Signs of embryo-maternal communication: miRNAs in the maternal serum of pregnant pigs.

Circulating miRNAs were proposed to be indicators of normal or complicated pregnancies. Based on this knowledge and our recent transcriptomic approach showing expression of miRNAs in the porcine endometrium, conceptuses and uterine extracellular vesicles during pregnancy, we have hypothesized that signs of ongoing local embryo-maternal crosstalk involving miRNAs can be detected in the circulation of pregnant gilts as early as a few days after maternal recognition of pregnancy. By applying several molecular biology techniques that differ in dynamic range and precision in maternal serum of Day 16 pregnant pigs, we were able to show for the first time increased levels of several miRNAs, previously reported to be expressed in either conceptuses and extracellular vesicles (miR-26a and miR-125b) or pregnant endometrium (miR-23b). Our results clearly showed that real-time RT-PCR and digital PCR are the most reliable methods, being able to detect small-fold changes of low-abundant circulating miRNAs. Further validation in a separate group of gilts confirmed an increase in miR-23b and miR-125b levels. In silico analyses identified pregnancy-related biological processes and pathways affected by these miRNAs. Target prediction analysis revealed hundreds of porcine transcripts with conserved sites for these miRNAs, which were classified into signaling pathways relevant to pregnancy. We conclude that a unique set of miRNAs can already be observed in the circulation of pigs during the first weeks of pregnancy, as a result of the initiation of embryo-maternal communication.

Comparison of accuracy of pregnancy-associated glycoprotein (PAG) concentration in blood and milk for early pregnancy diagnosis in cows.

The aim of this study was to compare two methods of early pregnancy diagnosis by determining pregnancy-associated glycoprotein (PAG) concentration in blood and PAG concentration in milk. Blood and milk samples were obtained on days 0 (AI day), 14, 21, 28, 35, 49, 63, 77, 91 and 105 of gestation from 60 lactating Holstein Frisian cows from one herd, carrying live fetuses. To determine PAG concentration a specific RIA system (RIA-706) was used. PAG concentration in blood and milk increased after 28 days of pregnancy, with blood concentrations being significantly higher than in milk. However, the accuracy of both tests at this time point was similar (sensitivity: 92 % in blood, 93 % in milk; specificity 53 % and 60 % respectively). None of the tests were able to detect open cows properly at this stage. On day 35 of gestation sensitivity (100 % for blood, 97 % for milk) and specificity (100 % for blood, 100 % for milk) were high enough to be used for reliable pregnancy diagnosis. The accuracy (sensitivity and specificity) for PAG concentrations in blood and milk for the rest of the study was 100 %. Our investigation shows that PAG determination in milk is a stress-free and non-invasive method for early pregnancy diagnosis in cattle.

Le but de cette étude était de comparer les concentrations de glycoprotéines associées à la gestation (PAG) dans le sang et le lait en vue d'un diagnostic de gestation précoce chez la vache. Des échantillons de sang et de lait ont été prélevés aux jours 0 (IA), 14, 21, 28, 35, 49, 63, 77, 91 et 105 de la gestation sur 60 vaches Holstein Frisonnes en lactation avec un fœtus vivant. On a utilisé pour mesurer la concentration de PAG une méthode RIA spécifique (RIA-706). Les concentrations de PAG dans le sang et le lait s'élevaient à partir du 28ème jour de gestation, les concentrations mesurées dans le sang étant nettement plus élevée que celles mesurées dans le lait. L'exactitude des deux tests était à ce moment-là similaires (sensitivité: 92 % dans le sang, 93 % dans le lait, spécificité: 53 % respectivement 60 %). Aucune des deux méthodes n'était, à ce stade, à même de distinguer une vache non portante avec justesse. Au 35ème jour de gestation, la sensitivité des deux méthodes (100 % dans le sang, 97 % dans le lait) et leur spécificité (100 % dans le sang, 100 % dans le lait) était assez élevées pour permettre un diagnostic de gestation sûr. L'exactitude (sensitivité et spécificité) des mesures de concentration de PAG dans le sang et le lait durant la suite de la gestation était de 100 %. Ces études montrent donc que la mesure de PAG dans le lait représente une méthode non-invasive et exempte de stress pour le diagnostic précoce de gestation chez la vache.

Chitosan -- a promising biomaterial in veterinary medicine.

Biomaterials originate from natural substances and are widely used in medicine. Although they have to satisfy many conditions to be useful for treatment, more and more research is carried out with new types of biomaterials that can help replace various tissues such as tendons and bones. Chitosan is a very promising material, revealing unique features, which makes it useful for veterinary medicine -- antimicrobial activity, biocompatibility, biodegradability. It is also known as good scaffold material, especially when combined with other polymers. This article describes chitosan as a biomaterial and tissue engineering scaffold with possible applications in veterinary medicine.

Short-term whole body vibration exercise in adult healthy horses.

The purpose of this study was to analyze the acute effect of whole body vibration exercise (WBVE) on clinical parameters and blood values in horses. Seven horses were exposed to a 10 min WBVE at a frequency of 15-21 Hz. Clinical parameters and venous blood samples were taken before and directly after WBVE. Acute short-term WBVE produced a decrease in serum cortisol (p = 0.02) and creatine-kinase (p = 0.02) values. Clinical parameters, hematology, fibrinogen, lactate, IGF-I, GGT, creatinine, myeloperoxidase activity and bone marker values were not significantly changed by WBVE. In adult sound horses WBVE was well tolerated and did not cause any sign of measured discomfort.

Biochemical markers of bone turnover during pregnancy in horses: a longitudinal study.

The effect of pregnancy on bone metabolism was investigated in healthy mares. Venous blood samples were collected 7 times from 19 multiparous mares starting at 20-weeks pre-parturition, continuing 6 times in 4-week intervals, including the week of parturition and one week after parturition. Serum concentrations of osteocalcin (OC) and carboxy-terminal cross-linking telopeptide of type I collagen (CTX-I) were determined. Measurement cycles and age had a significant (p < 0.01) influence on OC and CTX-I values. Pregnancy influenced bone metabolism with peak bone formation and resorption values around the time of parturition.

Mastitis and fertility disorders in cows.

Udder inflammations, independently of significant losses in milk yield and alteration of its quality, can negatively affect cows' reproduction efficiency. Mastitis causes changes in many active constituents, both in milk and blood. Pathogenic changes in existing constituents and new active chemical compounds, generated during disease, can affect other organs, particularly the reproductive system and its mechanisms. Fertility disorders in mastitic cows are mostly connected with the activity of cytokines (especially TNF-alpha), cortisol, prostaglandin F2-alpha, reactive oxygen species (ROS), and classic inflammatory mediators. The successful treatment of mastitis as well as protection against udder infections should be considered important methods for prophylaxis of fertility disorders in cows. The first Polish trials indicated that injection of supportive drugs (antioxidants or lysozyme dimer or flunixin meglumine) to intramammarily treated cows can increase fertility in cows with mastitis.

Pregnancy - associated glycoproteins as a new diagnostic tool in cattle reproduction.

Pregnancy-associated glycoproteins (PAGs) are powerful pregnancy markers in domestic cattle. These proteins are expressed in mono- and binucleate trophoblast cells from the first days of gestation until calving. Different molecules were identified as being expressed at various stages of pregnancy. However, up to date, their functions and activities during pregnancy were not yet established. Specific RIA tests were developed (classic and alternative RIA) and used to measure the concentration of these glycoproteins in blood during gestation and the postpartum period in cattle. In maternal blood, PAGs rise to detectable levels from days 24 to 28 after fertilization. A recent study indicated that PAGs can also be detected in milk samples. However, concentrations in milk are much lower when compared to those of plasma.

Alterations in the liver of intrauterine growth retarded piglets may predispose to development of insulin resistance and obesity in later life.

Intrauterine growth retardation (IUGR) leads to increased predisposition to metabolic syndrome in adult life but the mechanisms remain obscure. Considering a significant number of functional similarities, IUGR piglets appear to be a good model to study the development of this syndrome in humans. The aim of the present study was to investigate the ultrastructure and proteomic profile of the liver in IUGR pig neonates to discover early markers of predisposition to obesity and insulin resistance. In our study intestine and liver tissue samples were investigated in 7 day old IUGR and normal body weight (NBW) littermate piglets using histometry, mass spectrometry, in-tissue cytometry analysis and confocal microscopy. Compared to NBW, the liver in IUGR neonates was characterized by a significantly enhanced ratio of Kupffer cells to hepatocytes and insulin receptor abundance as well as higher percentages of cells expressing receptors for adipokines (resistin and adiponectin), increased expression of TNF-α (as marker of inflammation), and increased expression of insulin receptor and uncoupling protein 3 (UCP3). Moreover, NBW and IUGR differed in proteomic profile, including protein metabolism (proteasomes, cathepsin D, phermitin, phosphoglucomutase), carbohydrate metabolism (hexokinase 1, phosphoglucokinase, galactokinase, aldolase B, glucose-6-phosphate isomerase), oxidative stress and chromatin organization and DNA uptake (histones, lamin a/c). Reduction of hepatocyte numbers concomitant with significant modifications of expression of key hormones and enzymes for protein and carbohydrate metabolism in IUGR neonates may predispose to insulin resistance and obesity in adult life.

The effect of supplementing sow with bioactive substances on neonatal small intestinal epithelium.

Development of the small intestinal epithelium in early postnatal period has a significant influence on pig's survival rate and further productivity. The aim of this research was to verify whether the diet supplementation of pregnant and lactating sow with a blend of bioactive substances (flax seed, rapeseed, linden inflorescence, taurine, L-carnitine and tocopherol acetate) had an effect on the development of intestinal epithelium in their offspring. The doses of bioactive substances were calculated to meet the demands for optimal supply of the pig fetuses and newborns. Pig neonates from two groups of sows, control and supplemented, were sacrificed at the day 1, 2, 4, 7 and 14 of life. The samples taken from mid-jejunum were evaluated for mitosis (Ki67), apoptosis (active caspase 3), autophagy (MAP I LC3), and DNA damage (p53). Increase of mitotic index was noticed at day 1, 4 and 7 for supplemented group when compared to the control. Reduction of apoptotic index was observed at day 2 as compared to control. A tendency toward elevated autophagy was observed during the first 2-4 postnatal days in both groups. p53 expression was significantly lower in supplemented group as compared to control. Overall, the mitosis to programmed cell death ratio was increased and the maturation of epithelial cells quickened. We suppose that the supplementation of pregnant and lactating sow diet with bioactive substances enhanced maturation of the small intestinal epithelium in their offspring during the early postnatal period.

[Risk factors of increased abortions in dairy herds - a statistical analysis]. / Risikofaktoren für ein erhöhtes Abortvorkommen in Milchviehbetrieben - eine statistische Analyse.

OBJECTIVE: To examine whether dairy herds with and without abortion problems differ in their management and prevalence of antibodies against various pathogens, through collecting and analysing data from these herds. MATERIAL AND METHODS: Data on management parameters, clinical examinations and serological results were obtained from 48 dairy herds in middle Hessen. Information on the extent of stock renewal, form of animal housing, type of herd occupancy and use of a maternity pen was compiled. A random sample was taken from every herd, consisting of a minimum of three healthy non-lactating cows, three clinically sound puerperal animals, three animals having had an abortion and three clinically healthy pregnant heifers. In these animals, the body condition score was evaluated and a clinical gynaecological examination was performed. Additionally, these animals were serologically examined for the presence of antibodies against the bovine viral diarrhoea virus, Coxiella, Listeria and Neosporacaninum. A comparison between the herds with and without an abortion problem was performed. RESULTS: Data of 591 animals from 48 farms was compiled. Thirty-eight herds had an abortion problem. In the affected farms, a maternity pen was used significantly more often and cattle were significantly more frequently housed in free stalls when compared to farms without an abortion problem (p < 0.05). The number of animals serologically positive for Coxiella was significantly higher in herds with abortion problems (p = 0.03). No significant difference was found between the occurrence of antibodies against Listeria and Neospora caninum in herds with or without abortion problems, respectively (p > 0.05). CONCLUSIONS: It was identified that in addition to infectious agents, management/husbandry factors that promote the spread of disease are a risk factor for abortion in dairy cattle. Besides taking steps against pathogens, farms with abortion problems have to implement measures to minimize the spread of infection between animals. A critical control point is likely to be the maternity pen.

Characteristics and analysis of uterine electromyographic activity in pregnant cows.

The electromyographic activity of the cow uterus in the last trimester of pregnancy was investigated. The investigation was performed on 12 animals and the electrical activity was recorded in the last trimester of pregnancy during 11 different periods until the delivery. The duration of the action potential bursts (APB) recorded during the first 7 periods, was small. It did not exceed 2 seconds. A significant increase, however, was recorded at 7 to 9 days before the labor and it involved all the investigated areas in the uterus. The number of APB of the gravid horn was significantly higher than that recorded at other locations in the uterus with the exception of the day of delivery. A significant correlation was found between the number of APB and the level of magnesium in blood serum. The levels of 17-beta estradiol and progesterone were similar during all studied periods with the exception of the last week, in which a dramatic fall in estradiol level and a significant increase in the progesterone concentration were observed. The results showed that it is possible to distinguish three different phases of electric activity in the cow uterus during the last trimester of pregnancy. The features of these phases were discussed.

The effect of detomidine hydrochloride on the electrical activity of uterus in pregnant mares.

The effect of detomidine on the electrical activity of the uterus was studied during the last trimester of pregnancy in 6 mares. The effect was observed in 3-5 min after the i.m. injection and it lasted for 50-70 min. 20 and 40 micrograms/kg b.w. doses of detomidine decreased the myometrial electrical activity, whereas 60 micrograms/kg dose did not have any effect on the activity. The results suggested that 20, 40 and 60 micrograms/kg b.w. doses of detomidine can be administered to mares during the last trimester of pregnancy without the risk of abortion induced by increased uterine electrical activity.

[Strategic hormonal reproductive programs in cows. Part 1: overview, Ovsynch and its modifications]. / Strategische hormonelle Fruchtbarkeitsprogramme bei Kühen. Teil 1: Übersicht, Ovsynch und seine Modifikationen.

INTRODUCTION: Description of different hormonal reproductive programs in cows. MATERIAL AND METHODS: Literature review. RESULTS: The underlying physiological principles and the implementation of hormonal programs based on the use of prostaglandins and combinations of prostaglandins with GnRH analogues are presented. In this context, the advantages and disadvantages of each program are explained. Based on the Ovsynch program, its modifications, Cosynch, Select Synch, Heatsynch and Ultrasynch, are described. CLINICAL RELEVANCE: The description of the different reproductive programs is designed to assist the attending veterinarian in choosing the optimum strategy for each farm.

[Strategic hormonal reproductive programs in cows. Part 2: Presynchronization and resynchronization]. / Strategische hormonelle Fruchtbarkeitsprogramme bei Kühen. Teil 2: Präsynchronisation und Resynchronisation.

OBJECTIVE: Description of hormonal presynchronization and resynchronization methods in cattle. MATERIAL AND METHODS: Review. RESULTS: As the pregnancy rate of cows treated with the Ovsynch program primarily depends on the stage of the oestrous cycle at the time of program initiation, different methods of presynchronization based on the use of PGF2α and combinations of PGF2α and gonadotropin- releasing hormone analogues have been developed. An additional problem includes the earliest possible insemination of open cows following the Ovsynch program. As a solution to this problem a post- insemination treatment of all animals seven days before the scheduled date of the pregnancy examination is recommended.If a cow is found not to be pregnant by the pregnancy examination, it will be treated with PGF2α to enable a further insemination as soon as possible thereafter. CLINICAL RELEVANCE: By utilizing presynchronization and resynchronization programs, the reproductive performance of a dairy herd can be increased.

Current trends in follow-up of trophoblastic function in ruminant species.

During the pregnancy of ruminants, different hormones and proteins are secreted by placenta or corpus luteum allowing the follow up of gestation. Among them, progesterone (P4) and pregnancy-associated glycoproteins (PAG) were proposed as laboratory tools to establish or to confirm pregnancy diagnosis. In last years, PAG assay also provided useful information for researchers working in programs focused on the follow up of trophoblastic function. Concentrations of PAG appeared as altered after the use of embryo biotechnology (in vitro fertilization, cloning by nuclear transfer, inter-specific pregnancies), according to nutritional status of pregnant females (overnourished or undernourished), or consecutive to infectious diseases leading to pathologies affecting the pregnancy in cows (Actynomyces pyogenes and Neospora caninum) and goats (Toxoplasma gondii, Listeria monocytogenes and Trypanosoma congolense). As well, in numerous studies, the association of repeated ultrasound examinations with P4 and PAG determinations allowed a better understanding of mechanisms related to embryonic and fetal mortalities: failure after artificial insemination or embryo transfer techniques, large offspring syndrome after in vitro fecundation and cloning.