Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation.

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and ß4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and ß4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.

Advances in Organ and Tissue Xenotransplantation.

End-stage organ failure can result from various preexisting conditions and occurs in patients of all ages, and organ transplantation remains its only treatment. In recent years, extensive research has been done to explore the possibility of transplanting animal organs into humans, a process referred to as xenotransplantation. Due to their matching organ sizes and other anatomical and physiological similarities with humans, pigs are the preferred organ donor species. Organ rejection due to host immune response and possible interspecies infectious pathogen transmission have been the biggest hurdles to xenotransplantation's success. Use of genetically engineered pigs as tissue and organ donors for xenotransplantation has helped to address these hurdles. Although several preclinical trials have been conducted in nonhuman primates, some barriers still exist and demand further efforts. This review focuses on the recent advances and remaining challenges in organ and tissue xenotransplantation.

Maturation and development of fetal pig intestinal tissue in immunodeficient mice

Purpose: This study aimed to compare the degree of maturation and development of fetal pig segmental intestinal tissue with that of spheroids created by in-vitro reaggregation of dissociated fetal intestinal cells after transplantation into immunodeficient mice. Methods: Fetal pig small intestines were transplanted as segmental grafts into the omentum and subrenal capsules of immunodeficient mice or enzymatically treated to generate single cells. Spheroids made by in-vitro reaggregation of these cells were transplanted into the subrenal capsules of immunodeficient mice. The segmental grafts and spheroids were harvested four and eight weeks after transplantation, and the structural maturity and in-vivo development of these specimens were histologically evaluated. Results: The spheroids were engrafted and supplied blood vessels from the host mice, but an intestinal layered structure was not clearly observed, and there was almost no change in size. On the other hand, the segmental grafts formed deep crypts in the mucus membrane, the inner circular layer, and outer longitudinal muscles. The crypts of the transplanted grafts harvested at eight weeks were much deeper, and the smooth muscle layer and the enteric nervous system were more mature than those of grafts harvested at the fourth week, although the intestinal peristaltic wave was not observed. Conclusions: Spheroids created from fetal small intestinal cells could not form layered structures or mature sufficiently. Conversely, segmental tissues structurally matured and developed after in-vivo transplantation and are therefore potential grafts for transplantation.

Critical diagnostic and cancer stem cell markers in neoplastic cells from canine primary and xenografted pulmonary adenocarcinoma.

It is challenging to diagnose metastatic tumors whose cellular morphology is different from the primary. We characterized canine primary pulmonary adenocarcinoma (PAC) and its xenografted tumors by histological and immunohistochemical analyses for critical diagnostic and cancer stem cell (CSC) markers. To generate a tumor xenograft model, we subsequently transplanted the tissue pieces from the PAC into athymic nude mice. Immunohistochemical examination was performed for diagnostic (TTF-1, Napsin A, and SP-A) and CSC markers (CD44 and CD133). The use of CSC markers together with diagnostic markers can improve the detection and diagnosis of canine primary and metastatic adenocarcinomas.

Scan of the endogenous retrovirus sequences across the swine genome and survey of their copy number variation and sequence diversity among various Chinese and Western pig breeds.

In pig-to-human xenotransplantation, the transmission risk of porcine endogenous retroviruses (PERVs) is of great concern. However, the distribution of PERVs in pig genomes, their genetic variation among Eurasian pigs, and their evolutionary history remain unclear. We scanned PERVs in the current pig reference genome (assembly Build 11.1), and identified 36 long complete or near-complete PERVs (lcPERVs) and 23 short incomplete PERVs (siPERVs). Besides three known PERVs (PERV-A, -B, and -C), four novel types (PERV-JX1, -JX2, -JX3, and -JX4) were detected in this study. According to evolutionary analyses, the newly discovered PERVs were more ancient, and PERV-Bs probably experienced a bottleneck ~0.5 million years ago (Ma). By analyzing 63 high-quality porcine whole-genome resequencing data, we found that the PERV copy numbers in Chinese pigs were lower (32.0±4.0) than in Western pigs (49.1±6.5). Additionally, the PERV sequence diversity was lower in Chinese pigs than in Western pigs. Regarding the lcPERV copy numbers, PERV-A and -JX2 in Western pigs were higher than in Chinese pigs. Notably, Bama Xiang (BMX) pigs had the lowest PERV copy number (27.8±5.1), and a BMX individual had no PERV-C and the lowest PERV copy number (23), suggesting that BMX pigs were more suitable for screening and/or modification as xenograft donors. Furthermore, we identified 451 PERV transposon insertion polymorphisms (TIPs), of which 86 were shared by all 10 Chinese and Western pig breeds. Our findings provide systematic insights into the genomic distribution, variation, evolution, and possible biological function of PERVs.

Ovarian tissue features assessed in bovine fetuses after vitrification and xenotransplantation procedures.

Cryopreservation and transplantation of ovarian tissue are proposed methods for the restoration of endocrine function and reproductive potential. Therefore, this study aimed to evaluate the effects of vitrification and xenotransplantation on follicle viability, activation, stromal cell integrity, vascularization, and micronuclei formation. Bovine fetal ovaries were fragmented and assigned to the following groups: Fresh control (FC), ovarian fragments immediately fixed; Vitrified control (VC), ovarian fragments vitrified; Vitrified xenotransplanted (VX), ovarian fragments vitrified and xenotransplanted; and Fresh xenotransplanted (FX), ovarian fragments xenotransplanted. Ovarian fragments were grafted in female BALB/c mice and recovered after 14 days. Follicular viability was preserved (P > 0.05) in VC group. The rate of developing follicles was greater (P < 0.05) in the FX group compared to other groups. Follicular density was higher (P < 0.05) in the VC group than the FC, VX, and FX groups. A decrease (P < 0.05) of stromal cell density was recorded after vitrification (VC vs. FX). Blood vessel density decreased in VC, VX, and FX groups compared with the FC group, and blood vessel density was correlated with follicular viability (positively; P = 0.07) and developing follicles (negatively; P < 0.001). Both vitrification and xenotransplantation groups (VC, VX, and FX) had a greater (P < 0.05) number of cells with one MN compared to the FC group. In summary, our findings showed that both vitrification and xenotransplantation modified blood vessel, follicular and stromal cell densities, follicular viability and activation, and micronuclei formation in ovarian tissue.

The use of acellular porcine corneal stroma xenograft (BioCorneaVet™ ) for the treatment of deep stromal and full thickness corneal defects: A retrospective study of 40 cases (2019-2021).

OBJECTIVE: To document the effectiveness and outcome of corneal grafting using acellular porcine corneal stroma (APCS) for veterinary use (BioCorneaVet™ ) to restore corneal integrity in dogs. METHODS: A review of medical records of patients that underwent keratoplasty with APCS graft to repair deep corneal defects, descemetoceles, and perforations between 2019 and 2021 was carried out. Only animals with intact dazzle reflex, consensual PLR before the surgery and a minimum follow-up of four weeks were considered for the study, with forty dogs (1 eye each) meeting the inclusion criteria. RESULTS: Brachycephalic breeds were the most frequently represented, and 20 right eyes and 20 left eyes were affected with 25 perforations, 8 descemetoceles, and 9 deep stromal defects (1 eye had both perforation and descemetocele). Most of the patients had concurrent ocular diseases or had undergone previous surgery on the other eye. Two different thickness of xenograft was used (300 or 450 µm), and the diameter ranged from 3 to 10 mm. Postoperative complications included mild to severe corneal vascularization, partial dehiscence, melting, and glaucoma. Follow-up time ranged from 28 to 797 days (mean: 233 days). Ocular integrity was maintained in 37/40 cases (92.5%), and vision was preserved in 36 cases (90%). CONCLUSION: The use of APCS (BioCorneaVet™ ) is an effective surgical treatment for deep stromal defects, descemetocele, and perforations in dogs, providing a good tectonic support and preserving anatomical integrity and vision. The cosmetic appearance was considered good in all the cases and continued to improve with time.

Embryo production by intracytoplasmic injection of sperm retrieved from neonatal testicular tissue of Agu pigs after cryopreservation and grafting into nude mice.

The Agu is the only indigenous pig breed in Japan but its population is very small. In order to estimate the efficacy of testicular xenografting for the conservation of Agu pigs, we investigated whether neonatal testicular fragments would acquire the capacity to produce sperm after they had been cryopreserved and grafted into nude mice. Although on day 180 (day 0 = xenografting), grafts showed a low proportion of seminiferous tubule cross-sections containing sperm (0.1 ± 0.1%, mean ± SEM for four mice), the proportion reached 36.9 ± 16.7% (n = 4 mice) by day 240. When single sperm obtained on day 240 was injected into individual porcine oocytes, 28.2% of the oocytes were found to contain one male and one female pronuclei with the second polar body. Moreover, the blastocyst formation rate after injection of the xenogeneic sperm was 28.4%, whereas that in the absence of sperm injection (attributable to parthenogenesis) was 13.3%. These findings suggest that more than half of the blastocysts resulted from fertilization. Thus, testicular xenografting could assist the conservation of Agu pigs by salvaging germ cells present in neonatal testes even after cryopreservation.

Effective cryopreservation protocol for preservation of male primate (Macaca fascicularis) prepubertal fertility.

RESEARCH QUESTION: Can specimen types (cells versus tissues) and additive cryoprotectant agents contribute to efficient cryopreservation of primate spermatogonial stem cells (SSC)? DESIGN: Testicular tissues or cells from four prepubertal monkeys were used in this study. The freezing efficacy of testicular tissue was compared with cell suspensions using conventional freezing media (1.4 mol/l dimethyl sulfoxide [DMSO]) and the efficacy of cryoprotectant additives (1.4 mol/l DMSO combined with trehalose 200 mmol/l, hypotaurine 14 mmol/l, necrostatin-1 50 µmol/l or melatonin 100 µmol/l) was evaluated in testicular tissue freezing. RESULTS: The survival rate (46.0 ± 4.8% versus 33.7 ± 6.0%; P = 0.0286) and number of recovered cells (5.0 ± 1.5 × 106 cells/g versus 0.7 ± 0.8 × 106 cells/g; P = 0.0286) were significantly higher in frozen tissues than in frozen cell suspensions. After tissue freezing, a higher number of recovered PGP9.5+ cells were observed with 200 mmol/l trehalose treatment than in DMSO controls (2.4 ± 0.6 × 106 cells/g versus 1.1 ± 0.3 × 106 cells/g; P = 0.0164). Normal establishment of donor-derived colony was observed in SSC after tissue freezing with 200 mmol/l trehalose. CONCLUSIONS: Testicular tissue freezing is more effective than single cell suspension freezing for higher recovery of undifferentiated spermatogonia. Moreover, it was verified that slow freezing using 200 mmol/l trehalose, 1.4 mol/l DMSO and 10% KnockOut™ Serum Replacement in Dulbecco's phosphate-buffered saline is an effective cryopreservation protocol for primate testicular tissue.

Case Series Describing the Use of Low-Temperature Vacuum-Dehydrated Amnion (Omnigen) for the Treatment of Corneal Ulcers in Cats and Dogs: 46 Cases (2016-2017).

Amniotic membrane is widely used in the treatment of ocular surface disorders in human and veterinary patients. Preservation and storage of amnion has proven challenging, prompting the development of new preservation techniques. Omnigen, a novel low-temperature vacuum-dehydrated amnion, is reported to possess enhanced structural properties and biochemical stability in vitro, but its clinical use in veterinary patients is not well described. This study aims to document and describe the varied use of Omnigen for the surgical treatment of corneal ulceration in cats and dogs. A total of 45 patients (46 eyes) were recruited from the clinical record system of the Royal Veterinary College (London) between January 2016 and December 2017. Brachycephalic breeds were over-represented (37/45; 82.2%). Omnigen was used as a standalone graft in 5/46 (10.9%) eyes, as a supplementary graft in 29/46 (63.0%) eyes and as a patch in 12/46 (26.1%) eyes. Graft failure occurred in 10/46 eyes (21.7%). At final examination 43/46 eyes (93.5%) had healed and 31/33 eyes (93.9%) were visual. This study demonstrates the successful use of Omnigen for the surgical treatment of corneal ulceration in cats and dogs. Further studies are needed to clarify its properties and benefits in the clinical field.

Use of four-layer porcine small intestinal submucosa alone as a scaffold for the treatment of deep corneal defects in dogs and cats: preliminary results.

BACKGROUND: To describe the efficacy of four-layer porcine small intestinal submucosa (Vetrix BioSIS plus+) as single scaffold for the treatment of deep corneal lesions in dogs and cats. METHODS: 10 dogs and 3 cats with deep or full thickness corneal defects were treated surgically with BioSIS plus graft. Corneal transparency scores and vision were evaluated. RESULTS: Lesions in dogs were four perforations, three descemetoceles, two limbal melanocytomas and one deep corneal ulcer. In cats, there were one limbal melanocytoma and two perforations. The average length of the follow-up was 86 days. In all, 12 out of 13 eyes treated were visual at last recheck (92.3 per cent). The scars were mild eight cases (66.7 per cent), but denser in four cases (33.4 per cent). Complication were partial collagenolysis in three cases (25 per cent), which resolved with medical therapy, mild corneal pigmentation in one case (8.4 per cent) and anterior synechia in one case (8.4 per cent). One case experienced severe collagenolysis and was enucleated 21 days postoperatively. CONCLUSIONS: Four-layer porcine SIS graft was successfully used for surgical treatment of deep corneal lesions in selected corneal diseases in a small series of dogs and cats, with good results in terms of mechanic support and corneal transparency.

Efficient production of GGTA1 knockout porcine embryos using a modified handmade cloning (HMC) method.

Handmade cloning is a zona-free nuclear transfer approach and an economical, efficient, and simple micromanipulation-free alternative to dolly based traditional cloning (TC). In this study, based on handmade cloning with minor modifications, an optimized bi-oocyte fusion (BOF) cloning method was established to produce GGTA1 KO porcine embryos using the CRISPR/Cas9 gene editing system. The GGTA1 gene is responsible for the generation of Gal epitopes on the surface of porcine cells, triggering hyperacute immune rejection in preclinical porcine-to-human xenotransplantation. The purpose of the present study is to establish an efficient protocol for activation of porcine oocyte cytoplast-fibroblast fused constructs developed to GGTA1 KO blastocysts by the zona-free bi-oocyte fusion cloning method. High percentages of cleavage (90 ±â€¯2.6%) and blastocyst rates (39 ±â€¯4.0%) were achieved upon treatment with demecolcine-assisted oocyte enucleation followed by 6 V alternating current for proper alignment and single-step fusion technique using a single direct current pulse of 1.0 kV/cm for 9 µs duration, compared to the double-step fusion method with combined chemical activation using thimerosal and dithiothreitol. Overall blastocyst rate was higher for oocyte enucleation by demecolcine (0.4 µg/ml) and 45 min incubation (42 ±â€¯1.5%) compared to without demecolcine incubation followed by complete chemical thimerosal/dithiothreitol activation (33 ±â€¯1.1%). The blastocyst rate (39 ±â€¯1.0%) was found to be significantly higher 1 h post-electrofusion, compared to at 0 and 4 h (28 ±â€¯1.5 and 6 ±â€¯1.5%, respectively). Blastocyst development rates for GGTA1 knockout embryos (38 ±â€¯1.76%) were comparable to those obtained with wild-type embryos (41.1 ±â€¯0.67%). In conclusion, we achieved high overall efficiency in production of GGTA1 KO blastocysts by modified HMC protocol.

Genome and transcriptome analyses providing insight into the immune response of pearl oysters after allograft and xenograft transplantations.

The immune response after allograft or xenograft transplantation in the pearl oyster is a major factor that cause its nucleus rejection and death. To determine the mechanism underlying the immune response after allograft and xenograft transplantations in the pearl oyster Pinctada fucata martensii, we constructed two sets of transcriptomes of hemocytes at different times (6 and 12 h; 1, 3, 6, 12, and 30 d) after allograft and xenograft transplantations, in which the xenografted mantle tissue was from Pinctada maxima. The transcriptomic analysis reveals many genes are involved in the immune response to transplantation, such as transient receptor potential cation channel (TRP), calmodulin (CaM), DNA replication-related genes, and sugar and lipid metabolism-related genes. The expression of these identified genes was higher in the host pearl oyster transplanted with xenograft than that by allograft. The histological analysis of the pearl sac also confirmed that many hemocytes were still gathered around the transplanted nucleus, and no pearl sac was formed in the host pearl oysters at 30 d after xenograft transplantation. The genomic analysis indicated that pearl oysters evolved many copies of genes, such as TRP, CaM, and GST, to sense and cope with the immune response after transplantation. "Ribosome" and "Cytosolic DNA-sensing pathway" were specifically induced in the xenograft group, whereas "Notch signaling pathway" specifically responded to the allograft transplantation. These results can improve our understanding of the mechanism underlying the immune response of pearl oysters after allograft and xenograft transplantations.

Conservation of chicken male germline by orthotopic transplantation of primordial germ cells from genetically distant donors†.

Successful derivation and cultivation of primordial germ cells (PGCs) opened the way to efficient transgenesis and genome editing in the chicken. Furthermore, implantation of male PGCs from non-chicken galliform species into the chicken embryos resulted in cross-species germline chimeras and viable offspring. We have recently improved the PGC technology by demonstrating that chicken male PGCs transplanted into the testes of adult cockerel recipients mature into functional sperms. However, the availability of this orthotopic transplantation for cross-species transfer remains to be explored. Here we tested the capacity of genetically distant male PGCs to mature in the microenvironment of adult testes. We derived PGCs from the Chinese black-bone Silkie and transplanted them into infertile White Leghorn cockerels. Within 15-18 weeks after transplantation, we observed restoration of spermatogenesis in recipient cockerels and production of healthy progeny derived from the transplanted PGCs. Our findings also indicate the possibility of cross-species orthotopic transplantation of PGCs. Thus, our results might contribute to the preservation of endangered avian species and maintaining the genetic variability of the domestic chicken.

Xenotransplantation of goat ovary as an alternative to analyse follicles after vitrification.

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.

Donor sperm production in heterologous recipients by testis germ cell transplantation in the dromedary camel.

The object of this study was to investigate if testis germ cell transplantation (TGCT) into a heterologous recipient would result in donor-origin spermatogenesis in the dromedary camel. First, we investigated a workable protocol for TGCT in camels, including donor cell isolation, enrichment by density gradient centrifugation (Percoll and Bovicoll), rete testis injection and microsatellite detection of donor and recipient genotypes. Second, the effects of three doses of Dolichos biflorus agglutinin (DBA), a glycoprotein that specifically binds to gonocytes or Type A spermatogonia, on testis germ cell depletion were investigated by direct injection into the rete testis of a male camel. Seven recipients were prepared with DBA treatment, two males were castrated at 4 weeks for depletion assessment and the remaining five received donor cells 4-6 weeks after treatment. On average, ~17 million cells were isolated per gram of testis tissue, with 19.5±1.9% DBA-positive (DBA+) cells. Percoll centrifugation yielded a 1.5-fold increase in DBA+ cells while Bovicoll centrifugation produced a 2.5-fold increase from the input cells of 18.6±2.1% DBA+ cells. Semen was collected from the recipients 13-20 weeks after transfer and the presence of donor DNA in the samples was determined using microsatellite markers. In two of the five recipients, all semen samples were shown to be positive for donor-derived cells. These results demonstrate for the first time that: (1) heterologous testicular germ cell transplantation in camels is feasible and the recipients are able to produce spermatozoa of donor origin and (2) DBA can be used effectively to deplete endogenous stem cells.

Pre-clinical imaging for establishment and comparison of orthotopic non-small cell lung carcinoma: in search for models reflecting clinical scenarios.

OBJECTIVE:: Clinically relevant animal models of non-small cell lung carcinoma (NSCLC) are required for the validation of novel treatments. We compared two different orthotopic transplantation techniques as well as imaging modalities to identify suitable mouse models mimicking clinical scenarios. METHODS:: We used three genomically diverse NSCLC cell lines [National Cancer Institute (NCI)-H1703 adenosquamous cell carcinoma, NCI-H23 adenocarcinoma and A549 adenocarcinoma) for implanting tumour cells either as spheroids or cell suspension into lung parenchyma. Bioluminescence imaging (BLI) and contrast-enhanced cone beam CT (CBCT) were performed twice weekly to monitor tumour growth. Tumour histological data and microenvironmental parameters were determined. RESULTS:: Tumour development after spheroid-based transplantation differs probably due to the integrity of spheroids, as H1703 developed single localised nodules, whereas H23 showed diffuse metastatic spread starting early after transplantation. A549 transplantation as cell suspension with the help of a stereotactic system was associated with initial single localised tumour growth and eventual metastatic spread. Imaging techniques were successfully applied to monitor longitudinal tumour growth: BLI revealed highly sensitive qualitative data, whereas CBCT was associated with less sensitive quantitative data. Histology revealed significant model-dependent heterogeneity in proliferation, hypoxia, perfusion and necrosis. CONCLUSION:: Our developed orthotopic NSCLC tumours have similarity with biological growth behaviour comparable to that seen in the clinic and could therefore be used as attractive models to study tumour biology and evaluate new therapeutic strategies. The use of human cancer cell lines facilitates testing of different genomic tumour profiles that may affect treatment outcomes. ADVANCES IN KNOWLEDGE:: The combination of different imaging modalities to identify tumour growth with subsequent use in treatment planning and orthotopic transplantation techniques to develop initially single lesions to ultimate metastases pave the way towards representative pre-clinical NSCLC models for experimental testing of novel therapeutic options in future studies.

Xenotransfusion with packed bovine red blood cells to a wildebeest calf (Connochaetes taurinus).

A 4-month-old female blue wildebeest (Connochaetes taurinus) was presented for bilateral pelvic limb fracture repair. Clinical examination under anaesthesia revealed a water-hammer pulse and a haematocrit of 0.13. A xenotransfusion was performed using bovine (Bos taurus) erythrocytes because of inability to acquire a wildebeest donor. Clinical parameters improved following transfusion and the post-operative haematocrit value was 0.31. The wildebeest remained physiologically stable with a gradually declining haematocrit for the next three days. On the third post-operative day, the wildebeest refractured its femur and was humanely euthanised because of the poor prognosis for further fracture repair. Xenotransfusion using blood from domestic ruminants represents a life-saving short-term emergency treatment of anaemic hypoxia in wild ungulates. Domestic goats could be used as blood donors for rare ungulates where allodonors are not available.

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia-27%; grayling spermatogonia-28%; grayling oogonia-23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.