Search

Mares were inseminated with motile spermatozoa suspended in 30-150 microliters Tyrode's medium directly onto the uterotubal papilla at the anterior tip of the uterine horn, ipsilateral to the ovary containing a dominant preovulatory follicle of > or = 35 mm in diameter, by means of a fine gamete intrafallopian transfer (GIFT) catheter passed through the working channel of a strobed light videoendoscope. Insemination of 10, 8, 25, 14, 11 and 10 mares with, respectively, 10.0, 5.0, 1.0, 0.5, 0.1 or 0.001 x 10(6) motile spermatozoa resulted in conception rates of, respectively, 60, 75, 64, 29, 22 and 10%. Deposition of 1.0 x 10(6) motile spermatozoa onto the uterotubal papilla began to approach the limit of successful fertilization. These doses are far lower than the 3-15 x 10(9) spermatozoa normally ejaculated by fertile stallions during mating, and the accepted minimum dose of 500 x 10(6) spermatozoa used for conventional uterine body insemination in mares. The simplicity of the technique offers a practical means of exploiting new breeding technologies that require very small numbers of spermatozoa in horse breeding.

Aliquots of ejaculated boar semen containing known numbers of spermatozoa were deposited into the caudal isthmus or rostral ampulla of the Fallopian tubes of gilts at, or immediately after, ovulation to assess regional influences on the rate of capacitation. Eggs were recovered during a second intervention 4, 5, 6 or 7 h after surgical insemination and were examined by phase-contrast microscopy. Results were obtained from ten animals in each of the 4-, 5- and 6-h groups and from eight animals in the 7-h group. With two exceptions, fertilized eggs were not recovered until 6 h after insemination into the isthmus, the proportion (45.6%) being significantly greater than the corresponding figure (1.4%) for ampullary insemination (P < 0.001). Similarly, the proportion of fertilized eggs recovered 7 h after insemination into the isthmus (58.7%) was significantly greater than after ampullary insemination (21.9%; P < 0.01). Numbers of spermatozoa associated with the zona pellucida remained low in all these instances, with mean figures per egg ranging from 0.3 to 3.8. Insemination into the isthmus gave a 1-2 h advantage in fertilization compared with insemination into the ampulla. Although relative rates of sperm cell progression to the site of fertilization may have contributed to this, there is strong evidence that rates of capacitation differ significantly in the respective portions of the Fallopian tube. Therefore, attention was focused on: (1) the viscous glycoprotein secretion in the caudal isthmus acting to remove seminal plasma from the sperm surface; and (2) the phase of sperm head binding to the isthmus epithelium. Gradients in local endocrine modulation by the adjacent ovary offer one explanation for the functional specialization of different regions of the Fallopian tubes.

Hydrosalpinges were created to collect adequate volumes of fluid during pre-, peri- and postovulatory intervals from the ampulla, ampullary-isthmic junction and the isthmic-utero-tubal junction of the oviducts from Large White gilts that had exhibited at least two natural oestrous cycles. The accumulated fluids, follicular fluid and Butschwiler's medium were compared for their effects on various parameters of boar sperm motility using the CellSoft, computer-assisted, digital image analysis system. Sperm velocity (micron s-1 +/- SEM) was significantly higher (P < 0.05) in follicular fluid (84 +/- 3; n = 5) than in fluids from the ampulla during peri- and early postovulatory intervals, and from the isthmic-utero-tubal junction during pre- and early postovulatory intervals. It was also higher (P < 0.05) than in the fluid from the ampullary-isthmic junction during pre- and early postovulatory intervals; however, sperm velocity in follicular fluid was not significantly different from that in the periovulatory fluid from the ampullary-isthmic junction. The mean lateral head displacement (ALHmean) of spermatozoa was significantly greater in follicular fluid (3.9 +/- 0.3 microns; n = 5) than in fluid from the ampulla during peri- and early postovulatory intervals and from the isthmic-utero-tubal junction during pre- and early postovulatory intervals, and was also higher (P < 0.05) than in fluid from the ampullary-isthmic junction during the preovulatory period, but was not different from the peri- and postovulatory ampullary-isthmic junction fluids. The proportion of spermatozoa exhibiting circular motion was significantly higher (P < 0.05) in the periovulatory fluid from the ampullary-isthmic junction (24 +/- 3%) compared with fluids obtained during preovulatory and early postovulatory periods. Follicular fluid had no effect on the proportion of spermatozoa exhibiting circular motion. The average radius of sperm movement in circular trajectories was higher in follicular fluid than in the periovulatory fluids from the ampulla and ampullary-isthmic junction (P < 0.05). In hydrosalpingeal fluids collected 2-5 days after ovulation, the average radius of movement was greater in the ampulla fluid and ampullary-isthmic junction fluid than in fluid from the isthmic-utero-tubal junction (P < 0.05). These results demonstrate that follicular fluid and oviductal fluids have considerable influences on boar sperm motility. Furthermore, the immediate effect of periovulatory ampullary-isthmic junction fluid in increasing the percentage of spermatozoa swimming in circles (hyperactivated) is relevant, since it is at this time and within this region that fertilization occurs.

Large White gilts, 9 to 18 months old, that had exhibited at least two natural oestrous cycles were divided into three groups (phases): unmated pre-ovulatory, unmated post-ovulatory and mated post-ovulatory (n = 16, 20 and 18). Oviductal luminal fluid samples were collected under anaesthesia by micropipette from the ampulla and ampullary-isthmic junction and analysed by an ultramicrofluorometric technique. Glucose concentrations (mmol 1(-1), means combining regions; mean +/- SEM) were significantly higher in blood plasma than in oviductal fluid (4.56 +/- 0.20 versus 0.59 +/- 0.16; P < 0.0001; n = 27), whereas lactate was higher in the oviduct (5.71 +/- 0.53 versus 2.48 +/- 0.24; P < 0.0001; n = 27). No significant differences were found between the ampulla and the ampullary-isthmic junction. However, the concentration of glucose was significantly higher (P < 0.05) in the ampulla of the pre-ovulatory group (0.97 +/- 0.20; n = 13) compared with the mated group (0.25 +/- 0.05; n = 14) and its concentration in the ampullary-isthmic junction in the pre-ovulatory group (1.65 +/- 0.63; n = 13) was significantly greater (P < 0.05) than in the post-ovulatory (0.43 +/- 0.11; n = 11) or mated groups (0.17 +/- 0.02; n = 14). Lactate in the ampulla of mated animals was higher than in the pre-ovulatory group (6.83 +/- 0.70 versus 3.86 +/- 0.38; P < 0.05; n = 15 and 13), but neither was significantly different from the post-ovulatory group. Furthermore, no change was seen at the ampullary-isthmic junction in lactate concentration with phase.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Studies of 3 genetically female, intersex pigs revealed that only one showed oestrous cycles and lordosis, the other 2 exhibited various components of male sexual behaviour. Each uterus was a patent bicornuate structure, but 5 of the oviducts were incompletely formed. Two of the animals had bilateral ovotestes, whilst the third had a functional ovary on the right and an ovotestis on the left. The small ovarian portion of each ovotestis contained Graafian follicles which did not respond to a systemic injection of PMSG. The major portion of each ovotestis consisted of extensive interstitial tissue and seminiferous tubules devoid of germ cells. A structure closely resembling an epididymis adjoined the ovotestes. Concentrations of oestradiol and progesterone in plasma corresponded with those expected in the follicular phase of an oestrous cycle, but concentrations of testosterone represented approximately one-tenth of the values found in mature boars.

Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.

After challenge with oestradiol benzoate, the mean maximum LH concentration in 5 XX intersex pigs possessing ovarian and testicular tissue, or only testicular tissue, was 2.10 (+/- 0.41) ng/ml compared with 8.9 ng/ml in mature domestic gilts. These results indicate that exposure of the pig brain to testosterone before Day 30 of gestation is important, or that early testicular secretions other than testosterone are involved in the determination of brain gender. The observation that some intersex pigs show normal oestrous cycles implies that the response to these prenatal factors is primarily quantitative rather than qualitative.

No unusual steroid-binding proteins that might react with the oocyte or its investments could be detected in follicular fluid. Corticosteroid-binding globulin occurred in follicular fluid from pigs, sheep and cows, and sex hormone-binding globulin occurred in follicular fluid from sheep and cows. The bulk of the steroid in follicular fluid is bound to albumin with low affinity, indicating that steroid molecules can readily be released, and oestrogen can react with the oocyte and granulosa cells in a manner analogous to that demonstrated for target cells bathed with interstitial fluid. Pigs lack a sex-hormone binding globulin in blood plasma and, hence, in follicular fluid. Because no proteins exist in follicular fluid that would compete with antibodies to bind steroids, direct radioimmunoassay of follicular steroids appears to be a valid technique.

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.

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 degree C cooler than the ampulla (range 0.2-0.7 degree C) whereas in 10 mated animals the isthmus was 0.69 degree C cooler (range 0.2-1.6 degree C); 3 animals in the latter group had within-oviduct differences of greater than or equal to 1 degrees C. By contrast, in 12 animals that had recently ovulated, the isthmus was a mean of only 0.1 degree C cooler than the ampulla; there was no measurable temperature gradient in 3 of the animals, whilst the isthmus was 0.1 degree 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.

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.