Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A.

BACKGROUND: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. OBJECTIVES: To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. PATIENTS/METHODS: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. RESULTS AND CONCLUSIONS: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

Hybridization between wapiti (Cervus elephus manitobensis) and sika deer (Cervus nippon): a comparison of two artificial insemination techniques.

The present study compared pregnancy rates of sika deer (Cervus nippon) hinds artificially inseminated with frozen-thawed wapiti (Cervus elephus manitobensis) semen by laparoscopic intrauterine or transvaginal/cervical artificial insemination (AI) techniques. Estrous cycles of 59 sika hinds were synchronized with one-half of a norgestomet ear implant. Fourteen days after implant insertion, norgestomet ear implants were removed and hinds received 50 IU of PG-600 intramuscularly, a combination of 50 IU PMSG and 25 IU hCG. Hinds were then randomly allotted for laparoscopic (n = 25) or transvaginal/cervical (n = 34) AI. AI of the hinds with frozen-thawed wapiti semen (40 x 10(6) spermatozoa) was time-fixed to occur at 55 hr for transvaginal/cervical AI and 65 hr for laparoscopic AI postnorgestomet implant removal. Semen deposition for hinds inseminated by transvaginal/cervical AI occurred as follows: vagina. 8.8% (3/34); os cervix, 67.6% (23/34); intracervical, 20.5% (7/34); and uterus, 2.9% (1/34). On day 42 post-AI, transrectal ultrasonography was performed to determine pregnancy rates. Pregnancy rates were not significantly different (P > 0.10) between laparoscopic (8/25, 32.0%) and transvaginal/cervical AI (9/34, 26.4%), with an overall conception rate of 28.8% (17/59). These results indicate that transvaginal/cervical AI techniques can be as effective as laparoscopic AI under some circumstances. Further refinement of the transvaginal/cervical AI technique to improve pregnancy rates might lead to more widespread use of this technique when laparoscopic AI is not possible.

In vitro maturation of ovine oocytes in a portable incubator.

Immature ovine oocytes were collected from ovaries obtained from an abattoir and assigned to one of three treatment groups for in vitro maturation. For Treatment 1 (T1), oocytes were matured in a conventional incubator, in tissue culture wells in an atmosphere of 5% CO(2) and air. Maturation medium consisted of bicarbonate buffered Tissue Culture Medium 199 (TCM199) supplemented with fetal calf serum (FCS), follicle stimulating hormone (FSH), luteinizing hormone (LH), and penicillin/streptomycin (pen/strep). For Treatment 2 (T2), oocytes were matured in a portable incubator, in plastic tubes containing the same medium as T1. The medium was equilibrated with 5% CO(2) and overlayed with oil. For Treatment 3 (T3) oocytes were matured in the portable incubator without CO(2) equilibration, in tubes containing HEPES buffered TCM 199 supplemented as in T1. After 24 hours at 39 degrees C, the percentage of oocytes undergoing normal nuclear maturation was 72.55, 68.14 and 66.96% for T1, T2 and T3, respectively (P >0.05). In a second experiment oocytes were matured in the 3 treatments described, then fertilized in vitro using frozen-thawed ram sperm. Fertilization rates were 44.09, 58.62 and 55.69% for T1, T2 and T3, respectively. T1 and T2 were significantly different (P < 0.05). For Experiment 3, oocytes matured and fertilized as described were cultured in drops of Modified Brinster's Mouse Ova Culture (MBMOC) containing bovine oviductal cells. These were incubated at 39 degrees C in an atmosphere of 5% CO(2) and air for 7 days. T1, T2 and T3 resulted in 20.26, 16.94 and 24.43% development to morulae, and 4.01, 3.06 and 1.85% development to blastocysts, respectively (P >0.05). The results of these experiments indicate that maturation, fertilization, and developmental rates of ovine oocytes matured in the portable incubator are similar to those of oocytes matured in a conventional incubator. This technique shows promise for transportation of oocytes to laboratories where abattoirs are not in close proximity, and holds promise for transportation of oocytes from non-domestic animals collected in the field or remote locations, to facilities capable of utilizing and preserving the gametes.

Interspecific transfer of IVM IVF-derived red sheep (Ovis orientalis gmelini ) embryos to domestic sheep (Ovis aries ).

Two embryo production methodologies were investigated to generate Red sheep embryos for use in an interspecific embryo transfer program. In Experiment 1, 4 multiparous female Red sheep (Ovis orientalis gmelini ) were implanted with CIDR type G devices for 11 d. Forty-eight hours prior to CIDR removal, a total of 22.5 mg bid of FSH-P was administered over a 3-d period. Laparoscopic embryo collection was performed 5 d post breeding, and embryos were transferred to domestic recipient ewes (Ovis aries and Ovis orientalis musimon ). In Experiment 2, 7 nulliparous female Red sheep were implanted with CIDR devices and injected with 200 IU of PMSG and 25 mg of FSH-P on the 8th day of implant insertion. At 60 to 70 h post PMSG/FSH-P treatment, follicular oocytes were aspirated laparoscopically. The recovered oocytes were matured in M199 (with fetal calf serum, FSH, LH, penicillin and streptomycin) at 39 degrees C in a humidified atmosphere containing 5% CO(2). At 24 h oocytes were fertilized with frozen-thawed semen at a concentration of 1.6 x 10(6) sperm/ml. The ova/embryos were placed in CR2 or BOEC culture medium at 20-22 h post IVF. Following 3 to 4 d in culture, embryos were transferred laparoscopically to the uterine horn of synchronized recipients. In Experiment 1, 4 embryos and 6 UFO were collected from 2 embryo donors, respectively. Two embryos were transferred with the aid of a laparoscope to each of 2 Rambouillet recipients, one of which gave birth to a healthy Red sheep lamb at 158 d of gestation. In Experiment2, a total of 62 oocytes was collected from 7 oocyte donors; 16 developed to the 16- to 32-cell stage and were transferred to 8 recipients. Three of these IVM-IVF embryos were transferred laparoscopically to 2 Mouflon recipients, resulting in no pregnancies. Thirteen IVM-IVF embryos were transferred to 6 Rambouillet recipients. Each of these gave birth to a single healthy Red sheep lamb. Gestation lengths of the 3 IVM-IVF lambs ranged from 152 to 162 d. This research demonstrates that when using compatible species IVM-IVF technology in conjunction with interspecific ET can lead to the production of live offspring and can be used to propagate exotic ovine species.