The effect on the recovery profile of a change from enflurane to desflurane during the latter part of anaesthesia.

This study compared emergence and recovery characteristics after either enflurane anaesthesia or crossover from enflurane to desflurane anaesthesia. At an estimated 1 h prior to the end of operation, enflurane was either reduced (group E, n = 23) or replaced with desflurane (group X, n = 23). At the end of the operation, emergence and recovery characteristics of the two groups were compared. The crossover technique accelerated recovery compared with enflurane anaesthesia. The time taken for the eyes to open in response to painful pinching or a verbal command, and to regain awareness of age and name, were significantly shorter after crossover anaesthesia than after enflurane anaesthesia. The digit symbol substitution test and serial seven test scores were significantly better in patients subjected to crossover anaesthesia than in those subjected to enflurane anaesthesia. We conclude that, during surgery, the substitution of enflurane with desflurane in the latter part of anaesthesia can improve recovery.

Modulation of Chelidonii herba on GABA activated chloride current in rat PAG neurons.

Modulation of Chelidonii herba on gamma-aminobutyric acid (GABA) activated chloride current in the acutely dissociated periaqueductal gray (PAG) neuron was studied by nystatin-perforated patch-clamp technique. High concentrations of Chelidonii herba elicited ion current, that was blocked by bicuculline. Low concentrations reduced the GABA activated current in PAG. Two types of inhibitory action of Chelidonii herba on GABA activated current have been implicated in PAG. One is the inhibitory action of Chelidonii herba on GABA was abolished by naltrexone and the other is that of Chelidonii herba was potentiated by naltrexone. In addition, all of two types of action of Chelidonii herba are linked to pertussis toxin-sensitive GTP-binding proteins. These results suggest that the inhibitory modulation of Chelidonii herba on GABA activated current via G-proteins in PAG neuron is an important analgesic mechanism.

Immune enhancing effect of a growth hormone secretagogue.

Growth hormone (GH) has been known to enhance immune responses, whether directly or through the insulin like growth factor-1, induced by GH. Recently a nonpeptidyl small m.w. compound, a GH secretagogue (GHS), was found to induce the production of GH by the pituitary gland. In this study, we examined the effect of GHS in immunological functions of 5- to 6-wk-old and 16- to 24-month-old mice. In young mice, we observed a significant increase in PBLs, but T and B cell-proliferative responses were not consistently enhanced. The old mice, treated with GHS for 3 wk, did not show increases in peripheral lymphocytes, but they exhibited a statistically significant increase in thymic cellularity and differentiation. When inoculated with a transplantable lymphoma cell line, EL4, the treated old mice showed statistically significant resistance to the initiation of tumors and the subsequent metastases. Generation of CTL to EL4 cells was also enhanced in the treated mice, suggesting that GHS has a considerable immune enhancing effect, particularly in the old mice. We have also found that GHS promoted better thymic engraftment in bone marrow transplant of SCID mice. We found more cycling cells in the spleens of treated mice, suggesting that GHS may exert its immune enhancing effect by promoting cell division in lymphoid cells. These observations ascribe to GHS a novel therapy possible for aging, AIDS, and transplant individuals, whose immune functions are compromised.

HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine.

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

Correolide and derivatives are novel immunosuppressants blocking the lymphocyte Kv1.3 potassium channels.

The voltage-gated potassium channel, Kv1.3, is specifically expressed on human lymphocytes, where it controls membrane potential and calcium influx. Blockade of Kv1.3 channels by margatoxin was previously shown to prevent T cell activation and attenuate immune responses in vivo. In the present study, a triterpene natural product, correolide, was found to block Kv1.3 channels in human and miniswine T cells by electrophysiological characterization. T cell activation events, such as anti-CD3-induced calcium elevation, IL-2 production, and proliferation were inhibited by correolide in a dose-dependent manner. More potent analogs were evaluated for pharmacokinetic profiles and subsequently tested in a delayed-type hypersensitivity (DTH) response to tuberculin in the miniswine. Two compounds were dosed orally, iv, or im, and both compounds suppressed DTH responses, demonstrating that small molecule blockers of Kv1.3 channels can act as immunosuppressive agents in vivo. These studies establish correolide and its derivatives as novel immunosuppressants.

Characterization of a monoclonal anti-porcine CD3 antibody.

Partially inbred miniature swine have been developed in this laboratory as a large animal model for studies related to transplantation tolerance and as a source of hematopoietic cells and organs for xenotransplantation. The identification of swine CD3 specific mAbs capable of activating or depleting T cells in vitro and inducing an immunosuppressive state in vivo greatly facilitates studies of the swine immune system, transplantation tolerance and xenotransplantation research. Flow cytometry was used to determine the phenotypic profile of the swine specific mAb 898H2-6-15 (2-6-15). The specificity of 2-6-15 was further defined biochemically by surface labeling and immunoprecipitation. The ability of this mAb to activate pig T cells in vitro was examined by several criteria including proliferation assays, calcium flux analysis and detection of surface CD25 upregulation by fluorescence activated cell sorter (FACS) analysis. Monoclonal antibody 898H2-6-15 is specific for swine CD3 and is capable of inducing proliferation and CD25 upregulation in cultured swine peripheral blood lymphocytes. In addition, it induces calcium flux in purified pig T cells. Surprisingly, in contrast to described antibodies to CD3 in swine and other species, the binding of this antibody to porcine CD3 is dependent on the presence of extracellular calcium. Thus calcium was required in order to immunoprecipitate labeled surface molecules for biochemical analysis and to stain cell surfaces for FACS analysis of swine lymphocytes. In this paper, we describe a new swine CD3 specific mAb, 898H2-6-15 (2-6-15) the characteristics of which make it an extremely useful tool for in vitro and in vivo studies of the swine immune system and xenotransplantation. The availability of swine T cell specific reagents should facilitate the monitoring of swine T cell engraftment and/or release amongst xenogeneic mixed chimeras and thymic transplant recipients as well as provide a means to treat potential GvHD across xenogeneic barriers. We are currently evaluating the in vivo effects of 2-6-15 in the pig.

A tacrolimus-related immunosuppressant with reduced toxicity.

BACKGROUND: Tacrolimus (FK506) has potent immunosuppressive properties reflecting its ability to block the transcription of lymphokine genes in activated T cells through formation of a complex with FK506 binding protein-12, which inhibits the phosphatase activity of calcineurin. The clinical usefulness of tacrolimus is limited, however, by severe adverse effects, including neurotoxicity and nephrotoxicity. Although this toxicity, like immunosuppression, appears mechanistically related to the calcineurin inhibitory action of the drug, a large chemistry effort has been devoted to search for tacrolimus analogs with reduced toxicity but preserved immunosuppressive activity that might have enhanced therapeutic utility. METHODS: Here, we report on the identification of such an analog, which was synthetically derived from ascomycin (ASC), the C21 ethyl analog of tacrolimus, by introducing an indole group at the C32 position. The profile of biological activity of indolyl-ASC was characterized in rodent models of immunosuppression and toxicity. RESULTS: Indolyl-ASC was found to exhibit an immunosuppressive potency equivalent to that of tacrolimus in T-cell activation in vitro and in murine transplant models, even though indolyl-ASC bound about 10 times less to intracellular FK506 binding protein-12 than tacrolimus or ASC. Further evaluation of indolyl-ASC revealed that it is threefold less potent than tacrolimus in inducing hypothermia, a response that may reflect neurotoxicity, and in causing gastrointestinal transit alterations in mice. Moreover, indolyl-ASC was at least twofold less nephrotoxic than tacrolimus upon 3-week oral treatment in rats. CONCLUSIONS: Altogether, these data indicate a modest but definite improvement in the therapeutic index for indolyl-ASC compared with tacrolimus in rodent models.

Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.

The voltage activated K+ channel (Kv1.3) has recently been identified as the molecule that sets the resting membrane potential of peripheral human T lymphoid cells. In vitro studies indicate that blockage of Kv1.3 inhibits T cell activation, suggesting that Kv1.3 may be a target for immunosuppression. However, despite the in vitro evidence, there has been no in vivo demonstration that blockade of Kv1.3 will attenuate an immune response. The difficulty is due to species differences, as the channel does not set the membrane potential in rodent peripheral T cells. In this study, we show that the channel is present on peripheral T cells of miniswine. Using the peptidyl Kv1.3 inhibitor, margatoxin, we demonstrate that Kv1.3 also regulates the resting membrane potential, and that blockade of Kv1.3 inhibits, in vivo, both a delayed-type hypersensitivity reaction and an Ab response to an allogeneic challenge. In addition, prolonged Kv1.3 blockade causes reduced thymic cellularity and inhibits the thymic development of T cell subsets. These results provide in vivo evidence that Kv1.3 is a novel target for immunomodulation.

Association of serine protease with the rise of intracellular calcium in cytotoxic T lymphocytes.

The precise role of the granular enzyme A (granzyme A), a serine protease, in the lytic process of cytotoxic T lymphocytes (CTL) is not clear. We have recently constructed a CTL line transfected with the antisense gene of granzyme A (a-GrA). These a-GrA CTL had lower GrA activity as well as decreased lytic activities, as measured by 51Cr and by DNA degradation assays. Furthermore, at low effector:target ratio (1:8) in prolonged lytic assays, they could not lyse targets as rapidly as the control CTL. When we examined their ability to exocytose BLT (CBZ-L-lys-thiobenzyl)-esterase in the presence of anti-CD3 antibody, the a-GrA CTL exocytosed poorly compared to the parental CTL or control transfectant with a CAT gene. Most strikingly, a-GrA cells could not release intracellular stores of Ca2+ in response to anti-CD3 induction, although the Ca2+ flux was normal when they were stimulated with ionomycin. When the parental CTL was treated with a specific benzyllactam inhibitor of BLT-esterase or N-tosyl-L-phenylalanylchloromethyl ketone, the Ca2+ flux induced by anti-CD3 was also suppressed. We propose that granzyme A is involved in the signal transduction pathway that causes the rise of the intracellular calcium.

CTL lysis: there is a hyperbolic relation of killing rate to exocytosable granzyme A for highly cytotoxic murine cytotoxic T lymphocytes.

The lysis of susceptible targets by efficient cytotoxic T lymphocytes (CTL) increases both with time and with the ratio of CTL to target. Simple methods for calculating a killing rate constant from the time dependence of killing and for calculating the relation of the killing rate constant to the concentration of exocytosable granzyme A are given. Application of these methods to the killing of target cells by the highly efficient mouse CTL AR1 is presented. AR1 needed granzyme A for efficient killing. AR1 contained sufficient exocytosable granzyme A to kill at about 80% of the rate possible at infinite exocytosable granzyme A.

Anti-CD11b antibody prevents immunopathologic changes in viable moth-eaten bone marrow chimeric mice.

The effect of in vivo treatment with anti-CD11b (MAC-1) antibody (Ab) was examined in an inflammatory disease model, the viable moth-eaten (mev) mutant mouse. The autosomal recessive mev gene occurred spontaneously as a point mutation of the hematopoietic cell protein tyrosine phosphatase in C57BL/6 mice. Homozygotes (mev/mev) develop a chronic myelomonocytic inflammation, involving accumulation of myelomonocytic cells in lungs and skin, resulting in interstitial pneumonitis and severe edema in the paws. These mice also exhibit abnormalities in lymphoid development, thymic atrophy, with T cell and NK cell dysfunction. These inflammatory changes are transferrable by bone marrow cells of mev/mev mice, indicating that mev mutation is due to a stem cell defect in the myelomonocytic pathway. An anti-CD-11b (5C6) Ab inhibited the immunopathologic changes in the bone marrow chimeras, when the Ab treatment was initiated on day -1 or day 0 of the bone marrow transplant. The lungs, paws, and thymus all remained normal after treatment. Furthermore, the Ab also delayed the onset of the mev syndromes when the Ab was given 10 days after the bone marrow transfer. Therefore anti-CD11b Ab inhibited inflammation both prophylactically and therapeutically, and restored normal function of T and NK cells in this disease model. These results support the contention that CD11b molecules expressed in the myelomonocytic cells play a critical role in this naturally occurring inflammatory disease.

Prevalence of peripheral artery disease in patients with coronary artery disease.

The incidence of coronary artery disease in Singapore has shown a rapid rise in recent years, however, there is a subjective impression that the prevalence of atherosclerotic peripheral artery diseases, although sharing common aetiological factors with coronary artery disease, has not shown a similar rise. We set out to determine the incidence of occlusive peripheral artery diseases of the lower limb in a selected population of Asian patients using a non-invasive test and we analysed the risk factors. The overall prevalence of peripheral artery disease of the subjects as determined by an abnormal ankle brachial index less than 0.9 in at least one limb was 19.1%. Increasing age, hypercholesterolemia and smoking were risk factors. In patients with proven coronary artery disease, abnormal ankle brachial indices were present in 21.3% of patients with angiographic evidence of coronary artery disease but in only 13.0% of those without. In this study, the incidence of peripheral artery disease in this selected Asian population was high. With the increasing availability of vascular laboratories and awareness of the diseases, we can expect to see increasing numbers of patients presenting with these problems.

A single administration of LFA-1 antibody confers prolonged allograft survival.

C57BL/6 (B6) thyroid gland transplanted to the left kidney capsule of an allogeneic (BALB/c) host was typically rejected in 14 days. A single administration of 500 micrograms of an antibody to the adhesion molecule, leucocyte function-associated antigen (LFA-1, CD11a), prevented all thyroid allograft rejection for at least 70 days. Fifty percent of the treated recipients retained intact allografts for 470 days. However, the same treatment with anti-CD11a could not protect a sensitized BALB/c mouse from rejecting a second B6 thyroid allograft. Production of donor-specific alloantibodies elicited by allograft rejection was also inhibited in this system. In this transplant model, the Ab therapy is more efficacious than that of FK506, administered daily for 14 days at 15 mg/kg. These results demonstrate the remarkable effect of an anti-LFA-1 antibody in promotion of allograft survival.

FK-506 and cyclosporin A: selective inhibition of calcium ionophore-induced polymorphonuclear leukocyte degranulation.

This paper investigates the abilities of FK-506 and cyclosporin A (CsA) to inhibit human polymorphonuclear leukocyte (PMNL) degranulation. PMNLs, purified from human blood, were stimulated in vitro with A23187, ionomycin, the complement derived peptide C5a, formylmethionylleucinylphenylalanine (FMLP) or phorbol myristate acetate (PMA). Degranulation was assessed by measuring the release of either lactoferrin or N-acetyl-beta-D-glucosaminidase (NAG). Both FK-506 and CsA produced a concentration-related inhibition of degranulation induced by either A23187 or ionomycin but did not affect C5a-, FMLP- or PMA-induced degranulation. The IC50 values for inhibition of degranulation (approximately 0.7 nM for FK-506 and 33.7 nM for CsA) are very close to the published values for inhibition of human T-cell proliferation. Removal of calcium from the incubation medium with ethyleneglycolbis(aminoethylether)tetra-acetate (EGTA) totally inhibited calcium ionophore-induced degranulation but had no effect against C5a-, FMLP- or PMA-induced degranulation. Preincubation of PMNLs with actinomycin D or cycloheximide did not affect either A23187- or PMA-induced degranulation. Non-immunosuppressive analogs of CsA were ineffective at inhibiting degranulation. Rapamycin, a macrolide structurally related to FK-506, did not inhibit degranulation but it did antagonize the inhibition produced by FK-506. Given the similar profiles of activity of FK-506 and CsA in neutrophils and T cells, we conclude that similar activation or signal transduction pathways may be present in both T cells and neutrophils. Because A23187-induced PMNL degranulation was not sensitive to either actinomycin D or cycloheximide, it is apparent that the signal transduction pathways ultimately control different cellular functions.

Suppressive effects of monocytic cells and transforming growth factor-beta on natural killer cell differentiation in autoimmune viable motheaten mutant mice.

Viable motheaten (mev) mice are homozygous for a recessive single gene mutation at chromosome 6. These mice develop numerous inflammatory and arthritic syndromes and exhibit abnormal B cell functions as well as lower T and NK cell activity. In this study, the differentiation of NK cells in mev mice was examined to elucidate the underlying basis for decreased NK activity. Although NK cells appear to be present in mev mice, their activity was demonstrable only when the spleen cells were enriched by nylon wool passage. Similarly bone marrow cells from these mice could be shown to contain precursors of NK cells when they were passed over nylon wool and transplanted into irradiated recipients. The adherent cells from both the spleen and bone marrow of mev mice suppressed the differentiation of NK cells from normal splenic populations. These suppressive adherent cells were F4/80(+), AsGm-1(+), Qa-5(+), and NK-1.1(+). They were not cytolytic when cultured in IL-2. Antibodies to a number of cytokines, such as IFN-alpha, -beta, and gamma, or TNF-alpha, could not reverse the suppressive effect of the adherent cells. Addition of anti-TGF-beta antibody could, however, overcome the suppression, suggesting that TGF-beta was partly responsible for the defective NK differentiation in the mev mice.

Studies on murine natural killer (NK) cells. V. Genetic analysis of NK cell markers.

This paper attempts to clarify the number and nomenclature of murine natural killer (NK) cell specific alloantigens by defining the genetic relationships between them, that is, are they coded by loci which are independent, allelic, or linked. Strain typing and F2 analyses using five alloantisera (C3H X BALB/c)F1 anti-CE, CE anti-CBA, NZB anti-BALB/c, C3H anti-ST, and BALB/c anti-DBA/2 revealed that (a) the alloantigens NK-1.1 and NK-3.1 are determined by distinct loci which are linked on the same chromosomes, (b) the alloantigen NK-2.1 is determined by an independently segregating locus to those coding for NK-1.1 and NK-3.1, (c) the alloantisera, CE anti-CBA and NZB anti-BALB/c, which have been designated anti-NK-2.1 alloantisera recognize different alloantigens coded by independent genetic loci. Thus, these five alloantisera detect four NK cell specific alloantigens which, based on the chronology of their discovery, have been designated NK-1.1-(C3H X BALB/c)F1 anti-CE, NK-2.1-CE anti-CBA, NK-3.1-C3H anti-ST, and BALB/c anti-DBA/2 and NK-4.1-NZB anti-BALB/c.

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.

Immune regulation of metastasis from in vivo derived syngeneic murine melanoma.

In earlier work, we demonstrated that in vivo derived B16F10 tumor cells metastasize aggressively from intracameral (ic) and subcutanous (sc) sites, colonizing the lungs and lymph nodes. Natural killer (NK) cells play an important role in metastasis from ocular tumors. Treatment of mice with MoAb PK136, a highly specific anti-NK antibody, altered the pattern of metastasis; metastases appeared in the lungs, adrenal glands, liver, and spleen. Treatment with cyclophosphamide (Cy) did not affect survival or metastasis, but combined treatment with the immunomodulator Linomide (LS2616) and Cy decreased metastasis and increased survival. In the present study, we examine the role played by NK cells in regulating metastasis of sc tumors. Treatment of mice with LS2616 enhanced NK cell activity and decreased metastasis. Treatment with MoAb PK136 decreased survival and increased metastasis, but did not affect the pattern of metastasis. Treatment with 25 mg/kg Cy alone resulted in a decrease in growth of the primary tumor, increased survival, and decreased metastasis. Combined treatment with LS2616 and Cy was very effective in decreasing metastasis, increasing survival, and affecting cures (30%). In summary, our experiments demonstrate the importance of NK cells in regulating metastasis from sc tumors of in vivo derived B16F10 melanoma and demonstrate the effectiveness of LS2616 and low doses of Cy on metastasis and survival. In combination with earlier work, the present experiments demonstrate: 1) that modulation of NK activity differentially affects metastasis from sc and ic compartments, and 2) that regional differences in the location of the primary tumor may determine the effectiveness of treatment with Cy.

Demonstration of the antiviral role of natural killer cells in vivo with a natural killer cell-specific monoclonal antibody (NK 1.1).

A monoclonal antibody (NK 1.1) to mouse natural killer (NK) cells selectively depleted NK cell activity in virus-infected mice without significantly depressing other immune functions, including the development of virus-specific cytotoxic T cells. NK cell depletion with this antibody resulted in markedly enhanced plaque-forming unit titers of some (murine cytomegalo, Pichinde) but not other (mouse hepatitis, lymphocytic choriomeningitis) viruses. This confirms that NK cells do play a role in regulating certain infections and shows that this antibody provides a convenient tool for examining the role of NK cells in viral infections.

Enrichment of bovine X- and Y-chromosome-bearing sperm with monoclonal H-Y antibody-fluorescence-activated cell sorter.

Bovine spermatozoa were assessed indirectly for the presence of a Y chromosome by monitoring expression of the H-Y antigen. Spermatozoa labeled with a monoclonal H-Y antibody (MoAb) and fluorescein-conjugated goat antibody to mouse F(ab)2 were counted with both a fluorescent microscope and a fluorescence-activated cell sorter (FACS). Of ejaculated spermatozoa, 40% to 60% fluoresced by this procedure compared to 1% to 15% of sperm reacted with nonimmune serum. Semen from three bulls was exposed to nonimmune serum (control) or MoAb, sorted by FACS, and analyzed for DNA content with a scanning microdensitometer. Control samples showed two distinct peaks with a mean difference in DNA content of 3.95%; these peaks were assumed to represent Y- and X-chromosome-bearing spermatozoa populations, respectively. DNA analyses of the MoAb-treated sperm of three bulls that sorted positively for H-Y antigen (fluorescent sperm) yielded ratios of Y- to X-chromosome-bearing spermatozoa of 76 : 24, 88 : 12, and 77 : 23, and those sorted negatively for H-Y antigen (nonfluorescent sperm) yielded ratios of 26 : 74, 35 : 65, and 23 : 77. The proportions of Y- and X-chromosome-bearing spermatozoa in nonsorted samples were not different from 50 : 50. Suitable MoAbs can be used in conjunction with FACS to enrich the proportion of Y- or X-chromosome-bearing spermatozoa in bovine semen.