Short-term increases in stocking density affect the lying and social behavior, but not the productivity, of lactating Holstein dairy cows.

Reduced access to resources because of increased stocking density may have a detrimental effect on the behavior of the lactating dairy cow. The objective of this study was to determine the short-term responses in behavior, productivity, fecal cortisol metabolites, and udder and leg hygiene of lactating Holstein dairy cows housed at stocking densities of 100 (1 cow per freestall and headlock), 113, 131, and 142%. Multiparous cows (n=92) and primiparous cows (n=44) were assigned to 1 of 4 pens (34 cows per pen) in a 4-row freestall barn. Pens were balanced for parity, milk production, and days in milk. Stocking densities were imposed for 14 d using a 4 × 4 Latin square design. Time spent feeding and time spent ruminating were quantified by 24 h of direct observation of focal cows (n=12 per pen) beginning at 0800 h on d 11 of each period. Data loggers recorded lying behavior (time and bouts) from the same focal cows per pen at 1-min intervals during the final 5 d of each period. Fecal cortisol metabolites were quantified from samples collected on d 13 and 14 of each period from the same focal cows. Displacements from the feed barrier were recorded on a pen basis after 9 milkings over the last 4 d of each period. Productivity was assessed on a pen basis from milk yield (recorded from d 10 to 14 of each period) and milk components (quantified from composite samples collected on d 12 of each period). Milk composition was further analyzed for milk fatty acid profiles, which were determined from a subset (n=6 per pen) of the focal cows. Data were analyzed using the MIXED procedure of SAS, with the pen (n=4 per treatment, except displacements where n=3 per treatment) as the experimental unit. Feeding and ruminating (h/d) did not differ among treatments. Lying time was reduced at stocking densities of 131 and 142%, relative to 100 or 113%. Lying bouts were not affected by treatment. Stocking densities of 131 and 142% reduced the percentage of time cows spent ruminating within a freestall relative to 100%. Displacements from the feed bunk increased linearly across treatments. Fecal cortisol metabolites, udder hygiene score, milk yields, milk composition, and milk fatty acids did not differ among treatments. Decreased lying time and increased aggression at the feed bunk suggest that an alteration of the time budgets of lactating dairy cows may occur at higher stocking densities, but it is unclear at what point these changes might have further biological consequences.

Effect of alternative models for increasing stocking density on the short-term behavior and hygiene of Holstein dairy cows.

The primary objective of this study was to evaluate short-term responses in lying behavior and hygiene of Holstein dairy cows housed at a stocking density of 100 (1 cow per stall and headlock) or 142% imposed by 1) the denial of access to freestalls and headlocks, 2) the denial of access to freestalls, headlocks, and 26.6 m(2) of alley space, or 3) the addition of a rotating group of 14 cows to the resident group of 34 cows. The secondary objective was to determine the bioequivalence of the 3 methods of experimentally increasing stocking density. Cows (n=136) were assigned to 1 of 4 pens in a 4-row freestall barn and treatments were allocated using a 4×4 Latin square with 14-d periods. Lying time (h/d) and number of bouts/d for 12 focal cows per pen were determined using dataloggers recording at 1-min intervals during the final 5 d of each period. Dry matter intake (DMI) was established from the pen mean over the final 4 d of each period. Feeding and rumination activities on focal cows were determined by direct observation at 10-min intervals for 24h on d 11. Hygiene of focal cows was assessed from the difference in the scores after the legs and udder were cleaned on d 2 of each period and those on d 14. Lying time was greater for 100% stocking density (13.0 h/d) than the 142% stocking density treatments (11.8 h/d), which did not differ. Lying bouts (12.3/d) and bout duration (64.8 min/bout) did not differ among treatments. Short-term responses in DMI (24.6 kg/d) did not differ in response to the treatments. The 3 stocking density treatments decreased, or tended to decrease, the time spent feeding compared with 100% (4.4 versus 4.2 h/d). The stocking density treatments decreased the percentage of rumination occurring within a stall (92.3 versus 85.3%). A treatment effect on udder and leg hygiene scores was not evident on d 14 of each period or in the change from d 2 to 14 of each period. With the exception of rumination time (h/d), the 3 methods for experimentally imposing stocking density were bioequivalent for responses in behaviors, DMI, and hygiene. Future stocking density experiments in 4-row barns should simply deny resting and feeding space to simulate overcrowded housing conditions for lactating dairy cows because it is bioequivalent to more complicated, and potentially confounding, research models.

Detection of minimal residual disease and persistence of host-type hematopoiesis: a study in 28 patients after sex-mismatched, non-T cell-depleted allogeneic bone marrow transplantation for Philadelphia-chromosome positive chronic myelogenous leukemia.

We investigated the persistence of host-type hematopoiesis as defined by mixed chimerism (MC) in 28 male patients with chronic myelogenous leukemia (CML) who underwent opposite sex, non-T cell-depleted bone marrow transplantation (BMT) by amplification of Y-chromosome specific sequences, and correlated these results with the detection of minimal residual disease (MRD) by BCR/ABL mRNA amplification. Patients were studied at two time periods (> 3 months and > 24 months post-BMT). All but two patients were conditioned with total body irradiation (TBI) and cyclophosphamide (CY). One patient received busulfan (Bu), thiothepa (Thio) and CY, another patient CY and Bu. Detection of MRD occurred exclusively among patients with a MC (significance P < 0.04). Six of 18 patients with MC had detectable MRD, four of these consecutively developed cytogenetic and hematological relapse. Of 28 patients studied more than 3 months post-transplant, 18 (64%) had mixed chimerism and 10 (36%) had exclusively donor-derived blood cells. Nineteen patients were followed for their chimeric status between 24 months and 136 months post-BMT. Four patients converted from MC to complete chimerism and 10 patients (53%) remained mixed chimeric. The high incidence of MC in patients who underwent BMT without T cell depletion was measured by using a PCR assay with a sensitivity of 0.001%. As previously described by other investigators patients with complete chimerism developed more acute GVHD grade I-II (seven of 10 patients (70%) than patients with MC (nine of 18 patients (50%), not significant). This study also suggests that chimeric status might depend upon the regimen to prevent GVHD. One of four patients who received weekly methotrexate as GVHD prophylaxis developed MC, whereas in 11 of 18 patients receiving short course methotrexate and cyclosporine MC was detectable. All of six patients, prophylactically treated with a murine monoclonal antibody directed to the human alpha/beta T cell receptor in combination with cyclosporine, were mixed chimeras.

Bcr-abl-positive and -negative clonogenic cells in CML patients undergoing long-term interferon treatment.

Bone marrow (BM) and peripheral blood cell (PBC) samples of 11 Philadelphia (Ph) chromosome-positive chronic myelogenous leukemia (CML) patients in long-lasting hematologic remission induced by interferon (IFN) treatment were examined for the presence of leukemic hematopoietic precursor cells. Southern blot analysis revealed residual leukemic cells in BM samples of four patients, whereas seven patients showed no aberrant bands. Reverse transcription polymerase chain reaction (RT-PCR), however, amplified bcr-abl-specific cDNA in unfractionated BM or PBC samples in all 11 patients. The patients demonstrating bcr rearrangements in Southern blots had either a mosaic pattern (three patients) of bcr-abl-negative and positive colony-forming precursors (CFU-GEMM, BFU-E, CFU-GM, CFU-Mega), or all colonies were derived from leukemic precursors (one patient). However, in soft agar cultures of four patients without aberrant bands in Southern blots, only colonies without amplifiable bcr-abl transcripts were detectable. In another patient, few bcr-abl-positive colonies were found after 44 months of treatment, but not after 53 and 56 months of therapy. In these patients, therefore, residual disease detectable by PCR analysis of unfractionated cell samples does not appear to reside in the colony-forming cell compartment. The prognostic implications of these observations and the nature of the remaining bcr-abl-positive cells within unfractionated cell samples remain to be determined.

Presence of re-appearance of BCR-ABL-positive cells years after allogeneic bone marrow transplantation for chronic-phase chronic myelogenous leukemia in patients in hematological remission.

Allogeneic bone marrow transplantation (BMT) is considered to be the only curative therapy for chronic myelogenous leukemia (CML). The cytogenetic marker of CML, the Philadelphia (Ph) chromosome, or the molecular alterations caused by the BCR-ABL gene fusion can be used to monitor the success of treatment. A sensitive two-step reverse-transcription polymerase chain reaction (RT-PCR) was done to score BCR-ABL-mRNA-positive leukemic cells in frozen bone marrow samples of 15 CML patients retrospectively. These patients, 4 females, 11 males, had undergone BMT during the first chronic phase after a preparative regimen consisting of total body irradiation (TBI) and cyclophosphamide; median age at BMT was 38 years (range 20-49 years). At the time of this study, 8 patients were in cytogenetic and/or clinical remission. Seven patients relapsed after BMT; all presented with Ph-chromosome-positive metaphases and BCR-ABL-positive cells at the time of relapse. In only 1 patient in hematologic remission was no positive PCR analysis obtained in the two samples tested. However, 5 patients have remained or became Ph-chromosome and/or PCR-positive after BMT without clinical symptoms of disease. In samples from another patient, transient presence of leukemic cells was observed only early after BMT. Clinically, these patients were relapse free at days 3,055, 2,581, 2,252, 1,846, 1,839, 1,747, and 1,173 after BMT, respectively. Based on these data, the presence of single BCR-ABL-positive cells > 1 year after BMT has no prognostic significance.