leipzig, 8, 37.(1932). Landvj Pelztierz. 3, 113.

PARKES, A. S., ROWLANDS, I. W. and BRAMBELL, F W. R. (1932). Proc. Roy Soc B, 109, 425.PINCUS, G. (1930). Proc. Roy. Soc. B, 107, 132.QUINLAN, J., MARE, G S. and Roux, L. L. (1932). 18th Rep Div. Vet. Services and Anim. Ind.,

Umon of Sqftth Africa, Pt. II, 813.ROBINSON, A. (1918). Trans. Roy. Soc. Edinburgh, 52, 303.