Diagnosis of endometritis and cystitis in sows: use of biomarkers
A. Grahofer1,2,3, S. Sipos4 and W. Sipos11Clinic for Swine, VetmedUni Vienna, Austria; 2Animal Physiology, Institute of Agricultural Sciences, ETH Zurich, Switzerland;
3Clinic for Swine, Vetsuisse Faculty, University of Bern, Switzerland; 4Veterinary Practice Entenfellner, Stössing, Austria Email: alexander.grahofer@usys.ethz.ch
Introduction Reduced fertility in sows, often caused by urinary tract infection (UTI), plays a major role in sow mortality and their replacement, thus causing economic losses to piglet producers. The aim of this study was to analyse the prevalence of UTI in so ws with reproductive failure and to compare different sample sites and methods to diagnose UTI. Moreover the bacteriological aetiology of cystitis in sows located in Lower Austria and the correlation with urine analysis data was examined.
Materials and Methods Thirty-nine sows with reproductive disorders from 9 farms in Lower Austria were sampled. Midstream urine was examined macroscopically and physically-chemically one day before slaughter. Another urine sample was taken under sterile conditions directly out of the urinary bladder immediately after slaughter. Furthermore, swabs were taken of bladder and uterus mucosa. A bacterial investigation of all four matrices was conducted.
Results More than three fourths (84.6%) of the sows showed a UTI (Fig.1). Nearly identical culture results were obtained from urine collected immediately after slaughter and from swabs of bladder mucosa. Samples obtained after slaughter most often yielded pure cultures of one bacterium, whereas midstream urine samples often led to isolation of two or more different bacterial species (Fig.2).
The most frequently detected bacteria were Escherichia coli in 64.1% of the samples, α-hemolytic streptococci in 34.6% and Staphylococcus hyicus in 21.8%. No statistically significant correlation between the physical and chemical parameters and the bacteriological results were found.
Figure 1 Overview of most frequently detected bacteria in Figure 2 Differences between the number of bacteria per the collected samples. sample and the used sample.
Conclusions In this study also results with low quantity of bacterial agents (+ growth compared to ++ and +++ growth) were considered. So it was difficult to interpret culture results for midstream urine samples, likely due to environmental contamination.
Therefore, any utilization of a similar strategy of interpretation in practice may lead to an overestimation of UTIs. Apart from this, pathogens not specific for UTI were found and might have led to reproductive disorders in the sow herds. It was concluded that sterile urine or swabs from the bladder of culled sows should be tested, if there are problems with the genitourinary system, i.e. presumption of UTIs as a herd problem.
Session 12: Disease aspects in reproduction
Metabolism and host-pathogen interactions shape the development of uterine disease in dairy cows
I. M. SheldonSwansea University Medical School, Singleton Park, Swansea, United Kingdom Email: i.m.sheldon@swansea.ac.uk
Multiple pathogenic bacteria are present in the uterus of cattle after parturition. Controlling these bacteria to maintain uterine health depends on a robust immune response to resist the pathogens, and the ability to tolerate the damage that pathogens cause (Sheldon et al, 2019). Unfortunately, metabolic stress impairs resistance and tolerance, and 20 to 40% of modern dairy cattle develop some form of postpartum uterine disease.
Resistance to pathogens in the uterus is highly dependent on innate immunity, which relies on receptors on host cells binding to pathogen-associated molecular patterns, such as bacterial lipopeptides and lipopolysaccharide. Activation of innate immunity leads to the secretion of inflammatory mediators, including chemokines and cytokines. These inflammatory mediators attract and regulate the neutrophils that clear pathogens and damaged cells from the endometrium. Of course, neutrophils and damaged cells form the pus that is the cardinal sign of postpartum uterine disease.
The innate immune response scales to match the severity of the pathogen challenge and tissue damage. However, innate immunity is metabolically demanding, and glucose and glutamine are important to fuel the inflammatory response in the endometrium.
Conversely, metabolic stress, caused by limiting glucose or glutamine, impairs the ability to mount robust inflammatory responses.
The close integration of immunity and metabolism is highlighted by the regulation of endometrial inflammatory responses by AMP-activated protein kinase (AMPK), which is the most fundamental sensor of cellular energy homeostasis (Turner et al, 2016).
Tolerance is the ability to cope with the presence of pathogens by limiting the tissue damage they cause. Tissue damage in the endometrium is caused by peripartum trauma and by pore-forming toxins secreted by a wide range of pathogenic bacteria. For example, Trueperella pyogenes secretes pyolysin, which is a cholesterol-dependent cytolysin that forms pores in cholesterol-rich areas of the plasma membrane of cells. The impact of pore-forming toxins is dependent on the type of cell, with endometrial epithelial cells more tolerant to pyolysin than stromal cells. However, cell metabolism also modulates tolerance, with glucose or glutamine deprivation impairing the ability of cells to tolerate cholesterol-dependent cytolysins. Conversely, reducing cellular cholesterol increases cell tolerance to pyolysin, as does inhibiting enzymes in the cholesterol synthesis pathway (Griffin et al, 2018).
In conclusion, postpartum uterine disease is a consequence of compromised resistance and/or failure to tolerate the pathogens in the endometrium. The severity of disease is shaped by the inflammatory response to pathogenic bacteria and the damage pathogens cause.
Metabolic stress increases the risk of uterine disease by impairing innate immunity and tolerance. Enhancing the ability of animals to tolerate pathogens and controlling metabolic stress may help counter the impact of uterine disease on animal health and production.
References
Griffin S, Healey GD, Sheldon IM (2018) Isoprenoids increase bovine endometrial stromal cell tolerance to the cholesterol-dependent cytolysin from Trueperella pyogenes. Biology of Reproduction, 99(4), 749–760
Sheldon IM, Cronin JG, Bromfield JJ (2019) Tolerance and innate immunity shape the development of postpartum uterine disease and the impact of endometritis in dairy cattle. Annual Review of Animal Biosciences, 7, 361-384
Turner ML, Cronin J.G, Noleto PG, Sheldon IM (2016) Glucose availability and AMP-activated protein kinase link energy metabolism and innate immunity in the bovine endometrium. PLOS ONE, 11(3), e0151416
Repeat breeding: The cause and the possible treatment to restore fertility
S. Katagiri, Y. Yanagawa and M. NaganoLaboratory of Theriogenology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan Email: katagiri@vetmed.hokudai.ac.jp
Introduction Poor reproductive efficiency is a worldwide problem that has affected the dairy industry during the last several decades.
In an attempt to explain the changes in reproductive physiology caused by high milk production, a model of elevated steroid metabolism in lactating dairy cows has been proposed. A slow increase in levels and low peak levels of estradiol (E2) and progesterone (P4) characterize endocrine changes in high producing cows. Similar changes have been reported in repeat breeder cows.
The abnormal changes in E2 and P4 concentrations of these cows may cause an improper uterine environment due to disturbed expression of growth factors and cytokines in the endometrium. This presentation focuses on the alteration in epidermal growth factor (EGF) profile in the uterine endometrium during the estrous cycle.
Seeking hidden causes of infertility The reasons for the decline in fertility of modern dairy cows are multifactorial; thus, it is most important to exclude animals with any known causes of infertility when seeking hidden causes of infertility in repeat breeder cows.
The study should exclude cows with any signs of abnormality in the reproductive organs and function. Repeat breeder cows should be examined for inflammation of the uterus by using ultrasonography and endometrial cytology and for oviductal patency. Cows showing extended estrous periods, delayed ovulation and those that fail to show clear signs of estrus should also be excluded from the study. In addition, cows conceived by either 1 or 2 artificial inseminations (AI) immediately after the study might be used as the normal (fertile) control cows. The strict selection policy for recruitment of animals for a study may be essential to enabling the detection of a hidden difference between the repeat breeder and normal cows.
Dairy cows failed to conceive after 3 AIs In our study with about 2,500 cows in 18 farms, 397 cows (16%) failed to conceive after 3 AIs, but we found potential causes of infertility in about half of them (8.0%) after detailed examinations as above. The potential causes are an extended estrous period (9.1%, 36 cows), an irregular estrous cycles (11.6%, 46 cows), delayed ovulation (6.3%, 25 cows), periparturient disorders (4.5%, 18 cows), mastitis and foot diseases (6.3%, 25 cows), urovagina (4.5%, 18 cows), inflammation of the uterus (6.3%, 25 cows), abnormal oviductal patency (1.5%, 6 cows). When cows were treated to the identified causes of infertility, 40 to 66% of them, depending on the causes, conceived by 2 AIs. While we could not find any causes in the other half (198 cows) of cows. They were diagnosed as repeat breeders. We use the same strict selection of repeat breeder cows in all studies.
Endometrial EGF and fertility Growth factors and cytokines in the endometrium form a regulatory network of uterine function. The network is primarily regulated by E2 and P4. EGF is one of components of the network and has been shown to replace E2 action in the uterus. In cows, preimplantation embryos express EGF receptors, but not EGF ligand. This may indicate that EGF acts as a maternal signal to an embryo to support its development or survival. The normal cow has two peaks of endometrial EGF concentrations on Days 2-4 and 13-14. In fertile animals, peak levels of EGF concentrations were higher in heifers than cows and the concentrations decreased as the number of parities increased in Holstein cows. Low concentrations of EGF on these days distinguished both high-producing and repeat breeder cows from normal cows. Alteration of the EGF profile could be found in 70 and 25% of repeat breeder and high-producing Holstein cows between 50 and 90 days postpartum, respectively. In an embryo transfer experiment with 439 recipient cows, those with low endometrial EGF concentrations (5 mg/g tissue weight) on Day 3 showed a lower conception rate (33%) than controls (77%). Size of embryos on Day 16 of pregnancy was smaller in cows with low EGF concentrations on Day 3 than controls with the normal EGF profile. Further, an abnormal EGF profile persisted over the 3 estrous cycles, if cows were not treated.
This abnormality was found less frequently in Japanese Black (JB) repeat breeder cows (about 20%) than Holstein cows. However, many of JB repeat breeder cows with low EGF peaks were with high BCS.
Hormonal treatment to normalize EGF profile and fertility Treatment with a high dose (5 mg) of estradiol benzoate and an intravaginal progesterone-releasing device restored the normal EGF profile and restored fertility. In a controlled study, the treatment normalized the EGF profile in about 70% and restored fertility in about 60% of treated cows. However, efficacy of this treatment protocol differed between farms (25-70%) and decreased in the presence of some factors (high levels of lactation, obesity, heat stress).
Treatment with seminal proteins Recently, we have shown that seminal plasma (SP) contains an activity to normalize the endometrial EGF profile and restore fertility in repeat breeder cows. When SP was infused into the vagina, the concentrations of EGF in the uterus increased in about 50-70% of repeat breeder cows. The activity of SP on the EGF profile differed between bulls, but the activity was consistent between ejaculates from the same bulls. The effect of SP treatment on fertility was consistent between farms.
However, repeat breeder Holsten cows under heat stress and repeat breeder JB cows with high BCS (> 4.0) did not respond to SP treatment as well as hormonal treatment. Interestingly, repeat breeder JB cows became responders to treatment after reducing BCS by 0.75 or greater.
Identification of seminal proteins SP was separated by gel chromatography and SDS-PAGE. The activity was found in protein fractions with molecular weight of 16-29kDa with pI5.8-7.0. This protein fraction contains 14 protein spots on 2D-PAGE. Protein spots were subjected to TOF-MS and 11 protein spots have been identified. One of the 14 protein spots showed the activity to increase the EGF concentrations on Day 3 in a dose-dependent manner in repeat breeder and high-producing cows.
Conclusions We could identify a common abnormality in strictly-selected repeat breeder cows. This abnormality of the EGF profile in the uterine endometrium could be corrected by hormonal treatment and seminal proteins. We have identified seminal proteins in the protein fraction with the activity to normalize the EGF profile. Currently, we are preparing a field study to confirm the activity of the identified seminal proteins. This study will provide a new strategy to deal with repeat breeding in dairy cows.
Session 12: Disease aspects in reproduction
Factors affecting reproductive wastage in beef cattle herds
D. A. Kenny1,3, M. G. Diskin2,3 and P. Lonergan31Teagasc Animal and Bioscience Research Department, Grange Research Centre, Dunsany, Co. Meath, Ireland; 2Mellowes Research Centre, Athenry, Co. Galway, Ireland; 3Agriculture and Food Science Centre, University College Dublin, Belfield, Dublin, Ireland Email: david.kenny@teagasc.ie
Worldwide, a reproductively efficient beef cow herd is fundamental to meeting the protein and specifically, red meat demand of an ever increasing global human population. However, achieving a high level of reproductive efficiency is underpinned by producers recognizing and attaining many key targets throughout the production cycle and requires significant technical competency (Diskin and Kenny, 2014). While not receiving the same degree of attention as for their dairy contemporaries, the impact of poor fertility in beef cattle is arguably of greater economic significance, given that the production of a weaned calf is the primary output from beef cow based enterprises. Following insemination the greatest increment of cow reproductive wastage occurs in the form of early embryo mortality with approximately 80% of this occurring within 14-16 days (Diskin et al., 2016). Coincidently, during this period, embryo development is occurring at an exponential rate and is heavily reliant on its maternal uterine environment (Sánchez et al., 2018).
Although the extent of late embryo loss is numerically lower than early embryo mortality, it causes serious economic losses in all production systems, but particularly in seasonal calving herds because it is often detected after the end of the season breeding period resulting in increased culling rates.
The lifetime productivity of the beef bred female commences from the onset of puberty and is dictated by subsequent critical events including age at first calving, duration of the postpartum interval for each successive calving, conception and pregnancy rate and ultimately manifested as length of intercalving intervals and number of calves weaned over her lifetime. Puberty in heifers is a consequence of the interactive effects of genetics and both pre and post-weaning nutrition. Early onset of puberty is essential to achieving the first main reproductive target for beef cow herds; first calving at two years of age. Our work and others has now clearly shown that nutrition and in particular during calfhood, critically influence the rate of sexual development and in particular the ontogeny of the hypothalamic-pituitary-ovarian axis (Kenny et al., 2018). In calved heifers and mature cows, the onset of ovarian activity, postpartum is a key event dictating the calving interval. Again, this will be the product mainly of prepartum nutrition, manifested through body condition and the strength of the maternal bond between cow and calf, though there is increasing evidence of genetic influence, while modest, on this trait. Following the initiation of postpartum ovarian cyclicity, conception and subsequent pregnancy rate is generally a function of bull fertility in natural service herds and heat detection and timing of insemination in herds bred through AI. Where natural service is practiced producers must be cognisant of the potential for infertility and perhaps more worryingly, subfertility in their stock sires. Breeding females should be maintained on a steady plane of nutrition during the breeding season, but the contribution of significant excesses or deficiencies of nutrients including protein and trace elements is likely to be minor where adequate pasture is available. Many bacterial, viral, protozoan and fungal infections have been associated with early embryo, late embryo, fetal and abortion losses, as well as with perinatal losses in cattle. While the overall impact of pathogens on the extent of reproductive wastage in beef cattle is likely to be small, where such losses do occur, they are often incurred from mid to late gestation; therefore representing a potentially significant economic loss to the producer and highlighting the importance of control measures to prevent infection. Systemic concentrations of progesterone during both the cycle preceding and following insemination affect embryo survival, with evidence of either excessive or insufficient concentrations being negatively associated with survival rate.
Genomic technology will not only provide scientists with an improved understanding of the underlying biological processes involved in fertilisation and the establishment of pregnancy, but also, in the future, could identify genes responsible for improved embryo survival. Indeed, data from our own laboratory have revealed remarkable differences in uterine endometrial transcriptomes of high compared to low fertility heifers during both the mid and late luteal phases of the oestrous cycle. Such information could be incorporated into breeding objectives in order to increase the rate of genetic progress for embryo survival. While, increased efforts are being made internationally to genetically identify and select for more reproductively efficient beef cows, this is a more long-term strategy and will not replace the necessity for a high level of technical efficiency and management practice at farm level. The correction of minor deficits in several areas can have a substantial cumulative positive effect on herd reproductive performance.
References
Diskin M.G. and Kenny DA. 2014. Optimising reproductive performance of beef cows and replacement heifers. Animal. Suppl. 1:27-39.
Diskin MG, Waters SM, Parr MH and Kenny DA. 2016. Pregnancy losses in cattle: potential for improvement. Reproduction, Fertility and Development. 28: 83–93.
Kenny DA, Heslin J and Byrne CJ. 2018. Early onset of puberty in cattle: implications for gamete quality and embryo survival. Reproduction, Fertility and Development. 30: 101–117.
Sánchez JM, Mathew DJ, Passaro C, Fair T, Lonergan P. (2018). Embryonic maternal interaction in cattle and its relationship with fertility. Reprod Domest Anim.
Suppl 2:20-27.
Sensing sperm by maternal immune crosstalk: A potential mechanism for interfering with fertility in the cow
A. Miyamoto1, M. A. Ezz1,2, I. Akthar1, M. S. Yousef1,3, K. Imakawa1,4, M. Shimada1,5 and M. A. Marey1,61Obihiro University of Agriculture & Veterinary Medicine, Obihiro, Japan; 2Mansoura University, Mansoura, Egypt; 3Assiut University, Assiut, Egypt; 4Tokai University, Kumamoto, Japan; 5Hiroshima University, Higashi-Hiroshima, Japan; 6Damanhur University, Behera, Egypt
Email: akiomiya@obihiro.ac.jp
The female genital tract, one of the initial barriers for foreign antigens, orchestrates pivotal functions in reproduction such as sperm transport, fertilization, conceptus implantation, placentation, and maintenance of pregnancy until parturition. Concurrently with these functions, the uterus and oviduct require a defence system against invading pathogens as well as tolerating allogenic sperm and semi-allogenic embryo. Therefore, the uterine and oviduct milieu should be equipped with a well-developed and strictly controlled immune system that can respond effectively to various antigens to which it is exposed.
At artificial insemination (AI), millions of sperm are deposited into the bovine uterus, but most of these sperm are eliminated and only a few thousands reach the oviduct where fertilization takes place. Recently, we demonstrated that live, but not dead, sperm attach to bovine endometrial epithelial cells (BEECs) in vitro which subsequently produce an acute pro-inflammatory response [1]. Using a newly developed ex-vivo uterine explant culture model, it was shown that sperm immediately penetrate into endometrial glands, and activate a local innate immune response, suggesting that endometrial glands are working as a sperm-sensing system which triggers the
At artificial insemination (AI), millions of sperm are deposited into the bovine uterus, but most of these sperm are eliminated and only a few thousands reach the oviduct where fertilization takes place. Recently, we demonstrated that live, but not dead, sperm attach to bovine endometrial epithelial cells (BEECs) in vitro which subsequently produce an acute pro-inflammatory response [1]. Using a newly developed ex-vivo uterine explant culture model, it was shown that sperm immediately penetrate into endometrial glands, and activate a local innate immune response, suggesting that endometrial glands are working as a sperm-sensing system which triggers the