Assisted reproduction mediated resurrection of a feline model for Chediak-Higashi syndrome caused by a large duplication in LYST.

Chediak-Higashi Syndrome (CHS) is a well-characterized, autosomal recessively inherited lysosomal disease caused by mutations in lysosomal trafficking regulator (LYST). The feline model for CHS was originally maintained for ~20 years. However, the colonies were disbanded and the CHS cat model was lost to the research community before the causative mutation was identified. To resurrect the cat model, semen was collected and cryopreserved from a lone, fertile,  CHS carrier male. Using cryopreserved semen, laparoscopic oviductal artificial insemination was performed on three queens, two queens produced 11 viable kittens. To identify the causative mutation, a fibroblast cell line, derived from an affected cat from the original colony, was whole genome sequenced. Visual inspection of the sequence data identified a candidate causal variant as a ~20 kb tandem duplication within LYST, spanning exons 30 through to 38 (NM_001290242.1:c.8347-2422_9548 + 1749dup). PCR genotyping of the produced offspring demonstrated three individuals inherited the mutant allele from the CHS carrier male. This study demonstrated the successful use of cryopreservation and assisted reproduction to maintain and resurrect biomedical models and has defined the variant causing Chediak-Higashi syndrome in the domestic cat.

Learning outcomes: Exploring implications of adopting a different level of detail.

INTRODUCTION: Outcome-based programmes provide a framework to support educators and learners in understanding content and end points within taught courses. Management of these outcomes in the Dental Degree at Newcastle University has been a challenge in relation to quality assurance and enhancement, having over 1500 detailed student-level outcomes (SLO). This research aimed to explore the implications of adopting a more superficial "course" level of outcome (CLO), when reviewed against a reference set of external LO requirements. MATERIALS AND METHODS: A purposive sample of five courses within the undergraduate dental programme was selected. The mapping of both SLOs and CLOs was reviewed in relation to their total number and the mapping connections to the reference outcomes. RESULTS: There was a mean reduction of 79% in outcomes when comparing SLOs to CLOs. The number of mapping connections between CLOs and the reference set reduced in three courses and increased in two, when compared to SLOs. DISCUSSION: From a purely numerical perspective, changing the detail of learning outcomes has led to a change in mapping connections. As the delivered curriculum has remained unchanged, this demonstrates a potential impact of differing interpretations of learning outcomes. Further review of learning outcomes in relation to the domains categorised within the reference outcome document suggested more mapping links were obtained in clinically focused courses than academic or theoretical courses. CONCLUSION: A demonstrable impact in mapping connections was observed when the detail within the learning outcomes was changed. This has implications for programme leaders in structuring LOs for a curriculum.

Virtual courses: enhancing a curriculum.

INTRODUCTION: Providers of education programmes are required to demonstrate to students, staff and external regulators the content of the programme and how this aligns to key outcomes. Whilst many programme themes are straightforward to illustrate, other integral themes prove challenging to demonstrate independently. INNOVATION: A virtual course takes elements within the current programme relating to a specific theme, repackages and maps them to provide clear signposting by highlighting each occasion the theme is taught, experienced and assessed. The individual elements remain within their current course, so there is no disruption to the established curriculum, but there is an enhancement in terms of transparently displaying linkages within a theme. DISCUSSION: Using the concept of virtual courses we have found we can respond to new initiatives and requirements of external regulators, as well as providing improved signposting for staff and students. This can be undertaken without the need to redesign a whole curriculum. CONCLUSION: In well-designed and currently effective curricula, we would like to recommend that virtual course development can enhance programme transparency, demonstrate external regulatory requirements and promote quality assurance without disruption to current content.

Temporal changes in serum luteinizing hormone following ovariohysterectomy and gonadotropin-releasing hormone vaccination in domestic cats.

Measurement of circulating luteinizing hormone (LH) concentrations in cats and temporal changes following ovariohysterectomy (OHE) or possibly GnRH vaccination may be informative for assessing their fertility, contraception or sterilization status. In this study, serum LH concentrations were measured in domestic cats (n = 6) immediately prior to and up to 120 days post-OHE. Basal LH concentrations of females previously subjected to OHE (n = 4; ~1.5 years post-OHE) were compared pre- and post-vaccination with a GnRH immunocontraceptive, and to LH concentrations in intact females. Basal serum LH concentrations (2.67 ± 0.43 ng/ml; mean ± SEM) in intact females increased (p < .01) by 30 days post-OHE (5.65 ± 0.87 ng/ml) but then declined (p < .05) to pre-OHE levels (mean range, 3.26-3.62 ng/ml) at days 60-120 post-OHE. Serum LH (3.84 ± 0.51 ng/ml) in four females ~1.5 years after OHE tended to be higher (p = .10) than those of intact females prior to OHE. Three months following first or second GnRH immunocontraceptive vaccine treatment, serum LH values in females previously subjected to OHE decreased (p < .05) to concentrations similar to those observed in intact females. Our preliminary results suggest that OHE of domestic cats causes a marked increase in basal LH levels within the first few weeks after ovariohysterectomy followed by a return to pre-OHE basal values over the next several months. Reduced LH concentrations after GnRH vaccine may indicate the effectiveness of the immunocontraceptive in reducing the circulating levels of GnRH, thereby reducing secretion of LH.

Urethral catheterization and sperm vitrification for simplified semen banking in felids.

Semen banking of domestic cats and wild felids represents a vital resource for their long-term conservation, but current methods require access to advanced training and specialized equipment. A newer method of semen collection, urethral catheterization of medetomidine-treated cats, allows recovery of high sperm numbers, but it is unclear if this approach permits maximal sperm recovery or is feasible using less expensive alpha-2 agonists. Similarly, a newer sperm preservation approach, vitrification, offers advantages of simplicity and minimal equipment needs, but its efficacy in combination with urethral catheterization has not been investigated. Our specific objectives were to (i) evaluate sequential semen recovery with urethral catheterization and electroejaculation in domestic cats, (ii) assess the effectiveness of a weak (xylazine) versus strong (dexmedetomidine) alpha-2 agonist for inducing sperm release, and (iii) compare post-thaw sperm motility, acrosome status and fertilizing capacity of catheter-recovered samples after vitrification or straw freezing. Results indicated that electroejaculation following repeated catheterization allowed recovery of additional spermatozoa (range, 11-32 × 106  sperm/male) and that xylazine was ineffective for inducing meaningful sperm release (range, 0-0.4 × 106 sperm/male). Post-thaw motility and acrosome status of vitrified catheter samples did not differ (p > .05) from that of straw frozen samples. Preliminary results indicated that in vitro fertilization success (9/30, 30%) of vitrified catheter sperm did not differ (p > .05) from that observed with straw frozen samples (17/30, 57%). In conclusion, urethral catheterization of dexmedetomidine-treated cats allows recovery of substantial sperm numbers but electroejaculation still may be warranted for maximal sperm recovery. Xylazine is not suitable as an inexpensive alternative to dexmedetomidine for catheterization. Vitrification of catheter samples results in comparable post-thaw parameters to straw freezing and may be adequate for use with oviductal insemination procedures.

Improved cryopreservation of domestic cat sperm in a chemically defined medium.

The objective was to compare a proprietary egg yolk-based cryopreservation medium with a chemically defined soy-based medium, as well as to examine effects of temperature of glycerol addition on sperm parameters and IVF after freezing and thawing of domestic cat sperm. Semen was collected from adult cats (four males and three ejaculates per male), divided in four equal aliquots, and extended in either egg yolk with 4% glycerol added before (EYG) or after (EY) cooling to 5 °C, or soy-lecithin with 4% glycerol added before (SLG) or after (SL) cooling to 5 °C. Extended sperm were frozen in straws over liquid nitrogen vapor. Sperm progressive motility (%) and rate of progressive movement (scale of 0-5) were evaluated at 0, 1, 3, 6, and 24 h post-thaw. Sperm capacitation, acrosome integrity, and DNA integrity were assessed at 15 min post-thaw. Effects of media (EY or SL) on IVF success was also examined (three males and three ejaculates per male). Sperm motility was greater (P < 0.05) in soy-based compared with egg yolk-based media at 3, 6, and 24 h post-thaw. A higher (P < 0.05) percentage of noncapacitated sperm (pattern F) were present in soy-based (SLG, 63.7 ± 9.2%; and SL, 64.1 ± 9.2%) compared with egg yolk-based (EYG, 49.9 ± 7.9%; and EY, 52.4 ± 18.6%) cryopreservation media, regardless of temperature of glycerol addition. Addition of glycerol at 5 °C increased (P < 0.05) percentage of sperm motility at 6 h (EYG 16.3 ± 8.3% vs. EY, 24.0 ± 11.7%; SLG, 36.7 ± 6.5% vs. SL, 42.9 ± 10.1%) and 24 h (EYG, 2.1 ± 3.3% vs. EY, 8.3 ± 3.9%; SLG, 11.3 ± 8.3% vs. SL, 18.8 ± 7.4%) post-thaw in both media. There were no differences (P > 0.05) between cryodiluents in embryo cleavage, percentage of embryos reaching blastocyst, or cell number per blastocyst. The chemically defined, soy-based medium resulted in better preservation of long-term motility and capacitation status of frozen-thawed domestic cat sperm compared with a commercial egg yolk-based extender, without compromising fertilizing ability.

Comparative fertility of freshly collected vs frozen-thawed semen with laparoscopic oviductal artificial insemination in domestic cats.

Artificial insemination (AI) is potentially invaluable as an adjunct to natural breeding for the conservation management of non-domestic felid populations. The efficacy of AI, however, must be substantially improved for applied use, especially when using frozen semen. Our recent advances in using laparoscopic oviductal AI (LO-AI) with low sperm numbers and freezing of cat semen in a soy lecithin-based cryoprotectant medium suggest that combining these two approaches might improve pregnancy outcomes with frozen-thawed spermatozoa. In this study, our objectives were to (i) assess the effect of two gonadotropin dosages (100 vs 150 IU eCG) on ovarian response in domestic cats and (ii) compare the relative fertility of frozen-thawed and fresh semen in vivo following LO-AI. All 16 females ovulated after gonadotropin treatment and were inseminated with fresh semen from one male and frozen-thawed semen from a second male. There were no differences between gonadotropin dosages in CL number, pregnancy percentage or litter size. Half (8/16) of the females conceived, with seven females giving birth to a total of 36 offspring. Paternity analysis showed that more kittens resulted from LO-AI with fresh (28/36, 78%) than frozen-thawed (8/36, 22%) semen, possibly due to impaired motility and longevity of thawed sperm. These results demonstrated that viable offspring can be produced by AI using semen frozen in a soy lecithin-based medium. Insemination with greater numbers of frozen-thawed spermatozoa, combined with further refinement of cat sperm cryopreservation methods, may be necessary to optimize pregnancy success with LO-AI in domestic and nondomestic cats.

In vitro maintenance, cooling and cryopreservation of red wolf (Canis rufus) spermatozoa.

A current priority for the preservation of the endangered red wolf (Canis rufus) is the development of a sperm-based genome resource bank. The aims of this study were to examine the effects of (i) holding temperature on the motility of spermatozoa over time, and (ii) cooling methods on the characteristics of spermatozoa after cooling and cryopreservation. Electroejaculates (n = 11; fresh) were evaluated for the percentage of motile spermatozoa, cell and acrosome morphology (Spermac (Meditech 1st Canada Inc, Montreal, Ontario) and fluorescein isothiocyanate-labelled Pisum sativum agglutinin lectin (PSA/FITC; Sigma Diagnostics, Oakville, Ontario) staining), and zona penetration. Semen samples were then divided into two equal samples and centrifuged to remove seminal plasma. One half of the ejaculate sample was re-suspended in sperm-Tyrode's albumin lactate pyruvate (TALP), divided into three aliquots and maintained either at room temperature (approximately 21-23 degrees C), 0 degree C or 37 degrees C. Sperm motility was examined at 0.5 and 1.0 h, and subsequently every hour for 10 h. Motility of spermatozoa decreased after 2 h, but was consistently greater at room temperature than at 37 degrees C or 0 degree C. The other half of the ejaculate sample was re-suspended in an egg yolk-based extender and divided into two aliquots. One aliquot was cooled in a refrigerator (5 degrees C) for 30 min, whereas the second aliquot was put into a beaker containing water at 37 degrees C, which was then placed into an ice bath until the sample reached 0 degree C (approximately 120 min). Spermatozoa were evaluated after cooling and after freezing and thawing treatments. No differences were observed between cooling treatments either after cooling or freezing and thawing. However, marked decreases in intact acrosomes, post-thaw motility and normal morphology of spermatozoa after treatment demonstrate that further investigations are necessary to improve cryopreservation methods in this species.

Piecing together the puzzle of carnivore reproduction.

Recent advances in feline and canine reproductive studies demonstrate how methodically piecing this information together is beginning to reap rewards for wildlife conservation programs. Non-invasive endocrinology can be used to monitor female reproductive function, time con-specific introductions or AI, and diagnose pregnancy. Sperm morphology characteristics and cell membrane function may be genetically inherited and differ between genetically diverse and inbred species/populations in felids. It is not clear if the same is true for the endangered red wolf. While standards exist for freezing feline and canine sperm, new information using fluorescent staining and zona penetration assays (ZPA) indicates that significant damage can occur during pre-freeze cooling, and may also be related to a species' genetic diversity. Posthumous gamete salvage from genetically valuable animals not only provides a means to study sperm and oocyte physiology but also to assist with genetic management of populations. Using the knowledge gained, IVM/IVF and ICSI have been successful in the domestic cat and AI has resulted in offspring in numerous non-domestic felids. However, understanding the processes of IVM/IVF is still not well understood in canids. New information reveals that sperm and the cumulus cells may be integral to oocyte maturation and that canine epididymal sperm are not capable of undergoing fertilization. The acquisition of knowledge and application of biotechnologies lags behind for non-domestic canid conservation programs.