Addition of eCG to a 14 d prostaglandin treatment regimen in sheep FTAI programs.

In the present study, there was evaluation of the alternative of adding eCG as part of a long-interval prostaglandin-F2α (PG) treatment on the reproductive efficiency of Merino sheep during the breeding season. A total of 210 ewes and 182 ewe lambs were randomly assigned to three experimental groups to induce the timing of estrus among ewes in a: Long-interval PG, group being synchronized using two doses of PG 14 days apart; Long-interval PG + eCG group being synchronized using the same treatment regimen as Group PG with the addition of 200 IU eCG to the regimen, administered concomitantly with the second PG administration; and MAP + eCG group being synchronized with intravaginal progestin sponges for 14 days plus 200 IU eCG, administered at the time of sponge removal. The percentage pregnancy rate in ewes of the MAP + eCG group was greater than the ewes of the Long-interval PG and Long-interval PG + eCG groups (76.4 % compared with 52.0 % and 62.5 %, respectively; P < 0.05). The prolificacy rate was greater in the ewes of the Long-interval PG+eCG group compared with the other groups (114 % compared with 100 % and 103 %, respectively; P < 0.05). When considering the fecundity rate, ewes of the Long-interval PG+eCG and MAP+eCG groups had greater values than ewes of the Long-interval PG group (71.2 % and 78.8 % compared with 52.0 %, respectively; P < 0.05). The Long-interval PG+eCG is an alternative to the conventional progestin sponge plus eCG treatment regimen with there being a greater fecundity rate when this regimen is used compared with the Long-term PG and similar to MAP-eCG treatment regimens.

Effect of GnRH or hCG administration on Day 4 post insemination on reproductive performance in Merino sheep of North Patagonia.

Different therapeutic strategies have been used with the objective of improve luteal function to reduce embryonic losses. The objective of this work was to study the effect of the administration of GnRH or hCG at Day 4 post fixed time artificial insemination (FTAI) on reproductive efficiency in Merino sheep during the breeding season in North Patagonia. Estrus of multiparous Merino ewes (n = 288) was synchronized by two injections of prostaglandins (PG; 125 µg, Cloprostenol), 14 days apart. Cervical FTAI was performed 53-56 h after the second PG with a dose of fresh semen (100 × 106 spermatozoa) from five Merino rams. In all ewes body condition score (BCS) was determined at FTAI. At 4 days post FTAI ewes were randomly assigned into three experimental groups: GnRH group (4 µg, Buserelin; n = 99), hCG group (300 IU, hCG; n = 92) and Control group (1 ml, saline solution; n = 97). Pregnancy and pregnancy losses were evaluated by ultrasonography on Days 33 and 90 post FTAI. Additionally, embryo crown-rump length (CRL) was measured by ultrasonography (n = 12 single-pregnant ewes by experimental group) at the first ultrasound. Date of birth, litter size and lamb weight were recorded (n = 111 pregnant ewes). Pregnancy rate on Days 33 and 90 post FTAI did not differ among treatment groups (P > 0.05). Pregnancy losses at Day 33 post FTAI were lower in the hCG group compared to the GnRH and Control groups (0, 3, 7.2%, respectively; P < 0.05). Pregnancy losses between Days 33 and 90 after FTAI were negligible (P > 0.05). The embryo CRL at Day 33 post FTAI was not increased by the hormonal treatments (P > 0.05). Moreover, it was lower in GnRH group compared to Control group (P < 0.05). Litter size tended to be greater in the GnRH group compared to the hCG and Control groups (P < 0.1). The birth weight of twin lambs tended to be higher in the GnRH group compared to the Control group (P < 0.1). The birth weight of single lambs was not affected by treatments (P > 0.05). Ram fertility and BCS of ewes at FTAI influenced the effect of hormonal treatments on reproductive parameters. In conclusion, administration of hCG or GnRH at Day 4 post FTAI does not improve pregnancy rate but treatment with hCG reduces pregnancy loss on Day 33 post FTAI. GnRH treatment improves litter size and twin lambs birth weight.

Hormonal therapeutic strategy on the induction of accessory corpora lutea in relation to follicle size and on the increase of progesterone in sheep.

We determined the effect of GnRH or hCG treatment on day 4 post-time artificial insemination (FTAI) on the formation of accessory corpora lutea (acc-CL) and on the concentration of serum progesterone (P4) in sheep. Multiparous adult Merino ewes (n = 36) were synchronized for estrus using double injection of PGF2α agonist (125 µg Cloprostenol) with an interval of 14 days. At 53-56 h after the second PG application, FTAI was performed. On day 4 post FTAI, ewes were either treated with analogue of GnRH (4 µg buserelin; n = 12) or hCG (300 IU, hCG; n = 12) or saline solution (1 ml; Control; n = 12). Two laparoscopic ovarian examinations were performed on days 4 and 10 post FTAI. In the first observation, we determined the number of post ovulation corpora lutea (po-CL) and the site, number and diameter of follicles present in both ovaries. In the second laparoscopy, we observed the number of po-CL and acc-CL. The sizes of the follicles that generated the acc-CL were determined according to the position of the follicles observed in the first laparoscopy. Serum P4 concentration was determined on days 4, 7, 10, 13, 17 and 21 post FTAI by chemiluminescence. A similar follicular population in size and number was observed in the three experimental groups prior to the beginning of treatments (Follicles 2 mm: 6.4 ± 3.7, 3 mm: 3.0 ± 2.3, 4 mm: 1.1 ± 0.5, 5 mm: 1.4 ± 0.8; P Ëƒ 0.05). The formation of 1.0 ± 0.4 and 1.1 ± 0.3 acc-CL was observed in the GnRH and hCG groups, respectively (P Ëƒ 0.05), but was not observed in the Control group (P < 0.05). Follicle sizes from which acc-CL generated were 3, 4 and 5 mm and did not differ between hormonal treatments (P Ëƒ 0.05). The hCG group had higher mean concentrations of P4 on days 7, 10, 13 and 17 post FTAI compared with the GnRH group and the Control group (P < 0.05), while no differences were observed between these two latter groups (P > 0.05). Mean P4 concentrations in ewes treated with hCG showed no differences according to the size of the follicle from which acc-CL were generated (P Ëƒ 0.05). In conclusion, administration of hCG or GnRH on day 4 post FTAI induced the formation of one acc-CL from follicles of 3, 4 or 5 mm, indistinctly. However, serum P4 concentration increased significantly only in the hCG group. The serum P4 concentrations of acc-CL that originated from different follicle sizes did not differ.

Pre-Selection Test to Identify High Responder Donor Goats.

The aim of the study was to evaluate the feasibility of pre-selection of high or low responder does prior to the superovulatory protocols. Twenty Saanen does received 800 IU of equine chorionic gonadotropin (eCG) at the end of long-term progestogen treatment. Fourteen days later, a second progestogen protocol associated with a multiple-dose follicle stimulation hormone (FSH) treatment (5 IU/kg of FSH, in six decreasing doses between days 4 to 6 of the protocol) was administered. Transrectal ultrasound was used to assess the follicular status at the beginning of superovulatory treatments, at the oestrous onset and on the seventh day of the oestrous cycle for counting corpora lutea (CL). A significant lower number of CL was obtained in eCG-treated in comparision with FSH-treated does (p < 0.05). A quartic regression was able to explain the relationship between the number of CL in response to both treatments (r(2) =0.50; p < 0.05). Seventy per cent (14 of 20) of does maintained the same ovulatory response (high or low) after treatments. The Kappa (κ = 0.40; p < 0.05) and Spearman (rs = 0.39; p = 0.08) coefficients were able to show a relationship between treatments. Regarding the follicular status, there is a significant relationship between the number of small follicles (r = 0.71; r(2) =0.47; p < 0.01) and total follicles (r = 0.60; p < 0.01) at eCG and first FSH dose with the number of CL. Moreover, it was found a negative relationship between the presence of large follicles and the number of CL in response to eCG treatment (r = -0.44; p < 0.05), but not from FSH (p > 0.05). In conclusion, the screening test with eCG has the potential to identify Saanen does that will better respond to the superovulatory protocol with FSH. In addition, it highlighted the importance of an ultrasound evaluation prior to the beginning of superovulatory treatments with FSH to characterize the follicular status and identify the potential donors of high ovulatory response in MOET programmes in goats.

Effects of season and superovulatory treatment on embryo yields in fine-wool Merinos maintained under field conditions.

The aim of the study was to assess the effects of superovulatory treatment (multiple FSH-dose vs single-shot FSH treatment) and seasonality on embryo yields in fine-wool Merino ewes. Treatment based on multiple FSH-dose consisted of 200 mg of FSH (Folltropin(®)) administered in seven decreasing doses. Single-shot treatment consisted of a single dose of 70 mg of FSH + eCG. In ewes treated with multiple FSH doses, number of recovered embryos was higher (6.0 ± 0.5 vs 3.5 ± 1.0), while non-fertilization rate was lower (12.8 ± 3.9 vs 40.3 ± 9.5) during the breeding season when compared to the non-breeding season (p < 0.05); although similar values of recovered Grades 1-2 embryos were observed between seasons. During the breeding season, proportion of responding ewes (98.1 vs 57.1%), ovulation rate (13.9 ± 0.8 vs 3.2 ± 1.2), recovered structures (7.9 ± 0.6 vs 1.7 ± 0.7), total recovered embryos (6.0 ± 0.5 vs 1.2 ± 0.6) and good-quality embryos (5.1 ± 0.5 vs 0.9 ± 0.6) were higher for the multiple FSH-dose treatment than for the single-shot protocol. In a similar way, in the non-breeding season, ovulation rate (11.3 ± 1.8 vs 6.0 ± 1.1) and recovered structures (6.6 ± 1.2 vs 2.7 ± 0.6) were higher for the multiple FSH injections protocol than those for the single-shot treatment, resulting in higher recovered Grades 1-2 embryos (3.2 ± 0.9 vs 1.4 ± 0.5). Current results indicate that seasonal anestrus affected embryo yields when applying multiple FSH-dose superovulatory treatment in Merino ewes, by decreasing the number of recovered embryos although the number of recovered good-quality embryos was not affected. During both seasons, multiple FSH injections produced higher ovarian response and number of viable embryos than the single-shot treatment.

Correlation between natural killer cell activation in the bone marrow and haemopoietic dysfunction following cytomegalovirus infection of mice.

We describe here the activation of natural killer (NK) cells in the bone marrows and spleens of mice infected with murine cytomegalovirus (MCMV). NK activity at these sites peaked at day 2 to 3 post-infection (p.i.) and declined between days 6 and 10 p.i. in BALB/c and C57BL/6 mice. In BALB/c mice, the increases in NK activity coincided with depletion of colony-forming units of the granulocyte-monocyte lineage (CFU-GM) from the marrow. CFU-GM depletion in MCMV-infected C57BL/6 mice was less severe, despite the presence of activated NK cells in the marrow. Treatment of BALB/c mice with anti-asialo GM1 prior to MCMV infection resulted in less severe CFU-GM depletion at day 2 p.i. than infection with MCMV alone. When homozygous C57BL/6 or CBA/CaH bg/bg mice were infected with MCMV, depletion of marrow CFU-GM was more severe than in their heterozygous littermates. Finally, we observed some inhibition of colony formation when marrow cells from MCMV-infected and uninfected BALB/c donors were mixed and incubated prior to the CFU-GM assay. These results suggest that activated NK cells may contribute to depletion of haemopoietic cells soon after MCMV infection of BALB/c mice, but may limit the loss of these cells in C57BL/6 and CBA/CaH mice.

Analysis of hematopoietic stem and progenitor cell populations in cytomegalovirus-infected mice.

We have studied the effects of murine cytomegalovirus (MCMV) infection on bone marrow stem and progenitor cell populations to find an explanation for the defects in hematopoiesis that accompany CMV infections in patients. Sublethal MCMV infection of BALB/c mice resulted in a 5- to 10-fold decrease in the numbers of myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitor cells in the marrow, but not in primitive myeloerythroid progenitor cell (colony-forming unit-spleen [CFU-S]) numbers. In contrast, we observed a 10- to 20-fold reduction in CFU-S as well as CFU-GM and BFU-E in lethally infected mice. Depletion of marrow CFU-GM was less severe in C57BL/10 and C3H/HeJ mice, which are more resistant to the effects of MCMV infection. Treatment of bone marrow cells with MCMV preparations in vitro did not reduce the numbers of CFU-GM, although up to 10% of the cells were productively infected. This finding suggests that CFU-GM were not susceptible to lytic MCMV infection in vitro and are probably not eliminated by lytic infection in vivo. Increases in the frequencies of Sca-1+Lin- marrow cells, a population that includes cells with the characteristics of pluripotential stem cells, were observed in MCMV-infected BALB/c, C57BL/10, and DBA/2J mice. Increases in the frequencies of c-kit+Lin- marrow cells were only seen in DBA/2J mice. MCMV infection did not impair the function of pluripotential stem cells because transplantation of marrow from MCMV-infected donors into irradiated recipient mice resulted in successful reconstitution of the T, B, and myeloid cell lineages.

Bone marrow atrophy induced by murine cytomegalovirus infection.

Acute, sublethal infection of mice with murine cytomegalovirus (MCMV) resulted in up to 80% decreases in the number of cells recoverable from the bone marrow, and a decrease in peripheral blood leucocyte counts during the first week of infection. Depopulation of the leucopoietic areas of the marrow was evident from examination of histological sections. The severity of bone marrow atrophy in MCMV-infected mice of different strains correlated with previously described genetically determined sensitivity to MCMV disease. Although the phenomenon only occurred when mice were inoculated with infectious virus preparations, fewer than one in 10(5) marrow cells were productively infected, suggesting that atrophy was not due to lytic infection of large numbers of bone marrow cells. Interestingly, increases in serum colony-stimulating activity were observed and these were proportional to the severity of bone marrow atrophy. After MCMV infection, we observed increases in the proportions of cells expressing some B-cell and myeloid cell markers and a decrease in the proportion of cells expressing an erythroid cell marker. There was no change in the frequency of marrow cells expressing mature T-cell markers. The numbers of myeloid lineage-committed progenitor cells (GM-CFU) in the marrow decreased 10- to 20-fold in BALB/c nu/+ mice, while there was a threefold decrease in their numbers in BALB/c nu/nu mice. In addition, increases in serum colony-stimulating activity were greater in BALB/c nu/+ mice than in BALB/c nu/nu mice. Our results suggest that growth factors produced after MCMV infection may accelerate the maturation and migration of cells from the marrow to sites of virus replication and inflammation, thus accounting for the depletion in numbers of marrow cells observed soon after MCMV infection.

Characterization of thymic involution induced by murine cytomegalovirus infection.

Infection of BALB/c mice with murine cytomegalovirus (MCMV) decreased the numbers of cells recovered from the thymus by 80-90% after 4-7 days, although less than 10 thymocytes per million were productively infected with the virus. A loss of cortical thymocytes was evident in histologic sections and correlated with depletion of CD4+ CD8+ cells. Thymic involution was minimal in C57BL/6 mice. This resistance was not H-2b-associated, as BALB.B (H-2b) mice were severely affected. In CXB recombinant inbred mice, thymic involution and MCMV replication co-segregated with atrophy and infection of the spleen and bone marrow. This suggests common regulation by natural killer (NK)1.1+ cells, consistent with the enhanced thymic involution demonstrated in NK-deficient bg/bg mice. However, CD4- CD8- cells were not depleted, so bone marrow hypoplasia may not be the proximal cause of thymic involution. MCMV infection activated CD4+, CD8+ and CD4+ CD8+ thymocytes, as expression of MEL14, major histocompatibility complex class I (H-2) and Sca-1 antigens increased on these cells. In vitro lymphoproliferation and interleukin (IL)-3 release were enhanced in unseparated and CD4(+)-enriched thymus preparations. Maturation of the thymus population was also evident from the high frequencies of single positive CD4+ and CD8+ cells and the decline in Sca-2 expression. However, unlike peripheral T cells, thymocytes from infected mice did not release IL-2. The results suggest that thymic involution accelerates the transit of cells through the thymus. The possibility that this impairs the elimination of autoreactive T cells within the thymus and promotes the autoimmune manifestations of MCMV disease is discussed.

B-cell activation following murine cytomegalovirus infection: implications for autoimmunity.

Infection of susceptible mice with murine cytomegalovirus (MCMV) induces persistent inflammation, and the production of autoantibodies reactive with large numbers of proteins from all major organs. However the roles of polyclonal B-cell activation, autoreactive T-helper cells and host-virus cross-reactions in these phenomena have not been evaluated. The present study reveals six- to 20-fold increases in serum immunoglobulin levels in MCMV-infected BALB/c and CBA mice, with IgG3 and IgG2b most affected. Titres of antibodies reactive with autologous tissues and ovalbumin (OVA) also increased following MCMV infection, whilst responses to a synthetic antigen [polyvinyl pyrrolidone (PVP)] were unaffected or depressed. IgG2a was the isotype most affected in responses to OVA, MCMV antigens and autologous tissues, suggesting interferon-gamma (IFN-gamma) may contribute to responses induced in the presence of the relevant antigen. Increases in total and antigen-specific immunoglobulin levels were CD4 dependent, as they were reduced in infected mice depleted of these cells with anti-CD4 antibodies. Serological changes were preceded by B-cell expansion and activation evident from increased cell yields, frequencies of cells releasing immunoglobulin and proliferation of T-depleted spleen and lymph node preparations. Numbers of mature B cells and macrophages increased in the lymph nodes, but B-1a (CD5+ Ig+) cell counts remained low. Alterations in the B-cell phenotypic profiles were more complex in the spleen, but correction for increased cell yields revealed increases in some subpopulations.

The effect of the Cmv-1 resistance gene, which is linked to the natural killer cell gene complex, is mediated by natural killer cells.

The resistance of mice to lethal infection by murine CMV (MCMV) is under complex host genetic control with contributions from both H-2 and non-H-2 genes. We have previously shown that an autosomal, non-MHC encoded gene, Cmv-1, controls MCMV replication in the spleen. We have investigated the mechanism by which the Cmv-1 resistance gene confers protection against MCMV infection. Using H-2 compatible irradiation bone marrow chimeras, the enhanced resistance to MCMV infection that is associated with the Cmv-1l allele in the C57BL background was shown to be mediated by an irradiation-sensitive bone marrow-derived cell population, or a factor produced by these cells. The lack of correlation between serum IFN titers and the strain distribution pattern of Cmv-1 in CXB recombinant inbred mouse strains suggests that IFN does not mediate resistance conferred by this gene. Similarly, the lack of effect of in vivo depletion of mature CD4+ and CD8+ T cells on virus replication in C57BL/6J mice indicates that T cells are unlikely to be involved. In contrast, in vivo depletion of NK cells by injection of the anti-NK1.1 mAb PK136 abrogated restricted splenic virus replication in C57BL/6J----BALB.B chimeric mice and in the Cmv-1l CXB strains. These data indicate that the effect of the Cmv-1 gene is mediated by NK cells. The significant augmentation in NK cell activity after MCMV infection of the susceptible Cmv-1h strains (BALB/cBy), CXBG/By, CXBH/By, CXBI/By, and CXBK/By) indicates the existence in these mice of NK cells that are functionally and phenotypically distinct from those in Cmv-1l strains. NK cells present in the Cmv-1h strains are unable to restrict efficiently splenic MCMV replication in vivo, possibly due to a lack of specificity for virus-infected target cells. Finally, flow cytometric analysis of NK1-1 expression in CXB and BXD RI mice together with MCMV replication studies in the BXD RI strains indicate that Cmv-1 is closely linked to NK1.1 and other loci that reside on a distal segment of murine chromosome 6 in a region that has recently been defined as the natural killer complex.

Replication of murine cytomegalovirus in mast cells.

Mast cells purified from the peritoneal cell population and mast cells derived in culture from bone marrow cells were examined for their sensitivity to murine cytomegalovirus (MCMV) infection in vitro. While up to 70% of mast cells expressed viral antigens, less than 12% of the cells produced infectious virus. Transmission electron microscopy demonstrated nucleocapsids in the nuclei and in association with the cisternal elements of the Golgi apparatus. Some complete virions were found within small cytoplasmic vacuoles. In contrast with previous studies of macrophages and fibroblasts, the susceptibility of mast cells to MCMV infection in vitro was not influenced by the H-2 or non-H-2 genotype of the donor.

H-2 class I loci determine sensitivity to MCMV in macrophages and fibroblasts.

Peritoneal (PM) and bone marrow-derived (BMM) macrophages and lung fibroblasts (LF) from inbred, intra-H-2 recombinant, H-2 mutant, and hybrid mice were infected with murine cytomegalovirus (MCMV) under centrifugal enhancement. At the concentration of virus employed, peritoneal macrophages from strains carrying Kd, Kb, Dd, Ks and/or Ds, Kq and/or Dq alleles could be infected to a level of 80%-100%, as assessed by viral antigen expression or loss of Fc receptors. Cells lacking these haplotypes and carrying Kk, Kj, Dk, Dj, or Db were resistant, yielding levels of infection below 20%. The background (non-H-2) and class II genotype and the S allele did not influence the proportions of cells infected. Furthermore, sensitivity was dominant in the F1 progeny of H-2b X H-2k and H-2d X H-2k crosses, and was not compromised by the bm1, bm3, bm10, or bm14 mutations in the alpha 1 or alpha 2 regions of Kb or Db. The proportions of cells able to release infectious virus were low, but paralleled the frequencies of viral antigen expression. The class I genotype also determined susceptibility to MCMV infection in BMM and LF, although up to 35% of H-2k BMM and 46% of H-2k LF could be infected. The findings are consistent with an association between K and D antigens and a cellular receptor for MCMV on all three cell types.

The Eagle effect revisited: efficacy of clindamycin, erythromycin, and penicillin in the treatment of streptococcal myositis.

We investigated the relative efficacies of penicillin, clindamycin, and erythromycin in a mouse model of myositis due to Streptococcus pyogenes. Penicillin was ineffective unless given at the time of bacterial injection, and treatment delays of 2 h reduced its efficacy such that survival was no better than that of untreated control animals (P less than .05). Survival of erythromycin-treated mice was greater than that of both penicillin-treated mice and untreated controls, but only if treatment was begun within 2 h. Mice receiving clindamycin, however, had survival rates of 100%, 100%, 80%, and 70% even if treatment was delayed 0, 2, 6, and 16.5 h, respectively. Thus, clindamycin demonstrated superior efficacy to penicillin among all the various treatment groups (P less than .05). Our results corroborate the failure of penicillin in this model of streptococcal infection and suggest that, unlike penicillin, the efficacy of clindamycin is not adversely altered by the "Eagle effect."