New factors to consider before neutering your dachshund – reducing the risk of intervertebral disc disease

The UK’s dachshund population is increasing rapidly, with a 23% rise in 2019. This can be attributed to their presence throughout social media, television, and celerity ownership. As with any breed, the affectionately known sausage dog comes with its own list of risks and complications that owners and breeders should be aware of. One such disease is Intervertebral Disc Herniation (IVDH), a condition that affects the cartilaginous discs in the spinal column. The disease has several risk factors, but with 54% of dog owners in the UK choosing to the neuter their pet, it is important to assess what risk factor, if any, neutering poses on the likelihood of dachshunds developing IDVH.

What is IVDH?  

The dachshunds’ spinal column includes the bony vertebrae, and in between each body is a cushioning disc made up of a fibrous covering and a jelly like centre, with a total of 26 discs throughout the column. Over time the disc becomes less supple and its ability to withstand and absorb every day compressive forces reduces. The outer disc becomes hardened, with a gradual inhibition of cartilage cell production and deposition, the jelly centre, called the nucleus pulposus, is at higher risk of rupturing and herniating (protruding from its normal place). When this herniation due to disc degeneration occurs, it is classified as Hansen Type 1-disc disease, or IVDH. IVDH can vary in severity depending on the level of injury to the spinal column ranging from affected dogs suffering temporary or permanent gait abnormalities to being left partially or fully paralysed. Some cases can be fatal, as the herniated disc can cut the spinal cord.

The process of disc degradation can begin between 8-24 months of age, but there are factors than can influence and speed up this process. Dachshunds are the most likely breed to incur obesity, which puts excessive weight loads onto their small frame, compressing the discs and increasing the likelihood of IVDH.

What does neutering have to do with the spine?

Oestrogen, a hormone produced in the ovaries of bitches and the adrenal gland and testes of male dogs, influences blood fat levels, enzyme production and salt balance in both males and females. Testosterone in both sexes has a role in producing red blood cells, as female dogs can produce testosterone in small quantities too. Oestrogen plays a large role in protecting cartilage by encouraging the deposition of collagen and aggrecan, a protein that is vital in the health and strength of the nucleus polposus, the centre of the disc.  The neutering of both male and female dogs removes the largest production sources for these hormones reduces the available hormones to fulfil and maintain these processes, and the depth of this effect has been the focus of several studies.

Linking the sausage

Recently, a study of 1964 Dachshunds took place to determine the relationship between IVDH incidences and neutering. The group included dogs ranging from less than 12 months old, to dogs up to 15 years old. The study population included the 6 variations within the Dachshund species, with 1051 male and 951 participants, male and female retrospectively. From that, 672 males and 555 females Dachshunds had been neutered before 12 months of age, while the remaining participants had been neutered after this age. The study completed a statistical analysis to calculate the years that the animal would be at risk of experiencing a case of IVDH, using the time frame between 36 months of age and 10 years, and an average risk of first incidence between 5.4 and 11.4% per year.    

The risk of IDVH was significantly higher in neutered females than entire females, and those neutered before 12 months of age were twice as likely to develop it than late-neutered bitches. In males, the findings were similar in that those neutered before 12 months of age were at higher risk of an incidence of IDVH than those neutered after that time. This large study emphasises results of previous studies that suggest that removing the reproductive systems in females before their first ‘season’ can have detrimental effects on several systems and restricting the release of the respective hormones, which are then unable to perform protective and encouraging processes on the discs, which in turn would reduce the incidence rate of IDVH. Neutering was also found to influence body condition, with neutered males in the study being significantly more likely to be overweight than entire males, this difference was seen less significantly in females. This finding highlighted a relationship between neutering and obesity that had not been firmly consolidated in previous studies

The future of neutering  

The study did have certain limitations, mainly stemming from the data collection method: a survey. Many owns are not likely to report their pets’ true condition score, with only 6.6% of owners reporting their dogs were overweight or obese, which does not reflect previous studies performed in a standardised way. However, this particular study is representative of progression in the topic area as studies done previously did not look at the effects of neutering on each specific gender, and didn’t eliminate cases of IVDH that had occurred after neutering, which would rule it  out as a causative factor.

Overall, most animals included in the study were diagnosed with a case of IVDH between 2-8 years, with lowest incidence frequency in entire bitches, and highest case incidence in early-neutered bitches. Researchers of this study concluded that along with assessing factors such as unplanned mating and hormone related aggressions, the age at which to neuter the dachshund will have a strong influence on the development of IVDH. Breeders and owners should combine the information of the study with guidance from a veterinarian when discussing neutering.

This article was based on the following research article:

Dorn, M. Seath, I 2018, ‘Neuter status as a risk factor for canine intervertebral disc herniation (IVDH) in dachshunds: a retrospective cohort study’, Canine Genetics and Epidemiology, vol. 5.

Reference List

Agrawal, A, Guttapalli, A, Narayan S, Albert, TJ, Shapiro, IM & Risbud, MV 2007, ‘Normoxic stabilization of HIF-1alpha drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk’, American Journal of Physiology: Cell Physiology, vol. 293, no. 2, pp. C621-C631

Appalainen, AK, Maki, K & Laitinen-Vapaavuori, O 2015, ‘Estimate of heritability and genetic trend of intervertebral disc calcification in Dachshunds in Finland’, Acta Veterinaria Scandinavica, vol. 23, pp. 57-78.

Belanger JM, Bellumori TP, Bannasch DL, Faula TR & Oberbauer AM 2017, ‘Correlation of neuter status and expression of heritable disorders’, Canine Genetics and Epidemiology, vol. 4, no. 6.

Crusoe the Celebrity Dachshund n.d., IVDD Info, viewed 07 December 2020, <https://www.celebritydachshund.com/ivdd-info/&gt;.

Mogensen, MS, Karlskov-Mortensen, P, Proschowsky, HF, Lingaas, F, Lappalainen, A, Lohi, H, Jensen, VF & Fredholm, M, 2011, ‘Genome-wide association study in Dachshund: identification of a major locus affecting intervertebral disc calcification’, The Journal of Heredity, vol.1, S81-6.

Mort, JS, Geng, Y, Fisher, WD & Roughley, PJ 2016, ‘Aggrecan heterogeneity in articular cartilage from patients with osteoarthritis’, BMC Musculoskeletal Disorders, vol. 17.

Packer RMA, Hendricks A, Volk HA, Shihab NK, Burn CC 2013, ‘How long can you go? Effect of conformation on the risk of thoracolumbar intervertebral disc extrusion in domestic dogs’, PLOS One, vol 7.

Rusbridge, C 2015, ‘Canine Chondrodystrophic intervertebral disc disease (Hansen type 1-disc disease)’, BMC Musculoskeletal Disorders, vol. 16, no. S11.

Silver, G 2013, Dachshund and intervertebral disk disease, viewed 07 December 2020, <https://www.ethosvet.com/blog-post/dachshund-and-intervertebral-disk-disease/&gt;.

Smith, AN 2014, ‘The role of neutering in cancer development’, Veterinary Clinics of North America: Small Animal Practice, vol.44, no. 5, pp. 965-975.

Veterinary Practice 2011, The neutering of dogs and bitches in the UK and Europe, viewed 12 January 2020, <https://veterinary-practice.com/article/the-neutering-of-dogs-and-bitches-in-the-uk-and europe#:~:text=Britain%20has%20one%20of%20the,practices%20having%20been%20surgically%20neutered >.

Synopsis of systematic reviews and what they entail

Systematic reviews are regarded as high-quality evidence and have been a pillar in the development of evidence-based practice since the emergence of the reviews in the healthcare sector. Systematic reviews have been developed as a step up from the widely used, less specific narrative reviews and meta-analyses.  Systematic reviews aim to present a comprehensive and unbiased synthesis of existing data, in relation to answering a specific question (Sargeant and O’Connor, 2014).  The methods involved in carrying out a systematic review have been generalised across different regulating bodies including JBI and PRISMA. These generalised steps encourage practitioners of any field to be able to conduct a systematic review which as little risk and bias as possible.  The first step is to formulate a review question. The question should then be ran through search databases (Cochrane, PubMed and Prospero alike) to see if any similar studies have been conducted recently. If not, it is advised to register the title of the review to avoid duplications.    Next, the search protocol needs to be develop. This defines how studies will be searched for and selected, as well as defining exclusion and inclusion criteria. This is essential as it not only allows for the control of reporting bias but will allow for the transparency and how reproduceable the study is.  Once the search protocol have been developed, studies can be searched for and filtered down using objectives and exclusion criteria’s set beforehand. the selection and screening allows also for the removal of poor quality and irrelevant studies.  Afterwards, data can be extracted from the selected studies and then collated. The data can then be presented and interpretations made from it in relation to the topic in question. The certainty of the evidence can be determined using systems such as GRADE.  The finished review can then be published. It is important to think about the topic of the review when deciding where to publish it, as that would define the audience it reaches.

Becoming an evidence-based practitioner in Bioveterinary science

EBP magazine article PDF

Staphylococcus pseudintermedius

Using 3D biomaterial matrices as an alternative to using protected animals.

Abstract
The use of animals in scientific research has been prominent for the past 100 years (AMP, 2020) and it is widely recognized that many medical advances such as vaccinations as well as surgical innovations, are born out of scientific animal research (Festing and Wilkinson, 2007). The perpetual nature of medical, scientific, veterinary and environmental research means the use of animals in this process is continued. The number of animals used in the United Kingdom estimated at 3.52 million in 2018 (Understanding Animal Research, 2018). The use of animals in the UK is governed by several legislation and guidelines. The most predominant legislation is the Animals (Scientific Procedures) Act 1986, which regulates all procedures on protected animals. The Act incorporates the principles of the 3Rs – reduction, replacement and refinement which aims to regulate the use of animals in research (Balls, 1994). These principles prompt the development of alternatives to the use of protected species. One alternative to animals is 3D biomaterial cultures. This form of biomaterial is developed from cells from the target species, so in the case for human research, the absolute replacement of animals in research becomes more feasible. This review will assess the efficacy of 3D tissue models an alternative to using animals in research.

Introduction
From a historical perspective, a wide array of scientific breakthroughs has stemmed from animal research. One example of this is the development of deep brain stimulation to reduce the effects of Parkinson’s Disease, studies that worked on this use monkeys (Lozano et al, 2002). Other studies to produce biomedical interventions for patients with HIV have been developed using humanized mice (Dezzutti, 2015). A current perspective on animals being used for research is the COVID-19 pandemic, caused by a virus named SARS-CoV-2. The first vaccine was developed just four weeks after the virus’ genetic sequence was available from china, and its first tested on animals. Affected countries like Russia and multinational companies like Johnson and Johnson, used animals for testing including mice, rats, hamsters and lower primates in their studies (Chen et al, 2020). Recognition the importance of animal research is highlighted in global situations such as this where the continuity of scientific research is under threat. Organisations including the Max Planck Society in Germany, take several measures including stockpiling feed, bedding and veterinary drugs for the animal as well as the prioritising of species preservation (cryoconservation) and staff safety protocols to allow the use of animals in research to continue and not put either animal or human life at risk (European Animal Research Association, 2020).

The main legislation is the Animals (Scientific Procedures) Act, or ASPA, which came to into effect in 1986. It is implemented and regulated by the Home Office in England, Scotland and Wales and by the Department of Health, Social Security and Public Safety in Northern Ireland (Gov.co.uk, 2020). The preliminary of the Act starts with two definitions, the first being that a protected animal is a vertebrate in its foetal, larval or embryonic state from two thirds of its gestation if it is a bird, mammal or reptile or when, in any other case, it becomes capable of independent feeding, and that a cephalopod in its embryonic form is not a protected animal. The second defining what a regulated procedure is, being “any applied to a protected animal that may have the effect of causing the animal a level of pain, suffering, distress or lasting harm equivalent to, or higher than, that caused by the introduction of a needle in accordance with good veterinary practice” (Consolidated ASPA, 2013). This means that animals like the common fruit fly and worms are not protected in use for scientific research (MRC, 2020). When animals are used, it is important that they receive the best available animal husbandry and welfare, as this not only improves the quality of life for the animal, but inevitably improves the quality of the results.
Within the legislative guidelines of both the ASPA and the European directive, is the application of the 3 R’s – replacement, reduction and refinement. Replacement is a process that was initially discussed by Hume and Russell in 1957, and was further developed by Russel and Burch into a number of ways that research using animals could become more humane, which would later go on to be known as the 3 R’s (Doke & Dhawale, 2015, Russell & Burch, 1959).
The first principle of the three R’s, replacement, can be divided into two categories – partial and absolute. Partial replacement entails using animal derived cell lines instead of whole-animal studies or using an animal thought to be less likely to suffer under the required intervention such as replacing a protected animal to a non-protected animal including Drosophila and nematode worms (ASPA, 1986). Absolute replacement aims to completely remove animals from scientific procedures and experiments including using animals as study subjects, whole animal studies and sources of tissue (Parker and Browne, 2014). This would then turn researchers to use human tissues and cells, mathematical and computer models as well as developed cell lines (NC3Rs, 2020). Some studies suggest that the principle of absolute replacement may allow for discoveries not possible with animals, the possible socioethical pressures could deter scientists pursuing justifiable research that would not produce reliable data without the use of animals (MacArthur, 2018).
Refinement relates to the experience the animal has when using live animals in scientific research, taking an approach that avoids or minimises the pain, emotional stress and other adverse effects of its involvement and ultimately enhancing their wellbeing (Buchanan-Smith et al, 2005). For example, animals kept in cages without adequate stimuli may experience an imbalance in hormonal levels: refinement encourages scientists to accommodate animal participants as to not only improve their life or time spent in the laboratory, but also increase the validity of obtained results and reduce the number of times an experiment needs to be repeated, which would call for the use of more animals (Doke and Dhawale, 2015).
When looking at reduction, it is explicitly, using less animals in research. As with any study, it is never necessary to test the whole population. Instead, a large sample size promotes results that are more reflective and applicable to the existing population. When applying this to the principle of reduction, the aim is to use the minimum number of animals needed to be able to make a valid inference to the wider population. An important aspect of applying reduction into regulated procedures is experimental design, a researcher may make changes to the study design that will enable them to use less animals, or to use more animals in one experiment, instead of lots of smaller experiments (Hendriksen, 2009). Changing the design of an experiment also requires a change in the statistical analysis, but by making the changes would reap the benefits of producing high-quality data as well as reducing the use of animals.

Laboratory animals are usually obtained from purpose breeders. Included in the species laboratories use are humanized mice, those that have DNA engineered with human cytokine genes or those with a highly deficient immune system, replicating that of a human (Eswaraka and Giddabasappa, 2017). These lab animals, after the researchers gain their ethics approval, are situated in the environment relevant to the study design. Some animals are only used for a number of days to weeks but lower primates like chimpanzees, who have a life span close to that of a humans’, may live, and be experimented on for the majority of that (Knight, 2008). When it comes to the end of the animals use to the research, more often than not it is euthanized. In mice, CO2 is often used for euthanasia procedures, but studies are being done to assess if this is the best, humane and available option (Boivin et al, 2017, Marquardt et al, 2018)
The ASPA contains suitable methods of euthanasia determined by species and interventions used. Carcasses are then incinerated. The Act discuss the possibility of rehoming the animals, but it is not always possible to do this even, with larger, companion animals (namely dogs, cats and horses) due to either the biohazard risk they may pose or the quality of life they would have after the interventions of research (Franco and Olsson, 2016)
According to research and current events, a national survey conducted on the general public showed that 73% of participants accepted the use of animals including mice, dogs and monkeys would be pivotal in developing vaccines and treatments for the COVID-19 virus, but only in the case that there was no unnecessary suffering and no alternative to using animals (Understanding Animals in Research, 2020). But there is still little public understanding into animals in research, as depicted by ASPA, the minimum pain threshold of regulated procedures in the insertion of a needle. The high cost, skilled manpower and lengthy protocols could hinder the speed scientific developments such as developing a vaccine amidst a global pandemic (Doke and Dhawale, 2013). Additionally, the general public may also not consider the number of animals that are used in scientific procedures, an example being that 200,000 fish and 20,000 amphibians were used in the UK in 2004, predominantly zebra fish (Badyal and Desai, 2014). Furthermore, the genetic and cellular limitations of animal tissues limit the standardization and transferability of results (Parker and Browne, 2014).

Alternatives to using protected animals
Often, there are some differences between animal and human tissues that causes the use of animals to be poor predictors of the effect an intervention may have on human life, therefore the need to develop more applicable alternatives arises (Heinonen, 2015). One alternative to animal use are a biomaterial known as three-dimensional (3D) cell cultures, commonly referred to as a biomatrix. These bioengineered models allow for a micro-environment that mimics a real, living tissue (3D Biomatrix, 2011) and has been in clinical use for over 25 years (Tornello et al, 2016). The culturing of skin tissues for clinical use originated in the 1980s when Yannas and Bell created a dermal skin tissue from neonatal fibroblast in collagen gels, which was then expanded into the use of bioscaffolding and molecular ques to promote the growth of skin cells (Hodges and Atala, 2014). This clinical development has been prominent in medical settings for burn victims, as well as some cardiovascular, soft tissue and musculoskeletal uses (Jelinek, 2013) but its use in research is becoming more widespread.
The development of the 3D cell culture entails embedding the desired cells into a matrix of, often, collagen or gel and allowed to cultivate. There are several methods of embedded the cells into the matrix and forming the 3D tissue. Jelinek describes a method of pulsar laser deposition. Other methods described include the use of a human skin derived extracellular matrix as a bio-ink, which is then used to print and build up a tissue section (Kim et al, 2018). This study showed that in comparison to a collagen-based skin tissue, it was more stable, less susceptible to shrinkage and have better dermal secretions and barrier functions.
One the common uses for 3D biomaterials is in the pharmaceutical sector (Heinonen, 2015). One application is seen in the development of a human cornea to replace that of using an animal one and showed a higher reproducibility and less variability in results than the animal equivalent. Additionally, the study highlights the use of this type of 3D cell culture as a promising replacement to the previously used rabbit cornea (Hahne et al, 2012). Further applications include the research of stem cells, 3D scaffolding enables researchers to more accurately recreate the microenvironment that these cells would be in including the ability of cell to cell communications and morphology of the cells (Lui et al, 2018). Using 3D biomatrices allows for better control and grafting of the cells which will enhance the quality and validity of the research.
3D biomatrices are also used to research into human chronic liver disease. The continual progress in research observes a shift from using rat or porcine hepatocytes to using human primary hepatocytes (hPHs). This change, as well as seeing an adoption of replacement, offers a more biocompatible cell source. By using the hPHs in 3D models, confounding factors such as loss of function, necrosis and dedifferentiation can be controlled (Mirdamadi et al, 2020). Similarly, 3D cell cultures are also used in oncology research, by using a human-like microenvironment, it enables the study of proangiogenic factors, chemoattractants and angiogenesis of cancer cells (Katt et al, 2016).
One of the promising aspects of using 3D biomatrices as an alternative to animals is the ability to replicate interaction of multiple systems such as neural and blood vessels in one culture, or observe the interaction of cancer cells with the heart and muscles without the use of a whole animal study (Sung and Beebe, 2014, Arslan et al, 2019). Like the use, at the end of using the 3D biomatrix for research, they can be disposed of with other biohazardous tissues and materials through methods including incineration, but there is no need for euthanasia (VUMC, 2020).

In the UK, legislations exist for the use of human tissues in research, especially after an incident of inappropriate retention of organs from paediatric post-mortems was discovered in Britain. The case prompted the development of guidelines and legislations from bodies including the Royal College of Pathologists (Ananthamurthy, 2008). There are now two legislations that govern the use of human tissues for purposes other than diagnosis. The first is The Human Tissue (Scotland) Act 2006 that puts forward legislation for the use of tissues for research, post-mortem and transplant for those over 16, children ages 12 or older at the time of death and children aged 12 or under (Legislation.gov.uk, 2020). Section 6 of this Act sates that an adult can authorise the removal and use of parts of their body for the use of research, transplant, education or audit (OTD, 2020). The second piece of legislation is the Human Tissue Act 2004, which encompasses the use of both living and deceased tissue from donors. The use of tissue for the sole purpose of research from a living donor much be consented and reviewed by an ethics committee, prior consent is needed to obtain tissue from a deceased individual during post-mortem (Legislation.gov.uk, 2020). The Act contains Scheduled Purpose which lists all the uses of living or deceased tissue retrieval that are dependent on consent from the participant or family member (HTA, 2016).
There is a current lack of social engagement surrounding the topic of 3D cell cultures, however for its continued advancement, there is need for consideration. Some 3D cell cultures are made from neural and brain tissue, raising the issue of ownership as obtaining the cells or tissue culture. Consent is needed but public issues arise at the possibility of the tissue culture able to express neural activity, as seen in one study (Quadrato et al, 2017). Furthermore, there is the question of species boundary crossing, when the 3D tissue is cultivated, it may be transplanted into a living animal to observe further. The concerns here include whether the animal is able to obtain a degree of capacities such as pain, pleasure and a sense of self (Vermeulen et al, 2017, Farahany et al, 2018).
When constructing the 3D biomatrices, researchers should also consider the source of the tissues used and the 3Rs. One study using 3D matrices used from bovine spinal cord cells highlights the drawback of using non-human cells and the justifiability of obtaining them from animals (Arslan et al, 2019). Using a 3D cell culture as alternative to using a protected animal such as a rabbit, would be an efficient replacement if the cells being used are not derived from the slaughter of healthy animals.

Conclusion
The use of animals in research has been longstanding and its importance does not go unrecognized in science and the general public alike. Despite this, both researchers and the general public are aware of the ethics and legislation surrounding animal use, with studies being done in response to the effect laboratory animal euthanasia has on other subjects and personnel (LaFollette et al, 2020). However, the increased biotechnology has led to the development of promising alternatives like the 3D biomatrix. This biomaterials ability to create a microenvironment for research has already had positive impacts in reducing and replacing the use of animals in research.

Bibliography

o Americans for Medical Progress (2020) Supporting biomedical research [online]. Available at: https://www.amprogress.org/animal-research/. Accessed 05/02/2020.
o Ananthamurthy, A. (2008) Use of human tissues for research; Ethical concerns, Indian Journal of Medical Ethics, 5(3).
o Badyal, D.K; Desai, C. (2014) Animal use in pharmacology education and research: The changing scenario, idian Journal of Pharmacology, 46(3).
o Balls, M. (1994) Replacement of animal procedures: alternatives in research, education and testing, Laboratory Animals, 28, pp. 193-211
o Buchanan-Smith, H; Rennie, A.E; Vitale A; Pollo, S; Prescott, M.J; Morton, D.B. (2005) Harmonising the definition of refinement, Animal Welfare, 14, pp. 379-384
o Chen, W.H; Strych, U; Hotez, P; Bottazzi, M.E. (2020) The SARS-CoV-2 vaccine pipeline: an overview, Current Tropical Medicine Reports.
o Dezzutti, C.S. (2015) Animal and human mucosal tissue models to study HIV biomedical interventions: can we predict success, Journal of the International AIDS Society, 18(1).
o Doke, S.K. & Dhawale, S.C. (2015) Alternatives to animal testing: A review, Saudi Pharmaceutical Jounral, 23(3), pp. 223- 229
o European Animal Research Association (2020) Crisis plans help animal facilities respond to the corona virus [online] Available at: https://www.eara.eu/post/crisis-plans-help-animal-facilities-respond-to-the-coronavirus-pandemic. Accessed 15/04/2020.
o Farahany, N.A; Greely, H.T; Hyman, S; Kch, C; Grady, C; Pasca, S.P, Sestan, N; Arlotta, P; Bernat, J.L; Ting, J; Lunshof, J.E; Iyer, E.P.R; Hyun, I, Capestany, B.H; Church, G.M; Huang, H; Song, H. (2018) The ethics of experiementing with human brain tissue, Nature, 556, pp. 429-432.
o Franco, N.H; Olsson, I.A.S. (2016) The end of animal life: a start for ethical debate [online] Available at: https://www.wageningenacademic.com/doi/abs/10.3920/978-90-8686-808-7. Accessed on: 21/04/2020.
o Gov.uk (2020) Animal testing and research [online]. https://www.gov.uk/guidance/research-and-testing-using-animals. Accessed 12/03/2020.
o Heinonen, T. (2015) Better science with human cell-based organ and tissue models, Alternatives to Laboratory Animals, 43(1), pp. 29-39.
o Jelinek, H. (2013) Lasers for medical applications, diagnostics, therapy and surgery, 1st ed, England, Woodhead Publishing.
o Katt, M.E; Placone, A.L; Wong, A.D; Xu, Z.S; Searson, P.C. (2016) In vitro tumour models: advantage, disadvantages, variables, and selecting the right platform, Frontiers in Bioengineering and Biotechnology,4,
o Kim, B.S; Kwon, Y.W; Kong, J; Park, G.T; Han, W; Kim, M; Lee, H; Kim, J; Cho, D. (2018) 3D cell printing of in vitro stabilized skin model and in vivo pre-vascularized skin patch using tissue-specific extracellular matrix bioink: A step towards advanced skin tissue engineering, Biomaterials, 168, pp. 38-58.
o Knight, A. (2008) The beginning of the end for chimpanzee experiments, Philosophy, Ethics and Humanities in Medicine, 3(16).
o Lazano, A.M; Dostrovsky, J; Chen, R; Ashby, P. (2002) Deep brain stimulation for Parkinson’s disease: disrupting the disruption, The Lancet, 1(4), pp. 225 – 231.
o Legislation.gov.uk (2020) Human Tissue (Scotland) Act 2006 [online]. Available at: https://www.legislation.gov.uk/asp/2006/4/section/6D . Accessed 18/04/2020.
o Lui, Z; Tang, M; Zhao, J; Chai, R; Kang, J. (2018) Looking into the future: Toward advanced 3D biomaterial for stem-cell based regenerative medicine, Advanced Materials, 30(17).
o MacArthur Clarke, J. (2018) The 3R’s in research: a contemporary approach to replacement, reduction and refinement, British Journal of Nutrition, 120(s1), pp, S2-S7.
o Medical Research Council (2020) Regulation and policy [online] https://mrc.ukri.org/research/research-involving-animals/regulation-and-policy/. Accessed 18/03/2020.
o Mirdamadi, E.S; Kalhori, D; Zakeri, N; Azarpia, N; Solati-Hashjin, M. (2020) Liver tissue engineering as an emerging alrernative for liver disease treatment, Tissue Engineering Part B: Reviews, 26(2).
o Northern Star (2017) Testing on animals leads to important medical breakthroughs [online]. https://northernstar.info/opinion/columnists/testing-on-animals-leads-to-important-medical-breakthroughs/article_94ffd440-60af-11e0-ac68-001a4bcf6878.html. Accessed on 05/02/2020.
o OTD clinical (2020) The Human Tissue Scotland Act 2006 [online]. Available at: https://www.odt.nhs.uk/odt-structures-and-standards/regulation/the-human-tissue-scotland-act-2006/. Accessed on: 18/04/2020.
o Parker, R.M.A; Browne, W.J. (2014) The place of experimental design and statistics in the 3R’s, ILAR Journal, 55(3), pp. 447-458.
o Russell, W.M.S & Burch, R.L. (1959) The principles of humane experimental techniques, 1st ed, London, Methuen & Co LTD.
o Sung, K.E; Beebe, D.J. (2014) Microfluid 3D models of cancer, Advanced Drug Delivery Reviews, 79-80, pp. 68-78.
o Understanding Animal Research 2018) Number of animals [online]. http://www.understandinganimalresearch.org.uk/animals/numbers-animals/ . Accessed 12/03/2020.
o Vanderbilt University Medical Centre (2020) Environmental Health and Safety [online] Available at: https://www.vumc.org/safety/waste/biological-waste-guide. Accessed on 21/04/2020.
o Vermeulen, N; Haddow, G; Seymour, T; Faulkner-Jones, A; Shu, W. (2017) 3D bioprint me; a socioethical view of bioprinting human organs and tissues, Global Medical Ethics, 43, pp. 618-624.
o

Clinical case study – necrotising soft tissue infection in a dog caused by Staphylococcus pseudintermedius.

Introduction

Necrotising soft tissue infections, otherwise known as NSTI’s are brutal infections with the possibility of being fatal (Weese. et al, 2009) and affects a plethora of the living tissues, including all layers of the dermis, adipose tissue and muscle (Campos. et al, 2017). One bacterial species that is able to cause an NSTI is Staphylococcus pseudintermedius, a Gram positive, cocci bacterium, part of the Staphylococcus family, of which there are at least 22 species (Dubois. et al, 2009).

Figure 1. Staphylococcus pseudintermedius stained in crystal violet via Gram Staining (Willows, 2015)

S. pseudintermedius is a single celled bacterium, measuring 1 umhickness and are predominantly found in clusters shown in Figure 1 (Devriese. et al, 2005, Foster, 1996). Many species of Staphylococcus are found on the surface of the animals’ skin but a symptomatic infection is only caused when the bacteria enter the body, this could be from a cut allowing the bacteria enter the blood stream or if the bacteria are ingested from personal grooming or from breastfeeding young. S. pseudintermedius is a leading cause of skin, ear and post-operative infections in dogs and cats (Beale. et al, 2009).

 

Figure 2. S. pseudintermedius taxonomy (Devriese. et al, 2005)

Staphylococcus bacteria are either coagulase positive postie or negative, with S. pseudintermedius being coagulase positive. DNA-ribosomal RNA hybridisation show that the bacteria have several strains with varying number of alleles (Bannoehr. et al, 2007)

One case study (Campos. et al, 2017) shows a NSTI infection caused by S. pseudintermedius in a six – year – old female beagle. The dog was presented to the veterinary clinic after being found in boarding kennels in lateral decubitus with pale mucous membranes and apathetic. The patients’ clinical signs included intense signs of pain, oedema, erythema, loss of sensation and crepitations stretching from the dorso-lumbar region of the right flank to the mammary glands. On physical examination a small perforated wound was found, 1cm in diameter, possibly obtained during a fight in the kennels. The area surrounding the lesion, however, was inflamed and erythematous. One section, measuring 4cm in diameter, was found to be black in colour and had lost sensation completely. The areas showing oedema, towards the right mammary glands had changed colour and had decreased sensation after being palpated.

Figure 3. Irregularities in skin colour, especially near the right mammary gland indicated by the black arrow (Campos. et al, 2017).

Both normocytic and normochromic anaemia were displayed in the blood samples, and cell cultured biopsies showed necrotising S. pseudintermedius and S. agalacitate, leading to the diagnosis of a Type II NSTI, which is more commonly seen in younger animals. Type I is predominantly seen in older animals with a weaker immune system, the type of infection roughly dictates the treatment protocol or guidelines followed (Surgical Critical Care, 2014). Antibiotic treatment was administered using ceftriaxone, metronidazole and tramadol clorhidrate as an analgesic. Early diagnosis and regular wound cleaning allowed the case to have a good prognosis.

Pathogenicity and diagnosis

Replication and infection

S. pseudintermedius reproduces asexually by binary fission, the way that the majority of bacteria do. In binary fission, one bacterium separates into two new genetically identical cells – the genetic information is duplicated, and each new cell gains one copy (CALS, 2020). Generally, bacteria species have a FtsZ protein that during binary fission, allows the two cells to cleave from one another, but S. pseudintermedius produces a biofilm, a complex extracellular layer of polysaccharide biopolymers, that causes the cells to partially penetrate and stick to one another. This biofilm aids in creating clusters and has a degree of pathogenicity (Lopez. et al, 2017).

Infection can be brought on by the entrance of the bacteria into the bloodstream, in this case was likely to have been through the perforated wound, as S. pseudintermedius has little virulence on the skin surface. Once in the blood stream and tissues and replicates therefore increasing its pathogenicity. As seen in this case, the dog obtained a perforates wound, where the bacteria were most likely to have entered.

 

Diagnostics

Cutaneous fragments of 0.8 cm were collected from the patient, immediately afterwards, purulent and fetid material came from the openings and pus is more often than not indicative of a bacterial infection (Mayo Clinic, 2017). DNA sequencing via PCR is the most reliable method of distinguishing S. pseudintermedius due to its different phenotypic markers (Bannoehr. et al, 2008). The histopathology of the subcutaneous tissue cuts showed areas of prevalent neutrophilic or fibrinous infiltration, diagnosing the patient with necrotizing fibrino-purulent cellulitis. Additionally, PCR sequencing can identify S. pseudintermedius by the presence of the sodA gene (Hiramatsu. et al, 2007).

As the case study suggests, the most recommended diagnostic method is exploratory surgery, with a positive diagnosis being given with the presence of greyish, necrotic tissue, pus and lack of resistance to digital pressure. In some cases, such as this, surgical debridement is deemed unnecessary.

 

Pathogenicity

S. pseudintermedius has several virulence factors. The most predominant pathogenic factors in canines is Protein A (Garbacz. et al, 2013), a surface protein with 4 or 5 domains that bind to immunoglobulin G, when the two are bound together, the affected IgG molecules cannot be recognized by neutrophil receptors, allowing the infection to progress and not be targeted by phagocytes.

The bacterium also possesses a gene that produce an exfoliative toxin, known as S. pseudintermedius exfoliative toxin (SIET), this toxin is able to bind to MHC class II, making it less able to initiate an immune response, perpetuating the infection (Langley. et al, 2017). Moreover, the toxin is able to degrade cell walls – causing erythema, exfoliation and crusting of skin. This aids in explaining the patient’s clinical presentation with the discolouration seen on the affected areas.

The production of Enterotoxins produced by S. pseudintermedius can be linked to the erythema and loss of sensation seen in the patient. One of these toxins is. Luk-I, which destroys leukocytes and necrosis of tissues (Garbacz. et al, 2013). These toxins also cause the non-specific activation of polyclonal T cell proliferation and exponential cytokine release, which can cause systemic toxicity- increasing the rates of mortality. The regulation of this toxin is controlled by an expression gene that is upregulated in the initial infection (Futagawa-Saito. et al, 2006)

An additional pathogenic factor of the bacteria is the production of beta-hemolysin. Hemolysins are enzyme that act on lipids within the erythrocyte membrane (Foster, 1996). This causes the tissue to become necrotic as the oxygen supply has been cut off, and the lack resistance in the tissue due to burst cells, in addition to the grey/purple appearance of affected areas displayed in Figure 3.

Another pathogenic enzyme the bacteria produces is DNase, and it is quite potent (Beale, M. et al, 2005). DNase cleaves to DNA in a non-specific manner and breaks down 5’-phosphodiester bonds releasing dinucleotides trinucleotides and oligonucleotides (ThermoFisher, 2001).

One of the factors that causes inflammation in a S. pseudintermedius NSTI is its ability to produce a biofilm. The extracellular protein and polysaccharide matrices that exist in the colonies contribute to inflammatory responses which positively confers with the inflammation seen around the wound site and the surrounding tissue. Interleukin-1 beta and interleukin-6 mRNA are expressed in the grouped colonies and an active TLR signalling pathway is observed on animal derived agars and the Inflammatory response is increased if the relevant coding is upregulated (Arima. et al, 2018).

 

Figure 4. Biofilm production from cocci bacteria (Singh. et al, 2013).

Treatment

In the case, once the diagnosis was established, the treatment was as follows. 50 mg/kg IV 12/12h of Ceftriaxone was given alongside 25 mg/kg IV 12/12h of Metronidazole. This treatment was determined by doing an antibiotic susceptibility test by disc diffusion assays. The S. pseudintermedius isolates were found to be susceptible to the following antibiotics: Ceftriaxone, Enrofloxacin, Amoxicillin and Clavulanic Acid, Penicillin, Gentamicin, Sulfamethoxazole and Trimethoprim, and Azithromycin. Metronidazole also used due to the possibility of anaerobic microbes being present, despite these cultures not being collected. The importance of disc diffusion assays is that bacteria are becoming ever more antibiotic resistant and as a common infection, S. pseudintermedius already has a resistant strain, MRSP – methicillin resistant Staphylococcus pseudintermedius, including strains that test positive for Luk-I and SEIT (Ruscher. et al, 2010). The MRSP strains are resistant to beta-lactam antibiotics (The Bella Moss Foundation, 2020). Disc diffusion assays allow clinicians to choose the most effective antibiotic and reduce the risk of increasing resistance.

An additional reason antibiotic disc diffusion assays would have important before deciding on a drug therapy course is that Gram positive bacteria have different susceptibilities to antibiotics due to their cell structure, in conjunction to their ability to produce biofilms.

Gram positive bacteria have a thick outer membrane made up of layers of peptidoglycans, lipoproteins and other molecules (Microbeonlince.com, 2015), including multidrug efflux pumps that lie throughout the outer, as well as inner, membranes of the outer membrane of Gram-positive bacteria. These can pump out antibiotics using a proton-motive force and transport proteins (Kaatsz. & Shcindler. 2016), removing beta-lactams before they are able to take effect. This raises the minimum inhibitory concentrations needed for a drug to be effective (Nikaido. 1998). Beta-lactamases in antibiotics can also be degraded by the bacteria’s enzymes. In addition to having a role in the pathogenicity of S. pseudintermedius, the biofilm also plays a role in the microbes’ susceptibility to treatment: the biofilm produced by the bacteria also contributes to protecting the colonies from antibiotics due to the lipopolysaccharides.

Disc diffusion assays showed susceptibility to Ceftriaxone, so it was used in this case to control and inhibit further infection in the infection by inhibiting cell wall synthesis. Looking at the pharmacodynamics, the active substance in the drug binding to PBP’s, thus inhibiting the final transpeptidation of peptidoglycan in cell walls. This cases the cells to eventually lyse due to arrested cell wall assembly (Ravizzola. et al, 1985)

As well as receiving tramadol as an analgesic, the patient also received wound management, which included thorough cleaning every 12 hours with diluted iodopovidone (0.01%) as well as a silver sulfadiazine cream applied subcutaneously and to lesions, all in combination with compression bandages.

After 15 days, the biochemical test values were at normal levels however there was a high presence of immature and abnormally sized blood cells. After a month of continued treatment, there was considerable clinical improvement, with successful skin recovery and growth and a small area of scar tissue where the necrotized tissue had been and where the biopsy sample was collected. The patient persisted with some signs of anaemia but overall was cleared to discontinue treatment.

Discussion

Other cases of necrotising bacteria, such as described by Abma et al (2013), proposed that surgery and then the use antibiotics and regular wound cleaning as post-operative care, is the best treatment option as it more effectively eradicates the bacteria from the body and mortality rates are increased without aggressive surgical debridement, However, in this case the attending clinicians decided it would be too much skin and muscle to remove and opted for a non-surgical route.  Silver sulfadiazine also possesses immediate bactericidal effects and residual bacteriostatic effects (Atiyeh. et al, 2009). Bactericidal action as well as antibiotic treatment in this instance seemed to prove effective without causing the animal unnecessary pain and discomfort, as well as not compromising their quality of life by prolonged recovery.

It can be assumed that the approach taken in this case study was different to similar cases due to the rapid diagnosis, and the choice not to debride a large area of tissue. Intuitive understanding of the clinical signs prompted the appropriate diagnostic methods, aiding the prognosis of the patient

As well as necrotising tissue, S. pseudintermedius releases toxins that could cause the patient to suffer a systemic toxic shock, if not diagnosed and treated rapidly. As discussed, delayed immune responses from Protein A increase chances of STS occurring. Other methods of diagnosis, such as a urine analysis would be an adequate way to see if the bacteria have become systemic, making it easier to avoid or control a toxic shock occurring.

Conducting diffusion disc assays was a pinnacle part of treating bacterial infections as it reduces the possibilities of bacteria becoming resistant and ensures that the best possible medication is given on the first attempt. By using a bactericidal in conjunction with the antibiotic increases the chances of eradicating the bacteria. Awareness of appropriate antibiotic use was also shown in this case, as one case saw the appearance of MSRP in a site that was previously eradicated of S. pseudintermedius (Mayer. & Rubin, 2012).

Conclusion

As seen in this case study, the patient was subject to prompt diagnostic techniques and appropriate treatment protocols, showing the clinicians understanding of the importance of early and efficient diagnosis. The severity of S. pseudintermedius caused was able to be reduced, resulting in successful eradication of the bacteria and leaving the patient with a good quality of life.

Bibliography

Abma, E; Bosmans, T; Campos, M; Stock, E; de Rooster, H; Vandenabeele, S. (2013) Necrotising fasciitis in a dog, Vlaams Diergeneeskundig Tijdschrif, 82.

Arima, S; Kataoka, Y; Kibe, R, Mitsuhashi, M; Ochi, H. (2018) Staphylococcus pseudintermedius biofilms secrete factors that induce inflammatory reactions in vitro, Letters in Applied Microbiology, 67(3), pp. 214-219.

Atiyeh, B.S; Dibo, S.A; Hayek, S.N. (2009) Wound cleansing, topical anaesthetics and wound healing, International World Journal, 6(6).

Baele, M; Devriese, L.A; Haesebrouck, F; Hermans K (2009) Staphylococcus pseudintermedius versus Staphylococcus intermedius, Veterinary Microbiology, 133(1-2), pp.206-7.

Bannoehr, J; Battisti, A; Franco, A; Fitzgerald, J.R; Iurescia, M. (2008) Molecular diagnostic identification of Staphylococcus pseudintermedius, Journal of Clinical Microbiology, 47(2), pp. 469-471.

Campos, D.R; Laguna,  A.G.V; Lambert, M.M; Barros de Abreu, D.P; Soares de Souza, M; Fernandes, J.I. (2017) Necrotising Soft Tissue Infection in Dog – Case Report, Acta Veterinaria Brasilica, pp. 191-195.

College of Agriculture and Life Sciences (2020) Binary fission and other forms of reproduction in bacteria [online] https://micro.cornell.edu/research/epulopiscium/binary-fission-and-other-forms-reproduction-bacteria/. Accessed on 04/03/2020.

Department of Veterinary Disease Biology (2011) Staphylococcus pseudintermedius [online] https://atlas.sund.ku.dk/microatlas/veterinary/bacteria/Staphylococcus_pseudintermedius/. Accessed on 30/01/2020.

Devriese, L.A; Vancenneyt, M; Baele, M; Vaneechoutte, M; De Graef, E; Snauwaert, C; Cleenwerck, I; Dawyndt, P; Swings, J; Decostere, A; Haesebrouch, F. (2005) Staphylococcus pseudintermedius sp. Nov., a coagulase-positive species from animals, International Journal of Systematic and Evolutionary Microbiolgy, 55(4).

Drugs.com (2020) CefTRIAXone [online] https://www.drugs.com/ppa/ceftriaxone.html. Accessed on 29/01/2020.

Dubois, D; Leyssene, D; Chacornac, J.P; Kostrzewa, M; Shmit, P.O; Talon. R; Bonnet, R; Delmas, J. (2009) Identification of a variety of Staphylococcus species by matrix-assisted laser desorption ionization-time of flight mass spectrometry, American Society for Microbiolgy Journals, 48(3), pp.941-945.

Foster, T. (1996) Medical Microbiology, Texas, University of Texas.

Futagawa-Saito, K; Ba-Thein, W; Sakurai, N; Fukutasu, T. (2006) Prevalence of virulence factors in Staphylococcus intermedius isolates from dogs and pigeons, BMC Vet Res, 2(4).

Garbacz, K; Haras, K; Piechowicz, l; Zaranowska, S. (2012) Pathogenicity potential of Staphylococcus pseudintermedius strains isolated from canine carriers and from dogs with infection signs, Virulence, 4(3), pp.255-259.

Hiramatsu, K; Kamata, S; Kikuchi, K; Sasaki, T; Takahashi, N; Tanaka, Y. (2007) Reclassification of phenotypically identified staphylococcus intermedius strains, Journal of Clinical Microbiology, 45(9), pp. 2770-2778.

Kaatz, G.W; Schindler, B.D. (2016) Multidrug efflux pumps of Gram-positive bacteria, Drug Resistance Updates, 27, pp. 1-13.

Langley, R.J; Ting, Y.T; Clow, F; Young, P.G; Radcliff F.J; Choi, J.M, et al. (2017) Staphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors, PLOS.

Lopez, D; Kolter, R; Vlamakis, H. (2010) Biofilms, Cold Spring Harbor Perspectives in Biology, 2(7).

Mayer, M.N; Rubin, J.E (2012) Necrotizing fasciitis caused by methicillin-resistant ­Staphylococcus pseudintermedius at a previously irradiated site in a dog, The Canadian Veterinary Journal, 53(11), pp. 1207-1210.

Nikaido, H. (1998) Antibiotic resistance caused by Gram-negative multidrug efflux pumps, Clinical Infectious Diseases, 27(S1), pp. S32-S41.

Ravizzola, G; Bonfanti, C; Turano A. (1985) Ceftriaxone against gram-negative and gram-positve bacteria: bacteriacidal and post-antibiotic effect, Chemioterapia, 4(3), pp. 204-208.

Singh, A; Walker, M; Rousseau, J; Weese, J.S (2013) Characterization of the biofilm forming ability of Staphylococcus pseudintermedius in dogs, Veterinary Research, 9(93).

Surgical Critical Care (2014) Necrotizing soft tissue infections [online] http://www.surgicalcriticalcare.net/Guidelines/NSTI%202014.pdf?fbclid=IwAR0hQC8_jzGNKG_u1eQF2LokvnthD4U54jmB0bwtE1Bne6izwjYrGV5Xnlg. Accessed 04/03/2020.

The Bella Moss Foundation (2020) MRSP [online] http://www.thebellamossfoundation.com/pet-carers/treatment/mrsp/. Accessed 15/02/2020.

ThermoFisher (2001) DNase I demystified [online] https://www.thermofisher.com/uk/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/dnase-i-demystified.html. Accessed on 29/01/2020.

Tocris (2011) Toll-like receptor signalling pathway [online]. https://www.tocris.com/pathways/toll-like-receptor-signaling-pathway. Accessed on 28/01/2020.

Willows (2015) The concerning rise of the canine superbug – MRSP [online] https://www.willows.uk.net/news/general-practice-news/m/news/page/23/view/471

How does the Gibbs Reflective model integrate with Evidence based practice in veterinary medicine?

Professional thinking and clinical reasoning are underpinned by the integration of reflective practice into evidence-based practice. Its application in the field allows for professionals in the veterinary world to think in a well-rounded and rational way that revolves around a patient and has the best outcome in mind. Hand in hand with evidence-based practice, is usually reflective practice. The dynamics between the two allow for a practitioner to be retrospective of their current practice and be influenced by the relevant evidence to advocate for the best patient welfare.  There are several models of reflective evidence-based practice, in veterinary and healthcare the Gibbs reflective model is the one that seems the most prevalent (Jasper, M. 2013).

Figure 1. The Gibbs reflective model

Evidence in practice can come from a range of sources, which can be divided into roughly four groups, research, clinical experience, patient and experience and information from the local context such as national policy, social networks and performance data (Rycroft-Malone, J. et al, 2003). These all have their own impact into evidence-based practice, and subsequently reflective practice.

Figure 2. Evidence based medicine pyramid (Students for best evidence, 2014).

When combining reflective practice with EBP, it is necessary to utilise the best type of evidence to support your development, looking at figure 2, there is a range of different types of evidence and the quality of evidence used increases as you go up the pyramid. However, it is important to consider that even the highest quality evidence is subject to mistakes and publication bias, so appraising the evidence used will aid in better practice.

Evidence based practice may have its greatest effect at the analysis, conclusion and action plan stages of the Gibbs reflective model. When reflecting upon an event, it requires an individual to think in depth about the good and bad areas of it, and when moving on to evaluating this, looking at sources of evidence such as case studies may indicate how to handle a situation such as a treatment or drug prescription. One source of evidence that is commonly presented to practitioners is anecdotal evidence, a source of evidence which has little to no scientific base  and may be due to post-hoc fallacy, where a chance correlation causes the patient or client to believe that a second event was due to the first one (van Veggel, N. 2017). When presented with anecdotal evidence, the practitioner’s ability to conduct evidence-based practice is key as it promotes the use of research papers, case studies and other credible evidence to support decisions that ultimately benefit the patient.

In the analysis stage of the Gibbs reflective model, it requires you to think about what sense can be made from the event, assessing what lessons could be learned from it (Toolshero, 2018) and this is a good time to look at evidence such as case studies, a useful source for practitioners, to see how events similar to yours have been handled by others.  Additionally, evidence-based practice is not just about using sources of academic or government produced evidence, but also incorporating the views of the patient, and veterinary medicine, the clients needs. As well as using sources, it is important to reflect on whether the personal requirements of the client are met, for example prosthetics in animal amputees. An animals’ life may be thought to be sustainable with 3 legs but with what quality of life would it have. A highly active individual may not achieve the same emotional quality of life with less limbs despite there being there being a “good” prognosis physically (Mich, P. 2014). In this case, reflecting on what you would do in these situations by using both evidence and the needs of the client. As one article suggests, evidence-based practice shouldn’t be used as a blueprint for practitioners, but rather be combined with the clinician’s knowledge and experience as well as what is essential to the client.

In drawing a conclusion using the Gibbs reflective model, it is necessary to emphasise the variability of real-life practice to the training that is provided, and sometimes the need to turn to something other than the textbooks. One of the main points of making a conclusion in the Gibbs reflective model is deciding what could be done differently should a similar event occur again. Reflecting using sources of evidence including clinical trials, where new methods are developed, and clinical guidelines. Often it can be thought that reaching a conclusion using the Gibbs models a change, but as one paper suggests, using evidence to enhance clinical practice may allow for the affirmation that your current methods are correct and encourage you to continue using them. By going through the stages of the Gibbs reflective cycle, your conclusion may highlight a gap in research which can then be used as a prompt to other professionals that development in that section of the industry needs developing and/or updating.

One other important aspect that impacts professional thinking is continuing professional development (CPD). It aids in reflective processes as well as allowing for improved evidence-based practice. Similar to reflective practice, CPD activities, such as online courses and seminars, should be undertaken regularly and facilitate for skill development. One advantage of integrating CPD with EBP and the use of Gibbs is that evidence, such as textbooks, have the drawback of being current to the time of publication, but journals and clinical evidence that CPD providers use is up to date. In one paper, it is suggested that the interaction between EBP and CPD not only allows for increased accountability of the practitioner but also has the possibility to increase public confidence in judgement. This can be related back to the anecdotal evidence, and the value in reassuring clients of the reasoning behind your method of practice.

In conclusion, integrating reflective models like Gibbs with evidence based practice has the main advantage of allowing a practitioner to keep up to date with current developments in their relevant sector as well as being able to merge this evidence with the other aspects of the event they are dealing with to provide appropriately tailored care to a patient. It is important to use the right kind of evidence, appraising it appropriately though your reflection as not all evidence is good quality evidence.

 

Bibliography

Auldeen, A. (1997) Evidence-based practice and continuing professional development. British Journal of Occupational Therapy, 60(11), pp. 503-508.

Bannigan, K; Moores, A. (2009) A model of professional thinking: integrating reflective practice and evidence based practice. Canadian Journal of Occupational Therapy, 5(76), pp. 342-350.

Barredo, R.V. (2005) Reflection and evidence based practice in action: a case based application. The Internet Journal of Allied Health Sciences and Practice, 3(3).

E-learning network (2018) Gibbs reflective cycle model (1988) [online] https://www.eln.io/blog/gibbs-reflective-cycle-model-1988. Accessed on: 03/12/2019.

Jasper, M; Rosser; M; Mooney, G. (2013) Professional development, reflection and decision-making in nursing and healthcare, 2^nd edn, John Wiley and Sons, United Kingdom.

Kings College London (2003) Integrating evidence based practice with continuing professional development: a seminar report [online] https://www.kcl.ac.uk/sspp/departments/politicaleconomy/research/cep/pubs/papers/assets/wp15.pdf. Accessed on: 03/12/2019.

Mich, P.M. (2014) The emerging role of veterinary orthotics and prosthetics (V-OP) in small animal rehabilitation and pain management. Topics in Companion Animal Medicine, 29(1), pp.10-19.

Mulder, P. (2018). Gibbs Reflective Cycle by Graham Gibbs [online]. https://www.toolshero.com/management/gibbs-reflective-cycle-graham-gibbs/. Accessed 03/12/2019.

Rycroft-Malone, J; Seers, K; Titchen, A; Harvey, G; Kitson, A; McCormack, B. (2003) What counts as evidence in evidence-based practice? Journal of Advanced Nursing, 47(1), pp. 81-90.

Steves, R; Hootman, J.M. (2004) Evidence-based medicine: what is it and how does it apply to athletic training? Journal of Athletic Training, 39(1), pp.83-87.

Students 4 best evidence (2014) The evidence-based medicine pyramid! [online]. https://www.students4bestevidence.net/blog/2014/04/29/the-evidence-based-medicine-pyramid/. Accessed 05/12/2019.  The e-learning Network (2018) Gibbs – reflective cycle model (1988) [online] https://www.eln.io/blog/gibbs-reflective-cycle-model-1988. Accessed 23/11/2019.

van Veggel, V. (2017) “But it worked for my mother’s cat”. Some misconception about anecdotal evidence. Veterinary Nursing Journal, 32(8), pp. 239-240.

Thrombocytopenia in dogs

Introduction

When considering immune system itself it can be regarded as a complex system comprised of various cells and tissues that defend the body from pathogens. However, the body sometimes does not make the distinction of self from non-self and attacks itself, this is known as autoimmunity. The first spontaneous model of autoimmunity was presented in 1956 with the New Zealand black mouse, but in dogs, autoimmunity is more likely to be derived from the selective breeding process, inadvertently propagating autoimmune diseases (Gershwin, L.J. 2007).

Immune-mediated Thrombocytopenia is an autoimmune disease which can be primary or secondary and can be found in dogs. The disease destroys platelets due to misrecognition (Veterinary Health Centre, 2019). Primary ITP has an unknown cause but in secondary ITP, it is caused by a variety of things such as bacterial and viral infections. Platelets, which are fragments of megakaryocyte, are key in the mammalian body system as they circulate in the blood stream and facilitate clotting to repair damaged vessels. This is done by platelets, at a site of injury become active and bind to each-other to form a “platelet-plug” which closes the whole in the blood vessel. With an insufficient number of platelets, the body is not able to clot as quickly or efficiently, which puts the patient in danger of severe blood loss and reduced oxygen saturation of the tissues.

Pathophysiology

In both thrombocytopenia, auto antibodies are produced against platelet GP IIb/IIIa and Ib/IX, the former being the primary receptor in platelet adhesion. The auto antibodies coat the platelet which are detected by macrophages present in the spleen, or liver. Subsequently, the macrophages then phagocytose the platelet which can cause haemorrhagic episodes. In some cases of ITP, reduced platelet numbers are often in correlation to enlarged platelets. It is also characterized by purpura. Secondary immune thrombocytopenia can be caused due to several diseases including bacterial or viral infection, hereditary (in breeds like King Charles spaniels), brought on by certain antibiotics or other causes such as splenic enlargement or disseminated intravascular coagulation, where small blood clots develop in the blood stream, inadvertently decreasing platelets (MSD Manual, 2018).

Figure 1. Schematic showing the destruction of platelets in ITP (Labpedia, 2013).

Clinical signs/symptoms

ITP can present in a varying degree of clinical sings. Common presentations are those related to anaemia including lethargy, which is due to the reduced oxygen content of the blood, fever and heart murmur are also common signs. Like anaemia, dogs will have pale gums and a poor CRT. More severe cases of thrombocytopenia may see the dog have haematuria, excessive coughing and nasal mucus. Unprovoked internal bleeding may occur, and this is more likely when an undiagnosed patient is on medication such as heparin or pentoxifylline (Antimicrobe, 2017). In rare cases of ITP, collapse and intercranial bleeding may occur due to platelets not being able to repair blood vessels at an effective speed. Additional symptoms will include the patient showing easy and excessive bruising to minor injuries and abnormal amount of bleeding to cuts and grazes.

Diagnostics

Immune thrombocytopenia is diagnosed when other diseases that cause low platelet count are ruled out such as anaemia, infection or liver cirrhosis (Kelton et al, 1989). There are several diagnostic methods available, including complete blood count (CBC) or a mean platelet volume (MPV). In some cases, enlarged platelets can be indicative of ITP, this would be observed by a blood smear. A CBC and a blood smear should be used in conjunction to each other as a blood count alone may show a low platelet count, but platelets may coagulate affecting the result and therefore observing under a microscope will allow for the distinction of either a low platelet count or clumped together cells.

Previous diagnostic methods used to diagnose ITP was bone marrow aspiration. This was a favoured diagnostic tool as it would not be colluded by other diseases that may cause a low platelet count e.g. lupus erythematosus, however bone marrow aspirations are highly invasive and painful and with the advancement of diagnostic methods and technology, it is no longer classed as first-line procedure and is reserved for older patients and those with atypical features (Marsh, J.C. et al, 2003).

Where a normal platelet count would range between 130,000 to 400,000/mcL, the hallmark for immune related thrombocytopenia us a platelet count of <100,000/mcL which is often accompanied by mucocutaneous bleeding and petechiae, small red, purple or brown spots on the skin, but this is less noticeable in dogs than it is in humans (George, J.N, et al, 1998).

Where an internal bleed is expected, and in rare cases this can include inter cranial haemorrhages, an X-ray or ultrasound may be performed (Pet MD, 2019). This in combination with a CBC will aid the veterinarian in making the diagnosis.

Treatment/ prognosis

There are a range of treatments available for dogs affected by immune related thrombocytopenia, the most immediate to provide a way to deliver more platelets to the blood stream. One of these methods is a blood transfusion, it is commonly used and is the fastest way to increase the platelet count in the blood and may be used when haemorrhaging has occurred. This reduces the effects of anaemia also. More long-term treatments include immunosuppressive drugs including prednisone that act by blocking the cell mediated destruction of the platelets, but that can come with side effects of varying tolerability. Another treatment is chemotherapy, one drug involved is vincristine, that provokes the formation of platelets, but this is only temporary (DVM. 2007). Similarly, intravenous immunoglobin, which contains antibodies, that causes a short-lived reprieve from platelet destruction. One of the most long-lasting treatments is supportive care, this includes resting, exercise restriction and reducing activities that may cause bleeding. Regular transfusions may also be included in supportive care to replenish platelets.

Despite 20% of ITP patients are euthanized or die due to the complications surrounding the disease, the range of different treatments available, the prognosis 80% of dogs affected by ITP have a good prognosis with a normal life span (Jain, N.C & Switzer, J.W. 1981).

Bibliography

Antimicrobe (2017) Thrombocytopenia [online] http://www.antimicrobe.org/e24.asp. Accessed 22/11/2019.

DVM (2007). Overcoming the diagnostic and therapeutic challenges of canine immune-mediated thrombocytopenia [online] http://veterinarymedicine.dvm360.com/overcoming-diagnostic-and-therapeutic-challenges-canine-immune-mediated-thrombocytopenia. Accessed 22/11/2019.

George J.N; Raskob, G.E; Shah S.R, et al. (1998) Drug-induced thrombocytopenia: a systematic review of published case reports. Annals of International Medicine, 129(11), pp.886–890.

Gershwin, L.J. (2007) Veterinary autoimmunity: autoimmune diseases in domestic animals. Annals of the New York Academy of sciences, 1109(1).

Jain, N.C; Switzer, J.W. (1981) Autoimmune thrombocytopenia in dogs and cats. The Veterinary Clinics of North America: Small Animal Practice, 11(2), pp. 421-343.

Kaito, K; Ostubo, H; Usui, N; Yoshida, M; Tanno, J; Kurihara, E; Matsumoto, K; Hirata, R; Domitsu, K; Kobayashi, M. Platelet size deviation width, platelet large cell ratio, and mean platelet volume have sufficient sensitivity and specificity in the diagnosis of immune thrombocytopenia. British Jounral of Haematology, 128(5), pp. 698-702.

Kelton, J.G; Murphy, W.G; Luracelli, A; Garvey-Williams, J; Santos, A; Meyer, R; Powers, P. (1989) A prospective comparison of four techniques or measuring platelet-association IgG. British Jounral of Heamatology, 71, pp. 97-105.

Labpedia (2013) Platelets – part 1 – Idiopathic thrombocytopenic purpura (ITP), platelet antibody (anti-platelet antibody) [online] http://www.labpedia.net/test/215. Accessed 22/11/2019.

Latour, R.A; Sivaraman, B. (2011) Delineating the roles of the GPIIb/IIIa and GP-Ib-IX-V platelet receptors in mediating platelet adhesion to adsorbed fibrinogen and albumin. Biomaterials, 32(23), pp. 5365-5370.

Marsh, J.C; Ball, S.E; Darbyshire, P; Gordon‐Smith, E.C; Keidan, A.J; Martin, A; McCann, S.R; Mercieca, J; Oscier, D; Roques, A.W; Yin, J.A. British Committee for Standards in Haematology (2003) Guidelines for the diagnosis and management of acquired aplastic anaemia. British Journal of Haematology, 123, pp. 782–801.

Mitchell, O; Feldman D; Diakow, M, Signal, S. (2015) The pathophysiology of thrombocytopenia in chronic liver disease. Hepatic Medicine: Evidence and Research, 2016(8), pp. 39-50.

MSD Manual (2018) Disseminated intravascular coagulation (DIC) [online] https://www.msdmanuals.com/en-gb/home/blood-disorders/bleeding-due-to-clotting-disorders/disseminated-intravascular-coagulation-dic. Accessed 20/11/2019.

Pet MD (2019) Low platelet count in dogs [online] https://www.petmd.com/dog/conditions/cardiovascular/c_multi_thrombocytopenia?page=show. Accessed 21/11/2019.

Veterinary Health Centre (2019) Immune-Mediated Thrombocytopenia [online] http://vhc.missouri.edu/small-animal-hospital/small-animal-internal-medicine/diseases-and-treatments/immune-mediated-thrombocytopenia/. Accessed 22/11/2019.

Professional Practice in Bioveterinary Science – Part D, Sector Studies Exam Review

Upon reflection of the Sector Studies exam, as well as the module as a whole, I feel overall pleased with my current knowledge and understanding on statistics. After achieving 76% on the exam, I am more than confident that I have understood the concepts and been able to apply it in a context applicable to a bioveterinary science student. This combined with reflecting on my mathematics confidence at the beginning of the academic year, I feel reassured that my skill set in regard to statistics is at the required level at the end of the first year and the end of this module.

On of the main things I have identified on stating the Bioveterinary Science degree was my nerves about the time elapsed between starting and doing both my mathematics and statistics GCSE’s. Additionally, after personally disappointing result at A-Level Biology, where statistical tests such as Chi Squared were relevant, I was not confident about the statistics module. Despite this, when Chi Squared was covered in the lecture, and when it was asked in the exam, I didn’t struggle with the question and gained (most of/all) of the marks assigned to that question. Possibly, having the experience of doing these sort of tests at A-Level made it easier to succeed at this level. Studies looking at the development of the brain show that higher level circuits (and therefore concepts) are built upon lower level circuits and adapting higher level circuits is difficult if the lower level circuits are not present/developed properly. This is applicable to leaning (Harvard University, 2019).

An additional aspect I felt to have been instrumental in my understanding of the statistics and therefore doing well on the exam was that topics that may have seemed otherwise difficult or complex, were taught in a way that put them into context for the course. For example, when learning about experimental design, it was contextualised to pig enclosure and feeding and consequent agility which encouraged practical thinking. For me, this helped me get the right answer and approach the questions (that were asked in context also) with more confidence.

As I had identified early on in the course, periodically reviewing concepts within a module (either by reading/ re-writing notes or by the use of flashcards) increases the amount of content I retain and is generally helpful in revising, therefore reducing the need for cramming before the exam. By reflecting on this from the first reflective piece at the beginning of the academic year, I have seen that applying this technique across the different modules has improved my exam success.

Although the majority of the calculator functions (like variance and standard deviations) were provided on a sheet in the exam, the starter quizzes given at the majority of lecture, as well as personal revision, allowed for the need to use sheet to be minimal. This is reflected in a study that showed that constant use of a particular skill set reduced the likelihood of it being forgotten (Cahillane, M. 2015).

On looking at the results of the sector studies exams, the majority of the marks were lost on a general research methods questions. Some of the marks that were lost were due to small errors such as rounding too early during a calculation or misuse of decimal places and forgetting an error bars on the graph. This loss of marks could have been avoided by checking over my work again or trusting myself to have got it right the first time. Despite the loss of these marks, it is also reassuring as I now that I have grasped the main concepts and skills but, in the future, to take more time reviewing my answers as in real life these simple mistakes could have more dire effects in the future and, more presently, bring down my grade and do not reflect my capability.

Overall, during the exam I felt confident about tackling the topics covered and this is reflected in the result that I achieved. It is also encouraging that in both the Essential Laboratory module, where exam was maths based and for the Sector Studies exam, similar percentages where achieved indicating that my skill set, at the least hasn’t decreased. Furthermore, as statistics will be a large part of the Research Methods module in the second year, I feel confident that I have the level of skill to tackle it confidently and to achieve my desired result and feel as though I have learnt skills that can be both applied in the science field and be continually developed.

References

Harvard University , (2019). [ebook] Available at: https://developingchild.harvard.edu/wp-content/uploads/2015/05/Science_Early_Childhood_Development.pdf [Accessed 20 May 2019]

Cahillane, M, (2015). [online] Available at: http://www.icicte.org/ICICTE2015Proceedings(Papers)/7.2%20Cahillane_MacLean&Smy.pdf [Accessed 20 May 2019].

Meiosis and Meiotic Failures Causing Male Infertility

When considering cell division, there are a definitive two types, mitosis and meiosis. While mitosis is responsible for the cell division throughout the majority of cells within the body, meiosis is responsible for the formation of germ cells – sperm and ova (Gardener, A. 2018). Meiosis can be regarded as a complex two-stage process, but only in one stage does DNA replication occur. In the first stage, the homologous chromosomes of human cells (n= 46) pair up and exchange genetic information. At anaphase I the whole chromosomes migrate to the poles of the cell to produce haploid daughter cells (n=23). In the second stage, the chromatids become segregated to produce 4 haploid gametes (Aran, B. et al. 2000). Meiosis not only ensures an even number of chromosomes in offspring, allowing fertility, but also allows for genetic variation via the shuffling of DNA. When evaluating how integral meiosis is to molecular biology, it is necessary to emphasise that meiosis produces daughter cells with a different combination of genes to the parent cell. This in itself ensures genetic variation, which is important to human health, for example increased genetic variation can lead to reduced disease susceptibility (Brooks, L.D. et al, 2007). The variety of genetic variation can also influence factors such as common diseases, hereditary and even traits such as aggression (Frazer, K.A. et al, 2009). When meiotic failures occur, chromosomal diseases are facilitated. Examples of this are seen in Klinefelter Syndrome and the deletion of the Fkbp6 gene, which both result in male infertility.

Meiosis is a two-stage process, separated into meiosis I and meiosis II.

The first stage, meiosis I, entails 5 steps. The first is Prophase I. In a germ cell, the nuclear membrane dissolves and chromosomes condense, becoming visible. Homologous chromosomes pair up (synapsis), appearing as tetrad. Genetic variation via the crossing over of chromosomes is essential in most sexually reproducing organisms (Keeny, B. 2001) and occurs in prophase I, initiated in the zygotene sub phase, where the chromosomes align gene by gene and non-sister chromatids exchange genes at corresponding segments of DNA producing recombinant chromosomes. In the next stage, Metaphase I, the bivalent chromosomes align on the metaphase plate and kinetochores produced by centrosomes from opposite poles of the cell attach to the pair of homologous chromosomes. In Anaphase I, the homologous chromosomes separate by the retraction of the kinetochores. The sister chromatids remain attached at the centromere, causing them to move to the pole that the spindle fibre is attached to. In Telophase I, following that each half of the cell has a complete haploid set of chromosomes, the nuclear membrane reforms around 2 daughter nuclei. Chromosomes become less condense also and a cleavage furrow appears. Almost simultaneously, cytokinesis occurs, where a contractile ring forms near the cleavage furrow and pinches the cell, marking the end of meiosis I. The end result of meiosis I is 2 daughter cells with a half set of chromosomes.

The second stage, meiosis II, it is much similar to mitosis and occurs after Interkinesis, which is a period of rest for the cell before the next stage (Concepts of Biology, 2012). Immediately after cytokinesis, prophase II in initiated to prevent the elongation of the chromosomes. The nuclear membrane disappears, and chromosomes become compact, a spindle apparatus forms. The chromosomes are assembled as in meiosis I. Metaphase II then commences, the chromosomes align at the equator and microtubules from opposite poles of the spindle attach to the kinetochores of chromatids. In in this step, independent assortment occurs (Chinnici, J.P. et al. 2004). Independent assortment sees chromosomes move randomly to separate poles resulting in many different combinations of chromosomes in each gamete, ensuring genetic variation. The next step is anaphase II, the centromeres spilt causing the sister chromatids to separate and move to opposite poles. Telophase II follows where the nuclear membrane reappears, and 4 daughter nuclei are formed. Cytokinesis cleaves the cells apart to form a tetrad of 4 haploid daughter cells, each with 23 chromosomes.

Figure 1. 1. Prophase I, 2. Metaphase I, 3. Anaphase I, 4. Telophase I, 5. Cytokinesis, 6. Prophase II, 7. Metaphase II, 8. Anaphase II, 9. Telophase II, 10. Cytokinesis (Richardson, E. 2015)

At any stage within meiosis there is opportunity for failure which in turn could lead to chromosomal disease. Commonly, male infertility is associated with failure of spermatogenesis, specifically chromosome abnormalities (Anton, E. et al, 2005). Aneuploidy is a numerical aberration where there is a failure of chromosome division during meiosis causing an extra chromosome or a deficiency in chromosomes and is the common cause of male infertility causing Klinefelter Syndrome (Ramasamy, R. & Zafar, A. 2016) and is the most common sex chromosome disorder in men.

In those affected by Klinefelter Syndrome, there is an extra copy of the X chromosome, so where most males would have one X and one Y chromosome (46, XY), affected males will have 47, XXY (NIH, 2019). The syndrome is not inherited, but an addition of an extra X chromosome from an egg cell to a sperm cell with one Y cell will result in an offspring with Klinefelter Syndrome. The majority of KS patients have a 47XXY karyotype, but other variations include mosaic KS with a combination of 46XY cells and 47XXY cells, have higher grade aneuploidy (48XXXY, 49XXXXY). In mosaic patients, the phenotype and infertility presentations are less severe than the 47XXY karyotype (Dobs, S.A. et al 2018).

The main symptoms of Klinefelter Syndrome are infertility and reduced testes. Other symptoms include psoriasis and a lack of pubic, facial and underarm hair. The presence of the additional X chromosome reduced the ability to produce sperm, with biopsies in affected males often finding little to no sperm (Gies, I. et al 2015). The rapid deterioration in the production of germ cells is seen during puberty and may arise from aneuploidy induced non-homologous recombination and subsequent activation of apoptosis-related genes (Dobs, S.A. et al. 2018) that degrades the production and function of both spermatocytes and Sertoli cells. There are treatment options in males that have less severe symptoms such as hormone therapy to either harvest as many viable sperm as possible to preserve the sperm, or in some cases to give a chance to have normal fertility.

Not only can meiotic failures cause the addition of genetic information but may also cause the deletion of a gene. One particular chromosomal disease that causes male infertility is the deletion of the Fkbp6 gene, in human, the Fkbp6 gene is located on chromosome 7q11.23 and expresses in various tissues with the highest expression in testis and so the deletion of it is commonly linked to azoospermia or oligozoospermia (Xiao, C. et al, 2007). In several studies on rats and mice, it was observed that the deletion of the Fkbp6 exon 8 was the causative mutation for sterility as it blocked spermatogenesis from occurring. The loss of the Fkbp6 results in abnormal pairing and misalignments between homologous chromosomes as well as auto synapsis of X chromosome cores in meiotic spermatocytes. Such imbalances and mutations cause apoptosis of spermatozoa. The majority of the studies therefore conclude that Fkbp6 is a key component in sex specific fertility and for the fidelity in homologous pairing in meiosis (Crackower, M.A. et al, 2003). The micro-deletions causing male infertility (azoospermia factor, or AZF) are only currently found on the Y chromosome so when this region undergoes meiosis, spermatogenic failure occurs.

When summarising meiosis, it can be described as a complex two stage process that provides offspring with a varied set of genes to their parents. The process starts with one diploid cell that undergoes one round of cell division to produce 2 haploid cells that have undergone genetic shuffling in the zygotene subpages of prophase I. In the second cell division, there is no duplication of chromosomes, but the product is a tetrad of 23n daughter cells. This process is critical for the sexual stability of successive generations. Meiotic failures do occur and when they do it can have negative outcomes, such as infertility. Klinefelter Syndrome is chromosomal disorder causing infertility is aneuploidy, an additional X chromosome causing a 47XXY karyotype, instead of the normal 46XY in males. Comparatively, deletion of genes can also result in apoptosis of spermatozoa and impaired spermatogenesis as seen with the deletion of the Fkbp6 gene and its associated alleles.

References

Anton, E. Blanco, J. Egozcue, S. Egozcue, J. Sarrate, Z. Vidal, F. (2005) FISH Studies of Chromosome Abnormalities in Germ cells and its Relevance in Reproductive Counselling. Asian Journal of Andrology, 7(3), pp. 153-162.

Aran, B. Barri, P.N. Blanco, J. Egozcue, J. Egozcue, S. García, F. Veiga, A. Vendrell, J.M. Vidal, F. (2000) Human Male Infertility: Chromosome Anomalies, Meiotic Disorders, Abnormal Spermatozoa and Recurrent Abortion. Human Reproduction Update, 6(1), pp. 93-105.

Altshuler, D. Brooks, L. D. Bowcock, A. M., Carter, N.P. Church, D.M. Eichler, E.E. Felsenfeld, A. Guye, M. Lee, C. Lupski, J.R. Mullikin, J.C. Nickerson, D.A. Pritchard, J.K. Sebat, J. Sherry, S.T. Smith, D. Valle, D. and Waterston, R.H. (2007) Completing the Map of Human Genetic Variation. Nature, 447, pp. 161-165.

Chinnici, J.P. Torres, K.M & Yue, J.W (2004) Students as “Human Chromosomes” Role-Playing Mitosis and Meiosis. The American Biology Teacher, 66(1), pp. 35-39.

Crackower, M.A. Cohen, P.E. Hui, C. Kaneko, H. Kawai, Y. Kikuchi, K. Kobayashi, E. Kolas, N. K. Kozieradzki, I. Kunieda, T Landers, R. Mo, R. Moens, P.B. Noguchi, J. Nieve, E. Osborne, L.R. Penninger, J. M. Sarao, R. & Wada, T. (2003) Essential Role of Fkbp6 in Male Fertility and Homologous Chromosome Pairing in Meiosis. Science, 300(5623), pp. 1291-1295.

Dobs, A.S. Hawksworth, D.J. Herati, A.S. Jordan, P.W. & Szafran, A.A (2018) Infertility in Patients With Klinefelter Syndrome: Optimal Timing for Sperm and Testicular Tissue Cryopreservation. Reviews in Urology, 20(2), pp. 56-62

Frazer, K.A. Murray, S.S. Schork, N.J. Topol, E.J (2009) Human Genetic Variation and its Contribution to Complex Traits. Nature Reviews Genetics, 10, pp.241-251.

Gardener, A. Heim, M. López-Córdoba, A. Stauffer, S. & Ungu, D.A.K. (2018) Meiosis In: Labster Virtual Lab Experiments: Basic Biology. Springer Spectrum, pp. 27-41.

Gair, J. & Monlar, C. (2012) 7.2 Meiosis, Cell Division and Genetics, Concepts of Biology – 1st Edition. (24/03/2019) https://opentextbc.ca/biology/chapter/7-2-meiosis/

Gies, I. Oates, R. De Scheooer, J. & Tournaye, H. (2015) Testicular Biopsy and Cryopreservation for Fertility Preservation of Prepubertal Boys with Klinefelter Syndrome: a pro/con debate. Fertility and Sterility, 105(2), pp. 249-255

Harton, G.L. & Tempest, H.G. (2012). Chromosomal Disorders and Male Infertility. Asian Journal of Andrology, 14(1), pp. 32-39.

Keeny, B. (2001) Mechanism and Control of Meiotic Recombination Initiation. Current Topics in Developmental Biology, 52, pp. 1-53.

National Library of Medicine (2019) Klinefelter Syndrome, US National Library of Medicine (10/03/2019) https://ghr.nlm.nih.gov/condition/klinefelter-syndrome#sourcesforpage

Ramasamy, R.A. & Zafar, A. (2016) Management of Infertility in Klinefelter Syndrome. Male Infertility, pp. 135-144

Xiao, C. Yang, Y. Zhang, S. Zhang, W. & Zhoucun, A. (2007) Mutation Screening of the FKBP6 Gene and its Association Study with Spermatogenic Impairment in Idiopathic Infertile men. Reproduction, 133(2), pp. 511-516.