SUMMARY

A study was made of the effect of ligating the Fallopian tube or uterine horn near the tubouterine junction at specific time intervals after mating (½–6 hr) on fertilization in superovulated rabbits. A total of 140 does were used and altogether 2630 eggs were studied. Observations were made on the does' response to gonadotrophin treatment; the rate of egg development; the occurrence of abnormal eggs; the number of spermatozoa in fertilized eggs; the transfer of fertilized eggs to recipient does and the length of the female genital tract. Rabbit semen was collected by means of an improved type of artificial vagina.

The main results following ligation were as follows: when one Fallopian tube was ligated at the time intervals stated in parentheses, the mean proportion of eggs fertilized was 1·6% (1¼ hr), 8·8% (1¾ hr), 12·9% (2 hr), 16·1% (2¼ hr), 29·8% (2½ hr), 59·5% (2¾ hr), 85·9% (3 hr), 38·4% (3¼ hr), 73·7% (3½ hr), 50% (4 hr), 96·4% (5 hr) and 100% (6 hr). Out of a total of 1006 eggs from non-ligated (control) Fallopian tubes, 95·5% were fertilized. Following ligation of one uterine horn 30 min, 1 hr or 2 hr after mating, 9·1, 38·7 and 55·2%, respectively, of the eggs were fertilized.

It is concluded that although some spermatozoa may reach the tubo-uterine junction soon after mating, 2–5 hr are required before the number of spermatozoa entering the Fallopian tube is sufficiently high to produce maximum fertilization.

SUMMARY

Ovarian response (number of ovulations) to a single dose of 25 i.u. human chorionic gonadotrophin (HCG) given intravenously on the 5, 9, 12, 17, 21, 25 or 28th day of pregnancy or within 24 hr. post partum was examined in 67 does. HCG given post partum or early in pregnancy resulted in a normal number of ovulations but from mid-pregnancy to near term superovulation occurred in 50% or more of the does. Only one doe failed to ovulate. In similar, untreated does the mean number of Graafian follicles increased from 14 (6–23) on days 8–11 to 30 (9–52) on the 28th day of pregnancy. Egg transport through the tubes was significantly accelerated following ovulation induced during pregnancy.

In order to eliminate early luteal function it has been the practice either to remove surgically the whole or part of the ovaries (Fraenkel, 1903, 1910; Corner, 1928) or, in cases requiring the selective destruction of corpora lutea, to use thermocautery (Fraenkel, 1903, 1910; Bouin & Ancel, 1910; Joublot, 1927; King, Collins & Paterson, 1932; Brouha, 1934). Simple enucleation techniques have received little or no attention, except in the cow in which this procedure is carried out manually per rectum about mid-cycle (Hammond, 1927). The observations described below were made in order to assess the effectiveness of enucleation when performed shortly after ovulation.

Six adult does, mean weight 3·5 kg., were given intravenously 25 i.u. luteinizing hormone (LH) (Lutormone, Burroughs Wellcome and Co.) to induce ovulation, which occurs between 9½ and 14 hr. later (Harper, 1963). From 14½–18 hr. after the injection, laparotomy was performed under pentobarbitone and ether anaesthesia.

SUMMARY

The relationship between the number of fertilized eggs and prenatal mortality was studied in 160 rabbits by means of egg transfer. More than 3000 fertilized eggs were obtained 60 hr. post coitum from 118 donors, in which superovulation was induced with gonadotrophins. These eggs were transferred to the uteri of recipients which, 72 hr. previously, had been injected with luteinizing hormone. In seventy-one recipients, either 5, 10, 15, 20, 25 or 30+ eggs were transferred unilaterally; in seventy-seven recipients 5 eggs were transferred to one horn and either 5, 10, 15, 20, 25 or 30 + eggs to the other, and in twelve recipients, 10 eggs were transferred to each horn. A further seventeen does were mated with fertile males and used as controls. On day 10, laparotomy was performed in order to count and measure the implantation sites. The numbers of live and dead foetuses and condition of placentae were recorded at autopsy on day 28. Mortality was classified as follows: pre-implantation, days 2½–7; post-implantation, days 7–10 (early deaths); days 10–17 (middle deaths); days 17–24 (late deaths).

In recipients the mean number of ovulations was 10·4 (range 5–18). The proportion of recipients in which implantation occurred (pregnancy rate) was 86·9%, compared with 94·1% in controls. Pregnancy rate was not influenced either by number of eggs transferred, or whether the transfer was unilateral or bilateral.

With increasing numbers of eggs, the number of implantations also increased, reaching a maximum of twenty-one in one horn, but the proportion of eggs surviving showed a significant decrease. The mean yield of live foetuses showed little variation between groups, there being a marked rise in embryonic mortality as crowding increased. The maximum number of live foetuses observed in one horn was eleven.

A total of 124 recipients, containing 1495 implantations, maintained their pregnancy to day 28. Altogether 208 early deaths (13·9%) occurred, there being a slight tendency for the incidence of early deaths to increase with crowding. There were 367 middle deaths, equivalent to 28·5% of the embryos surviving into the middle period. Crowding had a striking effect on the middle death rate, which increased from 16·8% in horns with one to three living embryos to 60·7% in horns with sixteen to nineteen embryos. A further 141 deaths (15·3%) occurred from days 17–24. The incidence of late deaths remained almost constant in horns containing up to seven embryos surviving to day 17, but above this number it was influenced by crowding. Significantly different levels of mortality were observed between the two uterine horns in individual recipients, depending upon the number of eggs transferred.

It was concluded that the cause of mortality, associated with crowding and partial loss of litters, was related mainly to local environmental factors, e.g. inadequate uterine secretion (pre-implantation) and lack of placental development, rather than to systemic factors, which more probably accounted for complete loss of litters.

SUMMARY

The amount and distribution of prenatal mortality were studied in 126 first and second pregnancies in seventy-three rabbits, weighing 3·5–4 kg. The does were mated naturally, artificially inseminated to ensure against the failure of natural mating, and injected with 25 i.u. luteinizing hormone. The numbers of corpora lutea and implantation sites were counted at laparotomy 7–13 days post coitum (p.c.) and the number of live foetuses was recorded at autopsy 28 days p.c., or after parturition. The diameter of the uterine swellings was measured at laparotomy. In a further eight pregnancies only the litter size was recorded.

Ovulation occurred in every case, but in six (4·8%) of the 126 pregnancies all the ova (109, or 8·1%) were lost before implantation. In the remaining 120 pregnancies there was a total loss of 132 ova (or 154 if corrected for polyovuly), representing 9·7% of ovulations. In 64 out of the 120 pregnancies (53·3%) there was some loss of ova before implantation. The proportion of litters showing loss and the proportion of ova lost before implantation were significantly related to the number of ovulations.

After implantation, total resorption occurred in four pregnancies (3·3%) and in a further eighty-five pregnancies (70·8%) there was some loss of embryos, which altogether amounted to 18·3% of the ovulations. The proportion of litters suffering loss after implantation was significantly related to the number of implantations. It was concluded that 7% of the embryos died shortly after implantation, 66% died between days 8 and 17, and 27% were lost between days 17 and 23.

Data relating to the diameter of 1728 uterine swellings measured between days 7 and 20 p.c., counts of corpora lutea, the occurrence of corpora lutea atretica and polyovuly, the position of foetuses in utero, weights of placentae associated with resorbed embryos, foetal weights, birth weights, and the incidence of stillbirths are also presented.

SUMMARY

The effects of post-coital, unilateral ligation of the uterine tube and of bilateral ovariectomy on egg development have been studied in fifty rabbits. Ligation and ovariectomy were performed between 14 and 72 hr, and 11 and 30 hr, respectively, after mating. The animals were killed for examination of the eggs between 3 and 10 days after mating.

284 eggs from thirty-five operated rabbits and 214 fertilized eggs from twelve unoperated rabbits were measured with an eye-piece micrometer as fresh specimens. Until 72 hr after mating the zona pellucida and intra-zonal space were relatively constant at 20 and 125 μ, respectively, while the mucin increased in thickness from 11 μ at 24 hr to 103 μ at 72 hr. The diameter of the intra-zonal space increased to 168 μ at 84 hr and 273 μ at 96 hr, whereas the zona pellucida showed a corresponding decrease to 14 and 5 μ, respectively, but it was still present as a very thin membrane on 6-day blastocysts. In most eggs the mucin began to thin between 72 and 84 hr and apparently disappeared before 120 hr.

Ten does in which one uterine tube had been ligated were autopsied 10 days after mating. On the non-ligated, control side, 67·5% of the ovulations (fifty-two eggs) were represented as conceptuses, whereas thirty-six eggs (85·7% of the ovulations) recovered from the ligated tubes were degenerated. When twenty similarly treated does were examined between 3 and 5 days after mating, it was found that egg development continued normally for some 84 hr after mating and then collapse of the early blastocyst occurred. Following ovariectomy in fourteen does, egg development was arrested at approximately 84 hr after mating. In the majority of ovariectomized does, the rate of egg transport to the uterus was normal. Treatment of six ovariectomized does with progesterone prevented degeneration only when the eggs reached the uterus; in three of the six progesterone-treated does some of the eggs were retained in the uterine tubes and degenerated.

SUMMARY

Either one or two 60-h morulae were transferred to one uterine horn of Polish, Dutch or Strain A rabbits in which the mean number of ovulations was 4·3, 6·4 and 10·8. The pregnancy rate, as determined by palpation on day 10, varied from 45–65% (mean 53%) in recipients of one egg to 69–71% with two eggs. Only 20% of the does carrying one foetus maintained pregnancy to term compared with 87% of those with two implants. If pregnancy failed this usually occurred between days 15 and 20. In a further experiment, in which additional eggs were transferred to the contralateral horn, it was shown that all single conceptuses were inherently capable of surviving to term.

It is concluded that irrespective of breed and ovulation rate, two conceptuses are normally required to prevent regression of corpora lutea on about day 17. Treatment with follicle-stimulating hormone in mid-pregnancy or removal of the barren uterine horn on day 13 failed to maintain pregnancies involving only one conceptus.

SUMMARY

In an attempt to establish the measurement of uterine carbonic anhydrase activity as a quantitative test for progestationally active substances, the activity of carbonic anhydrase in the rabbit endometrium has been correlated with the conventional histological method of assaying progestational activity. The following steroids have been tested in this manner with special regard to the duration of their effect: (a) progesterone, deoxycorticosterone (DC), deoxycorticosterone acetate (DCA), 3-acetoxy-pregna-3,5-diene-20-one, 17α-hydroxy-progesterone, 17α-hydroxyprogesterone caproate (Delalutin); (b) 17α-ethinyl-19-nortestosterone, 17β-acetoxy-isoprogesterone, 3α-hydroxy-pregnane-12:20-dione, 12α-hydroxy-pregnane-3:20-dione, pregnane-3:12:20-trione, 2β-acetoxy-progesterone, 2 α-acetoxyprogesterone, pregna-5-ene-20-one.

The action of DCA was weak and transient; that of progesterone and 3-acetoxy-pregna-3,5-diene-20-one was marked but of limited duration; Delalutin was powerful as well as sustained; DC and 17α-hydroxyprogesterone failed to produce true progestational changes.

Ethinyl-19-nor-testosterone was the only steroid in group (b) endowed with progestational potency.

Stilboestrol (2 mg) administered a day before or simultaneously with DCA (16 mg), progesterone (4 mg) or Delalutin (2 mg), suppressed the endometrial response for 5 days but did not interfere with the late (8-day) progestational effect of the long-acting progestogen Delalutin.

Small doses of purified FSH induced a few ovulations in the rabbit when injected intravenously, the endometrium showed some proliferative changes by bioassay but the activity of carbonic anhydrase was not appreciably increased. Subcutaneous injections had no effect.

When progesterone was administered subcutaneously with FSH its effect was curtailed.

With few exceptions, there was satisfactory agreement between the enzymic and histological method of determining progestational activity.

SUMMARY

The behaviour of rabbit blastocysts and their uterine environment after ovariectomy was studied; the experimental period after ovariectomy varied from 17 to 26 hr. Most of the experiments were concerned with blastocysts recovered from rabbits spayed at 6 days; a few relate to embryos from animals spayed on day 5 or 7 of gestation.

Ovariectomy resulted in variable, often considerable, embryonic loss. Following bilateral ovariectomy at 6–6½ days, implantation on day 7 was completely prevented; those blastocysts which survived showed evidence of expansion and differentiation during the interval after ovariectomy. This provided an opportunity (1) to assess the stage of development, and mitotic activity, as well as certain biochemical characteristics, in embryos surviving ovariectomy, and (2) to use the free-lying 7-day-old blastocysts for culture experiments.

It was found that mitotic activity went on in blastocysts after ovariectomy. Upon transfer to suitable culture media the unimplanted 7-day-old blastocysts continued to grow and differentiate, the most advanced stage observed at the end of a 24 hr. period of incubation in vitro being the development of blood islands, primitive groove, and head process.

The fresh weight, and the content of bicarbonate and glucose of the 7-day-old unimplanted blastocysts differed little from those of normally implanted blastocysts of the same age; their content of lactate, however, was distinctly lower.

Carbonic anhydrase activity after spaying was fully maintained in the endometrium which showed little involutional change either on gross or on microscopical examination.

SUMMARY

In order to determine what part the level of oestrogen in the body might play in the passage of ova through the female reproductive tract, 1249 freshly ovulated ova from donor rabbits were transferred into the uterine tubes of seventy-four ovariectomized recipients, fifty-three of which had previously been treated for 5–18 days with small daily injections of oestradiol benzoate. From 10 to 78 hr after transfer, 41% of the ova were recovered from the uterine tubes, 9% from the uterine horns, and 23% from the vaginae of the recipient animals. Twenty-seven% of the ova were lost. The rate of ovum transport varied widely between similarly treated animals, and between the right and left sides of the reproductive tract of the same animal. Larger proportions of ova were retained in the uterine tubes, and smaller proportions in the uterine horns as the oestrogen dose was increased.

The great variability in the stage of cleavage and in the thickness of the mucin coat of ova recovered from the uterus and vagina suggested that the ova might be widely dispersed through the uterine tubes and that they probably pass out of the uterine tube at widely different periods of time. Evidence is presented that ovum transport in ovariectomized rabbits with or without oestrogen treatment is very irregular, that ova may be ejected from either end of the uterine tube at almost any time after transfer, and that ova are not normally retained in the uteri of such animals.

Approx. 1 μg oestradiol benzoate administered daily for 5–10 days was necessary to maintain the uterine weight of ovariectomized rabbits at about the same level as that in intact oestrous rabbits, and also to reduce the variability in egg transport observed in control and ovariectomized animals.