Effects of denervation and colchicine treatment on the chloride conductance of rat skeletal muscle fibers

Membrane potentials, cable parameters, and component resting conductances were measured in extensor digitorum longus (EDL) muscle fibers from adult rats in vitro at 24°C, after 15 to 18 days of denervation by nerve section, and at seven to ten days following epineural injection of 100 to 450 μg of colchicine in the peroneal nerve. The denervated muscles were paralyzed throughout the experimental period, whereas the colchicine-treated preparations showed no clinical paralysis except for the first day or two. The EDL from the untreated side served as a control. Both the denervated and colchicine-treated fibers were depolarized, showed signs of fibrillation, had tetrodotoxin-resistant action potentials, and membrane resistance was increased two- to sevenfold. In the denervated fibers, mean chloride conductance G_Cl dropped from a control value of 3196 to 596 μmhos/cm² while mean potassium conductance G_K showed a tendency to rise from 260 to 332 μmhos/cm². Colchicine-treated fibers while showing a similar fall in mean G_Cl from 2993 to 1066 μmhos/cm², also showed a significant fall in mean G_K from 213 to 116 μmhos/cm². It was concluded that factors transported by the microtubular system are important for the maintenance of the high resting G_Cl of mammalian skeletal muscle fibers.     

Intraventricular Infusions of Anti-Neural Cell Adhesion Molecules in a Discrete Posttraining Period Impair Consolidation of a Passive Avoidance Response in the Rat

Intraventricular infusions of anti-neural cell adhesion molecule (anti-NCAM) are demonstrated to inhibit consolidation of a passive avoidance response when administered in the 6-8 h posttraining period. Anti-NCAM was ineffective when administered during training or at any other time up to 10 h thereafter, and no amnesic effects were observed with absorbed anti-NCAM or anti-neurofilament protein. Amnesia was observed only at the 48-h recall time, and this could not be attributed to poor antibody penetration or a prolonged residence time, as studies with 125I-labelled anti-NCAM in trained animals demonstrated a rapid accumulation into all brain regions, and this was marked in the olfactory bulb and hippocampus, areas showing an inherent and paradigm-specific increase in NCAM sialylation state, respectively. The lack of an amnesic action at the 24-h recall time is attributed to anti-NCAM-impaired synapse structuring becoming apparent following the paradigm-specific increases in NCAM sialylation state.     

Fertility of sows injected with exogenous oestradiol and/or gonadotrophins to control post-weaning oestrus

In the first of two experiments 28 multiparous sows were allocated to one of the following treatments 2 days after weaning at approximately 35 days post partum: (1) untreated; (2) i.m. injection 10 μg oestradiol benzoate (OB)/kg body weight (b.wt.); and (3) i.m. injection 20 μg OB/kg b.wt. Sows were bred at first post-weaning oestrus and ovulation rate assessed at slaughter. The mean interval from weaning to oestrus in each group was: (1) 5.6 ± 0.2; (2) 4.7 ± 0.2; and (3) 4.7 ± 0.2 days; the mean ovulation rates in groups 1 and 2 (18.7 ± 0.6 and 17.4 ± 1.8, respectively) were significantly higher (P < 0.01) than that of 12.0 ± 1.7 for treatment 3 sows. Two untreated and one each of the treated sows were not cycling at slaughter.In the second experiment 75 multiparous sows weaned at 28 ± 3 days post partum (day 0) were evenly allocated with respect to parity to one of four treatment groups: (1) untreated; (2) i.m. injection 10 μg OB/kg b.wt. on day 2; (3) PG600 (400 iu PMSG + 200 iu hCG) injection subcutaneous day 0; and (4) combined PG600/OB treatment as in (2) and (3) above. Sows were bred naturally at the first post-weaning oestrus and fertility assessed at farrowing. Control animals had a significantly longer (P < 0.05) weaning to oestrus interval (4.53 ± 0.25 days) compared to treatment 2 (4.03 ± 0.13) treatment 3 (3.97 ± 0.12) and treatment 4 (3.81 ± 0.07) sows. Sows treated with PG600 alone showed a significant increase (P < 0.05) in numbers born live compared to pre-treatment values. A smaller and non-significant increase in numbers born live in control sows (probably related to increasing parity) was not observed in either OB- or PG600/OB-treated animals.These results suggest that with further modification of the treatments, a system may be developed for introducing fixed-time artificial insemination (AI) or mating as a means of controlling the reproductive performance of the weaned sow.     

Evidence for heterophilic adhesion of embryonic retinal cells and neuroblastoma cells to substratum-adsorbed NCAM

The adhesion of embryonic chicken retinal cells and mouse N2A neuroblastoma cells to purified embryonic chicken retinal NCAM adsorbed on a solid substratum was examined using a quantitative centrifugal adhesion assay. Both cell types adhered to NCAM and the adhesion was specifically inhibited by monovalent anti-NCAM antibody fragments. N2A cell adhesion depended on the amount of NCAM applied to the substratum, was cation independent, and was insensitive to treatment with the cytoskeletal perturbing drugs colchicine and cytochalasin D. These results indicated that the tubulin and actin cytoskeletons were not critically required for adhesion to NCAM and make it unlikely that the cell surface ligand for NCAM is an integrin. Adhesion was however temperature dependent, strengthening greatly after a brief incubation at 37 degrees C. CHO cells transfected with NCAM cDNAs did not adhere specifically to substratum-bound NCAM and pretreatment of N2A cells and retinal cells with anti-NCAM antibodies did not inhibit adhesion to substratum-bound NCAM. These results suggest that a heterophilic interaction between substratum-adsorbed NCAM and a non-NCAM ligand on the surface of the probe cells affects adhesion in this system and support the possibility that heterophilic adhesion may be a function of NCAM in vivo.     

Characterization of the kinetics of neural cell adhesion molecule homophilic binding

A solid-phase assay has been developed for the investigation of the kinetics of neural cell adhesion molecule (NCAM) binding. Using this assay we can show that NCAM binds to itself in a time-dependent and saturable manner. Binding constants (KB values) of 6.9 x 10(-8) M and 1.23 x 10(-6) M, respectively, were obtained for adult and newborn rat NCAM homophilic binding. Binding is specifically inhibited by Fab' fragments of polyclonal anti-NCAM antibodies but is unaffected by heparin or chondroitin sulphate. This indicates that the NCAM homophilic binding site is separate from and independent of the heparin-binding site and that a developmental modification, probably polysialation, gives rise to marked differences in the adhesive properties of NCAM.     

The behavioral and somatic effects of ovariectomy and replacement therapy in female collared lemmings (Dicrostonyx groenlandicus)

Reproductive behavior and organ weights in intact estrous and diestrous, and in ovariectomized treated and untreated female collared lemmings (Dicrostonyx groenlandicus) were examined. Sexual behavior of intact diestrous and untreated ovariectomized females was similar, and females in both groups behaved differently from intact estrous females in scores for lordosis and mounting the male. Receptivity was observed in some females within 3 days after the first daily dose of as little as 0.1 μg of estradiol benzoate (EB) and in 83% of all females treated with 0.1 μg or more of EB for 5 days. Vaginal perforation and cornification were related to the dose of EB and length of treatment. The doses of EB used were not related to the lordosis quotient. Ovariectomy and subsequent EB treatment influenced uterine but not preputial gland weight. Adrenal weight was not influenced by ovariectomy or EB treatment, but the increased adrenal weights observed may have been related to the daily pairing of females with males.     

A synthetic peptide ligand of neural cell adhesion molecule (NCAM) IgI domain prevents NCAM internalization and disrupts passive avoidance learning

The neural cell adhesion molecule (NCAM) mediates cell adhesion and signal transduction through trans-homophilic- and/or cis-heterophilic-binding mechanisms. Intraventricular infusions of anti-NCAM have revealed a functional requirement of NCAM for the consolidation of memory in rats and chicks in a specific interval 6-8 h after training. We have now extended these studies to a synthetic peptide ligand of NCAM (C3) with an affinity for the IgI domain and the capability of inhibiting NCAM-mediated neurite outgrowth in vitro. Intraventricular administration of a single 5 microg bolus of C3 strongly inhibited recall of a passive avoidance response in adult rats, when given during training or in the 6-8-h posttraining period. The effect of C3 on memory consolidation was similar to that obtained with anti-NCAM as the amnesia was not observed until the 48-h recall time. The unique amnesic action of C3 during training could be related to disrupted NCAM internalization following training. In the 3-4-h posttraining period NCAM 180, the synapse-associated isoform, was down-regulated in the hippocampal dentate gyrus. This effect was mediated by ubiquitination and was prevented by C3 administration during training. These findings indicate NCAM to be involved in both the acquisition and consolidation of a passive avoidance response in the rat. Moreover, the study provides the first in vivo evidence for NCAM internalization in learning and identifies a synthetic NCAM ligand capable of modulating memory processes in vivo.     

Distinct roles for adhesion molecules during innervation of embryonic chick muscle

In vitro studies have suggested that the cell adhesion molecules NCAM and G4/L1 contribute to a variety of events during neural development. We have directly tested the role played by these molecules in the process of initial nerve ingrowth and ramification in the embryonic chick iliofibularis muscle by in ovo injections of specific adhesion-blocking antibodies and analysis of the resultant nerve branching pattern in muscle whole mounts. Antibodies against both molecules produced axonal defasciculation, which resulted in an enhanced transverse projection to the fast region of the muscle. In the case of anti-G4/L1, we also observed a large increase in the number of side branches that form from nerve trunks in the slow region and an enhancement of nerve branching in the fast region. Conversely, anti-NCAM produced a striking decrease in both the number and length of side branches in the slow region, and a reduction in nerve branching in the fast region. A similar reduction of nerve branching was obtained following injection of an endosialidase, which removes sialic acid from NCAM, and which was observed to enhance fiber-fiber apposition, presumably by increasing cell adhesion. Based on their biochemical properties in vitro and their in vivo distribution, both NCAM and G4/L1 are in a position to contribute to axon-axon adhesive interactions, whereas NCAM would be expected to also promote axon-myotube interactions. Our observations in fact indicate that these two adhesion molecules play different but complementary roles during muscle innervation and, specifically, that axon-axon fasciculation is influenced by both NCAM and G4/L1 in an anatomically distinct manner to regulate the overall pattern of nerve branching and that NCAM-mediated axon-myotube interactions are necessary for the attainment of the normal stereotyped pattern of nerve branching in both fast and slow regions of this muscle.     

Effect of cytostatics on the development of denervation disorders in skeletal muscles

Experiments on rats were made to study the effect of cytostatics on the rest membrane potentials (RMP) of muscle fibres and chemosensitivity of the botulinum toxin (BT) poisoned m. soleus. Intramuscular injection of the sublethal dose of BT on the 5th day evoked the blockade of the synaptic neuromuscular transmission, depolarization of the muscle cells and the decreased sensitivity to acetylcholine. Daily intraperitoneal injections of vincristine (25 micrograms/100 g) and fluorouracil (5 mg/100 g) to rats did not affect the development of the neuromuscular transmission blockade induced by BT. The cytostatics did not change the RMP of the myocytes or chemosensitivity of the normal muscles. However, both the drugs prevented the depolarization of myocytes and the decreased chemosensitivity of the muscles paralyzed with BT. It is assumed that the delayed appearance of the cytostatic-induced denervation is a consequence of the suppressed division of the satellite cells.     

Soluble forms of NCAM and F3 neuronal cell adhesion molecules promote Schwann cell migration: identification of protein tyrosine phosphatases zeta/beta as the putative F3 receptors on Schwann cells

Neural cell adhesion molecule (NCAM) and F3 are both axonal adhesion molecules which display homophilic (NCAM) or heterophilic (NCAM, F3) binding activities and participate in bidirectional exchange of information between neurones and glial cells. Engineered Fc chimeric molecules are fusion proteins that contain the extracellular part of NCAM or F3 and the Fc region of human IgG1. Here, we investigated the effect of NCAM-Fc and F3-Fc chimeras on Schwann cell (SC) migration. Binding sites were identified at the surface of cultured SCs by chimera coated fluorospheres. The functional effect of NCAM-Fc and F3-Fc binding was studied in two different SC migration models. In the first, migration is monitored at specific time intervals inside a 1-mm gap produced in a monolayer culture of SCs. In the second, SCs from a dorsal root ganglion explant migrate on a sciatic nerve cryosection. In both systems addition of the chimeras significantly increased the extent of SC migration and this effect could be prevented by the corresponding anti-NCAM or anti-F3 blocking antibodies. Furthermore, antiproteoglycan-type protein tyrosine phosphatase zeta/beta (RPTPzeta/beta) antibodies identified the presence of RPTPzeta/beta on SCs and prevented the enhancing effect of soluble F3 on SC motility by 95%. The F3-Fc coated Sepharose beads precipitated RPTPzeta/beta from SC lysates. Altogether these data point to RPTPzeta/beta is the putative F3 receptor on SCs. These results identify F3 and NCAM receptors on SC as potential mediators of signalling occurring between axons and glial cells during peripheral nerve development and regeneration.     

Comparison of the effects of botulinum neurotoxin types A and E at the rat neuromuscular junction

Local blockade of transmitter release was produced by s.c. injection of purified botulinum neurotoxin (NT) types A or E above the tibialis anterior muscle of adult male rats. Extensor digitorum longus nerve-muscle preparation was examined for toxin-induced alterations in single twitch and tetanic tension (in situ) or transmitter release (in vitro). For both single twitch and tetanic tension, muscles treated with type E NT recovered from an initial partial paralysis (induced with 56 mouse LD50) or full paralysis (induced with 565 mouse LD50) by 7 days after NT injection, while those treated with only 5 mouse LD50 of type A remained either fully or partially paralysed through 10 days. Also, miniature end-plate potential frequency and mean quantal content were reduced for a longer period of time and/or to a greater extent for muscles treated with type A NT than for those treated with type E. The present results are consistent with the observed higher specific toxicity (i.p. injections in mice) for type A NT than for type E, although these differences may be exaggerated after s.c. injections. The differences in the paralytic effect between types A and E may be determined by differences in amino acid sequence, which causes type E to dissociate more easily from its site of action and/or be detoxified more rapidly. The clinical implications of these findings are discussed.     

The importance of prolactin for initiation of lactation in the pregnant ewe

A single injection of ergocryptine (0.5 mg/kg liveweight) given to ewes 0.5-20 days prepartum or two injections (0.5 mg/kg liveweight per injection) given c. 30 and 10 days prepartum reduced concentrations of plasma prolactin to negligible (less than 5 ng/ml) values for 4 weeks after parturition, but did not affect concentrations of growth hormone and placental lactogen. Milking of treated ewes had no effect on concentrations of plasma prolactin during the first 4 weeks of lactation, but concentrations of growth hormone were increased during the 10-20 min period after milking. The half-life of prolactin in plasma was estimated as 21 min. In spite of the dramatic effect of ergocryptine on plasma prolactin all treated ewes secreted copious quantities of milk of normal composition. Mean daily yields of ewes treated with ergocryptine were not significantly different (P greater than 0.05) from those of untreated control ewes, but the mean +/- s.e.m. of total milk production over the first 3 weeks of lactation for ergocryptine-treated ewes was significantly lower (P less than 0.05) than that of control ewes (9.5 +/- 1.11 v. 14.1 +/- 1.20 kg milk). The results suggest that prolactin is not an essential component of the lactogenic and galactopoietic complexes of hormones in the ewe.     

Induction of parturition in crossbred sows with cloprostenol,an analogue of prostaglandin F2α

Forty-six crossbred sows were treated with a single intramuscular injection of 175 μg of cloprostenol on day 110, 112 or 113 of gestation and compared with 46 control animals farrowing naturally during the same period. Of the treated animals, 96% farrowed within 48 h. The mean time to the induced farrowing was 28 ± 1.2 h with 64% of the farrowings occurring during daylight hours. Piglet mortality was greater for farrowings induced at day 110 than for 112 or 113 days of gestation. Since the mean length of gestation for natural farrowings for untreated control sows was 115 ± 0.9 days, it may be concluded that farrowings could safely be induced within 2–3 days of the average expected farrowing date for the herd. For precise timing of treatment, the average gestation length for individual herds should be pre-determined and accurate breeding records are needed.     

Neural cell adhesion molecule regulates cell contact-mediated changes in choline acetyltransferase activity of embryonic chick sympathetic neurons

E10 chick sympathetic ganglion cells display a cell contact-dependent rise in choline acetyltransferase (ChAT) specific activity over the first several days in culture. This effect can be mimicked by addition of crude membrane fractions prepared from E10 retina and adult chicken brain, but not by those from E10 brain. The effects of both cell-cell and membrane-cell contact are inhibited by the addition of anti-NCAM Fab fragments. The membranes capable of increasing ChAT and those which are ineffective all contain NCAM, however their relative levels of NCAM polysialic acid differ. Whereas membranes with high polysialic acid NCAM are ineffective, selective enzymatic removal of polysialic acid renders them capable of producing an increase in ChAT. The inhibition of NCAM-mediated adhesion produced by Fab fragments can be compensated for by addition of wheat germ agglutinin, but only with membranes whose NCAM has low levels of polysialic acid. Taken together, these data suggest that NCAM can regulate cell contact-mediated increases in ChAT activity. We propose that NCAM-mediated adhesion promotes contact between cell membranes to allow the transmission of an otherwise NCAM-independent signal. In addition, NCAM's polysialic acid moiety appears to influence the ability of cells to transmit this signal, even in the presence of an alternative adhesion mechanism.     

Developmental Expression of Neural Cell Adhesion Molecules of Oligodendrocytes In Vivo and in Culture

Abstract: Previously, we have shown that oligodendrocyte adhesion molecules are related to the 120,000–M_r neural cell adhesion molecule (NCAM-120). In this report, we present further evidence that the oligodendrocyte adhesion molecule is NCAM-120. Studies on the expression of NCAM-120 and other molecular forms of NCAM in vivo in rat brain, in vitro in primary mixed cultures, and in cultures enriched for oligodendrocytes are described. Western blot analysis of rat brain using anti-NCAM showed that NCAM-120 first appears at postnatal day 7 and increases in quantity thereafter, coincident with the development of oligodendrocytes in vivo and comparable to the expression of myelin basic protein. Purified oligodendrocytes from 4-week-old rat brains expressed only NCAM-120. Quantitation of various forms of NCAMs in rat brain showed marked age-related differences in the expression of three molecular forms of NCAM. Immunofluorescence analysis showed that oligodendrocytes, at all ages tested, expressed NCAM, but in older oligodendrocytes, the intensity of staining was less. Western blot analysis of oligodendrocyte-enriched cultures showed that from day 1 after isolation (12 days of age) through day 7 after isolation (18 days of age) only NCAM-120 is seen. A possible role for NCAM in myelination and remyelination is discussed.     

Effects of denervation and colchicine treatment on the chloride conductance of rat skeletal muscle fibers.

Membrane potentials, cable parameters, and component resting conductances were measured in extensor digitorum longus (EDL) muscle fibers from adult rats in vitro at 24 degrees C, after 15 to 18 days of denervation by nerve section, and at seven to ten days following epineural injection of 100 to 450 mug of colchicine in the peroneal nerve. The denervated muscles were paralyzed throughout the experimental period, whereas the colchicine-treated preparations showed no clinical paralysis except for the first day or two. The EDL from the untreated side served as a control. Both the denervated and colchicine-treated fibers were depolarized, showed signs of fibrillation, had tetrodotoxin-resistant potentials, and membrane resistance was increased two- to sevenfold. In the denervated fibers, mean chloride conductance GC1 dropped from a control value of 3196 to 596 mumhos/cm2 while mean potassium conductance GK showed a tendency to rise from 260 to 332 muhos/cm2. Colchicine-treated fibers while showing a similar fall in mean GC1 from 2993 to 1066 mumhos/cm2, also showed a significant fall in mean GK from 213 to 116 mumhos/cm2. It was concluded that factors transported by the microtubular system are important for the maintenance of the high resting GC1 of mammalian skeletal muscle fibers.     

NCAM in the differentiation of embryonic lens tissue

The role of the neural cell adhesion molecule (NCAM)2 in ocular lens differentiation was investigated in chicken embryos. Changes in expression of NCAM were documented by immunohistology of frozen sections. This analysis revealed that NCAM diminished during lens fiber differentiation, in contrast to the gap junction-associated protein MP26 which became more abundant. The form of NCAM expressed was determined by Western blot analysis of proteins extracted from the different regions of the Embryonic Day 6 lenses. All regions expressed NCAM with an apparent molecular weight of 140 kDa and relatively low levels of polysialylation. The function of NCAM in lens differentiation was investigated using antibodies that inhibit NCAM-mediated adhesion. Two parameters that change during maturation of the lens epithelial cells were monitored: the thickness of the tissue, indicating the length of lens cells, and the particle arrangement of gap junctions, reflecting the state of junctional differentiation. When epithelial cell explants of Embryonic Day 6 lenses were cultured for 5 days, the cells elongated and displayed an increase in the loose, random intramembranous particle arrangements characteristic of maturing lens fiber gap junctions. When the explants were cultured in the presence of anti-NCAM Fabs, the epithelia were thinner than in matched controls and had particle arrangements characteristic of a less mature state. The expression of NCAM during lens differentiation and the effects of attenuating NCAM function suggest that adhesion mediated by NCAM is an essential event in lens cell differentiation.     

Nerve sprouting in innervated adult skeletal muscle induced by exposure to elevated levels of insulin-like growth factors

Partial denervation or paralysis of adult skeletal muscle is followed by nerve sprouting, probably due to release of diffusible sprout-inducing activity by inactive muscle. Insulin-like growth factors (IGF1 and IFG2) are candidates for muscle-derived sprouting activity, because (a) they induce neurite growth from peripheral neurons in vitro; and (b) their mRNA levels in adult skeletal muscle increase severalfold after denervation or paralysis. We sought to determine whether the presence of elevated levels of IGFs in innervated adult skeletal muscle was sufficient to produce intramuscular nerve growth. Low concentrations of IGFs induced massive neurite growth from enriched embryonic chick motoneurons in vitro. Half-maximal responses required 0.2 nM IGF2 or IGF1, or 20 nM insulin. Similar hormone binding properties of motoneuron processes in vitro were observed. Exposure of adult rat or mouse gluteus muscle in vivo to low quantities of exogenous IGF2 or IGF1 led to intramuscular nerve sprouting. Numbers of sprouts in IGF-exposed muscles were 10-fold higher than in vehicle-exposed or untreated muscles, and 12.2% of the end plates in IGF-exposed muscle (control: 2.7%) had sprouts growing from them. The nerve growth reaction was accompanied by elevated levels of intramuscular nerve-specific growth-associated protein GAP43. Additional properties of IGF-exposed muscle included modest proliferation of interstitial cells and elevated interstitial J1 immunoreactivity. These results suggest that elevated levels of IGFs in denervated or paralyzed muscle might trigger coordinate regenerative reactions, including nerve sprouting and expression of nerve growth-supporting substrate molecules by activated interstitial cells.     

DYNAMIC HISTOLOGY OF A RAT HEPATOMA AND THE RESPONSE TO 5-FLUOROURACIL*

The cellular response of the rat hepatoma 3924A to a single intraperitoneal injection of 5-fluorouracil has been measured in respect of the spatial relationship of the cells to the tumour microvasculature. In this tumour the parenchyma is arranged in cords approximately 150 μm thick around central capillaries. For untreated tumours, those cells at distances less than 80 μm from the capillary had a mean [³H]TdR labelling index of 39% and a mitotic index of 2·1%, while for those cells more than 80 μm away the values were 14% and 0·8% respectively. Two days after 150 mg/kg of 5-fluorouracil, mean cord thickness was reduced by 25% and did not recover to the control level until 11 days after treatment. This was also true for the mitotic index. Recovery of the labelling index was complete 2 days earlier. Although absolute values of parameters were different in the populations adjacent to and remote from the capillary, the time course of recovery was similar, with a ‘growth spurt’ 7 to 9 days after treatment. The results from this histologically-based assay have been compared with those from biochemical/biophysical assays that sample the overall tumour population.     

The effect of treatment with a GnRH analogue on postpartum reproductive performance in Friesian cows

Three trials were conducted to examine the effect of GnRH injection on the reproductive performance of Friesian cows. In the first trial 100 μg gonadotrophin releasing hormone synthetic analogue (GnRH) was given at the time of A.I. to 32 cows while a control group received a saline placebo injection. Conception rate to first insemination was significantly higher in treated than control cows (81.3 v. 54.8%, P < 0.05).In the second trial, two groups of 19 cows each received either 100 μg GnRH or saline at 15 days postpartum. A total of 60 cows was used in the third trial in which GnRH was given at either 50 or 100 μg dose level on either days 7 or 15 postpartum while control cows were untreated. In both trials GnRH treatment reduced the intervals from calving to complete uterine involution, first ovulation and first detected oestrus. The effect was most pronounced when 100 μg GnRH was given on day 7 postpartum. Number of days open and number of services per conception were appreciably reduced when 100 μg GnRH was given at either 7 or 15 days postpartum with maximal effect when given 15 days after calving.