Ideal Donor Research Articles

Construction and Characterization of a cDNA Library from Liver Tissue of Chinese Banna Minipig Inbred Line

A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a λ: phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 × 10 6 pfu/mL and 5.0 × 10 9 pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.

An Alternative Dressing Material for the Split-Thickness Skin Graft Donor Site

The split-thickness skin graft (STSG) donor sites have been treated with various and plenty of dressing techniques and materials. An ideal STSG donor site dressing should have antibacterial, hemostatic, and promoting epidermal healing properties. We have performed a prospective study to evaluate the effect of the oxidized regenerated cellulose on STSG donor site healing. Between January 2002 and January 2005, 40 patients who were operated in any kind of reconstructive operations with STSG donor sites were included in the study. One half of the wound was covered with oxidized regenerated cellulose and the other half of the same wound of the same patient was covered with fine mesh gauze treated with Furacin (nitrofurazone). The patients were grouped into 2 depending on the dressing technique: group I, semiclosed and group II, closed. The wounds were evaluated for healing time, infection, pain perception of the patient, and final esthetic results. The oxidized regenerated cellulose side of the group I was healed in a mean of 6.5 +/- 0.51 days; in group II, 5.4 +/- 0.50 days (range, 5-6 days). The fine mesh gauze treated with Furacin in group I was healed in a mean of 9.9 +/- 0.97 days (range, 8-11 days); in group II, 8.4 +/- 0.99 days (range, 7-10 days). There was a statistical significance between the oxidized regenerated cellulose side and the fine mesh gauze side (P < 0.001) in group I and group II separately. The difference between group I and group II was statistically significant in the oxidized regenerated cellulose side (P < 0.001), and the difference between group I and group II was statistically significant in the fine mesh gauze side (P < 0.005). The antibacterial, hemostatic, and absorbable property of the oxidized regenerated cellulose could ensure the utilization as an alternative STSG donor site dressing, especially because the positive influence over the wound healing was proven.

Recovery and Utilization of Deceased Donor Kidneys from Small Pediatric Donors

The optimal use of kidneys from small pediatric deceased donors remains undetermined. Using data from the Scientific Registry of Transplant Recipients, 2886 small (< 21 kg) pediatric donors between 1993 and 2002 were identified. Donor factors predictive of kidney recovery and transplantation (1343 en bloc; 1600 single) were identified by logistic regression. Multivariable Cox regression was used to assess the risk of graft loss. The rate of kidney recovery from small pediatric donors was significantly higher with increasing age, weight and height. The odds of transplant of recovered small donor kidneys were significantly higher with increasing age, weight, height and en bloc recovery (adjusted odds ratio = 65.8 vs. single; p < 0.0001), and significantly lower with increasing creatinine. Compared to en bloc, solitary transplants had a 78% higher risk of graft loss (p < 0.0001). En bloc transplants had a similar graft survival to ideal donors (p = 0.45) while solitary transplants had an increased risk of graft loss (p < 0.0001). En bloc recovery of kidneys from small pediatric donors may result in the highest probability of transplantation. Although limited by the retrospective nature of the study, kidneys transplanted en bloc had a similar graft survival to ideal donors but may not maximize the number of successfully transplanted recipients.

Combined Liver-Kidney Transplantation: The Experience of the University of Bologna and the Case of Preoperative Positive Cross-Match

Combined liver and kidney transplantation (CLKT) has been increasingly used in recent years: 13 of our 19 cases were performed in the last 2 years being 3.2% of our liver transplantation (LT) and kidney transplantation (KT) activity. Only three of them were not on hemodialysis and the scheduling of a CLKT meant being at the top of the waiting list. We accepted only ideal donors and had no case of liver and only one case of kidney delayed graft function. Two deaths occurred during the first postoperative month, due to acute respiratory distress syndrome and multiorgan failure, both in patients with adult polycystic disease who were in poor nutritional condition due to a late indication for CLKT. We had two late deaths, one due to a native kidney tumor at 7 years and one at 8 years due to alcoholic cirrhosis recurrence. The late survival of our patients was 77.3% with all surviving patients showing good liver and kidney function. We planned not to do the KT in the case of a positive preoperative cross-match; but the only positive case became negative 8 hours after LT when we performed the KT. The patient is well after 2 years. The liver does not always protect the kidney if there are preformed antibodies, but we should try every possible technique not to lose the possibility of doing both transplants, because in case of LT alone the patients loses his top position on the CLKT waiting list and often waits years for a kidney.

Use of Marginal Donors in Kidney Transplantation: Our Experience

The use of elderly donors has been advocated to expand the organ donor pool because of increased needs and the organ shortage. The aim of this study was to analyze whether the use of elderly donors and marginal kidneys affected the outcome of renal transplantations. Herein we presented data on 126 kidney transplantations performed from January 1996 to September 2003 using 32 marginal donors (group A) and 94 ideal donors (group B). We analyzed the medical and surgical complications and the graft survivals at a median follow-up of 18 months. Medical and surgical complications occurred in 22% and 5% versus 7% and 4% in groups A and B, respectively. The mean cold ischemia time and the mean age were greater for patients undergoing kidney transplantations from marginal donors. No differences were observed in graft survival in groups A and B. In conclusion, our data suggested that with an appropriate strategy and a correct selection of patients, marginal kidneys can be safely used to decrease the gap between demand and supply.

Using the Descending Branch of the Lateral Femoral Circumflex Vessel as a Source of Two Independent Flaps

Simultaneous reconstruction of two separate defects by two free flaps is time consuming and often requires two donor sites. The anterior and lateral aspect of the thigh is an ideal donor site for free tissue harvest without incurring significant morbidity. In this article, the authors describe their recently developed technique that allows for harvesting two independent fasciocutaneous free flaps from the same descending branch of lateral circumflex femoral vessel as a new clinical application of the versatile anterolateral thigh flap. A total of eight flaps were developed from left thighs of four patients. There were two male and two female patients, with a mean age of 42.0 years. The defects were either on the lower extremity (n = 2) or in the oral cavity (n = 2). Each anterolateral aspect of the thigh was used to develop two perforator flaps, based on one perforator each. The anterolateral thigh flap was elevated in the standard manner based on at least two cutaneous vessels of the descending branch of the lateral circumflex femoral artery. The skin flaps were tailored to the recipient site requirement and the main descending branches of the lateral femoral circumflex vessels were divided in between to be used as the pedicle of both flaps. The mean flap dimensions ranged from 2.5 x 4 cm to 8 x 12 cm (mean, 5 x 8 cm). The mean operation time was 11 hours 30 minutes. The mean length of the pedicle was 9 cm (ranged, 8 to 11 cm). The mean external diameter of the descending branch in the mid thigh and where it joined to the lateral circumflex femoral artery was 1.75 mm and 3 mm, respectively. Three donor sites were closed directly and one was grafted. All flaps survived completely. No complications were encountered. Donor-site morbidity was negligible. Hospitalization averaged 9.9 +/- 1.7 days. Two fasciocutaneous flaps based on independent skin vessels can be reliably harvested from the same descending branch of the lateral femoral circumflex artery for simultaneous reconstruction of two separate defects.

Autologous Punch Grafting for Vitiligo of the Palm

Vitiligo of the palm can be resistant to conventional treatments, and grafting is not routinely attempted because of some difficulties. To do a autologous minigraft test on a vitiligo patch on the palm using skin of the instep as the donor. Under local anesthesia, two 2 mm minigrafts were transplanted into the vitiliginous skin of the palm of a 28-year-old female. Psolaren with ultraviolet A therapy followed the surgery for 12 weeks. Excellent repigmentation was seen around the grafts at the end of 12 weeks. There were no complications. The skin of the instep is an ideal donor site for palmar vitiligo.

Utilization of Elderly Donors in Living Related Kidney Transplantation

Kidney transplantation has become the treatment of choice for end-stage renal disease. However, its application is limited due to inadequate organ supply, mainly because many dialysis patients do not have suitable living donors. The increasing discrepancy between organ supply and demand has forced many transplant centers to consider using organs procured from marginal donors. The aim of this study was to investigate whether utilization of kidneys from living related elderly donors is safe for the recipients in the long term. We analyzed the clinical results of 296 consecutive recipients of living related renal transplants, among whom 44 recipients received kidneys from donors over 60 years of age. By the end of 12 months, the mean serum creatinine level of the recipients who were transplanted from the older donors was higher (1.55 ± 0.45 mg/dL) than that from other donors (1.21 ± 0.3 mg/dL), but the difference was not significant ( P = .08). In the long term (60 months), the graft function was similar (1.88 ± 0.55 vs 1.52 ± 0.38) for both groups. The similarity in outcomes of ideal versus older donors as shown less in the present series has encouraged us to utilize elderly living donors. We concluded that transplantations performed from the elderly donors yielded similar results to those of conventional donors. The long waiting list for transplantation, the treatment of choice for end-stage renal disease, should encourage us to be more flexible about donor selection.

Human Embryonic Stem Cell-Derived Cardiomyocytes: Inducing Strategies

Cell-based therapy is emerging as an innovative approach for the treatment of degenerative diseases, and stem cells appear to be an ideal source of cells for this. In cardiology, in particular, human embryonic stem cell (hESC)-derived cardiomyocytes theoretically fulfill most, if not all, of the properties of an ideal donor cell, but several critical obstacles need to be overcome. Many research projects are focusing on set-up strategies for directing hESC differentiation toward the cardiac lineage. It is one of the main difficulties in the search to provide a valuable source of cells to effect regeneration of myocardial tissue in patients with severe heart failure. To date, there are no easy and efficient protocols for the induction of hESC differentiation toward the cardiac lineage. Discovering new molecules or tools capable of doing this is imperative.

Advances in HLA: Practical Implications for Selecting Adult Donors and Cord Blood Units

HLA plays a critical role in hematopoietic cell ransplantation, but a full understanding of this role is onstantly in flux because of changing technologies nd burgeoning amounts of clinical transplantation esults. Today the clinician considering unrelated donor ransplantation must also consider stem cell source: an dult donor or an umbilical cord blood unit (CBU). In he context of cord blood, the task is further compliated by the strong interaction of cell dose. Each individual carries from 10 to 12 genes that ncode the HLA-A, HLA-B, HLA-C, HLA-DR, HLAQ, and HLA-DP molecules. Most of these genes are ighly polymorphic, ranging from 13 (HLA-DRB4) to 99 (HLA-B) alleles at a locus [1]. Most of the allelic ifferences result in changes in the HLA protein seuences, thus producing potential targets for allorecogition [2]. The frequency of alleles differs in different opulations: some are common in most populations, nd others are found predominantly in only 1 or a few opulations. The same is true for HLA haplotypes—the ollection of multiple HLA alleles within a short reion of chromosome 6—which are typically inherited s a single block of genetic information. Table 1 proides examples. DNA-based testing methods provide an accurate nd powerful approach to identify HLA diversity, and hese are replacing serologic testing [3,4]. DNA testng detects specific nucleotide differences that distinuish alleles or sets of alleles. The ability to distinuish among particular alleles at a locus (ie, the esolution) depends on the methodology, the set of ligonucleotide reagents used in the assay, or both. he choice of assay depends on the purpose of the yping, eg, initial testing of newly recruited volunteers r CBUs for a registry (low to intermediate resoluion) compared with testing of transplantation candiates and a few selected potential donors or CBUs high or allele-level resolution). C

Variable band gap conjugated polymers for optoelectronic and redox applications

We report here on the utilization of variable band gap conjugated polymers for optoelectronic redox applications comprising organic photovoltaics, color tunable light emitting diodes, and electrochromics. For the evaluation of morphology in photovoltaicdevices, atomic force microscopy, and optical microscopy provided direct visualization of the blend film structure. The evolution of the morphology in two and three component blends incorporating poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenlenevinylene] (MEH-PPV), poly(methylmethacrylate) (PMMA), and [6, 6]-phenyl C61-butyric acid methyl ester (PCBM) was investigated. It was found that while insulating PMMA can be used to modulate the phase separation in these blends, a bicontinuous network of donor and acceptor was required to achieve the best device results. Similarily, a MEH-PPVcopolymer with a decreased conjugation length has been used for investigating inter- and intramolecular photoinduced charge transfer in the presence of PMMA and PCBM.We fabricated MEH-PPV/PCBM solar cells that have power conversion efficiencies up to 1.5% with a range of 0.7–1.5%, dependent on the nature of the MEH-PPV used. This further indicates that in addition to blend morphology, polymer structure is critical for optimizing device performance. To this end, the concept of an ideal donor for photovoltaic devices based on poly[2,5-di(3,7-dialkoxy)-cyanoterephthalylidene] is described and two donor-acceptor polymers based on cyanovinylene (CNV) and dioxythiophene are discussed as representative examples of soluble narrow band gap polymers synthesized in our group. For light emitting applications, utilization of two blue emitting conjugated polymers poly (9,9-dioctylfluorene) (PFO) and poly[(9,9-dihexylfluorenyl-2,7-diyl)-co-(9,ethyl-3,6-carbazole)] (PFH-PEtCz)is presented for a color tunable polymer light emitting diode that emits orange, green, and blue light with a voltage range of 7–10 V as a function of the total conjugated polymer content in PMMA and is attributed to the phase separation between the conjugated polymers. Finally, the narrow band gap conjugated polymer, poly[bis(3,4-propylenedioxythiophene-dihexyl)]-cyanovinylene has been characterized for its electrochromic properties, illustrating the multifunctional nature of variable band gap conjugated polymers.

Treatment goals during care of adult donors that can influence outcomes of heart transplantation

Myocardial dysfunction during care of adult donors can result from injury occurring before hospital admission or during the progression of brain death. Few evidence-based data correlate specific hemodynamic goals during donor care with outcomes of heart transplantation, although many recommendations exist. Spontaneous reversal of early heart damage or correction of poor cardiac performance can yield outcomes equivalent to outcomes in recipients who had ideal donors. Hemodynamic goals developed in the operating room can be applied in intensive care to improve outcomes of transplantation. These goals include maintenance of mean arterial pressure greater than 60 mm Hg, central venous pressure less than 12 mm Hg, cardiac output greater than 3.8 L/min, cardiac index greater than 2.1, and systemic vascular resistance between 800 and 1200 dyne x sec x cm(-5). The ejection fraction and other echocardiographic data also provide helpful guidance when determining whether a heart is suitable for transplantation and during therapy. Titration of cardiovascular variables often requires invasive monitoring to ensure that cardiac preload, afterload, and contractility are optimal.

Treatment Goals during Care of Adult Donors That Can Influence Outcomes of Heart Transplantation

Myocardial dysfunction during care of adult donors can result from injury occurring before hospital admission or during the progression of brain death. Few evidence-based data correlate specific hemodynamic goals during donor care with outcomes of heart transplantation, although many recommendations exist. Spontaneous reversal of early heart damage or correction of poor cardiac performance can yield outcomes equivalent to outcomes in recipients who had ideal donors. Hemodynamic goals developed in the operating room can be applied in intensive care to improve outcomes of transplantation. These goals include maintenance of mean arterial pressure greater than 60 mm Hg, central venous pressure less than 12 mm Hg, cardiac output greater than 3.8 L/min, cardiac index greater than 2.1, and systemic vascular resistance between 800 and 1200 dyne x sec x cm(-5). The ejection fraction and other echocardiographic data also provide helpful guidance when determining whether a heart is suitable for transplantation and during therapy. Titration of cardiovascular variables often requires invasive monitoring to ensure that cardiac preload, afterload, and contractility are optimal.

Reverse-Flow First Dorsal Metacarpal Artery Flap for Index Fingertip Reconstruction

Dorsum of the hand is an ideal skin-flap donor site for coverage of hand or finger defects. We elevated 8 reverse-flow first dorsal metacarpal artery flap in 9 patients for soft-tissue cover of index fingertip injuries distal to distal interphalangeal joint with exposed bone. In 3 patients, the digital branch of the superficial radial nerve was coapted to the digital nerve. All flaps healed uneventfully, except for 1 patient in which partial necrosis of the flap occurred. Postoperative follow-up was 4-12 months (mean, 8 months). Reverse-flow first dorsal metacarpal artery flap is a good alternative for reconstruction of index fingertip injuries.

Tracheal wall for the reconstruction of extended hemilaryngectomy in the rabbit model

To evaluate reconstruction of the surgical wound of extended hemilaryngectomy using the tracheal wall. Prospective, nonrandomized experimental study in a rabbit model in which cricothyroid hemilaryngectomy was performed in 10 rabbits. The proximal trachea was tailored to cover the surgical wound and sutured to it. The ipsilateral lobe of the thyroid gland and the medial thyroid vascular pedicle were conserved so as to assure vascular supply to the tracheal flap. After 4 weeks the animals were sacrificed and pathology analysis conducted. Eight of the 10 rabbits survived without respiratory or deglutitory alterations. Pathologic analysis showed well-healed suture lines together with normal mucosa and cartilage rings in the tracheal flap. The tracheal wall appears to be the ideal donor tissue for repair of the larynx. This simple, 1-session surgical procedure demonstrates an effective reconstruction of an extended hemilaryngectomy wound.

Impact of the Expanded Criteria Donor Allocation System on the Use of Expanded Criteria Donor Kidneys

The U.S. Organ Procurement and Transplantation Network recently implemented a policy allocating expanded criteria donor (ECD) kidneys by waiting time alone. ECD kidneys were defined as having a risk of graft failure > or = 1.7 times that of ideal donors. ECDs include any donor > or = 60 years old and donors 50 to 59 years old with at least two of the following: terminal creatinine >1.5 mg/dL, history of hypertension, or death by cerebrovascular accident. The impact of this policy on use of ECD kidneys is assessed. The authors compared use of ECD kidneys recovered in the 18 months immediately before and after policy implementation. Differences were tested using t test and chi2 analyses. There was an 18.3% increase in ECD kidney recoveries and a 15.0% increase in ECD kidney transplants in the first 18 months after policy implementation. ECD kidneys made up 22.1% of all recovered kidneys and 16.8% of all transplants, compared with 18.8% (P<0.001) and 14.5% (P<0.001), respectively, in the prior period. The discard rate was unchanged. The median relative risk (RR) for graft failure for transplanted ECD kidneys was 2.07 versus 1.99 in the prepolicy period (P=not significant); the median RR for procured ECD kidneys was unchanged at 2.16. The percentage of transplanted ECD kidneys with cold ischemia times (CIT) <12 hr increased significantly; the corresponding percentage for CIT > or = 24 hr decreased significantly. The recent increase in ECD kidney recoveries and transplants appears to be related to implementation of the ECD allocation system.

Extended donor lungs: eleven years experience in a consecutive series☆

The aim of this study was to delineate the profile of extended donor lungs in comparison to ideal donor lungs and to analyse their outcome. Particular attention was given to donor lungs with a low PaO(2) (<250mmHg) before harvesting or with multiple extended criteria. Between 1993 and 2003, 148 patients (79 women, 69 men, mean age 39.9 years) underwent lung transplantation. Indications were cystic fibrosis in 35.8%, emphysema in 26.4%, pulmonary fibrosis in 12.2%, pulmonary hypertension in 9.5%, and others in 16.1%. Donor data and recipients medical files were reviewed. Criteria for donor lungs were considered extended if one or more of the following criteria were met: age >55 years, smoking >20 pack-years, PaO(2) before harvesting <300mmHg, pathologic chest X-ray, and purulent secretion at bronchoscopy. A comparison between recipients from ideal and from extended donor lungs was performed with respect to the median duration of mechanical ventilation, the median length of stay at the intensive care unit, postoperative complications, the 30-day and the 1-year survival, and the 6-month follow-up spirometry. Sixty-three (42.6%) donor lungs were considered extended and 20 (31.7%) met more than one criteria. Outcome comparison between recipients from ideal (I) and extended (II) donor lungs did not statistically differ in postoperative complications (18.8% (I) vs. 26.9% (II), P=0.32), mean duration of mechanical ventilation (d) (4.4+/-2.7 (I) vs. 2.6+/-2.1 (II), P=0.2), mean length of stay at the ICU (d) (11.5+/-8.8 (I) vs. 9.2+/-6.9 (II), P=0.4), 6-month pulmonary function (FEV1=83+/-23% of the predicted value (I) vs. 82+/-18% (II), P=0.81), 30-day survival (90.6% (I) vs. 93.7% (II), P=0.56), 1-year survival (83.5% (I) vs. 81% (II), P=0.83). Thirty-day survival was also comparable even in recipients from donor lungs with PaO(2)<250mmHg (n=8) (90.6% (I) vs. 87.5%, P=0.57). The number of extended criteria had no impact on the outcome. The combination of PaO(2)<300mmHg with purulent secretion at bronchoscopy seemed to influence the early outcome of recipients from extended donor lungs negatively. Our results suggest that the use of selected extended donor lungs does not compromise the outcome after transplantation. PaO(2) <250mmHg before harvesting of the lungs is not an absolute contra-indication for transplantation.

Use of Skin Grafting to Demonstrate Tolerance Before Kidney Transplantation Without Immunosuppression in the Recipient of a Previous Bone Marrow Transplant

Chronic renal insufficiency is a rare complication of bone marrow transplantation (BMT), occurring in 0.8% to 9.5% (1, 2) of patients and may be of sufficient severity to require renal replacement therapy. When BMT has been used as treatment for hematologic malignancy, an immune-mediated graft-versus-leukemia effect may be needed to maintain remission because systemic immunosuppression after cadaveric renal transplantation could cause risk of relapse of leukemia. The ideal kidney donor would be the same individual who donated the bone marrow. The recipient’s immune system could be identical or chimeric with that of the donor. It is difficult to be certain that 100% of the recipient immune system has been eradicated, and a small residual could proliferate to reject a donated kidney. Ethically, to submit a live donor to nephrectomy, a high level of assurance of success is needed. We present a case in which tolerance using a skin graft was demonstrated before successful kidney transplantation without immunosuppression in a BMT recipient. A 64-year-old male was referred for assessment of chronic renal insufficiency in January 2001. Four years previously, he had had chronic myeloid leukemia (CML) treated successfully with an allogenic BMT from his human leukocyte antigen (HLA)-identical (HLA-A, B, and DR only tested) sister. Two episodes of graft-versus-host-disease (GvHD) were treated with short courses of steroids and cyclosporine. In April 2001, relapse of CML was diagnosed on the basis of break-point cluster region Ableson leukemia transcripts found by polymerase chain reaction in peripheral blood and by 11 of 200 cells being of recipient (male) origin on bone-marrow examination. The relapse was successfully treated with donor lymphocyte infusion. PreBMT he had long-standing hypertension controlled by a single agent and normal kidney function. At referral, his serum creatinine had been raised for more than 24 months. An ultrasound examination revealed an absent left kidney and 9 cm right kidney. Renal insufficiency was progressive, and he was begun on peritoneal dialysis. The sister who had donated the bone marrow offered to donate a kidney. A full-thickness skin graft from the sister was performed in July 2002. One month after skin grafting, the graft had been accepted with no clinical or histologic evidence of rejection (Fig. 1). Kidney transplantation then took place without any induction or maintenance immunosuppression. Two years posttransplant, the patient remains well without episodes of transplant kidney rejection or recurrence of leukemia, with a serum creatinine of 1 mg/dL (89 μmol/L).FIGURE 1. Histology of full-thickness skin graft 1 month postgrafting showing no features of rejection.In a patient with prior documented GvHD, skin grafting provided a simple, practical, in vivo immunologic challenge to reassure us that live-donor renal allografting could be successful without immunosuppression. To our knowledge, there have been only two similar cases reported previously (3, 4). Even though the paradigm of solid-organ transplantation from the same donor in previous BMT recipients is not applicable to the vast majority of organ recipients, it offers a unique opportunity to realize the benefits of tolerance and avoid deleterious effects of immunosuppressive medication, including the risk of relapse of the primary hematologic malignancy. Rommel Ravanan Christopher R. K. Dudley Richard M. Smith Christopher J. Burton Richard Bright Renal Unit Southmead Hospital Bristol, UK Paul A. Lear Department of Surgery Southmead Hospital Bristol, UK David J. Unsworth Department of Immunology Southmead Hospital Bristol, UK

41 EFFECT OF CELL TYPES AND PASSAGES ON DEVELOPMENT AND APOPTOSIS OF PORCINE CLONED EMBRYOS

The purpose of this study was to improve the efficiency of somatic cell nuclear transfer (SCNT) in pigs by assessing the development, cell numbers and apoptosis when using different cell types as nuclear donors and different numbers of passages. Primary cultures of the donor cells, porcine fetal fibroblasts (pFFF) from a female fetus at ∼30 days of gestation and adult female ear skin cells (pAESC), were established in DMEM + 15% FCS. For nuclear donor, cells at different passages were cultured for 5 days until confluent. Cumulus-oocyte complexes were matured and fertilized in vitro as controls by the following methods (2000 Theriogenology 54, 787–797). Following enucleation, oocytes were reconstructed by transfer of donor cells and fusion with two DC pulses (1.4 kV/cm, 50 μs) in 0.28 M mannitol containing 0.01 mM CaCl2 and MgCl2. Eggs were then cultured in NCSU23 + 1.9 mM 6-dimethylaminopurine for 3 h. SCNT and IVF embryos were cultured in NCSU23 for 54 h and subsequently in the same medium with 5.55 mM glucose for 90 h at 38.5°C in 5% CO2 in air. In Experiment 1, when the rates of development between IVF and SCNT embryos constituted with cells at 5–7 passages were compared, no significant (P &lt; 0.05) differences were observed in the cleavage rates. The rates of blastocyst formation were significantly (P &lt; 0.05) higher in IVF than in SCNT embryos with pFFF and pAESC (21% vs. 15% and 10%), but it did not differ between SCNT embryos. Total cell numbers in IVF blastocysts (35.4 ± 12) were significantly (P &lt; 0.05) higher than in SCNT blastocysts with pFFF and pAESC (28.4 ± 8 and 26.2 ± 10, respectively). The apoptosis signal by TUNEL was initiated at Day 3 in IVF and SCNT embryos. Apoptosis rates in SCNT blastocysts with pFFF and pAESC (13.1 ± 2.5 and 16.6 ± 4.3, respectively) were significantly (P &lt; 0.05) higher than in IVF embryos (3.6 ± 1.4). As the embryos developed, the rates of apoptosis were increased. On Day 6, the rates of apoptosis in IVF (4.8%) were significantly (P &lt; 0.05) lower than those in SCNT embryos with pFFF (13.1%) and pAESC (16.6%). However, both total cell number and apoptosis in SCNT embryos with pFFF and pAESC revealed no significant differences. In Experiment 2, SCNT embryos with pFFF in different cell passages were compared for the development and apoptosis. No significant (P &lt; 0.05) differences were observed in the cleavage rates of SCNT embryos among different cell passages. The rates of blastocyst formation were significantly (P &lt; 0.05) higher in SCNT embryos with 5–7 passages than those with other numbers of passages (14% vs. 6–8%, respectively). Although total cell numbers of SCNT blastocysts did not differ among different cell passages, apoptosis rates were significantly (P &lt; 0.05) higher when the number of cell passages was increased. These results suggest that fetal fibroblasts at 5–7 passages are ideal nuclear donor cells for obtaining high-quality porcine SCNT embryos. This work was supported by grant No. 1000520040020000 from Biogreen 21, Republic of Korea.

218 EJACULATE TRAITS OF THE NAMIBIAN CHEETAH (ACINONYX JUBATUS)—INFLUENCE OF ANIMAL AGE, SEASON, AND CAPTIVITY

Wild cheetahs are threatened with extinction, and ex situ populations are not self-sustaining due to poor reproductive efficiency. Sperm cryo-preservation is a valuable tool for genetic management; however, increased knowledge of ejaculate traits is essential to improve cryopreservation protocols. The objective of this study was to characterize ejaculate traits of wild-born cheetahs in Namibia, Africa. Specifically, the influences of animal age, season and captive status on electroejaculate volume, sperm concentration, motility, forward progressive status (FPS scale 0-5, 5 = best), morphology, and acrosomal integrity were evaluated. Animal age was divided into categories: juvenile (14-24 mo; n = 16 males, 23 ejaculates), adult (25-120 mo; n = 76 males, 175 ejaculates), and aged (over 120 mo; n = 5 males, 5 ejaculates). Namibian seasons were divided into hot-wet (Jan-Apr), cold-dry (May-Aug) and hot-dry (Sep-Dec). Cheetahs were considered wild-caught (n = 29 males; 44 ejaculates) if trapped on farmland d30 days before semen collection. Raw ejaculates contained 69.0 � 1.1% motile sperm (mean � SEM) and 73.6 � 1.5% sperm with intact acrosomes. Overall, 18.4 � 0.9% of sperm were morphologically normal, with midpiece abnormalities being the most prevalent defects (?39%). To determine treatment differences, data were analyzed by General Linear Model procedures and means were separated with Duncan's multiple-range test. Juvenile cheetahs produced ejaculates with reduced (P &lt; 0.05) sperm motility (56.7 � 3.3%) and FPS (2.9 � 0.1) compared to adult (69.8 � 1.4% and 3.4 � 0.1, respectively) and aged (78.9 � 6.7% and 3.7 � 0.3, respectively) animals. Ejaculates from juvenile animals also had reduced (P &lt; 0.05) volume (0.69 � 0.3 mL) and fewer (P &lt; 0.05) total motile sperm (7.1 � 9.3 � 106) compared to adult (2.2 � 0.1 mL and 42.3 � 4.1 � 106) and aged (2.3 � 0.6 mL and 23.5 � 20.0 � 106, respectively) males. For all ejaculates combined, seminal quality was poorest during the hot-dry season with lower (P &lt; 0.05) sperm motility and intact acrosomes as well as an increased (P &lt; 0.05) percent of sperm with head abnormalities. Ejaculates from captive cheetahs (n = 68 males, 159 ejaculates) had increased (P &lt; 0.05) volume (2.0 � 0.2 mL) and intact acrosomes (80.1 � 3.6%), but lower (P &lt; 0.05) sperm density (14.3 � 3.9 � 106/mL) than wild-caught animals (1.5 � 0.3 mL, 71.9 � 4.6%, and 24.1 � 5.1 � 106/mL, respectively). These are the first large-scale data acquired to examine the reproductive biology of male cheetahs in Namibia, including those recently captured from the wild. Results reveal that this species demonstrates seasonal and age-based variations in ejaculate quality, and that all individuals (including those recently derived from the wild) produce unusually high proportions of pleiomorphic spermatozoa. These data are being used to select the ideal donor age and season during which spermatozoa should be collected for addition to a genome resource bank, thereby enhancing effective genetic management for cheetahs propagated ex situ.