Abnormalities of foetal fluids in sheep: two case reports

SUMMARY Two cases of abnormal accumulation of foetal fluids in sheep are described. In the first case, a ewe, at 64 days of pregnancy, had 2 sacs of fluid, one at each of the tips of the pregnant and non-pregnant horns. Neither sac contained a foetus. The composition of the fluids differed from those of the amniotic and allantoic fluids associated with a single foetus. The composition resembled that of foetal plasma. We conclude that the 2 sacs were amniotic sacs from conceptuses that had been resorbed, and that at this early (0.4) stage of pregnancy, amniotic fluid can accumulate in the absence of a viable foetus.
The second case was a rare case of hydrallantois in a ewe at 100 days of pregnancy. The volume of allantoic fluid (1800 mL) was more than 10 times normal. The composition was normal, except for high chloride and calcium concentrations and a very low creatinine concentration. The foetus and the volume and composition of the amniotic fluid were normal. The hydrallantois might have occurred as a result of some alteration in function of the allantoic membrane, perhaps as a result of altered hormonal status of the ewe.     

Anatomy, physiology and pathology of the amniotic and allantoic compartments in the sheep and cow

In both cows and sheep the allantoic fluid is confined to 2 sacs connected by a flattened isthmus, one in the non-pregnant horn and one in the upper part of the pregnant horn. The chorion encloses the amniotic and allantoic compartments, forming the amniochorion and chorioallantois, respectively. In the last third of gestation the compositions of both amniotic and allantoic fluids differ substantially from each other and from those of foetal urine, and maternal and foetal plasma. There is less variation in composition than in volume for a given gestational age. Abnormalities of volume are more common in cows than sheep, and hydrallantois is more common than hydramnois. Data obtained from both physiological experiments and pathological cases suggest that the foetal membranes play an important role in the regulation of composition and volume of foetal fluids. Evidence is presented that the permeability of the membranes to various solutes, as well as their capacity to produce and respond to a number of hormones, can affect the foetal fluid composition and/or volume. Progesterone, oestrogens and prolactin are some of the hormones known to affect foetal fluids. Foetal adrenal insufficiency has been associated with hydramnios implying a lack of hormones from this gland in this disease. The changes in allantoic fluid composition from normal to that closely resembling maternal/foetal extracellular fluid, in hydrallantois, suggests an alteration of membrane function as an aetiology and the continued production of fluid, after removal of the foetus in some cases, favours this hypothesis.     

Chemotherapy during pregnancy: a review of the literature

Although diagnosing cancer during pregnancy is uncommon in veterinary medicine, when it occurs, chemotherapy may represent a reasonable treatment option. A major consideration is that physiological changes associated with pregnancy affect drug pharmacokinetics and complicate correct dosing of chemotherapy agents. Additionally, most antineoplastic drugs are able to cross the placenta thus adversely affecting the foetus. However, favourable outcomes have been observed in human beings when chemotherapy has been administered after organogenesis. Conversely, chemotherapy should be avoided during the early embryonic and organogenesis periods as it might lead to foetal death and/or major malformations.     

The host-parasite relationship in bovine neosporosis

Infection with the protozoan parasite Neospora caninum is thought to be a major cause of reproductive failure in cattle worldwide. Cattle infected with the parasite are three to seven times more likely to abort compared to uninfected cattle. The parasite may be transmitted to cattle through the ingestion of oocysts that are shed in the faeces of acutely infected dogs (definitive host of N. caninum) or by congenital infection from mother to foetus via the placenta. Interestingly, transplacental transmission can occur over consecutive pregnancies and congenitally infected heifers can transmit the parasite to their own offspring. This repeated vertical transmission observed in naturally infected cattle suggests that cattle do not easily develop effective immunity to the parasite, presenting a significant challenge to the development of a control strategy based on vaccination. Neosporosis is a disease of pregnancy and studying the bovine maternal and foetal immune responses during pregnancy will help us to understand the change in the balance between the parasite and the host that may result in disease of the foetus. Studies in non-pregnant cattle and in murine models of infection have shown the importance of T-helper 1-type immune responses involving pro-inflammatory cytokines, such as IFNgamma and IL-12, in limiting intracellular multiplication of the parasite. During pregnancy, changes occur in the immune system allowing the mother to accept the foetal allograft. Research in other species has stressed the crucial role of T-helper 2-type cytokines at the materno-foetal interface in maintaining the pregnancy and regulating the potentially damaging effect of Th-1 responses. Studies in cattle have shown that cell proliferation and IFNgamma responses may be significantly down-regulated around mid-gestation. This may mean that cattle are less able to cope with N. caninum infection at this time and are more likely to transmit the parasite to the foetus. Another important factor is the gestational age and hence immuno-competence of the foetus at the time of infection. Early in gestation, N. caninum infection of the placenta and subsequently the foetus usually proves fatal, whereas infection occurring in mid to late pregnancy may result in the birth of a congenitally infected but otherwise healthy calf. Studies of foetal immune responses have shown that at 14 weeks of gestation, lymphocytes only respond to mitogen, while by 24 weeks (mid-gestation), they respond to antigen by proliferating and releasing IFNgamma. Clearly, there are several factors influencing the outcome of N. caninum infection in pregnancy: the timing, quantity and duration of parasitaemia, the effectiveness of the maternal immune response and the ability of the foetus to mount an immune response against the parasite. The challenge is to design a vaccine that will prevent foetal infection by N. caninum. This is likely to involve a fine balancing act with the immune system that will allow intervention in a manner that will tip the host-parasite balance in favour of the host without compromising the pregnancy.     

Aspects of prenatal development of muscle and adipose tissue: principles, regulation, and influence of maternal nutrition

During pregnancy the developing embryo/foetus is completely dependent on the supply with nutrients and the removal of metabolic by-products through the maternal organism. Therefore, each lasting inadequate nutrient supply may have serious consequences for foetal development. As a kind of "nutritional programming" resulting adaptive changes may be maintained until or manifested at adult age. Intrauterine growth retardation (IUGR) may cause problems in animal health and result in poor animal performance. The relationships between prenatal development and the postnatal phenotypic appearance of muscle and fat are insufficiently investigated. The present paper provides selected aspects of the prenatal development of skeletal muscle (myogenesis) and adipose tissue (adipogenesis), refers to the importance of interactions between both tissues and is focussed on the influence of maternal nutrition on these processes.     

实时B型超声扫描在绵羊妊娠监测中的应用

随着超声诊断技术的不断发展，它在兽医领域的应用也越来越广泛。目前，B型超声主要用于对动物妊娠期子宫和胎儿的监测，关于牛、羊、猪、马、犬、等动物都有应用B型超声监测妊娠的大量报道。本文仅就B型超声在绵羊早期妊娠诊断、怀胎数的判断及判断胎龄三方面进行综述。     

Foetal anasarca in Awassi sheep

Two anasarcous foetuses of Awassi sheep are described. The foetuses were removed from the dams by caesarean section because of dystocia due to failure of cervical dilation. Uterine incision was made in situ because uteri were so distended they could not be brought out from the site of incision. Large quantities of uterine fluids and abnormal thick placentas were found. One foetus weighed about 7 kg and the other 13 kg. The foetal heads were deformed: the upper jaw was prognathic and the left ear of the small foetus was cystic. Necropsy revealed subcutaneous musculature was soft and flabby and abdominal and thoracic cavities contained serosanguinous fluid. Histopathological examination revealed that only the larger foetus had focal aggregates of basophilic nucleated red blood cells and scattered megakaryocytes in the liver. We conclude that anasarca can occur in Awassi sheep, with and without associated extramedullary haematopoiesis.     

Experimental Infection of Cows with Bovine Viral Diarrhoea Virus in Early Pregnancy – Findings in Serum and Foetal Fluids

Nineteen pregnant cows were experimentally infected with bovine viral diarrhoea virus (BVDV) between day 74 and 81 of pregnancy. All cows became infected and developed serum antibodies. Sixteen of the cows delivered persistently infected (PI) offspring, whereas the remaining three gave birth to calves with detectable serum antibodies and free from BVDV. The 16 cows with PI foetuses developed higher levels of antibodies in serum during pregnancy than did their three peers carrying non‐PI calves. Multivariate analysis showed that the antibody levels in these two groups of cows were significantly different from day 135 of pregnancy. Foetal fluid was successfully collected from 18 of the 19 infected cows and from five uninfected control cows between 10 and 24 days before delivery by use of a percutaneous, blind puncture technique. No negative effects were observed in the cows or their offspring. BVDV was isolated and detected with an immunoperoxidase test in foetal fluid from 13 of the 16 cows carrying PI foetuses, and from 15 of the cows when a quantitative fluorescent polymerase chain reaction (PCR) technique was used. The negative sample in the PCR assay was positive for BVDV antibodies. The number of viral copies per microlitre in foetal fluids varied between 103 and 1080 in the positive samples. All samples taken from the cows carrying non‐PI foetuses were negative for BVDV in both assays. In this experiment, examination of either serum or foetal fluids could identify the cows carrying a PI foetus. Examination of serum for BVDV antibodies was a reliable indicator of a PI foetus if the serum was collected during the last 2 months of pregnancy. For examination of foetal fluids, both viral and serological analyses should be performed. For viral analysis, PCR should be the test of choice. High levels of BVDV antibodies in conjunction with a negative result in the PCR may be indicative of a false‐negative virus result. Further experience with the method of collection of foetal fluids is necessary for evaluation of its safety. Investigation of pregnant cows in order to discover a PI offspring before it is born could be a useful tool in control and eradication of BVDV.     

Experimental infection of cows with bovine viral diarrhoea virus in early pregnancy - findings in serum and foetal fluids

Nineteen pregnant cows were experimentally infected with bovine viral diarrhoea virus (BVDV) between day 74 and 81 of pregnancy. All cows became infected and developed serum antibodies. Sixteen of the cows delivered persistently infected (PI) offspring, whereas the remaining three gave birth to calves with detectable serum antibodies and free from BVDV. The 16 cows with PI foetuses developed higher levels of antibodies in serum during pregnancy than did their three peers carrying non-PI calves. Multivariate analysis showed that the antibody levels in these two groups of cows were significantly different from day 135 of pregnancy. Foetal fluid was successfully collected from 18 of the 19 infected cows and from five uninfected control cows between 10 and 24 days before delivery by use of a percutaneous, blind puncture technique. No negative effects were observed in the cows or their offspring. BVDV was isolated and detected with an immunoperoxidase test in foetal fluid from 13 of the 16 cows carrying PI foetuses, and from 15 of the cows when a quantitative fluorescent polymerase chain reaction (PCR) technique was used. The negative sample in the PCR assay was positive for BVDV antibodies. The number of viral copies per microlitre in foetal fluids varied between 103 and 1080 in the positive samples. All samples taken from the cows carrying non-PI foetuses were negative for BVDV in both assays. In this experiment, examination of either serum or foetal fluids could identify the cows carrying a PI foetus. Examination of serum for BVDV antibodies was a reliable indicator of a PI foetus if the serum was collected during the last 2 months of pregnancy. For examination of foetal fluids, both viral and serological analyses should be performed. For viral analysis, PCR should be the test of choice. High levels of BVDV antibodies in conjunction with a negative result in the PCR may be indicative of a false-negative virus result. Further experience with the method of collection of foetal fluids is necessary for evaluation of its safety. Investigation of pregnant cows in order to discover a PI offspring before it is born could be a useful tool in control and eradication of BVDV.     

Effects of dietary L‐arginine supplementation to early pregnant mares on conceptus diameter—Preliminary findings

The importance of the amino acid L‐arginine (ARG) for conceptus growth and litter size has been demonstrated in various species. L‐arginine is part of embryo‐derived polyamines, a substrate for nitric oxide synthase and stimulates protein synthesis by the embryo. In the present study, we have investigated whether dietary L‐arginine supplementation stimulates early conceptus growth in mares. Warmblood mares with singleton pregnancies received either an arginine‐supplemented diet (approximately 0.0125% of body weight, n = 12) or a control diet (n = 11) from days 15 to 45 after ovulation. Diameter of the embryonic vesicle (days 14, 17, 20 of pregnancy) and size of the embryo respective foetus (length and maximal diameter, days 25–45 of pregnancy at 5‐day intervals) were determined by transrectal ultrasound. At foaling, weight and size of the foal and the placenta were determined. Blood for determination of equine chorionic gonadotrophin (eCG) and progestin concentrations was collected repeatedly. Neither eCG nor progestin concentration in plasma of mares differed between groups at any time. No effects of arginine treatment on diameter of the embryonic vesicle between days 14 and 20 of pregnancy were detected. Diameter of the embryo/foetus on days 40 to 45 of pregnancy strongly tended to be enhanced by arginine supplementation (p = 0.06). Weight and size of neither the foal nor placenta at birth differed between groups. In conclusion, L‐arginine supplementation was without negative effects on early equine embryos and may support embryonic growth at the beginning of placentation.     

Ultrasonographic examinations of fetal growth of sheep between day 20 and day 50 of gestation

Previous studies on embryonic and fetal growth in sheep were mostly transversal using animals killed at various stages of gestation. Until now it was difficult to monitor the development of individual embryos/foetuses during pregnancy, especially during the first and second pregnancy month. Real-time ultrasound as a non invasive method could be an appropriate method for examination of embryonic and early foetal development in sheep. The aim of this study was to determine the embryonic and foetal development of the crown-rump-length (CRL) in pregnant ewes in relation to the number of fetuses and/or the breed. Between the 20th and 50th day of pregnancy the embryos/foetuses showed an exponential growth which can be best described by the equation of the form CRL (mm) = W * exp (k * day of pregnancy). The individual variability in embryofetal growth is in part due to the number of embryos per sheep and the sheep breed.     

Effect of decorin and selected glycosylation inhibitors on the adhesion of caruncular epithelial cells of pregnant cows—part I

Adhesion process ensures the formation of the appropriate connection between mother and foetus during placentation and further placental development, which determines physiological pregnancy course. Extracellular matrix of foetal membranes are a rich source of biologically active proteins, the synthesis of which is regulated by hormones. Depending on the stage of pregnancy, the protein profile of the placenta changes, thanks to which its remodelling is possible. The aim of the study was to evaluate the effect of decorin, as well as selected glycosylation inhibitors on the adhesion of caruncular epithelial cells derived from cows during pregnancy. Placental cells were isolated from healthy, pregnant (2nd and 4th month) cows after slaughter, which allowed for the establishment of 4 primary cell cultures without visible cells of fibroblast morphology. The presence of decorin in cell monolayer and cell lysates was determined by the use of immunocytochemistry and Western blotting, respectively. The viability of cells was evaluated by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. Protein N-glycosylation and O-glycosylation have a modulating effect on the adhesion and viability of placental cells during early–mid pregnancy. Decorin and tunicamycin were shown to have anti-adhesive properties with respect to caruncular cells of the pregnant bovine uterus.     

Serial investigations of early pregnancy in pony mares using real time ultrasound scanning

The uteri of 13 pony mares were examined daily by ultrasound during the first two months of gestation. The conceptus was first identified between 12 and 16 days after ovulation and the embryo was seen on the ventral surface of the conceptus after Day 21. The foetal heart could be visualised after Day 22 and spontaneous movement of the foetus occurred after Day 39. No consistent pattern was seen in the development of the foetal membranes, although attachment of the umbilical cord to the allantochorion was always on the dorsal aspect of the conceptus. Daily measurements were made of the diameter of the conceptus and the length of the embryo and foetus.     

High Periconceptional Protein Intake Modifies Uterine and Embryonic Relationships Increasing Early Pregnancy Losses and Embryo Growth Retardation in Sheep

The effects of supplemented protein level (PL) during the periconceptional period and their interaction with body condition were evaluated in sheep. Multiparous Rambouillet ewes (n = 12) received two PL of rumen undegradable protein (UIP) during a 30‐day pre‐mating and 15‐day post‐mating period: low [LPL, 24% crude protein (CP), 14 g UIP and 36 g/CP animal/day] and high [HPL, 44% CP, 30 g UIP and 50 g/CP animal/day]. While ovulation rate (OR) did not differ between treatments (1.6 ± 0.5, mean ± SEM), a lower fertility rate, a decreased embryo number and a reduced uterine pH (UpH) was observed in the HPL group (p < 0.05), irrespective of BC. Luteal tissue weight, volume and progesterone secretion did not differ among treatments. Sheep with lower UpH also had lower conceptus weight (Cwt; p < 0.05, r = 0.65) and conceptuses with lower mass tended to secrete less INF‐τ and IGF‐1, and the correspondent endometrial explants had a higher basal PGF_2α release. Current study indicates that high protein diets during the periconceptional period in sheep modify uterine and embryonic relationships, increasing early pregnancy losses and inducing embryo growth retardation. Surviving embryos were affected by weight reductions, which could compromise later foetal growth and birth weight. Results evidence the key role of a balanced diet in reproductive success and indicate that the quality and nutrient composition of the maternal diet are essential for an adequate establishment of pregnancy, having paramount effects on the interplay of the embryo and the uterus.     

Teratogenicity of a mutagenised Rift Valley fever virus (MVP 12) in sheep

A 5-fluorouracil mutagenised Rift Valley fever virus strain, which was shown to be attenuated and immunogenic in cattle and sheep, was evaluated for its ability to cause teratogenic effects in pregnant sheep. A group of 50 sheep at various stages of pregnancy was inoculated with the virus and the pregnancies followed to term. There were two abortions and 14% of the lambs produced by vaccinated ewes showed teratogenic effects, the most prevalent being spinal hypoplasia, hydranencephaly, brachygnathia inferior and arthrygryposis. The foetal malformations of the central nervous and musculo-skeletal systems were mostly consistent with those observed in sheep vaccinated with the attenuated Smithburn RVF strain. The teratogenic effects of MVP12 were not seen in previous experiments by other authors as immunisation of sheep took place in the second to third trimester of pregnancy, when the foetal brain tissue has completed most of its cell division.     

The effect of an intrauterine viral infection on the skin follicles of sheep

Border disease of sheep is a transplacental viral infection of the foetus. In medium and fine fleeced breeds, the most consistent of the pathological changes is permanent primary follicle enlargement, first apparent at 115 days gestation and accompanied by increased fibre size and frequency of fibre medullation. A tendency for fewer secondary follicles to develop in Border disease is probably an indirect consequence of infection.Primary follicles are only susceptible to the effects of Border disease following maternal inoculation with standard inoculum up to 80 days gestation. The subsequent resistance of primary follicles may be related to a loss of susceptibility with differentiation or, more probably, to the development of a foetal immunological response to the agent.In coarse fleeced breeds, the skin appears to be unaffected by Border disease. It may be that Border disease de-inhibits follicle growth limiting mechanisms in finer fleeced breeds, causing reversion to a more primitive, coarser fleece type.     

In utero programming of sexually differentiated gonadotrophin releasing hormone (GnRH) secretion

It has long been recognised that steroids can have both organisational and activational effects on the reproductive neuroendocrine axis of many species, including the sheep. Specifically, if the ovine foetus is exposed to testosterone during a relatively short 'window' of in utero development (from approximately day 30-90 of a 147 day pregnancy) the neural mechanisms regulating gonadotrophin releasing hormone (GnRH) secretion become organised in a male-specific manner. In post-natal life the consequences of foetal androgen exposure are sexually differentiated responses of the GnRH neuronal network to activation by factors such as photoperiod and ovarian steroid hormones. Studies in the gonadectomized lamb have demonstrated that elevated concentrations of oestrogen (E) are unable to trigger a preovulatory-like GnRH surge in the male and the androgenized ewe lamb. Further, these animals have markedly reduced sensitivity to the inhibitory actions of progesterone on tonic GnRH release compared with normal ewes. The reasons for these abnormal steroid feedback mechanisms may reside in sexually dimorphic inputs to the GnRH neurone, including those from oestrogen-receptive neurones in the arcuate nucleus that synthetize the neuropeptide, neurokinin B (NKB). The consequences of in utero androgen exposure are reflected in a progressive and dramatic impairment of fertility in the ovary-intact ewe.     

Arginine for gestating sows and foetal development: A systematic review

The use of functional amino acids during pregnancy has been linked to improved reproduction in mammals. In this context, arginine is a precursor in the synthesis of numerous molecules, such as nitric oxide and polyamines, which play an important role during reproduction. However, contradictory studies are found in the literature, particularly regarding the amount of supplementation and the period of pregnancy in which it is used. The objective of this study was to evaluate the effects of dietary arginine supplementation for pregnant sows on foetal development via a systematic review. The search for papers was performed during the month of December 2015, in the databases ISI Web of Science, Science Direct, Scopus, and SciELO. From a total of 5675 returned studies, only 13 papers were selected after applying selection criteria. Most (47%) of the studies that evaluated the effects of dietary arginine supplementation on foetal development in pigs used 1% arginine. Supplementation was initiated in the first third of pregnancy in 47% of tests, including in both primiparous and multiparous sows. These studies showed positive results for embryo survival and foetal development, evidenced by the increase in placental weight and the number and weight of piglets born alive. Of all evaluated studies, 53% showed benefits on foetal development. It is concluded that supplementing dietary arginine in gestating sows can benefit embryo survival and foetal development. However, to establish a supplementation plan with this amino acid, aspects related to the period of pregnancy, supplementation levels, and source of arginine must be well defined.     

Effect of Gestational Age and Fetal Position on the Possibility and Accuracy of Ultrasonographic Fetal Gender Determination in Dairy Cattle

The possibility and accuracy of transrectal ultrasonographic determination of foetal gender were studied by identifying and locating the genital tubercle, scrotum or mammary gland in 15 dairy cows at 49-168 days post-breeding. Ultrasound examinations were performed weekly. Each examination was made without references to the results of the examinations in the previous weeks. At each examination, an attempt was made to view the foetus in frontal, cross-sectional, and sagittal planes. The expected foetal gender was compared with that observed at birth. Gestational age affected significantly the possibility of foetal gender determination. The possibility of foetal gender determination increased from 26.7% at day 49 to 100% at day 56 of gestation. Between days 56 and 98, foetal gender determination was possible in all cases. Thereafter, the ability to determine foetal gender decreased with development of pregnancy and was impossible in all cases by days 161 and 168. The overall accuracy of foetal gender determination was 97.3%. Foetal gender determination between days 49 and 63 depended on the location of the genital tubercle in a frontal view of the foetus. By day 70, all foetal positions could be used for foetal gender determination. However, by progress of gestation, the usefulness of frontal view for sex determination decreased, while that of cross-sectional view increased. Accordingly, the best time for foetal gender determination in dairy cattle is between days 56 and 98 of pregnancy.