David S. Thain

Four-year contraception rates of mares treated with single-injection porcine zona pellucida and GnRH vaccines and intrauterine devices

We evaluated the multiyear contraceptive efficacy of the gonadotrophin-releasing hormone (GnRH) vaccine GonaCon, the porcine zona pellucida (PZP) vaccine SpayVac and the human intrauterine device (IUD) 380 Copper ‘T’ in mustang mares provided by the State of Nevada. Eight untreated control mares were compared with 12 mares treated with SpayVac, 16 mares treated with GonaCon and 15 mares treated with the copper-containing IUD. Rates of contraception for Years 1, 2, 3 and 4 respectively for SpayVac were 100% (12 of 12), 83% (10 of 12), 83% (10 of 12) and 83% (10 of 12), rates for GonaCon were 94% (15 of 16), 60% (9 of 15), 60% (9 of 15) and 40% (6 of 15) and rates for IUD-treated mares were 80% (12 of 15), 29% (4 of 14),14% (2 of 14) and 0% (0 of 14). Antibody titres against PZP and GnRH declined over the four-year study. For mares given SpayVac, uterine oedema was commonly observed. IUDs were visible by ultrasonography in non-pregnant mustang mares, suggesting that pregnant mares did not retain their IUD. IUD retention may be a function of uterine size: pony mares with IUDs had high retention and contraception rates for 4–5 years. We conclude that long-term contraception of mustang mares with a single shot of either the SpayVac or GonaCon vaccine is possible.

Multi-year fertility reduction in free-roaming feral horses with single-injection immunocontraceptive formulations.

Context. Contraception is increasingly used as a management technique to reduce fertility in wildlife populations; however, the feasibility of contraceptive formulations has been limited until recently because they have required multiple treatments to achieve prolonged infertility. Aims. We tested the efficacy and evaluated potential side effects of two contraceptive formulations, a porcine zona pellucida (PZP) formulation, SpayVac ® and a gonadotrophin-releasing hormone (GnRH) formulation GonaCon-B� ,i n a population of free-roaming feral horses (Equus caballus). Both formulations were developed to provide several years of infertility with one injection. Methods.FemalesweretreatedinJune2005witheitherGonaCon-B(n=24),SpayVac(n=20),adjuvantonly(n=22),or received no injection (n=18). Females were monitored for fertility status year round for 3 years after treatment. Keyresults.Bothcontraceptivetreatmentssignificantlyreducedfertilityfor3years.FertilityratesforGonaCon-Bmares were39%,42%and31%,respectively,and37%,50%and44%forSpayVacmares.Duringthesameseasons,61%,67%and 76% of control females were fertile. We found no significant effects from contraceptive treatment on the sex ratio of foals, birthing season or foal survival. Conclusions.Theseresultsdemonstratedthatbothvaccinesarecapableofsignificantlyreducingfertilityforseveralyears without boosters. Implications. Contraceptive vaccines examined in the present study represent a useful tool for the management of feral horses, because of their being efficacious for 3 years in the absence of booster immunisations.

Development and characterization of a novel CD34 monoclonal antibody that identifies sheep hematopoietic stem/progenitor cells

OBJECTIVE We and many others have long used sheep as a predictive model system in which to explore stem cell transplantation. Unfortunately, while numerous markers are available to identify and isolate human hematopoietic stem cells (HSC), no reagents exist that allow HSC/progenitors from sheep to be identified or purified, greatly impeding the application of this well-established large animal model to the study of autologous or allogeneic HSC transplantation. The current studies were undertaken to create a monoclonal antibody to sheep CD34 that would enable isolation and study of sheep HSC/progenitors. MATERIALS AND METHODS A partial cDNA to the extracellular domain of the sheep CD34 antigen was polymerase chain reaction cloned, characterized, and used to genetically immunize mice and create hybridomas. RESULTS The resultant monoclonal antibody to sheep CD34 allows flow cytometric detection of sheep HSC/progenitors present within bone marrow, cord blood, and mobilized peripheral blood. Moreover, this antibody can be used to enrich for HSC/progenitors with enhanced in vitro colony-forming potential, and also identifies endothelial cells in situ within paraffin-embedded tissue sections, similarly to antibodies to human CD34. CONCLUSIONS The availability of this monoclonal antibody recognizing the stem cell antigen CD34 in sheep will greatly facilitate the study of autologous and allogeneic HSC transplantation using this clinically relevant large animal model.

Persistent circulating human insulin in sheep transplanted in utero with human mesenchymal stem cells

OBJECTIVE To determine if mesenchymal stem cells (MSC) derived from human fetal pancreatic tissue (pMSC) would engraft and differentiate in sheep pancreas following transplantation in utero. MATERIALS AND METHODS A three-step culture system was established for generating human fetal pMSC. Sheep fetuses were transplanted during the fetal transplant receptivity period with human pMSC and evaluated for in situ and functional engraftment in their pancreas, liver, and bone marrow. RESULTS Isolation and expansion of adherent cells from the human fetal pancreas yielded a cell population with morphologic and phenotypic characteristics similar to MSC derived from bone marrow. This putative stem cell population could undergo multilineage differentiation in vitro. Three to 27 months after fetal transplantation, the pancreatic engraftment frequency (chimeric index) was 79%, while functional engraftment was noted in 50% of transplanted sheep. Hepatic and marrow engraftment and expression was noted as well. CONCLUSION We have established a procedure for isolation of human fetal pMSC that display characteristics similar to bone marrow-derived MSC. In vivo results suggest the pMSC engraft, differentiate, and secrete human insulin from the sheep pancreas.

In vivo generation of beta-cell-like cells from CD34(+) cells differentiated from human embryonic stem cells.

OBJECTIVE CD34(+) cells, present within the bone marrow, have previously been shown to possess pancreatic endocrine potential. Based on this observation, we explored the capacity of CD34(+) cells derived in culture from the differentiation of human embryonic stem cells (hESC), for their in vivo pancreatic endocrine capacity. MATERIALS AND METHODS Sheep were transplanted with hESC-derived CD34(+) cells, as well as nonsorted differentiated cultures. Transplantations were carried out with in utero intraperitoneal injections prior to development of the immune system in the fetus so that tolerance toward foreign antigens was acquired during gestation and persisted in the adult. RESULTS All cell populations that were tested demonstrated human cellular activity and long-term presence up to 5 years. However, the in vivo beta-cell-like activity achieved from the transplantation of the sorted CD34(+) cell population was not augmented by transplanting the entire cell population from which the CD34(+) cells were isolated. Human DNA and insulin messenger RNA were detected in sheep pancreases. An average of 1.51 ng/mL human C-peptide was detected in serum from eight animals transplanted with differentiated cell populations and assayed up to 55 months posttransplantation. Transplantation of as few as 23,500 cells resulted in long-term sustainable beta-cell-like activity. Teratomas were absent in the transplanted animals. CONCLUSION Our data suggest that hESC-derived CD34(+) cells have a potential for long-term in vivo endocrine cellular activity that could prove useful in regenerative medicine. Because the same cell population has previously been shown to contain hematopoietic potential, it could be used for the induction of immunological tolerance and bone marrow chimerism prior to cellular therapy for diabetes.

Immune Ontogeny and Engraftment Receptivity in the Sheep Fetus

Objective: The biologic explanation for fetal receptivity to donor engraftment and subsequent long-term tolerance following transplantation early in gestation is not known. We investigated the role fetal immune ontogeny might play in fetal transplantation tolerance in sheep. Methods: Engraftmentof allogeneic and xenogeneicHSC was determined 60 days following transplantation at different time points in sheep fetal gestation. Parallel analysis of surface differentiation antigen expression on cells from lymphoid organs of timed gestational age fetal sheep was determined by flow cytometry using available reagents. Results: An engraftment window was identified after day 52 gestation lasting until day 71 (term gestation: 145 days). This period was associated with the expression of the leukocyte common antigen CD45 on all cells in the thymus. Double-positive and single-positive CD4 and CD8 cells began appearing in the thymus just prior (day 45 gestation) to the beginning of the engraftment window, while single-positive CD4 or CD8 cells do not begin appearing in peripheral organs until late in the engraftment period, suggesting deletional mechanisms may be operative. In concert, surface IgM-positive cells express CD45 in the thymus at day 45, with a comparable delay in the appearance of IgM/CD45 cells in the periphery until late in the engraftment window. Conclusions: These findings support a central role for the thymus in multilineage immune cell maturation during the period of fetal transplantation receptivity. Further, they suggest that fetal engraftment receptivity is due to gestational age-dependent deletional tolerance.

Adaptability of pregnant Merino ewes to the cold desert climate in Nevada.

Grazing ability is difficult to record in animals under free-ranging conditions without sophisticated methods. Alternatively, grazing ability may be indirectly inferred from changes in BW and production characteristics during the grazing period. The present study investigated the effect of grazing on resource-limited rangelands on BW, wool characteristics, and offspring weaning weights in nine hundred five 5/8, 7/8, and fullblood Merino ewes of 2 to 7 yr of age during a grazing period of approximately 2.5 mo (between January and March). A total of 469 ewes gave birth to a single lamb, 248 to twin lambs, and 188 did not give birth. Body weights were measured and wool samples taken before and after the ewes were allowed to graze freely on the rangelands; absolute change in BW and change in BW as a percentage of initial BW were estimated. On average, grazing on resource-poor rangelands resulted in BW loss, a reduction in fiber diameter and its CV, and increased staple length. Animals with finer wool at the start of the grazing period lost phenotypically (r = -0.07, P < 0.05) and genetically (r = -0.23, P < 0.05) less BW during the grazing period and had a greater probability to carry 1 lamb (or 2) to term (P < 0.05). Animals that lost less BW produced more greasy fleece (r = 0.09, P < 0.01). Body weight change did not significantly influence offspring weaning weights. Change in BW was moderately heritable at h(2) = 0.29; fiber diameter was strongly heritable at h(2) = 0.51. Animals with the least inclusion of Merino genetics lost more BW (P < 0.01) during the grazing period and had a more uniform fiber diameter (P < 0.05) but shorter staples (P < 0.05) and less fleece (P < 0.0001) than animals with a greater level of Merino genetics. Our results indicate that animals with finer wool appeared to be better adapted to the cold Nevada desert. Thus, selection for finer wool may positively influence adaptability to resource-limited cold climate conditions; alternatively, BW change may be selected for directly. Because nutritional deficiencies during pregnancy can have adverse consequences for the offspring, indirect selection for grazing ability would foremost result in healthier ewes that can produce lambs and wool without compromising their welfare.

Are low infidelity rates in feral horses due to infanticide

A growing number of studies conducted on diverse taxa have shown that extra-pair/group paternity is higher than what would be predicted from behavioral observations alone. While it may be beneficial for females to mate with multiple males, this often results in offspring not sired by the behavioral father, which could influence offspring survival, especially in social mammals. Feral horses (Equus caballus) maintain stable social relationships over several years, usually with one stallion defending a harem band of unrelated mares against other males. Sneak copulations by subordinate males have been observed and mares sometimes change bands, both of which can result in foals sired by males other than the dominant band stallion. We measured female fidelity in free-ranging feral horses in 23 bands, with 51 foals over four foaling seasons and tested offspring paternity against parental behaviors. We used 12 polymorphic microsatellite loci and the program CERVUS 2.0 to determine and exclude potential sires. The majority of mares remained in the band with the sire of their foal resulting in most foals being sired by the band stallion. Most foals that were not sired by the band stallion were born in the year after a round-up and we could not determine if they were the result of band changing or sneak copulations. Foals born into a band without their sire had lower survival rates and mothers were significantly more protective of foals not sired by the band stallion. These findings suggest that band stability increases the reproductive success of mares and support the importance of infanticide risk in equid social structure.

Haplotype phasing after joint estimation of recombination and linkage disequilibrium in breeding populations

A novel method for haplotype phasing in families after joint estimation of recombination fraction and linkage disequilibrium is developed. Results from Monte Carlo computer simulations show that the newly developed E.M. algorithm is accurate if true recombination fraction is 0 even for single families of relatively small sizes. Estimates of recombination fraction and linkage disequilibrium were 0.00 (SD 0.00) and 0.19 (SD 0.03) for simulated recombination fraction and linkage disequilibrium of 0.00 and 0.20, respectively. A genome fragmentation phasing strategy was developed and used for phasing haplotypes in a sire and 36 progeny using the 50 k Illumina BeadChip by: a) estimation of the recombination fraction and LD in consecutive SNPs using family information, b) linkage analyses between fragments, c) phasing of haplotypes in parents and progeny and in following generations. Homozygous SNPs in progeny allowed determination of paternal fragment inheritance, and deduction of SNP sequence information of haplotypes from dams. The strategy also allowed detection of genotyping errors. A total of 613 recombination events were detected after linkage analysis was carried out between fragments. Hot and cold spots were identified at the individual (sire level). SNPs for which the sire and calf were heterozygotes became informative (over 90%) after the phasing of haplotypes. Average of regions of identity between half-sibs when comparing its maternal inherited haplotypes (with at least 20 SNP) in common was 0.11 with a maximum of 0.29 and a minimum of 0.05. A Monte-Carlo simulation of BTA1 with the same linkage disequilibrium structure and genetic linkage as the cattle family yielded a 99.98 and 99.94% of correct phases for informative SNPs in sire and calves, respectively.

Hematologic and IgG responses of heifers experimentally infected with the agent of epizootic bovine abortion

BACKGROUND Epizootic bovine abortion (EBA) is a tick-transmitted abortive disease of beef cattle in the western United States. Infected cattle do not have clinical signs until abortion occurs, usually within the last trimester of gestation. There is little information on the hematologic response of the dam following infection. OBJECTIVE The purpose of this study was to determine if changes in blood leukocytes and serum IgG concentrations could be detected following experimental infection of pregnant heifers with the etiologic agent of EBA (aoEBA). METHODS Twelve Angus heifers were infected during gestation with the aoEBA using an inoculum prepared from the thymus of an infected fetus. Five pregnant heifer controls were given an inoculum prepared from the thymus of an aoEBA-negative calf. PCVs, total and differential leukocyte counts, and serum IgG concentrations were measured weekly following administration of the inocula until abortion or calving. Gross and microscopic examinations were performed on all aborted fetuses to confirm infection. RESULTS Eleven of 12 heifers in the treatment group aborted, and significant findings were decreased lymphocyte counts at weeks 1 and 14 postinoculation and increased monocyte counts at week 4 compared with control animals. Serum IgG concentrations were significantly increased at weeks 6-8 and 11 in the treatment group. CONCLUSIONS Leukogram changes are subtle in infected cattle. Future research efforts should be aimed at development of an antibody test specific for detection of previously infected animals, which could graze safely on EBA-endemic pastures.