A colony of woodchucks were to be used on studies which involved repeated IV dosing and/or multiple blood collection time points. Some of the animals were wild-caught, most were aggressive, and all were challenging to handle or restrain for venipuncture. Therefore, all venipuncture had to be performed under anesthesia. Because of the frequency of vascular access required for the studies, we decided to place vascular access ports (VAPs) to allow IV dosing and blood collection without anesthesia. We incorporated knowledge and experience obtained in-house from implanting ports in other species and information from collaborators who worked with ported woodchucks to develop a successful Woodchuck VAP model. We placed arterial and venous ports in 70 woodchucks with patency extending up to 23 months. This poster will provide an overview of the surgical procedure and some unique aspects of this procedure in woodchucks.

The presentation will very briefly cover the regulatory system for lab animals in China, as well as the regulations and guidelines as related to education and qualification requirement for lab animal users and practitioners. A newly issued national standard entitled requirement for lab animal practitioner will be reviewed in detail and its implementation plans discussed. 

Learning Objectives:

• A basic understanding of the Chinese regulatory system for lab animals
• Be aware of the basic requirement for lab animal technicians, scientists, and veterinarians

The presentation will develop the FELASA (Federation of the European Laboratory Animal Science Association) views on education and training in an international setting, more specifically in Europe. Since 1995, date of the first publication on education of people involved in animal experimentation, FELASA has been dealing with this issue on one hand by introducing a taxonomy of roles in animal experimentation, the so-called FELASA categories A, B, C & D and on the other hand by proposing an accreditation scheme to ensure high quality education. 

With the introduction of the European Directive 2010/63/EU on the protection of animals used for scientific purposes, it is mandatory that any person working with animals in a scientific set-up has to be educated and trained beforehand. We will briefly explain how the FELASA accreditation process adapts to the new regulatory landscape. 

To the contrary to education, continuing professional development (CPD) is for many European countries not a legal requirement. However, FELASA has given some guidance on this crucial component of the acquisition and maintenance of competences of persons involved in animal experimentation. Guidance on how a CPD scheme could be introduced will be developed on the basis of seven principles. An example will be given with Switzerland which has made CPD mandatory  for any person working in animal experimentation, from animal care takers to members of regional ethical boards.

Learning Objectives:

• To be able to describe the European education and training system and to understand how the FELASA education and training accreditation process works 
• To acquire some knowledge of what are the basic ingredients for implementing a CPD scheme in an institution
   

During the last 50 years the claims for continuous development and training of laboratory animal staff has been progressively developing. In the same period competent veterinary support has become an increasingly integrated part of running high-tech laboratory animal facilities and performing state-of-the-art experiments in respect of the 3Rs. At present, the laboratory animal science community is faced with new challenges to meet the objectives of the new Directive 2010/63/EU. Although, the more detailed demands issued on the basis of the directive is generally to regarded as a positive input, because it has created an opportunity to further advance laboratory animal science training and education, it is also a challenge, because time and resources put a natural limit on the possibilities for lab animal vets to receive the proper training. Already today, not enough LAS programs are available across Europe, and new courses with a more flexible content need to be developed on the various levels of animal experimentation jobs. Online courses can help dealing with these challenges, as they are available anywhere at any time and are not limited to a certain number of participants. Theoretical knowledge and practical aspects of LAS can be taught online line at a students’ own study pace. Web-based programs offer the advantage of the option of multi-lingual training approaches within the same platform. Also it offers more flexibility for both teacher and student. For vets, it is obvious that there is also a need for practical training. However, if the hiring policy of the company or institution is directed towards securing a mixture of generations among vets, or if smaller organisation collaborate to secure this, the practical training is far more easy and probably also wiser to establish on-site rather than leaving this part to larger educational institutions such as the universities. In the future, the universities should, therefore, offer easily accessible online teaching programmes available globally to cover the needed curriculum, while the facilities themselves should collaborate on a more local scale to establish the practical training. The large non-governmental organisations in laboratory animal science and medicine, such as the veterinary unions, the laboratory animal science societies and the diplomat collages should then take on them to quality assure and accredit these programs. 

Learning Objectives:

• A basic understanding of the need for post graduate education of laboratory animal veterinarians
• An introduction to the potentials of blended learning for global teaching programs

The last few years have led to great progress in Chile in terms of laboratory animal care and use. There are new legal mandates to protect laboratory animals, and funding agencies now require all protocols to be evaluated by an ethics committee. Both academic and public institutions that conduct animal research have established ethical review bodies (IACUCs), and IACUCs come together for a yearly national meeting. A recent national survey evaluated rodent housing, in order to estimate the variability of vivaria conditions and their potential effect on experimental results. Some of these results will be discussed during this webinar. The second topic of discussion will be related to surgery: Among the experimental procedures performed on animals, surgery is perhaps at the top of severity levels. Before the existence of IACUCs in Chile, it was up to the researchers as to whether to use analgesics or not, as well as where  surgery should be performed, type of anesthesia, personnel conducting surgery and the level of asepsis, if any. Ethics committees must now be informed in detail of all procedures carried out with research animals: The surgical site, previous training of the person performing surgery, type and dose of anesthesia and analgesia, intra-operative support, post-operative care and daily monitoring of animals. In terms of anesthesia, there is progressive use of inhaled anesthesia due to technical advice from Vets and because researchers have realized that gas anesthesia equipment is an investment rather than a cost: mortality rates drop drastically when changing from injected to inhaled anesthesia.  Hands-on training surgical courses have become valuable tools to gradually change investigators’ outdated and tradition-based surgical practices. An additional effective measure has been to demonstration of evidence-based information: For example, necropsies performed on animals at the end of protocol to show the effect of poor aseptic technique in organs that were not the target of the experimental protocol, and hence go unnoticed too frequently. Lastly, emphasis has been placed on euthanasia methods: which methods are acceptable, acceptable with conditions and unacceptable. All of these advances require strong institutional support and the certainty that research quality improves when animal welfare is brought into the equation.   

Learning Objectives:

• Provide an overview of the main challenges and advances of animal research in a developing country with evolving regulations. 
• Increase awareness of the need of standards’ harmonization  in laboratory animal research between developed and developing countries.
   

Traditional models of liver fibrosis in rodents involve many weeks of induction, with frequent injections of a pro-fibrotic agent. The most commonly used end-point in these studies is immuno-histochemical analysis of markers of fibrosis in the liver. This whole process is time-consuming, very labor-intensive in terms of animal handling, processing of the tissues and produces only a semi-quantitative measure of fibrosis. In addition, if pharmacological agents are to be explored, this means more ‘hands-on’ work for the experimentalist due to the likelihood of the need for daily dosing and adds substantially to the cost of the study because of the amounts of compound required. We have modelled the inflammatory component which drives liver fibrosis, by using the concanavalin A (conA) challenge to evoke an acute liver inflammation and the Vium ‘Digital Vivarium’ as a way of monitoring mouse behavior in response to the challenge. Acute liver inflammation evokes measurable changes in mouse behavior over the course of 24hrs and we believe these behavioral measures will be useful in testing the efficacy of pharmacological agents which suppress the inflammation.  By focusing on the inflammatory drivers of the pathology we dramatically reduce the duration of the studies, obtain a quantitative measure of the effect of the conA whilst minimizing handler intervention and reduce compound demand. Moreover, we believe the value of efficient in vivo testing of hypotheses enables us to embrace ‘systems biology’ in a way that is entirely compatible with aspects of the ‘3Rs’. Using a behavioral assessment of inflammation enables us to make rapid decisions on mechanisms and targets of interest. This reduces costs and time but maybe more importantly, promotes improved iterative decision-making regarding targets and compounds.

The University of Michigan’s Unit for Laboratory Animal Medicine (ULAM) has a diverse Training Core that teaches a wide variety of techniques on all common laboratory species. The most common species used are mice, rats, guinea pigs and rabbits; for this reason, the Training Core holds small populations of these species for training classes with hands on technique demonstration and practice. With a small population that needs to train basic skills for competency to a rotating schedule of students, we acclimate our animals to handling and restraint as well as reward for participation in our regular workshops. To do this we spend time handling and practicing injections in our laboratory setting while modifying techniques in class to ease stress for animals and participants. Extra enrichment and treats are used to associate the training room and staff with positive interactions. Primary housing enclosures include social housing and additional enrichment to help lower stress in their environment. New enrichment devices are introduced to Training Core animals before moving forward into the larger animal population. An online database for the training core population allows for a systematic process in using all of our animals for greater efficiency. We have observed animals that are easier for new laboratory staff to handle and practice techniques as well as an improved staff bond with long term training animals.

There is no professional area in which CPD is more necessary than in the field of laboratory animals. The continuous advancement of science provides us with continuous opportunities to improve our performance, improving both the welfare of the animals and the scientific achievements obtained with them.

CPD must not only be a priority for high qualified professionals such as veterinarians or scientists but for all categories of personnel. Some countries, such as those in the European Union, have made CPD in Laboratory Animal Science (LAS) compulsory in order to maintain the competence of personnel working with animals. Several systems to recognize CPD have been developed by some organizations, such as the Federation of European Laboratory Animal Science Associations (FELASA). 

One characteristic of a CPD recognition system must be flexibility and adaptation to each person's needs and career. To this end, a diverse variety of educational activities must be included in CPD, for instance, attending a scientific meeting in the field of interest, participating in a course, but also learning new techniques that will allow us to refine our performance.

Due to this diversity, CPD recognition presents some difficulties in the validation and harmonization of systems. However, all of them require that each person is aware of the need to keep records for all CPD activities. A personal CPD port-folio needs to include: basic training records, such as basic courses in LAS, attendance at scientific meetings and a record of new techniques learned with dates and a validation signature. In the session some examples for keeping CPD records will be given.

For veterinarians there are several international recognized programs providing both LAS specialization and CPD. The Laboratory Animal Medicine Colleges in America, Asia and Europe and most recently the VetCEE LAS & Medicine program in Europe, require a 5 year CPD re-evaluation to keep the Specialist status.

Learning objectives:

• To be aware of how CPD is important for all categories of personnel working with experimental animals and how CPD can be recorded and evaluated by several organizations around the world.
• To be familiar with some of the Specialization and CPD international programs for veterinarians.
   

The Eastern woodchuck (Marmota monax) is a large, burrowing rodent with native territories throughout parts of the US and Canada. Woodchucks are susceptible to infection with woodchuck hepatitis virus (WHV), a hepadnavirus that is endemic in certain wild populations. The epidemiology and course of disease of WHV infection demonstrates numerous similarities with another member of the hepadnaviridae family, human hepatitis B virus (HBV), including a high rate of progression to hepatocellular carcinoma (HCC). These characteristics have led to the use of WHV–infected woodchucks as a valuable and highly translatable animal model of viral hepatitis and oncogenesis. Despite the clear utility of this species for these and other research applications, housing and maintenance of woodchucks in a laboratory setting is relatively uncommon and information regarding their care requirements and technical procedures is limited. In this presentation, we will give an overview of our facility’s approach to husbandry, management, health care, and experimental methods in this unique laboratory rodent.

Perlara PBC, is a public benefit company committed to discovering small molecule therapeutics for rare genetic diseases. Our approach consists of the use of simple, whole animal models such as yeast, worm, flies in drug discovery screens. Molecules that pass successfully through our screens are validated in patient cells before advancing into higher animal models of the disease.  Niemann Pick Type C (NPC) is a devastating lysosomal storage disease with high mortality rates. As part of our pipeline for advancing promising candidates for NPC disease from our screen into NPC mutant mice, Perlara partnered with Vium Inc. to validate an NPC knockout mouse model (NPCnih) for preclinical studies on their proprietary automated platform. Vium incorporates sensorized cages, automated image capture and processing algorithms to deliver high resolution data with minimal human intervention. 

The first goal of this project was to confirm that automated recording could be used to recapitulate previously published findings on NPC mutant mice. Using this approach, we validated the following previously published findings: a) NPC KO mice show significant weight loss relative to age-matched WT littermates, b) once weekly subcutaneous dosing of 4000mg/kg cyclodextrin normalized the weight loss observed in NPC KO to WT levels. Previous studies have also highlighted that NPC KO mice suffer from compromised pulmonary function. Unexpectedly, the automated data capture methods of Vium enabled us to identify two secondary endpoints of the disease, namely decrease in respiration and circadian motion. These metrics were correlated to the onset of disease pathology. We also compared NPC KO mice with WT littermates on traditional behavioral assays such as balance beam tests and accelerated rotarod assays. While baseline performance on these assays on postnatal day 35 did not differ between age-matched WT and KO, NPC KO mice showed significant decreases in performances on both behavioral assays by postnatal day 50. This is consistent with previously reported data. In conclusion, using automated data capture methods, Perlara was able to validate previously published data on NPC KO mice as well as identify two new metrics by which to benchmark disease progression. Future studies will focus on assessing the effect of Perlara’s ‘hit’ compounds on NPC KO mice. 

The powerful and expanding use of genetically modified mice introduces important aspects of genetics in managing these strains for breeding and experimental usage. This management takes on increasing importance within the current backdrop of focus by NIH on “rigor and reproducibility”, which means that increasingly grant and paper reviewers will expect specific data to support the stated genotype of an experimental mouse strain. We should recognize that this genotype includes both the specific genetic modification in the mice as well as the overall genetic background of these mice. The first issue is the need to genotype many of these strains for the desired genetic modification and the challenges that involves.  While genotyping of some genetically modified mouse strains is not particularly difficult, some strains can be quite challenging, and any genotyping is exacerbated when a lab is not accustomed to basic molecular biology studies.  

Specific challenges of genotyping each strain is unique to that strain but fundamentally it involves developing and utilizing a genotyping strategy that is most often dependent on polymerase chain reaction (PCR) for the assay.  Those who have worked with PCR know that each reaction has its own unique challenges of dependability that are impacted by many variables such as DNA prep, buffer, PCR machine parameters (temperature time and number of cycles), appropriate gel system to visualize the data if the assay is scored visually as opposed to a quantitative PCR.  Against the basic challenge of genotyping are unique aspects of each strain such as whether it is homozygous, hemizygous or heterozygous, and whether the modification is on a sex chromosome and whether more than one modification is involved and whether they are genetically linked in close physical proximity or unlinked on different chromosomes. A recent complexity in genotyping is the development of CRISPR knockout strains in which the mutation is different in each founder and each founder’s offspring have different challenges in genotyping.  Every strain of genetically modified mouse is unique and must be specifically genotyped appropriately every time or the genetics of the colony are disrupted and experiments fail after significant investment.  Another important issue in rigor and reproducibility is genetic background.  Genetically modified mice are made in a mixed or inbred background but for experimental reasons they are often backcrossed into other inbred backgrounds.  The genetic background has a major impact on results of many experiments.  However, genetic background is rarely directly assessed because historically it has been very difficult.  There now exists a shared resource at Dartmouth known as “DartMouse” that is under my direction, which is able to efficiently validate the genetic background of individual mice.  
 

In rabbits, buprenorphine instant release (IR) is commonly used to alleviate pain, especially in surgical models. However, maintenance of IR therapeutic levels requires redosing which significantly increases unnecessary animal handling, restraint and non-compliance issues post-operatively. The development of sustained-release (SR) formulations of buprenorphine in rabbits to deliver constant levels of the drug with adequate plasma concentration and satisfactory analgesia would greatly improve standard of care. In here, we hypothesized that single subcutaneous administration of at least one of three SR (Buprenorphine SRTM Lab, ZooPharm) concentrations (SRLow - 0.06 mg/kg, SRMedium - 0.15 mg/kg, SRHigh – 0.3 mg/kg - n = 3 animals per group) is associated with up to 72 hours of pain relief and more predictable behavior and pain-associated parameters than IR (0.05 mg/kg BID) in a bilateral rabbit knee defect model. In addition to body weight, fecal output, grimace and lameness (0-4, where 0 = continuous weight bearing, 1 = single limb lameness, 2 = multiple limb lameness, 3 = toe touch – non-weight bearing, and 4 = completely non-weight bearing) scales were assessed and scored before and for 72 hours after surgery. No skin reaction to any of the tested SR concentrations was observed within the study phase. Post-operative assessment also revealed no significant changes in body weight across groups. Compared to IR and SRHigh, daily fecal output assessment was higher in rabbits treated with SRLow and SRMedium. Single limb lameness and signs of pain (grimace scale) were observed only in rabbits treated with IR (24 hours) and SRLow (24-48 hours). In fact, rabbits treated with SRLow also received meloxicam (0.3 mg/kg PO SID) within the first 48 hours. Compared to IR, SRLow and SRHigh single administration of SRMedium had little effect in the fecal output and behavior of rabbits enrolled in the selected surgical model. Rabbits treated with IR, SRHigh and SRMedium had similar PK values during the first post-operative hours. In SRHigh and SRMedium, the plasma concentration of the drug remained within the 1ng/ml mark for at least 48 hours. In conclusion, rabbits treated with one dose of SRMedium at 0.15 mg/kg had similar post-operative outcome to the observed in animals treated twice a day with the gold standard drug (IR) and significantly less adverse effects when compared to the observed in the SRLow and SRHigh groups. Further investigation is needed to validate this pain management protocol in other rabbits orthopedic models.

Learning objectives: 

• Provide an overview of buprenorphine SR pharmacokinetics at different doses and over time.
• Provide an overview of adverse effects and clinical findings after buprenorphine SR administration.

Large animal models have recently become a staple in biomedical research.  The pig’s similarities to humans in terms of genetics, anatomy, and physiology have allowed the pig to gain momentum as a biomedical model.  On a nucleotide basis, the pig genome is 3 times closer to the human than rodents are to humans.  Although there are some anatomical differences between the human and pig, the pig is still becoming the model of choice for many human diseases.  In 2003, the National Institutes of Health developed the infrastructure for a central resource center for swine models for human health and diseases.  The National Swine Resource and Research Center (NSRRC) was developed to provide valuable swine models to investigators across the country.  Currently, the NSRRC has 60 models available to the scientific community with an additional 15 models in various stages of production and characterization.   Management of genetically modified swine models can range from standard housing to highly specialized housing which may be necessary for the disease model.   Maintenance or breeding of genetically modified swine models also ranges from strains being maintained in the heterozygous/monoallelic state while others can be maintained in the homozygous/biallelic state.  Although there may be specialized needs for some models, in general genetically modified swine models can be maintained as any other research pigs.  Biomedical research for human health and disease has begun to incorporate the swine model as the large animal of choice.  

Learning Objectives:

• To determine the availability of swine models for investigators to request
• To determine specific management skills for genetically modified swine models

In-vivo surgical models are becoming more prevalent and more critical to the drug development process.  As the vast majority of individuals performing these procedures have entered the field without any formal training in aseptic principles and surgical techniques, their instruction falls to trainers at their institutions.  Inadequate training programs lead to poor surgeons and study outcomes, which lead to flawed data and may also compromise animal welfare.  This presentation will offer strategies for evaluating a nascent program’s needs and resources, ways to utilize electronic media and other applications to maximize the use of training personnel, and methods to continually evaluate and improve the training program over time.

The cat remains an important comparative species for studying the long-term safety and efficacy of emergent medical modalities, such as gene and stem cell therapies for correcting degenerative diseases. Many spontaneous mutations have been characterized in breed cats, and some key feline genetic disease models are now actively maintained in research colonies. Accurate diagnosis and control of endemic feline infectious diseases is important to reduce morbidity and mortality, as well as to limit confounding factors that could compromise research studies. The impacts of common feline pathogens on cat colony health will be discussed, as well as management options for controlling and eliminating endemic diseases. The use of assisted reproductive technologies in cats to prevent and break cycles of transmission of some pernicious viral agents will be covered. The collection and cryopreservation of viable gametes and embryos also provides an important protection against loss of important models due to disease or disaster. In addition, measures to accommodate the specific social and behavioral needs of cats will be presented both in the context of reproductive management and social well-being. Group housing designs and management options to maximize social interactions and minimize opportunities for aggression and injury will be discussed as well. This presentation should benefit participants with an interest in comparative laboratory animal medicine who are establishing or working with feline research models.

Learning Objectives:

• Participants should become familiar with the impacts of specific feline pathogens on cat colony health; diagnostic tests and treatment options available; and methods of elimination.
• The audience should retain an understanding of available management tools and assisted reproductive technologies that can be used to eliminate endemic pathogens from closed cat colonies.
   

The utility of animal models in drug development is limited, in part, by the measures available to researchers for monitoring animals. In life measures are often subjective, lack clinical relevance and can be hard to reproduce.  Conventional approaches to collecting in-life measures are labor-intensive and may introduce experimental variability, as they require frequent handling of the animals during the course of the study.  Vium’s technology platform addresses these essential problems by continuously collecting physiologically relevant data, providing researchers greater insights and consistency for in vivo studies.
 
We present Vium’s Digital Vivarium, which combines HD video cameras and digital sensors with computer vision and machine learning to enable automatic, continuous assessment of disease progression for study animals simultaneously in the home cage.  The system measures physiologic, behavioral, environmental, husbandry-related, and disease-specific parameters.  Within specific animal models, novel metrics are identified that track disease severity and efficacy of reference compounds automatically and objectively.
 
We have applied this method across a spectrum of disease models.  This presentation will show how novel Vium metrics can be used to assess disease in a model of lung injury and in the MRL/lpr model of Systemic Lupus Erythematosus. The result is a new paradigm for profiling the safety and efficacy of pharmacological agents that is not limited in scale and throughput by individual technicians.
 
Learning Objectives:

• Describe the Vium Platform and demonstrate how novel insights can be derived from non-invasive digital metrics in preclinical rodent studies
• Continuous digital readouts of physiologic parameters provide reproducible, previously unattainable tracking of disease in Lupus and Lung Injury models as examples.
   

Training programs for animal care and use personnel are integral to both animal welfare and quality research outcomes. There are a variety of means of providing training to animal users, however ensuring the effectiveness of training can't be overlooked. This presentation will review  methods for training and for evaluating the effectiveness training of programs for animal care and use personnel.

There are many factors involved in rabbit breeding that impact performance.  In this evaluation of a Dutch Belted rabbit breeding colony, the environment, breeding techniques, and socialization all contribute to an overall goal of producing rabbits that may be utilized for biomedical research. Diligent record keeping of breeding activities and resulting production is imperative for successful colony management and animal welfare.   Challenges with pregnancy determination by breeding staff also effect general productivity.  The methods of palpation versus the use of ultrasound are evaluated in their effectiveness to determine pregnancy.  Once gestation comes to an end, strategies of fostering and re-breeding must be evaluated.  Successful fostering and timely re-breeding of does are tools to maximize production, post kindling.  Daily nest box checks to observe kit health and development is essential.  Early socialization of kits, starting as soon as day 1, may be incorporated into these nest box checks as a means to improve future temperament.  Pair housing upon weaning is a valuable introduction to a group housed setting, increasing the potential for successful long term pair housing.

Many of us who believe in the importance of aseptic rodent surgery have lived through the frustration of trying to implement rodent aseptic surgery in our programs because it seems that application of aseptic technique is so difficult to illustrate and implement.  For years, I have in vain searched the internet and other sources for easy, well-illustrated materials to teach our investigators proper technique.  The search is over!  My frustration has led us to create easy to follow illustrations to help investigators, veterinarians, trainers and IACUCs everywhere to develop sound rodent aseptic surgical skills.   

This presentation will rely heavily on images and videos.  The presenter will walk the audience through very simple and fun steps in the application of aseptic technique.

Other practical rodent surgery tips will be discussed as time allows.

Learning objectives:

• Learn easy to apply aseptic techniques of rodent surgery.
• Learn where to point others to sources where these techniques can be learned.

Several regulatory bodies require that animals used in biomedical and behavioral research exposed to procedures that may cause more than momentary or slight pain or distress should be provided with appropriate sedation, analgesia, or anesthesia. However, use of analgesics in certain murine models is a topic of debate, as analgesics have the potential to have an effect on biological responses of an animal.  A common analgesic used in research is the synthetic opiate buprenorphine HCL. Sustained release Buprenorphine (Bup SR) is a sustained-release injectable formulation of buprenorphine that has grown in popularity due to its extended duration of action compared to standard buprenorphine. This presentation will explore the findings of several research reports comparing buprenorphine HCL to sustained release formulations of buprenorphine with regards to effects on experimental data and postsurgical condition in mice.

Learning Objectives:

• Identify the benefits and disadvantages of the use of buprenorphine (HCL and sustained release) analgesia in a murine model
• Describe methods to evaluate postsurgical condition in mice

Development of assisted reproductive technologies in the dog has lagged behind many other domestic and laboratory animal species, due in part to their unique reproductive biology. Here, we will discuss the challenges with dog ART and the technologies associated with the recent successful development of in vitro fertilization in this species, including estrus hormone monitoring, gamete collection, sperm capacitation, fertilization, and embryo culture, cryopreservation and transfer. 

Learning Objectives:

• Describe unique aspects of female canid reproductive biology and how they influence development of ART
• Identify modifications in dog IVF protocols that led to the successful development of this technique