Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca²⁺ concentration ((Ca²⁺)_i), the present study aimed to determine the changes in Ca²⁺ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca²⁺ store of freshly prepared milk cells was estimated from the elevation of (Ca²⁺)_i after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca²⁺ store in milk cells after intracellular Ca²⁺ depletion by Bapta-AM followed by spiking with 2.5 mM Ca²⁺ for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca²⁺ store in milk cells was much less than blood PMNs. The production of superoxide anion (O₂^?) in vitro in response to (Ca²⁺)_i-dependent or (Ca²⁺)_i-independent modulators was used to evaluate the relevance of altered Ca²⁺ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O₂^? as blood PMNs when treated with ionomycin. However, the amount of O₂^? produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O₂^? production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca²⁺ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O₂^–production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca²⁺, but decreased O₂^? production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca²⁺ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.     

Lactation curves of friesian-bunaji crosses in NigeriaCourves de lactation de croisées frisonne-bunaji au NigeriaLaktationskurven von friesian-bunaji rinderkreuzungen in Nigeria

The validity of the gamma function to describe lactation curves of $\text{1}\text{2}$ and $\text{3}\text{4}$ bred Friesian-Bunaji crosses was investigated. The function explained 71% of the variation in yield in lactations which differed in duration, parity and season of calving. The effect of these variables on the components of the lactation curve was analysed by least-squares procedures. The goodness of fit of the function did not differ between classes of varying duration of lactation; short lactations, however, in addition to a lower persistence, also showed a low level of production. Lactation curves of first parity were flatter and also had a greatly reduced level of production compared to higher parities.Multiplying factors for estimating total lactation yield from part records were obtained from the fitted curves. The accuracy of prediction was greater when separate factors were used for each class of lactation length. The usefulness of part records in progeny testing is also discussed.     

Half-life,clearance and production rate for oxytocin in cattle during lactation and mammary involution I

Basal concentration, production rate, metabolic clearance rate and $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for oxytocin were measured in Holstein cows at three lactational stages and during mammary involution. At each lactational stage, oxytocin was given intravenously at .5 IU/min or 1.0 IU/min for 60 min. Infusions were preceded by priming. During involution, the high dose was used. Mean basal concentrations of oxytocin ranged from 8.7 to 21.4 uU/ml. Mean basal values at early, middle, late lactation and involution were 17.57, 12.33, 15.15, 21.13 uU/ml, respectively and differed significantly. The mean “rapid” $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 3.87 ± .1 min. Early, middle, late lactation and involution $\text{T}\text{1}\text{2}\text{s}$ were 4.2, 3.7, 4.0 and 3.5 min, respectively. The rapid $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was not affected by lactational stage. The mean “slow” $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 25.53 ± 1 min. Early lactation and dry period means differed significantly. The overall mean oxytocin clearance rate was 8.41 ± .1 ml/kg^·min. Clearance rate declined through lactation and into the dry period. Mean values of 9.3, 8.8, 7.8 and 7.1 ml/kg^·min were obtained at early, middle, late lactation and involution, respectively. Clearance rates at late lactation and involution differed significantly from one another and from the early and middle stages. Mean entry rates for oxytocin at early, middle, late lactation and involution were 168.79, 106.03, 111.1 and 146.8 uU/kg^·min, respectively. Measurements at early lactation and involution were greater than values for middle and late lactation. To summarize, basal oxytocin concentrations can be measured in cows that are lactating or undergoing mammary involution and changes in concentrations during lactation are related to hormone production (entry rate) and metabolic clearance rates.     

Single step purification procedure for the rapid separation of equine leucocytes

Percoll gradients have been used to separate relatively pure populations of viable equine polymorphonuclear (PMN) and mononuclear (MN) cells. In preliminary studies, a continuous density gradient of 70% Percoll solution was used to separate two distinct leucocyte-rich bands. After measurement of the density of each band on the continuous gradient, discontinuous Percoll gradients, using 60% and 75% Percoll solutions, were used to provide a rapid means of separating PMN and MN cells. The yield of viable cells per ml of blood was 3.0×10⁶ and 3.2×10⁶ for MN and PMN cells, respectively. Corresponding values for recovery were 45% and 72%. The purity was 94% for PMNs and 99% for MNs.     

The phagocytic cell response of the ovine lung to Pasteurella haemolytica

Conventionally-reared sheep were inoculated with (3.0 ± 0.6 × 10⁷) viable Pasteurella haemolytica type A1 by the intratracheal route and were killed immediately (0-time) or 2, 4, 12, 16, 24, 48 or 72 h later. Lung-wash cells and free bacteria were recovered by pulmonary layage.The number of recoverable bacteria tended to increase between 0-time and 4 h post-in-oculation (p.i.) then decline rapidly over the next 8 h. However, the rate of clearance was extremely variable and viable bacteria were recovered from $\text{3}\text{5}$ animals at 48 h p.i. and from $\text{1}\text{5}$ at 72 h p.i.In parallel with the clearance of the majority of the bacteria, total neutrophil numbers in the lung-wash rose to a peak of (36 ± 6) × 10⁸ cells/lung, which was, on average, 70-fold higher than 0-time levels. Their numbers remained constant from 12 to 24 h p.i. then fell to be 5-fold above 0-time levels at 72 h p.i. Macrophage numbers rose slowly throughout the experiment but most of the increase occurred between 24 and 48 h p.i. They reached a peak of (17 ± 11) × 10⁸ cells/lung at 48 h i.p. which was 3-fold higher than 0-time levels.     

Effect of Rhodococcus equi on equine polymorphonuclear leukocyte function

A procedure was developed for isolating large numbers of purified polymorphonuclear leukocytes (PMNs) from the peripheral blood of horses. Equine PMN function was evaluated by three procedures: 1) Staphylococcus aureus ingestion, 2) nitroblue tetrazolium reduction, and 3) iodination. Four preparations of R. equi were added to polymorphonuclear leukocytes (PMNs) in each test system. Live bacteria, heat-killed bacteria, the washed pellet from heat-killed bacteria, and the supernatant fluid from heat-killed bacteria were evaluated for effects on equine PMN function. None of the R. equi preparations had an effect on S. aureus ingestion by equine PMNs. Nitroblue tetrazolium reduction by PMNs, a measure of oxidative metabolism, was suppressed by pellet and supernatant fractions. Values for the iodination reaction were depressed by all R. equi preparations, indicating decreased activity of the myeloperoxidase-H2O2-halide system of the PMN. Further evaluation of the supernatant from heat-killed R. equi showed that it retained its inhibitory effect on iodination following autoclaving and/or passage through a 10,000 MW filter. R. equi fractions did not alter the enzymatic conversion of 125I to a protein-bound form in a PMN-free assay developed to evaluate this reaction. The presence of a surface component capable of inhibiting bactericidal mechanisms of the PMN may play an important role in intracellular survival of R. equi.     

Application of indomethacin as a potentiator of lymphocyte blastogenesis in Brucella abortus exposed cattle

Lymphocytes from $\text{Brucella abortus}$ field strain infected, strain 19 vaccinated, non-exposed and field strain infected, but immunologically unresponsive cattle were incubated with $\text{B. abortus}$ antigen and indomethacin. There were significant increases (P < 0.005) in the blastogenic responses, as measured by [³H] thymidine uptake, in cultures with indomethacin as compared to cultures without indomethacin. Lymphocyte blastogenic responses to $\text{B. abortus}$ antigen were potentiated by indomethacin in both $\text{B. abortus}$ exposed and non-exposed cultures. However, potentiation of sensitized lymphocyte blastogenic responses by indomethacin was significantly greater (P < 0.005) than that in non-exposed lymphocytes. Additionally, indomethacin significantly potentiated Brucella-induced lymphocyte blastogenic responses in lymphocytes from anergic cattle.     

Disease,production and culling in Holstein-Friesian cows VI. Effects of management on disease rates

Effects of management practices on rates of disease in Holstein—Friesian cows in 32 commercial dairy herds located within a 50-mile radius of Guelph, Ontario were investigated in a $\text{2}\text{1}\text{2}\text{year}$ study.A personal survey questionnaire was used to collect information on management factors related to housing, nutrition and disease control procedures. The logit transform was applied to the herd disease rates and multiple linear regression used to identify management factors related to disease rates. Large values of the variable “frequency of ration balancing” identified larger more progressive farms which may have had superior disease recording systems. In a second regression analysis, “frequency of ration balancing” was entered into the regression equation prior to step-wise selection of the other variables.The ability of the selected variables to predict herd rates of disease was only moderate (r² = 0.24 to 0.57). However, of the variables available for selection, those potentially reflecting the farmers attitude toward the dairy herd (e.g., amount of attention paid to individual cows) appeared to be important.     

Roles of pattern of secretion of luteinizing hormone and the ovary in attainment of puberty in ewes lambs

Four experiments were conducted to examine (1) how patterns of luteinizing hormone (LH) change as lambs approach first estrus, (2) whether mimicry of these changes by exogenous LH will initiate events of the pubertal process, (3) aspects of possible roles of the ovary and estradiol in the initiation of an ovulatory surge of LH in prepubertal lambs and (4) the time requirements for the presence of the ovary in induction of that surge of LH.In 19 lambs (Exp. 1), concentrations of LH in plasma were higher at puberty than 7 weeks earlier. Increased concentrations of LH in samples taken every 20 min for 6 hr during the luteal and follicular phases preceding first estrus were attributed to increased amplitude, but not increased frequency, of episodic pulses of LH. In Exp. 2, endocrine events which are included in the natural onset of ovarian cyclic activity (i.e. surge of LH and subsequent rise in progesterone) were initiated in $\text{5}\text{6}$, $\text{4}\text{6}$, $\text{1}\text{5}$, $\text{1}\text{6}$, and $\text{3}\text{6}$ lambs, receiving either injections of 7.5 μg/hr, 15 μg/hr, 30 μg/2hr, 45 μg/3hr or constant infusions of 15 μg/hr of purified ovine LH, respectively. However, these lambs did not exhibit estrus or cyclic ovarian activity. In Exp. 3, the mechanism by which hourly injections of LH prompted surges of LH was determined to be mediated through ovarian stimulation and not through a direct effect of exogenous LH on the hypothalamo-pituitary axis. All the intact (n=5) but no acutely (two weeks) ovariectomized (n=5) lambs had a preovulatory-like surge in response to exogenous LH. Further, (Exp. 4) it was shown that the ovarian signal must be maintained or evoked within 6 hr preceding the surge of LH, because lambs (n=20) ovariectomized at or 9, 18 or 27 hr after initiation of the exogenous LH with the one exception failed to show a surge of LH. It could not be demonstrated that the ovarian signal involved changes in peripheral concentrations of androstenedione, testosterone or estradiol-17β, although patterns of LH in ovariectomized lambs were responsive to both negative and positive feedback effects of estradiol (n=6, Exp. 3) and in general estradiol levels were increasing prior to the induced surge of LH.     

In Vitro Effects of Acellular Milk on the Bactericidal Components of Caprine Polymorphonuclear Neutrophils

The effects of acellular milk on the activity of the microbicidal cationic enzymes of the polymorphonuclear cells of goats were studied in an attempt to explain the phenomenon by which PMN functions fail in mastitis. Assays were undertaken on the myeloperoxidase, lysozyme and elastase activities in a polymorphonuclear cell (PMN) lysate, both in the presence and absence of acellular milk from homologous species. There was a significant decrease (p<0.05) in the activity of lysozyme, myeloperoxidase and elastase in the presence of acellular milk. Superoxide and H₂O₂ production following activation of caprine PMNs by lipopolysaccharide (LPS) was significantly reduced (p<0.05) in the presence of acellular milk. Thus, the microbicidal function of PMNs is significantly impaired in the presence of acellular milk and this may contribute to the development of mastitis in dairy animals.     

Evaluation of immunomodulatory effect of recombinant human granulocyte‐macrophage colony‐stimulating factor on polymorphonuclear cell from dogs with cancer in vitro

The objective of this in vitro study was to evaluate the immunomodulatory effects of recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF) on polymorphonuclear cell (PMN) function in dogs with cancer. PMNs were harvested from dogs with naturally developing cancer as a pre‐clinical model to evaluate the immunomodulatory effects of rhGM‐CSF on PMN phagocytic and cytotoxic functions, cytokine production and receptor expression. Some aspects of cancer‐related PMN dysfunction in dogs with cancer were restored following incubation with rhGM‐CSF including PMN phagocytosis, respiratory burst and LPS‐induced TNF‐α production. In addition, rhGM‐CSF increased surface HLA‐DR expression on the PMNs of dogs with cancer. These data suggests that dysfunction of innate immune response in dogs with cancer may be improved by rhGM‐CSF. The results of this study provided a pathophysiologic rationale for the initiation of clinical trials to continue evaluating rhGM‐CSF as an immunomodulatory therapy in dogs with cancer.     

The use of a cathodic antigen in the immunoelectrophoretic serodiagnosis of Echinococcus granulosus in sheep

An antigen was prepared from purified sheep hydatid-cyst fluid by gelfiltration on Sephadex G-200 followed by chromatography on DEAE-cellulose. In each case the first-peak material was used. This antigen, which migrated cathodically, was concentrated and used in immunoelectrophoretic analyses of 4X concentrated sera from sheep experimentally and naturally infected with $\text{Echinococcus granulosus}$ and from uninfected sheep. Of 34 sheep with $\text{E. granulosus}$ infection 31 were positive with the cathodic antigen while of 85 sheep without $\text{E. granulosus}$ 8 were (falsely) positive. Many false positives appeared to be associated with heavy infections of $\text{Taenia ovis}$ or $\text{T. hydatigena}$ larvae. The “arc 5” immunoelectrophoresis test, which is the most specific immuno-diagnostic test for echinococcus infection in humans, was not able to specifically identify $\text{E. granulosus}$ infections in sheep.     

Glycogen in Leukocytes from Bovine Blood and Milk

Glycogen content was determined quantitatively by the Anthrone reagent method in leukocytes obtained from blood and milk of five cows. Distribution of glycogen in leukocytes was studied by microscopic examination of slides stained by Periodic acid-Schiff (PAS) reaction. Blood glucose concentrations were investigated in these animals by standard procedures. In two of five cows both blood glucose levels and blood leukocyte glycogen levels on the same day were determined for six consecutive days. One hundred and two blood leukocyte samples from five cows had a mean glycogen content of 1.32 ± 0.04 (S.E.) mg/10⁹ WBC, and 6.11 ± 0.17 (S.E.) mg/10⁹ PMNs. Leukocyte preparations from 80 samples of milk comprising 97 to 98% PMNs contained 3.81 ± 0.18 (S.E.) mg glycogen/10⁹ milk leukocytes. In PAS preparations of blood and milk leukocytes glycogen was found almost exclusively in PMNs. Glycogen granules, present frequently in PMNs and occasionally in monocytes and large lymphocytes from blood, were not observed in those from milk. The glycogen level in milk leukocytes was significantly lower (P = <0.01) than that of the blood PMNs in every cow, and the overall mean difference between levels for milk leukocytes and blood PMNs was highly significant (P = <0.001). Mean blood glucose concentration in the five cows was 44.46 ± 0.66 (S.E.) mg%. There was no significant relationship between blood glucose and blood leukocyte glycogen levels in the five corresponding cows; nor between blood glucose and blood PMN glycogen levels on the same day in either of two cows investigated. Leukocyte preparations from milk samples obtained on the second day following intramammary infusion of endotoxin consistently contained markedly less glycogen than the leukocyte preparations from first day post-infusion samples.

These tended to level off and became intermediate between first and second day levels. It is postulated that the poor phagocytic competence of leukocytes from bovine mammary glands compared to their counterparts in blood observed by various workers may be due partially to low energy reserves in these cells.

     

Defences of the bovine mammary gland against infection and prospects for their enhancement

The equine alternative complement pathway has been partially characterized and compared to the equine classical activation pathway. A dose-dependent lysis of RbRBC was observed with peak lytic values noted within 10 minutes at 37°C when rabbit red blood cells (RbRBC) were used as an alternative pathway activator. Sheep red blood cells (SRBC) sensitized with rabbit hemolysin or partially purified equine IgM antibodies were equally sensitive to lysis. Dilution of the commercial hemolysin by $\text{1}\text{5}$ reduced lysis from 90% to 38% in the presence of constant cell numbers. Hemolysis of SRBC peaked at 10 minutes and the majority of lysis occurred within 10 minutes. Dilution of equine sera by as little as $\text{1}\text{5}$ decreased hemolytic activity for SRBC to 21.5% from greater than 90% with undiluted sera. The alternative pathway protein, equine factor B, was tested using RbRBC and monitored by its differential susceptibility to heat treatment at 50°C. This treatment led to almost complete inactivation after a 15-minute incubation. An apparent heat-dependent decay of certain classical pathway components was also observed after 50°C treatment. This sensitivity was indicated by a reduction in the lytic activity for sensitized SRBC. Treatment for 15 minutes at 56°C with either RbRBC or SRBC was sufficient to abolish hemolytic activity in all equine sera tested. Chelation of cations with 0.04 M EDTA blocked expression of alternative and classical pathway activation; however, chelation of Ca⁺⁺ ions with 10 mM EGTA containing 1 mM Mg⁺⁺ ions permitted lysis of the RbRBC but not the SRBC. A dose-related Mg⁺⁺-ion dependence for RbRBC hemolytic activity was observed as the concentration of Mg⁺⁺ was increased to 1.0 mM. In addition, our results obtained with pre-colostral foal serum strongly suggest that natural antibody to RbRBC was of little importance in the lysis observed with these cells. These results also show that the equine alternative pathway activation may require Ca⁺⁺ ions. If Ca⁺⁺ ions are required, the equine alternative pathway is quite different from any other mammalian complement system so far described. Our results suggest that the alternative pathway of activation is of major importance in the equine complement system. Confirmation of this hypothesis requires both purification of the components involved as well as further characterization.     

A radiolabeled staphylococcal Protein A assay for detection of anti-erythrocyte IgG in warm agglutinin autoimmune hemolytic anemia of dogs and man

A cell wall protein from $\text{Staphylococcus aureus}$, Protein A, (SpA) has been shown to have the ability to bind the Fc region of most mammalian IgG molecules. This study uses this unusual property as the basis for a quantitative assay for erythrocyte (RBC) bound antibodies. Test serum is incubated in a suspension of normal RBC's. The cells are then washed and incubated with ¹²⁵Iodinelabeled SpA (¹²⁵I-SpA). After incubation cells are pelleted and bound radiolabeled SpA counted. This procedure has been performed using canine anti-goat RBC (D$\text{a}$gRBC) serum and human anti-D serum (positive controls) to establish the kinetics of the SpA reaction in the above system. The results indicate that SpA binds to red blood cells as a function of membrane bound antibody. RBC's incubated with indirect Coombs positive sera bound 42.6% and 43.3% of the ¹²⁵I-SpA, as compared to 19.2%, the upper limit of the 95% confidence interval (n=9) for normal sera. Furthermore, significant binding was observed for certain indirect Coombs negative (direct Coombs positive) sera indicating that the SpA assay is more sensitive than the indirect Coombs test.The SpA system should provide the clinician with an inexpensive, sensitive, quantitative assay for the diagnosis of warm agglutinin autoimmune hemolytic anemia, as well as other autoimmune disorders involving membrane bound IgG.     

Lack of reactivity of sera from Theileriaparva-infected and recovered cattle against cell membrane antigens of Theileria parva transformed cell lines

Sera from $\text{Theileria}$$\text{parva}$ infected, recovered and rechallenged cattle were tested in complement-dependent cytotoxicity, membrane immunofluorescence and antibody-dependent cellular cytotoxicity assays for the presence of antibodies against cell membrane antigens of $\text{T}$. $\text{parva}$ transformed cell lines.In the complement-dependent antibody-mediated cytotoxicity assay, sera from lethally infected animals were negative. Some recovered cattle showed a positive reaction, but such reactions were also observed when an eland cell line infected with $\text{T}$. $\text{taurotragi}$, and bovine lymphoblastoid cells were used as targets. Reaction was less against Ig-negative peripheral blood lymphocytes.Evidence is presented that these reactions could be evoked by attachment of immune complexes to Fc-receptors. It is concluded that cattle exposed to $\text{T}$. $\text{parva}$ infection do not develop antibodies against specific $\text{T}$. $\text{parva}$ (or $\text{T}$. $\text{parva}$-induced) cell surface antigens.     

The irradiation of Babesia bovis. II. The immunogenicity of irradiated blood parasites for intact cattle and splenectomised calves

Intraerythrocytic forms of $\text{B. bovis}$ were exposed to 350 Grays (Gy) γ irradiation and were then injected intravenously into intact two and three year old Hereford steers. One of 15 steers died on initial infection and subsequently six steers were given a virulent heterologous challenge three weeks after recovery; all six animals were highly immune. The remaining eight animals were kept under quarantine conditions for 10 months and were then challenged with a different virulent heterologous strain of $\text{B. bovis}$. Seven of eight were highly immune, but one animal died. Subsequently a further 12 steers were injected intravenously with 1 × 10⁸ irradiated organisms. All showed only mild transient clinical signs. After 12 months quarantine in a tick-free area these animals were then challenged with a virulent heterologous strain and all 12 were shown to be highly immune. Irradiation reduced the infective dose from 1 × 10⁸ to 2.5 × 10³ parasites. These parasites multiplied at the same rate, and achieved the same maximum parasitaemia as the parent non-irradiated strain, but the disease produced by them was not severe. A dose of 2.5 × 10³ non-irradiated paasites was lethal to all of the four animals which received it. It was concluded that irradiation had produced a predominantly avirulent parasite population.     

Progesterone and the surge release of gonadotropins in the ewe

On day 12 of an estrous cycle, 4 groups of ewes were treated with either blank (no steroid) or 3 different sizes of progesterone-containing rubber implants to study the effect of maintained progesterone levels on preovulatory events. Following luteolysis progesterone levels were 0.55 ± 0.13 ng/ml in control ewes and 0.62 ± 0.10 ng/ml, 0.99 ± 0.09 ng/ml and 1.85 ± 0.04 ng/ml in the 3 groups of progesterone-treated ewes. Preovulatory surges of LH and FSH occurred in $\text{5}\text{5}\text{,}\text{5}\text{5}\text{;}\text{4}\text{5}\text{,}\text{3}\text{5}\text{;}\text{4}\text{5}\text{,}\text{3}\text{5}$ and $\text{0}\text{5}\text{,}\text{0}\text{5}$ ewes in these 4 groups respectively. Eleven days after implant insertion, all ewes responded to a GnRH challenge. The height of all FSH peaks was depressed by progesterone treatment (P<0.05). Three groups of ewes were ovariectomized at day 6 of a cycle and treated with estradiol-17β and progesterone-containing implants. After 8 days of treatment, progesterone implants were removed in sections to give 3 different rates of decline in serum progesterone levels. The gonadotropin surges occurring following progesterone removal were delayed by the slower rates of progesterone decline. Another group of ewes was treated as in experiment 2, but the progesterone implants were all removed together after 8 days. Subsequent replacement of progesterone implants, after 12 or 18 hours, blocked the gonadotropin surges in all, or 2 of 6 ewes respectively. When replaced after 24 hours, implants producing low progesterone (1.38 ± 0.22 ng/ml) did not block gonadotropin surges, but in 2 of 5 ewes high progesterone did (2.87 ± 0.22 ng/ml). Removal of progesterone implants, 12 or 24 hr after replacement, produced secondary gonadotropin surges of smaller magnitude than the initial peaks (P<0.01).