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Gene Expression Microarray technology was used to compare oviduct transcriptome between inseminated and non-inseminated pigs during spontaneous oestrus. We used an in vivo model approaching the study from a physiological point of view in which no hormonal treatment (animals were in natural oestrus) and no artificial sperm selection (selection was performed within the female genital) were imposed. It is therefore emphasised that no surgical introduction of spermatozoa and no insemination at a site other than the physiological one were used. This approach revealed 17 genes that were two-fold or more up-regulated in oviducts exposed to spermatozoa and/or developing embryos and 9 genes that were two-fold or more down-regulated. Functional analysis of the genes revealed that the top canonical pathways affected by insemination were related to the inflammatory response and immune system (Network 1) to molecular transport, protein trafficking and developmental disorder (Network 2) and to cell-to-cell signalling and interaction (Network 3). Some of the genes in network 1 had been previously detected in the oviduct of human and animals, where they were over-expressed in the presence of spermatozoa or pre-implantation embryos (C3, IGHG1, ITIH4, TNF and SERPINE1) whereas others were not previously reported (SAA2, ALOX12, CD1D and SPP1). Genes in Network 2 included RAB1B and TOR3A, the latter being described for the first time in the oviduct and clearly expressed in the epithelial cells of the mucosa layer. Network 3 integrated the genes with the highest down-regulation level (CYP51, PTH1R and TMOD3). Data in the present study indicate a change in gene expression during gamete encounter at the site of fertilization after a natural sperm selection within the female genital tract. These changes would indicate a modification of the environment preparing the oviduct for a successful fertilization and for an adequate embryo early development.

To avoid inappropriate conclusions being drawn from the extensive use of in vitro preparations of sperm-oviduct epithelial binding, it is recalled that events in the genital tract of mammals are regulated by the gonads, primarily by their changing secretion of steroid hormones. Key observations from in vivo models are used to emphasise the dynamic interactions between viable sperm cells and the caudal (distal) portion of the oviduct isthmus, the site of the functional sperm reservoir. These include (1) pre-ovulatory arrest and epithelial binding of intact sperm cells and thereby suppression of completion of capacitation, (2) peri-ovulatory activation and release from binding of discrete sub-populations of competent spermatozoa, and (3) post-ovulatory liberation of large numbers of spermatozoa. These observations underline the influence of endocrine regulation of sperm binding and release by peri-ovulatory Graafian follicles, a point brought out by the enhanced sperm release prompted by diverse treatments with solutions of progesterone. In the light of this evidence, the suitability of in vitro preparations for clarifying physiological events should be questioned, especially if myosalpingeal catecholamines diffusing out of the autonomic nervous system contribute to sperm activation and/or release. None of this is to infer that sperm cells themselves are without influence on their epithelial binding reaction(s). Nor is it to suggest that in vitro models of sperm-oviduct binding are without relevance to the development of sperm evaluation technologies. However, pre-ovulatory sperm-epithelial binding and a regulated peri-ovulatory release should be seen as vital tactics in the overall strategy of achieving successful monospermic fertilisation., ((c) 2007 Wiley-Liss, Inc.)

Whole boar semen or seminal plasma has been demonstrated to advance the time of ovulation in gilts. As a means of clarifying this influence, the contribution of uterine lymphatics and their white cell populations has been examined. After duct visualisation with Evan's blue, lymph was sampled from a mesometrial vessel in eight pre-ovulatory gilts whose uterine lumen was infused simultaneously with whole semen in one ligated horn and saline in the contralateral ligated horn. Lymph was collected from cannulated vessels for periods of up to four hours under general anaesthesia. Thereafter, mesometrial lymph nodes, utero-tubal junction and uterine wall tissues were sampled. The proportion of nucleated cells in the sampled lymph increased towards the end of the collection period, but erythrocytes were found in all instances preventing a meaningful differentiation and identification of leukocytes. Prominent uterine lymph nodes were present in the mesometrium on both sides of the reproductive tract in 7 of 10 gilts. Differences in cellular contents were demonstrated between the side of the tract infused with semen and that infused with saline control. Two of 4 gilts had lower values for CD4 (Cluster Differentiation) and 3 of 6 gilts higher values for MHC II (Major Histocompatibility Complex) markers on the side challenged with semen. In contrast, values remained constant for CD8 but ranged widely for CD18. Immunohistochemical analysis of uterine tissue samples for MHC II+ cells revealed significant differences (P < 0.05) between the control and semen-treated ligated portions of the horns, as well as between the tissue sample of uterine wall and that from the utero-tubal junction, but there were no significant differences for CD4+ cells. It therefore remains plausible that semen-induced cytokines in the uterine lymph undergo counter-current transfer to the ipsilateral ovary and accelerate the final maturation of pre-ovulatory Graafian follicles.

Ovulation failure and double ovulation rates were examined in 1917 inseminations performed in high-yielding dairy cows under standard commercial conditions. The ovulation rate was determined 11 days post-insemination by ultrasound detection of at least one corpus luteum in the ovaries. Analyzing the double ovulation and pregnancy rates, the study population consisted only of ovulated cows (n = 1792). Data were analyzed using logistic regression methods. A failure to ovulate was recorded in 125/1917 (6.5%) services: 82/663 (12.4%) during the warm and 43/1254 (3.4%) during the cool period. Based on the odds ratios, the risk of ovulation failure was 3.9 times higher for inseminations performed during the warm period. No significant effects of estrous synchronization, milk production and days in milk at AI, and service and lactation number on ovulation failure were found. Double ovulation was recorded in 277/1792 (15.5%) services: 146 (52.7%) unilateral double ovulations (42.5% left versus 57.5% right); 115 (41.5%) bilateral double ovulations; and 16 (5.8%) triple ovulations. Double ovulation was recorded in 72 (12.4%) and 205 (16.9%) AI during the warm and the cool period, respectively. The percentages of double ovulation for first, second and third or more lactations were 6.7, 16.6 and 25%, respectively. Double ovulation rates for early (less than 90 days), mid- (90-150 days) and late (more than 150 days) lactation periods were 13, 20.7 and 14.2%, respectively. Reaching estrus during the warm period decreased the likelihood of double ovulation by a factor of 0.86; the risk of double ovulation was lower in cows with higher milk production (a 1 kg increase in milk yield led to a 0.97-fold reduced risk of double ovulation); cows in their second and in their third or more lactations showed a likelihood of double ovulation (using the first lactation as reference) increased by factors of 3.4 and 5.6, respectively; and reaching estrus during the early and late lactation period was related to a decreased probability of double ovulation (using the mid-lactation period as reference) by factors of 0.56 and 0.84, respectively. No significant effects of synchronization and service number on the double ovulation rate were found. Pregnancy was recorded in 914/1792 (51%) services: rates of 53.5% (811/1515) were recorded for single ovulations; 37.2% (103/277) for double ovulations: 28.8% (42/146) for unilateral double ovulations; 45.2% (52/115) for bilateral double ovulations; and 56.3% (9/16) for triple ovulations. The likelihood of pregnancy diminished in cows: inseminated during the warm period (by a factor of 0.5); inseminated by one particular bull (by a factor of 0.33); with higher milk production (a 1 kg increase in milk yield decreased the probability of pregnancy by a factor of 0.98); or undergoing unilateral (by a factor of 0.31) and bilateral (by a factor of 0.64) double ovulation. Logistic regression analysis indicated no significant effects of synchronization, days in milk, lactation number and service number on pregnancy rate. Collectively, our results indicate that cows showing estrus in conditions of heat stress had a high risk of ovulation failure. The effect of milk production on double ovulation was negative, whereas lactation number was positively correlated with this factor; the highest incidence of double ovulation occurring during the mid-lactation period.

In cows undergoing spontaneous oestrous cycles and mated during the first 6 hours of oestrus, the distribution of spermatozoa in the oviduct isthmus and changes in their surface membranes and neighbouring epithelium have been examined shortly before and after ovulation. In agreement with previous histological studies, relatively few spermatozoa were detected in the oviduct lumen: most were located in the caudal isthmus before ovulation, frequently among folds and in the presence of a viscous secretion. A majority of spermatozoa in this region showed strands and droplets of secretory material distributed over the anterior portion of an intact head before ovulation, whereas distribution of material over the post-nuclear cap of spermatozoa close to vesiculation or already acrosome-reacted was characteristic of the post-ovulatory situation. These changes in sperm head membranes were viewed as an expression of the completion of capacitation, and seemingly permit microvillous engagement with the rostral tip of the head. In conjunction with a narrow lumen and viscous secretions in the caudal isthmus, microvilli may thus serve to regulate periovulatory sperm progression towards the site of fertilisation, and be the basis of intermittent phases of adhesion to the oviduct epithelium as seen by phase-contrast microscopy. Although cilia do not similarly engage the heads of bull spermatozoa (cf. boar spermatozoa), they may act to regulate progression of capacitated spermatozoa by contacting the principal piece of the flagellum. In the light of these observations, changes in the molecular composition of sperm surface domains during the process of capacitation in vivo now require specific definition.

Ovulation of 97 follicles was recorded in a gilt that had not received any hormonal treatment, and circumstantial evidence adduced for a rapidly proliferating tumour of the adenohypophysis. A lesser degree of superovulation had occurred in the previous oestrous cycle, whilst the rate of follicular development in the current cycle was exceptional. None of the 93 eggs recovered from the oviducts was a primary oocyte, but three were polyspermic pronucleate eggs. The most advanced stage of embryonic development was that of 4-cells. Such a degree of spontaneous superovulation has never been observed before during careful examination of more than 1200 pairs of ovaries from gilts in the same herd.

Using sexually mature animals, the distribution of spermatozoa has been examined at the utero-tubal junction and in the distal and proximal portions of the oviduct isthmus. Mating occurred during early oestrus and, with one exception, specimens were prepared shortly before or after ovulation. Distinct reservoirs of spermatozoa were identified in furrows between the terminal folds of the isthmus, and particularly within the troughs and transverse ridges of this region. The density of spermatozoa diminished steeply from the utero-tubal junction towards the isthmus, especially in the pre-ovulatory specimens. The membranes of most spermatozoa in the isthmus were intact up to the time of ovulation, suggesting that the acrosome reaction is a peri- or post-ovulatory event. Whilst the flagella of spermatozoa in the reservoirs were usually straight or only slightly curved, those on the surface of the epithelial folds were undulating (S-shaped). Specific microenvironments may therefore exist in the distal portion of the isthmus to regulate sperm motility; droplets of secretion were a notable feature in this region. In specimens prepared 24 hr after ovulation, spermatozoa were almost absent from the utero-tubal junction and isthmus. However, denuded eggs were observed in the proximal portion of the isthmus in this animal, and they had spermatozoa associated with the zona pellucida. Arguments are presented for a peri-ovulatory endocrine regulation of sperm redistribution and capacitation.

Progression of ram spermatozoa within the sheep oviduct has been studied in relation to the time of ovulation. Ewes were mated at the onset of estrus and, at increasing intervals thereafter, the oviduct isthmus was transected 1.5-2.0 cm proximal to the utero-tubal junction. The incidence of fertilization and number of accessory spermatozoa were examined in eggs recovered 1 to 3 days after surgery. Ovulation occurred 24-26 hours after the onset of estrus. None of 52 eggs recovered from 48 oviducts transected up to 21 hours after mating was fertilized, nor were spermatozoa associated with the zona pellucida. Two of 33 eggs (6.1%) from 29 oviducts transected between 22 and 24 hours after mating were fertilized, while 3 of 14 eggs (21.4%) and 13 of 16 eggs (81.3%) were fertilized with transection at 25 and 26 hours, respectively. The mean numbers of accessory spermatozoa on eggs in the latter two groups were 0.7 (range 0-2) and 7.9 (range 0-26). Results from transection at 27-30 hours after mating indicated a progressive release and adovarian movement of spermatozoa. Viable spermatozoa are apparently sequestered in the caudal isthmus for as long as 17-18 hours, until shortly before ovulation, when a local transfer of ovarian follicular hormones is thought to facilitate their redistribution. The adrenergic stimulation of powerful adovarian waves of contraction in the isthmus at this time may also act to provoke the capacitation and hyperactivation of ram spermatozoa.

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 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.