A simple and economic protocol for efficient in vitro fertilization using cryopreserved mouse sperm

The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. Large-scale comparison of fertilization rates from independent lines of genetically engineered C57BL/6 mice upon cryopreservation and in vitro fertilization with our approach demonstrated equal or significantly superior fertilization rates to current protocols. Our novel SEcuRe (Simple Economical set-up for Rederivation) method provides an affordable, easily adaptable and harmonized protocol for highly efficient rederivation using cryopreserved C57BL/6 sperm for a broad application of colony management in the sense of the 3Rs.

Knowledge of Biobehavioral Organization Can Facilitate Better Science: A Review of the BioBehavioral Assessment Program at the California National Primate Research Center

Animals vary on intrinsic characteristics such as temperament and stress responsiveness, and this information can be useful to experimentalists for identifying more homogeneous subsets of animals that show consistency in risk for a particular research outcome. Such information can also be useful for balancing experimental groups, ensuring animals within an experiment have similar characteristics. In this review, we describe the BioBehavioral Assessment Program at the California National Primate Research Center, which, since its inception in 2001, has been providing quantitative information on intrinsic characteristics to scientists for subject selection and balancing, and to colony management staff for management purposes. We describe the program and review studies relating to asthma, autism, behavioral inhibition, etc., where the BBA Program was used to select animals. We also review our work, showing that factors such as rearing, ketamine exposure, and prenatal experience can affect biobehavioral organization in ways that some investigators might want to control for in their studies. Attention to intrinsic characteristics of subject populations is consistent with the growing interest in precision medicine and can lead to a reduction in animal numbers, savings in time and money for investigators, and reduced distress for the animals.

Screening and validation of genome-edited animals

The emergence of an array of genome-editing tools in recent years has facilitated the introduction of genetic modifications directly into the embryo, increasing the ease, efficiency and catalogue of alleles accessible to researchers across a range of species. Bypassing the requirement for a selection cassette and resulting in a broad range of outcomes besides the desired allele, genome editing has altered the allele validation process both temporally and technically. Whereas traditional gene targeting relies upon selection and allows allele validation at the embryonic stem cell modification stage, screening for the presence of the intended allele now occurs in the (frequently mosaic) founder animals. Final confirmation of the edited allele can only take place at the subsequent G1 generation and the validation strategy must differentiate the desired allele from a range of unintended outcomes. Here we present some of the challenges posed by gene editing, strategies for validation and considerations for animal colony management.

Archiving genetically altered animals: a review of cryopreservation and recovery methods for genome edited animals

With the ever-expanding numbers of genetically altered (GA) animals created in this new age of CRISPR/Cas, tools for helping the management of this vast and valuable resource are essential. Cryopreservation of embryos and germplasm of GA animals has been a widely used tool for many years now, allowing for the archiving, distribution and colony management of stock. However, each year brings an array of advances, improving survival rates of embryos, success rates of in-vitro fertilisation and the ability to better share lines and refine the methods to preserve them. This article will focus on the mouse field, referencing the latest developments and assessing their efficacy and ease of implementation, with a brief note on other common genetically altered species (rat, zebrafish, Xenopus, avian species and non-human Primates).

Retrospective Analysis of the Effectiveness and Reversibility of Long-Acting Contraception Etonogestrel (Implanon®) in Common Marmosets (Callithrix jacchus)

Contraception is an important population control method for the colony management of primates housed in captivity. Etonogestrel (ENG) implants (i.e., Implanon®) are a widely used progestin-based contraceptive in common marmosets (Callithrix jacchus) with the theoretical advantages of being reversible and long-acting. However, no dose and efficacy data are available yet. Therefore, data from 52 adult female marmosets contracepted with ENG (one-fourth or one-third of an implant) housed at the Biomedical Primate Research Centre (BPRC, Rijswijk, The Netherlands) over the past 18 years were analyzed. Using an electronic database, a retrospective longitudinal cohort study was conducted to calculate the reproductive data before, during and after ENG use. The data show an effectiveness in preventing pregnancy of 99%. The implant was effective within one week after insertion. Unintended pregnancies did occur, but in 60% of these cases, the animals were already pregnant at the time of implant insertion. In these cases, healthy offspring were born despite the use of the implant. No stillbirths, neonatal deaths or maternal deaths could be linked to ENG use. After implant removal, 83% of the animals delivered healthy offspring. No difference in contraception efficacy was observed between the use of one-fourth or one-third of an implant. ENG achieved a contraceptive protection exceeding 99% and was shown to be reversible concerning fertility. To our knowledge, this is the first detailed analysis on the use of ENG in marmosets.

Geographical Distribution and Selection of European Honey Bees Resistant to Varroa destructor

Developing resistance to the varroa mite in honey bees is a major goal for apicultural science and practice, the development of selection strategies and the availability of resistant stock. Here we present an extended literature review and survey of resistant populations and selection programs in the EU and elsewhere, including expert interviews. We illustrate the practical experiences of scientists, beekeepers, and breeders in search of resistant bees. We describe numerous resistant populations surviving without acaricide treatments, most of which developed under natural infestation pressure. Their common characteristics: reduced brood development; limited mite population growth; and low mite reproduction, may cause conflict with the interests of commercial beekeeping. Since environmental factors affect varroa mite resistance, particular honey bee strains must be evaluated under different local conditions and colony management. The resistance traits of grooming, hygienic behavior and mite reproduction, together with simple testing of mite population development and colony survival, are significant in recent selection programs. Advanced breeding techniques and genetic and physiological selection tools will be essential in the future. Despite huge demand, there is no well-established market for resistant stock in Europe. Moreover, reliable experience or experimental evidence regarding the resistance of stocks under different environmental and management conditions is still lacking.

Monitoring of Honey Bee Colony Losses: A Special Issue

In recent decades, independent national and international research programs have revealed possible reasons for the death of managed honey bee colonies worldwide. Such losses are not due to a single factor, but instead are due to highly complex interactions between various internal and external influences, including pests, pathogens, honey bee stock diversity, and environmental change. Reduced honey bee vitality and nutrition, exposure to agrochemicals, and quality of colony management contribute to reduced colony survival in beekeeping operations. Our Special Issue (SI) on ‘’Monitoring of Honey Bee Colony Losses’’ aims to address specific challenges facing honey bee researchers and beekeepers. This SI includes four reviews, with one being a meta-analysis that identifies gaps in the current and future directions for research into honey bee colonies mortalities. Other review articles include studies regarding the impact of numerous factors on honey bee mortality, including external abiotic factors (e.g., winter conditions and colony management) as well as biotic factors such as attacks by Vespa velutina and Varroa destructor.

PRODUCTION AND MARKETING ECONOMICS OF HONEY FROM APIS CERANA IN DANG DISTRICT OF NEPAL

Beekeeping with Apis cerana is a vehicle to run livelihood of rural farmers in Nepal. Honey production is major source of cash income for them. This study was conducted in Dang, Nepal, with the aim to analyze production and marketing economics of honey form A. cerana. Total 55 beekeepers, two beekeeping groups, two collectors and five retailers were selected randomly for interview. The results showed that on average farmers holding 14.55 bee hive with productivity of honey 6.12 kg per hive. The benefit cost ratio was 3.71 and average annual net profit was NRs. 2,646.96 (USD 23.06) per hive. Human labour hours in colony management, number of beehives and number of honey harvest per year were significant factors affecting honey production. Ninety two percent of honey produce was actually marketed through three major marketing channels. Most of the producers directly sold honey to consumer and had higher farm gate price. Overall farm gate price, price spread, producer’s share and marketing efficiency index of honey was NRs. 671.06/kg, NRs. 93.15/kg, 84.12 percent and 5.30 respectively. Absconding behavior of A.cerana was the top production problem and lack of wholesale marketing points was top most marketing problem perceived by farmers.

Honey Bee Queen Replacement: An Analysis of Changes in the Preferences of Polish Beekeepers through Decades

We conducted a survey on honey bee (Apis mellifera) queen management. Data were collected every year from 1980 to 2018. In total, 2964 questionnaires were collected from all over Poland. We examined the trends by decade timeslot, apiary size, and geographical location. Regardless of the decade and the size of the apiary, on average, above 90% of Polish beekeepers replace old queens with new ones in their colonies. In general, during the observed period, beekeepers replaced almost 52% of their queens, 21% of which were purchased. In the last decade, there was an upward trend in the percentage of beekeepers replacing queens throughout the country. The involvement of purchased queens in colony management is associated with the size of the apiary, and it significantly grows with the number of colonies in the apiary. The percentage of purchased queens went up in all the voivodeships over time. Research and education in this area are needed in order to track the trends and further improve Polish beekeepers’ practices.