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Summary.

Capacitation in the golden hamster was studied by means of surgical insemination of two populations of spermatozoa into the independent uterine horns of individual animals, followed by examination of eggs 3, 4, 5 or 6 hr later. When epididymal spermatozoa were compared with ejaculated cells that had been incubated for 4 hr in an oestrous uterus, the temporal advantage of such incubation in terms of the subsequent penetration of eggs was less than 1 hr. Because some eggs were activated by epididymal spermatozoa as rapidly as any that had been exposed to the incubated sample in the contralateral tube, and because at least 3 hr was required following a period of uterine incubation before any eggs were activated, capacitation in the hamster would appear to take place principally in the Fallopian tube. The fact that incubation for ½ hr conferred some advantage, though less than that following 4 hr of incubation, indicates that not only is a fairly rapid change initiated in the uterus, but that this is of a progressive nature over the interval examined. The temporal advantage derived from uterine incubation of ejaculated spermatozoa did not depend on the passage of constituents of the follicular or tubal fluids into the uterus, nor on the presence of seminal plasma. Incubations of epididymal spermatozoa to establish this point were not very successful, for such cells exhibited extremely poor motility after even a brief exposure to the uterus, thereby emphasizing the importance of the male secretions as a substrate and/or protective agent.

Summary.

Microdroplets of progesterone were injected unilaterally beneath the serosa of the tubal isthmus and uterotubal junction in thirty pigs, a total of 1 mg in 0·1 ml of oil being deposited 8 to 12 hr before ovulation. A similar volume of the vehicle was injected as a control into the contralateral tubal structures. All animals were inseminated intracervically approximately 2 hr before these surgical procedures and slaughtered between 6 hr 20 min and 14 hr 30 min after completion of ovulation.

Polyspermic fertilization was found in 32·3% of the eggs recovered from the progesterone-treated tubes compared with 9·7% of those from the control tubes (P<0·01). The number of tubes yielding polyspermic eggs was also significantly higher on the progesterone-treated side (P<0·01). Injection of progesterone 8 hr before ovulation resulted in 38·2% polyspermic eggs from the treated tubes, dispermy being the principal anomaly of fertilization. In both dispermic and trispermic eggs, the female and a single male pronucleus were uniting, the accessory male element(s) remaining in the opposite hemisphere of the egg.

It is concluded that relaxation of the oedematous tissues of the isthmus and uterotubal junction occurs after local injection of progesterone. In these circumstances, polyspermy is thought to originate principally from increased numbers of spermatozoa reaching the site of fertilization, although a direct effect of progesterone on the membranes of the gametes cannot be excluded. The disposition of the accessory male pronuclei in the polyspermic eggs raises the question whether polyploids usually arise from polyspermy or whether delayed cleavage and cytoplasmic fragmentation are more frequent sequelae to this condition in pigs.

Summary.

The timing of sperm penetration, pronuclear formation and syngamy was examined in twenty-four gilts in which ovulation had been controlled precisely by means of an injection of hcg given during prooestrus. Animals were mated at the time of ovulation and eggs recovered at autopsy exactly 3, 4, 5 or 6 hr later.

Twenty of the twenty-four animals (83 %) yielded some penetrated eggs, a total of 234 eggs being examined. The mean number of spermatozoa attached to or within the zona pellucida increased progressively from 6·8 (range 1 to 25)/egg at 3 hr to 109·2 (range 1 to 466) some 6 hr after mating. Likewise, the proportion of eggs classified as fertilized increased from 23·7% at 3 hr to 71·9% by 5 hr after mating. Developing pronuclei were observed at 4 hr and apposing pronuclei within 5 hr of mating, but most of the penetrated eggs examined at these intervals were in anaphase or telophase of the second meiotic division.

The results emphasize that under conditions of induced ovulation, a population of capacitated spermatozoa first becomes available in the Fallopian tubes of oestrous pigs within 2 to 3 hr of semen deposition by way of the cervix. It is suggested that the sequence of events leading to sperm penetration may be accelerated following injection of hcg during pro-oestrus and mating at the time of ovulation.

Summary. Using the surgical approach of post-coital ligation and transection of the distal oviduct at different times relative to ovulation, together with subsequent recovery of the eggs, gilts mated at the onset of oestrus were studied for progression of viable spermatozoa within the isthmus. Results are derived from 76 animals and examination of 1047 eggs.

Transection of the isthmus 1·5–2·0 cm proximal to the utero-tubal junction at intervals from 3 to 36 h after mating prevented fertilization in 269 of 270 eggs, whereas 98% of 223 eggs were fertilized in the control oviducts. Transection at 38 h (pre-ovulatory), 40 h (peri-ovulatory) and 42–44 h (post-ovulatory) after mating yielded, respectively, 5%, 40% and 100% fertilization. The mean number of spermatozoa associated with the zona pellucida increased in a parallel manner. These results, and those obtained with ligatures placed closer to the site of fertilization just after ovulation, indicate a pre-ovulatory arrest of viable spermatozoa in the caudal region of the isthmus for 36 h or more followed by an active ad-ovarian redistribution.

The effect of post-ovulatory ageing of mammalian eggs before mating or insemination has been extensively studied, and the variable though relatively short fertilizable life of the egg has been documented for a considerable number of species (for review see Blandau, 1961). It is not clear, however, to what extent the fertilizable life is an intrinsic property of the egg or is under the influence of the conditions prevailing in the Fallopian tubes. Nor is it clear whether the abnormalities associated with ageing eggs, such as polyspermy, spontaneous activation, fragmentation and loss of fertilizability, are to some degree determined by the nature of the tubal secretions.

In the short interval during which post-ovulatory ageing occurs, the secretions of the Fallopian tube are subjected to the influence of ovarian progesterone rather than to that of oestrogen which predominated at the normal time of mating and ovulation. Two recent experiments in the

Summary. The rate of establishment of a population of viable spermatozoa in the oviducts was studied using a technique of post-coital transection in conjunction with subsequent examination of the proportion of eggs fertilized. Gilts were mated early in oestrus (before ovulation) or on the 2nd day of oestrus (after ovulation), and 30, 45 or 60 min later the reproductive tract was sectioned just above or below the utero-tubal junction in a total of 48 animals; these were slaughtered 1 or 2 days after the operation.

Some fertilized eggs were recovered from 40 animals, and 72·3% of the 679 eggs examined were fertilized. Mean percentage fertilization increased overall (a) with the time elapsing from mating to transection, (b) with transection below the utero-tubal junction compared with in the caudal isthmus, and (c) with a post-ovulatory versus pre-ovulatory mating. In a further 6 gilts, the results of transection in the lower third of the oviduct 3 h after mating at the onset of oestrus indicated that spermatozoa were initially sequestered in the caudal portion of the isthmus.

It is concluded that a population of spermatozoa sufficient to give maximum fertilization is established in the oviducts within 1–2 h of mating, thereby affording protection from the uterine invasion of polymorphonuclear leucocytes.

Summary.

Eggs were examined from a control group of gilts (Group 1) which were inseminated at least 6 hr before ovulation, and from five other groups (Groups 2 to 6) in which the postovulatory age of the eggs was estimated to be 4, 8, 12, 16 or 20 hr at the time of sperm penetration. Penetrated eggs were obtained from twelve animals in each group.

The conception rate fell from 100% in the control group to 66·7% in Group 6, whereas the incidence of unilateral fertilization increased from 8·3% in the controls to 33% in Group 6. Within the seventy-two animals with some penetrated eggs, the mean percentage of unfertilized eggs increased from 1·3% (Group 2) to 23·9% (Group 6), but unfertilized eggs occurred principally as a result of unilateral fertilization.

No decrease in the mean proportion of fertilized eggs developing normally was found until the eggs were 12 hr old at penetration (Group 4), when the figure fell to 73·6%. A further decrease occurred in Groups 5 and 6, means of 55·4% and 66·9% respectively, being obtained. The mean percentage of polyspermic eggs increased from 1·3% in Group 2 to 15·4% in Group 5, and the proportion of eggs showing varying degrees of fragmentation also increased from 3·8% in the control group to 29·2% in Group 5.

The results indicate that a very high proportion of eggs can be fertilized up to 8 hr after ovulation and undergo apparently normal development at least until the 4- to 8-cell stage.

Summary.

Ten sows, three entire and seven which had been ovariectomized at different stages of late gestation, were observed for post-partum oestrus. Serial blood samples were collected from six sows during the pre- and post-partum periods, and plasma oestrogen concentrations determined by radioimmunoassay. Morphological aspects of ovarian activity in the entire sows were examined at laparotomy and at autopsy.

Although peak values of oestrogen concentration in the plasma varied from 3·9 to 8·0 ng/ml between individuals, the pattern of oestrogen levels was similar for control and ovariectomized sows. Peak concentrations occurred just before parturition, and the timing of ovariectomy did not affect the incidence of the oestrogen peak. Oestrus was detected in one control and one ovariectomized sow at 46 and 40 hr post partum respectively, there being no evidence of ovarian activity in the entire sows.

The occurrence of post-partum oestrus in a sow ovariectomized at Day 108 of gestation indicates that this phenomenon is not directly connected with ovarian secretion of oestrogens. The post-partum oestrus is apparently a result of the peak of feto-placental oestrogens that occurs at parturition.

Summary. Fine thermistor probes positioned in each end of the same oviduct and connected to the same scale were used to measure temperature gradients in the lumen before and after spontaneous ovulation in normally-cyclic gilts. Readings were taken after full surgical closure of a mid-ventral incision and a subsequent period of stabilization, but whilst animals remained under general anaesthesia.

A small but consistent difference in temperature was recorded between the proximal ampulla and distal isthmus of the same oviduct in each of 20 preovulatory gilts. In 10 of these animals that had not mated, the isthmus was a mean of 0·43°C cooler than the ampulla (range 0·2–0·7°C whereas in 10 mated animals the isthmus was 0·69°C cooler (range 0·2–1·6°C 3 animals in the latter group had within-oviduct differences of ≥ 1°C. By contrast, in 12 animals that had recently ovulated, the isthmus was a mean of only 0·1°C cooler than the ampulla; there was no measurable temperature gradient in 3 of the animals, whilst the isthmus was 0·1°C warmer in 2 animals. The preovulatory temperature differences are thought primarily to reflect the extent and activity of the vascular and lymphatic beds in the oviduct tissues and, together with specific chemical microenvironments, may facilitate the relatively prolonged period of sperm storage in the distal portion of the isthmus.

Summary.

Exposure of rabbit semen to X-irradiation did not reduce the fertilization rate until doses of >25,000 r were used, although motility was noticeably affected at 15,000 r. Evidence of a depressant effect of much lower doses of X-irradiation on the fertilizing ability of rabbit spermatozoa was obtained, however, by using a more sensitive system in which irradiated aliquots from an ejaculate were inseminated mixed together with an exactly equivalent volume of untreated semen from the same ejaculate. Under these competitive conditions the fertilizing ability of spermatozoa was found not to be affected by a dose of 10,000 r, but was significantly depressed (P<0·01) by 15,000 r, as judged by an increase in the proportion of normally developing to retarded ova, recovered 50 hr after an ovulation injection of hcg. It appears that this depressant effect is exerted at the site of fertilization rather than on sperm transport.

X-irradiation of spermatozoa did not increase polyspermic or delayed fertilization, neither did irradiation have any significant effect on the formation of the male pronucleus. A dose response effect was seen at the time of the first cleavage division, for at 24 hr the cleavage rate of control ova (68·5%) was similar to that of ova fertilized by spermatozoa treated with 10,000 r (68%), but only 33% of ova fertilized by spermatozoa treated with 20,000 to 25,000 r had cleaved at this time compared with 78% of their controls. At 29 and 50 hr several ova fertilized by irradiated spermatozoa remained at the pronuclear stage or at metaphase of the first cleavage division, and even at 50 hr the great majority had only reached the 2-cell stage. Thus, although it is known that cleavage can occur in the absence of the male elements, the first cleavage division does not appear to follow activation of the egg automatically and independently since it has been shown that the state of the male elements introduced at fertilization can influence the rate and ultimate success of the changes which take place at this time.