Pregnancy recognition signaling mechanisms in ruminants and pigs

Maternal recognition of pregnancy refers to the requirement for the conceptus (embryo and its associated extra-embryonic membranes) to produce a hormone that acts on the uterus and/or corpus luteum (CL) to ensure maintenance of a functional CL for production of progesterone; the hormone required for pregnancy in most mammals. The pregnancy recognition signal in primates is chorionic gonadotrophin which acts directly on the CL via luteinizing hormone receptors to ensure maintenance of functional CL during pregnancy. In ruminants, interferon tau (IFNT) is the pregnancy recognition signal. IFNT is secreted during the peri-implantation period of pregnancy and acts on uterine epithelia to silence expression of estrogen receptor alpha and oxytocin receptor which abrogates the oxytocin-dependent release of luteolytic pulses of prostaglandin F2-alpha (PGF) by uterine epithelia; therefore, the CL continues to produce progesterone required for pregnancy. Pig conceptuses secrete interferon delta and interferon gamma during the peri-implantation period of pregnancy, but there is no evidence that they are involved in pregnancy recognition signaling. Rather, pig conceptuses secrete abundant amounts of estrogens between Days 11 to 15 of pregnancy required for maternal recognition of pregnancy. Estrogen, likely in concert with prolactin, prevents secretion of PGF into the uterine venous drainage (endocrine secretion), but maintains secretion of PGF into the uterine lumen (exocrine secretion) where it is metabolized to a form that is not luteolytic. Since PGF is sequestered within the uterine lumen and unavailable to induce luteolysis, functional CL are maintained for production of progesterone. In addition to effects of chorionic gonadotrophin, IFNT and estrogens to signal pregnancy recognition, these hormones act on uterine epithelia to enhance expression of genes critical for growth and development of the conceptus.     

Intraluminal Pressure Variations in the Isthmus of the Porcine Oviduct after Intrauterine Insemination with Saline,Oestrogen Solution or Boar Seminal Plasma

The purpose of this study was to investigate if an intrauterine deposition of saline, boar seminal plasma, or an oestrogen solution containing 11.5 µg oestrogens affected the intraluminal pressure in the isthmus of the oviduct in unrestrained gilts. In order to monitor variations in intraluminal pressure, 2 ultra-miniature pressure sensors, located at 2 different points along the same isthmus were used. After an initial recording period, either saline, boar seminal plasma, or the oestrogen solution was deposited directly into the uterus. Intraluminal pressure recordings were conducted up to 6 h after insemination, and blood samples, for monitoring oestradiol-17B and 15-ketodihydroprostaglandin F_2α levels, were collected. None of the inseminates had any consistent effect on the intraluminal pressure in the porcine oviduct. After deposition of the oestrogen solution, increases in the circulating levels of both oestradiol-17B and 15-ketodihydropro- staglandin F_2α were seen.     

Inhibition of lipopolysaccharide-induced suppression of luteal function in isolated perfused bovine ovaries

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF_2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF_2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 μg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 μg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF_2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF_2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF_2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge.     

Sequential Hormonal Changes in the Postpartum Dairy Cow

Peripheral plasma levels of 15-keto-13,14-dihydro-PGF_2α, progesterone, Cortisol, LH and prolactin were studied in 6 primiparous postpartum dairy cows. The cows were followed by hormone measurements and clinical examinations from parturition until pregnancy was established. Blood was collected 3 times per day. The cervix, uterus and ovaries were examined by rectal palpation at 6–10 days intervals. The cows were observed for signs of oestrus twice daily and were additionally teased with a bull to provoke standing heat.Four cows had a normal parturition and dropped their fetal membranes shortly afterwards. (NR group). The remaining 2 retained their fetal membranes for more than 24 h following parturition (RFM group). One out of 6 cows showed standing oestrus at the first ovulation, 4 animals were in oestrus at the second ovulation and all cows showed signs of oestrus at the third ovulation. Although the length of the first luteal phase varied from 9 to 22 days a corpus luteum was in all cases palpated. The secretion of progesterone during the first luteal phase was terminated by a PGF_2α release.A significant difference in 15-keto-13,14-dihydro-PGF_2α levels between the 2 groups was found on days 0–4 (2.39 vs 6.87 nmol/1 at Ρ < 0.06). Postpartum prostaglandin F_2α release as reflected by the level of 15-keto-13,14-dihydro-PGF_2α lasted shorter in the NR group than in the RFM group (15–17 vs 21 days). Significant positive correlations between 15-keto-13,14-dihydro-PGF_2α and Cortisol as well as between prolactin and Cortisol during the first 24 days postpartum were noted only in cows having normal parturition. The most pronounced daily prolactin variations occurred during the second luteal phase (NR group), when a significant difference between the times 8.00, 12.00 and 15.00 was recorded (14.7, 31.5 and 19.7 μg/l, respectively). Moreover, a partial negative correlation between log value of prolactin and arithmetical value of LH was found in these cows only during the first luteal phase after parturition.     

The Effect of Induced Standing Reflex,Cervical Stimulation and Insemination on Intraluminal Pressure Variations in the Isthmus of the Oviduct in Unrestrained Gilts

Four gilts were each equipped with 2 ultra-miniature pressure sensors, placed at 2 different points along the same isthmus of the oviduct, on the morning of the first day of standing oestrus (Day 1). Intraluminal pressure recordings were started the same afternoon. After an initial recording period, intraluminal pressure was recorded while the gilts showed standing oestrus and during cervical stimulation followed by insemination with either 100 ml saline or 100 ml boar semen. Monitoring of the pressure variations in the isthmus was continued for up to 6.5 h after the last insemination. Blood samples for monitoring oestradiol-17B, progesterone and 15-ketodihydroprostaglandin F_2α were collected before and after each manipulation of the gilt and every 30 min during the rest of the test period. None of the above manipulations had any consistent effect on the intraluminal pressure in the porcine isthmus, although, a clear 15-ketodihydroprostaglandin F_2α peak could be seen after insemination with boar semen.     

Changes in the C-reactive protein and 13,14-dihydro-15-keto-prostaglandin F2α concentrations of uterine lavage samples after the administration of povidone-iodine in cows

Changes in the C-reactive protein (CRP) and 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) concentrations of uterine lavage fluid were examined in cows given an intrauterine povidone-iodine (PI) infusion. The mean polymorphonuclear leukocyte (PMN) ratios (the ratio of PMN to total cells) and CRP concentration of uterine lavage fluid on the day after the treatment were significantly (P<0.05) greater in the PI infusion group (PMN: 53.0 ± 32.7%, CRP: 50.2 ± 32.3 ng/mL) than in the non-treatment control group (PMN: 7.9 ± 21.9%, CRP: 17.2 ± 5.9 ng/mL), whereas there was no significant difference in the mean PGFM concentration between the two groups. The present findings suggest that the uterine CRP level is a useful biomarker of local uterine inflammation in cows.     

Some Reproductive and Clinical Aspects of Endotoxins in Cows with Special Emphasis on The Role of Prostaglandins

Endotoxin from Salmonella typhimurium was administered intravenously to 3 cows. It produced an immediate increase in blood prostaglandin metabolite levels causing either luteolysis or a temporary drop in progesterone concentrations depending on the degree of prostaglandin synthesis. The clinical effects on heart rate, respiratory rate, ruminai contraction rate, body temperature and other general signs were studied. The cows showed a transient increase in heart and respiratory rate and a decrease in ruminai activity. The temperature responded by a small initial decrease and a secondary increase. Other symptoms included muscular weakness and shivering, excessive salivation and diarrhoea. Plasma Ca levels showed a direct decrease and then slowly increased to initial levels in the course of several days. The role of endotoxins in Gram-negative infections and their mode of action on the body are discussed with special emphasis on the role of prostaglandins on reproductive function.     

Effect of fractionated weaning on hormonal patterns and weaning to oestrus interval in primiparous sows

The effect of weaning the 4-5 heaviest piglets in the litter on day 33 of lactation and the remainder 2 days later (fractionated weaning) on plasma levels of prolactin, cortisol, oestradiol-17 beta (E2), progesterone (P4) and LH, as well as on the weaning to oestrus interval in primiparous sows was studied. Twelve crossbred sows were grouped into 6 pairs according to farrowing date and litter size. The litter of 1 sow in each pair (F) was weaned in 2 stages, and the other conventionally weaned at 35 days (C). Blood samples were collected via a permanent jugular vein catheter every 3 h from 9 a m to 9 p m daily throughout the experimental period, and intensively at 15 min intervals for 12 h on the day of first and final weaning and for 6 h on the day after each weaning. All sows were slaughtered following their first post-weaning oestrus and the reproductive organs were macroscopically examined. Lactational oestrus was not observed in any of the sows. Sows from 5 out of 6 pairs showed oestrus within 8 days of weaning and post-mortem examination showed normal ovulation. There was a tendency for the F sows to have a shorter weaning to oestrus interval, as compared with the C sows (5 of 6 pairs, 4.8 days v 5.6 days). The plasma levels of prolactin around weaning were not significantly different between the 2 groups. Within 6 h after final weaning, the prolactin concentrations decreased gradually from 7.6 and 8.7 to 1.6 and 1.7 microgram/l in the control and treatment groups, respectively. The plasma levels of cortisol, showing a diurnal rhythm (with the lowest level at 6 and/or 9 p m), did on no occasion differ between the 2 groups. On the day of final weaning, no diurnal rhythm was observed, with cortisol remaining high at 6 and 9 p m. The plasma levels of E2 and P4 were low until final weaning in both groups. After final weaning the E2 levels rose faster in the F sows than in the C sows, to 44.3 and 34.8 pmol/l, respectively, on day 2 (p less than 0.01). No significant differences in levels of plasma LH and the number of LH pulses were observed between the groups. After final weaning the average and base levels of LH and the number of LH pulse(s) increased significantly.     

Effect of transportation and relocation in post-weaning anoestrous primiparous sows

The object of this investigation was to study the clinical and endocrine responses to transportation and relocation in 8 post-weaning anoestrous sows. They had been anoestrous for at least 24 days after weaning before transportation/relocation was performed. Laparoscopy, performed at the beginning of the experiment, revealed that the ovaries contained many follicles (less than or equal to 6 mm in diameter), but no corpora lutea. Blood samples, taken before and after transportation/relocation, showed that LH activity was low at the beginning of the experiment and increased after transportation/relocation in the majority of the sows. Peripheral plasma concentrations of of oestradiol-17 beta increased 1-4 days after transportation/relocation in 6 out of 8 sows which was followed by oestrus and ovulation. Progesterone concentrations were also below the practical detection limit until the end of oestrus. This study has demonstrated that a change in environment by transportation and relocation can induce oestrus by increasing the LH activity in post-weaning anoestrous sows.     

Clinical and endocrinological studies in primiparous post partum sows. Effects of lactation length and litter size

The object of this investigation was to determine the relationships between clinical findings and hormonal patterns in primiparous sows with different lactation length and litter size during lactation, weaning and to the first oestrus. Seven pairs of primiparous full sib sows were used to determine the effect of lactation length with normal litter size. One sow of each pair was assigned to nurse the piglets for 3 weeks (group A) while the other nusred for 5 weeks (group B). Another 8 primiparous sows (group C) were assigned to nurse 2–4 piglets during a 5-week lactation period. Oestrus detection was performed twice daily and laparoscopic examination every 2 weeks. If the sows did not come in oestrus within 3 weeks after weaning they were slaughtered. Peripheral plasma levels of progesterone, oestradiol-17β and LH were estimated by radioimmunoassays throughout the experimental period.     

Presence of a Temperature Gradient Among Genital Tract Portions and the Thermal Changes Within These Portions Over the Estrous Cycle in Beef Cows

The aim of the present study was to describe the temperature of the different portions of the female genital tract and their relation to rectal temperature and to investigate the effect of steroid hormones profiles on these variables over the estrous cycle in cattle. Four nonpregnant Japanese Black cows were investigated daily over two successive estrous cycles using a digital thermometer with a long probe and rounded-end sensor to record the temperature of the rectum (RT), vagina (VT), cervix (CT), uterine body (UBT) and uterine horns (UHT). Blood samples were collected immediately before temperature recording to assay peripheral levels of progesterone (P₄) and estradiol-17β (E₂). Moreover, transrectal ultrasonography was carried out after temperature recording to monitor the ovulatory follicle and track ovulation. During the experiment, the ambient temperature and relative humidity were recorded for further calculation of the temperature humidity index (THI). The temperature within the genital tracts in these cows progressively increased towards the uterine horns from the vagina. The VT, CT, UBT and UHTs were significantly higher in association with peripheral P₄ concentrations greater than 4 ng/ml (mid-luteal phase) when compared with lower peripheral P₄ concentrations. The VT was more significantly (P<0.01) correlated to the CT, UBT and UHTs than RT. In conclusion, a temperature gradient was present among the vagina, cervix and uterus over the estrous cycle, and changes in peripheral P₄ concentrations were associated with the thermal variations within these portions. The VT could be more beneficial than RT in monitoring temperature of deeper portions of the female genital tract in bovine.     

Local regulation of corpus luteum development and regression in the cow: Impact of angiogenic and vasoactive factors

The corpus luteum (CL) of the estrous cycle in the cow is a dynamic organ which has a life time of approximately 17-18 days. The main function of the CL is to secrete a large amount of progesterone (P) thereby supporting the achievement of pregnancy. As the CL matures, the steroidogenic cells establish contact with many capillaries and the matured CL is composed of many vascular endothelial cells that account for up to 50% of all CL cells. The bovine CL produces several major angiogenic and vasoactive foctors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-1 and -2 (ANPT-1 and -2), prostaglandin F_2α (PGF_2α), endothelin-1 (EDN1), angiotensin II (Ang II) and nitric oxide (NO). These factors regulate P secretion directly and/or indirectly within the CL. Moreover, different actions of PGF_2α in the early cycle CL (non-luteolytic) and the mid cycle CL (luteolytic) may provide insight into the luteolysis cascade in the cow. The aim of the present review is to describe the current concepts of the local mechanisms for the cascade of development and regression of the bovine CL as regulated by luteal angiogenic and vasoactive factors.     

Regulation of matrix metalloproteinase-9 protein expression by 1α,25-(OH)2D3 during osteoclast differentiation

To investigate 1α,25-(OH)₂D₃ regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1α,25-(OH)₂D₃ during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1α,25-(OH)₂D₃ inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1α,25-(OH)₂D₃ administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.     

Intrafollicular Concentrations of Steroid Hormones and PGF2α in Relation to Follicular Development in the Mares during the Breeding Season

The concentrations of androstenedione, estradiol-17β, progesterone and PGF_2α contained in the follicular fluid produced by the follicles in collected ovaries of mares that have had estrous phase during the breeding season were measured and analyzed the relation between the growth stage of follicles and the hormone levels in the follicular fluid. An ultrasonographic diagnostic instrument was used to measure the diameter of the follicles in order to categorize the follicles into three groups the following: 8 small follicles (from 1.0 to less than1.5 cm), 8 medium follicles (from 1.5 to less than 3.0 cm), and 8 large follicles (from 3.0 to 5.0 cm), respectively. The analysis of the follicular fluid in ovaries of estrous mares showed that the concentrations of androstenedione were significantly higher in the medium or large follicles than in the small follicles and the concentrations of estradiol-17β were significantly higher in larger follicles than in the small or medium follicles (P<0.05). The concentrations of progesterone and PGF_2α, on the other hand, did not significantly vary regardless of follicluar size. In the follicles within the mare ovaries that have had estrous stage, the concentrations of the hormones related the ovulation, namely androstenedione and estradiol-17β, were higher with larger follicles.     

Phylogenetic groups and cephalosporin resistance genes of Escherichia coli from diseased food-producing animals in Japan

A total of 318 Escherichia coli isolates obtained from different food-producing animals affected with colibacillosis between 2001 and 2006 were subjected to phylogenetic analysis: 72 bovine isolates, 89 poultry isolates and 157 porcine isolates. Overall, the phylogenetic group A was predominant in isolates from cattle (36/72, 50%) and pigs (101/157, 64.3%) whereas groups A (44/89, 49.4%) and D (40/89, 44.9%) were predominant in isolates from poultry. In addition, group B2 was not found among diseased food-producing animals except for a poultry isolate. Thus, the phylogenetic group distribution of E. coli from diseased animals was different by animal species. Among the 318 isolates, cefazolin resistance (minimum inhibitory concentrations: ≥32 μg/ml) was found in six bovine isolates, 29 poultry isolates and three porcine isolates. Of them, 11 isolates (nine from poultry and two from cattle) produced extended spectrum β-lactamase (ESBL). The two bovine isolates produced bla_CTX-M-2, while the nine poultry isolates produced bla_CTX-M-25 (4), bla_SHV-2 (3), bla_CTX-M-15 (1) and bla_CTX-M-2 (1). Thus, our results showed that several types of ESBL were identified and three types of β-lactamase (SHV-2, CTX-M-25 and CTX-M-15) were observed for the first time in E. coli from diseased animals in Japan.     

15-ketodihydro-PGF2 alpha, progesterone and cortisol profiles in heifers after induction of parturition by injection of dexamethasone.

In order to study rapid changes in 15-ketodihydro-PGF2 alpha, cortisol and progesterone in the period preceding parturition in cattle, pre-term parturition was induced in 4 late pregnant heifers. Parturitions were induced by 2 intramuscular injections of 20 mg dexamethasone with a 24-h interval. The first injection was made on days 254, 258, 264 and 265 in gestation, respectively. Twenty-four h before the first injection an intravenous polyurethane cannula was inserted. Blood samples were collected at least every hour until 12 h after parturition and during the second stage of labour at least 6 times per hour. Plasma was analysed for 15-ketodihydro-PGF2 alpha and progesterone by radioimmunoassays, and for cortisol by an ELISA. The average time from injection to parturition was 7.7 (6.6-8.9) days (mean (range)). Two of the heifers had retained foetal membranes (RFM). At the start of the experiment the levels of PGF2 alpha metabolite were low (< 300 pmol/L) and increased slowly to levels between 1000 and 2000 pmol/L at one day before parturition. During the last day, however, the levels increased rapidly and the highest levels (> 10,000 pmol/L) were reached at the time of delivery. No pulsatile release was seen. Immediately after foetal expulsion the PG-metabolite levels decreased rapidly in all animals. In the 2 animals with RFM, however, this decline ceased within a few h. The PG-metabolite levels in these animals then started to increase and reached levels as high as during parturition. Luteolysis occurred between 1.6 and 0.4 days before parturition in all animals. The cortisol profile showed a distinct peak at the time of parturition in the RFM heifers. This peak was absent in the non-RFM heifers. This study shows that the PGF2 alpha release at prepartal luteolysis and parturition is not pulsatile in cattle and that cortisol profiles in heifers with retained foetal membranes might differ from the profiles in non-RFM heifers at the time of parturition.     

Endotoxin and Arachidonic Acid Metabolites in Portal,Hepatic and Arterial Blood of Cattle with Acute Ruminai Acidosis

Andersen, P. Haubro, M. Hesselholt and N. Jarlev: Endotoxin and arachidonic acid metabolites in portal, hepatic and arterial blood of cattle with acute ruminai acidosis. Acta vet.scand.1994,35,223-234.– Ruminai acidosis was induced experimentally with 70 g barley / kg body weight in 2 rumen fistulated cows with chronic indwelling catheters in the portal vein, in a hepatic vein and the carotid artery. The cows were followed for 24 and 20h after the overfeeding and evaluated clinically and by clinical chemistry. The 2 cows exerted different responses to the treatment. Both cows showed signs of severe ruminai acidosis. Both cows had endotoxin in portal and hepatic vein blood, but only 1 of the cows convincingly developed a systemic endotoxaemia. A pre-hepatic release of the stable prostacyclin and thromboxane metabolites, 6-ketoprostaglandin F_lα and thromboxane B₂ was demonstrated in this cow. The results of the present study show that endotoxin and arachidonic acid metabolites of pre-hepatic origin may be factors involved in the pathogenesis of ruminai acidosis, and that investigation of the factors affecting translocation of ruminai endotoxin and subsequent clearing in the liver, will be of importance.