Urine and fecal excretion patterns of dairy cows divergent for milk urea nitrogen breeding values consuming either a plantain or ryegrass diet.

Environmental degradation has been attributed to inefficient nitrogen utilization from pastoral dairy production systems. This degradation has especially been associated with the urine patch, which has been identified as a key component of nitrate leaching to waterways. However, a lack of information exists regarding the pattern of urination events and individual urination characteristics across the day, which would help inform strategic management decisions. The aim of this study was therefore to evaluate and report the patterns and characteristics of fecal and urination events throughout the day for cows divergent for milk urea nitrogen breeding values (MUNBV) on either a plantain [Plantago lanceolata L. (PL)] or ryegrass [Lolium perenne L. (RG)] diet as ways to reduce environmental impact. Sixteen multiparous lactating Holstein Friesian × Jersey cows divergent for MUNBV were housed in metabolism crates for 72 h, with all excretion events captured and analyzed. Cows selected as low for MUNBV consistently had a 65.2-kg lower urinary urea nitrogen (UUN) load (kg/ha) than high MUNBV cows for all hours of the day when consuming RG. The association between lower urinary urea loading rates and less N leaching implies a reduced environmental impact from low MUNBV cows consuming RG. When cows consumed PL, regardless of MUNBV, they had on average a 137.5-kg (UUN/ha) lower loading rate compared with high MUNBV cows on RG and a 72.2-kg (UUN/ha) lower loading rate compared with low MUNBV cows consuming RG across the day. Cows on PL also exhibited a different diel pattern of UUN load compared with cows consuming RG. Differences in the diel pattern of N excreted in feces were also detected based on MUNBV and by diet, with low MUNBV cows excreting on average 3.06 g more N in feces per event for the majority of the day compared with high MUNBV cows when consuming RG. Lower UUN loading rates and more N excreted in feces indicate a potentially lower environmental impact from low MUNBV cows when consuming RG compared with high MUNBV cows. The use of the PL diet also resulted in lower UUN loading rates and greater levels of N excreted in feces compared with RG, therefore also indicating its ability to reduce environmental impact compared with RG.

Grazing dairy cows with low milk urea nitrogen breeding values excrete less urinary urea nitrogen.

There is an increasing pressure on temperate pastoral dairy production systems to reduce environmental impacts, coming from the inefficient use of N by cows in the form of excessive urinary N excretion and subsequent N leaching to the waterways and NO2 emissions to the atmosphere, these impacts have spurred research into various mitigation strategies, which have so far overlooked animal-based solutions. The objectives of this study were first, to investigate the relationship between MUN breeding values (MUNBV) and urinary urea N (UUN) concentrations and total excretion in grazing dairy cows; and secondly, to evaluate such a potential relationship in the context of different sward compositions and stage of lactation. Forty-eight multiparous, lactating Holstein-Friesian dairy cows genetically divergent for MUNBV were strip-grazed on either a ryegrass-white clover (24 cows) or ryegrass, white clover and plantain sward (24 cows), during both early and late lactation. Cows were fitted with Lincoln University PEETER sensors to evaluate urination behaviour by measuring frequency and volume of urination, as well as daily urine excretion. Urine and faeces were sampled for urea N content. Milk yield and composition were measured for individual cows in both periods. There was a positive relationship between MUNBV and MUN (R2 = 0.67, P ≤ 0.05), with MUN decreasing 1.61 ± 0.19 mg/dL per unit decrease in MUNBV across both sward types and stages of lactation. Urinary urea N concentration decreased 0.67 ± 0.27 g/L (R2 = 0.46, P ≤ 0.05) per unit decrease of MUNBV, with no effect on urine volume or frequency (number of urination events per day), which resulted in a 165.3 g/d difference in UUN excretion between the animal with the highest and the lowest MUNBV. At the same milk yield, percentage of protein in milk increased by 0.09 ± 0.03 (R2 = 0.61, P ≤ 0.05,) per unit decrease in MUNBV. Our results suggest that breeding and selecting for dairy cows with low MUNBV can reduce urinary urea N deposition onto pasture and consequently the negative environmental impact of pastoral dairy production systems in temperate grasslands. Moreover, reducing MUNBV of dairy cows can potentially increase farm profitability due to greater partitioning of N to milk in the form of protein.

Expression of regulatory neuropeptides in the hypothalamus of red deer (Cervus elaphus) reveals anomalous relationships in the seasonal control of appetite and reproduction.

Red deer are seasonal with respect to reproduction and food intake, so we tested the hypothesis that their brains would show seasonal changes in numbers of cells containing hypothalamic neuropeptides that regulate these functions. We examined the brains of male and female deer in non-breeding and breeding seasons to quantify the production of kisspeptin, gonadotropin inhibitory hormone (GnIH), neuropeptide Y (NPY) and γ-melanocyte stimulating hormone (γ-MSH - an index of pro-opiomelanocortin production), using immunohistochemistry. These neuropeptides are likely to be involved in the regulation of reproductive function and appetite. During the annual breeding season there were more cells producing kisspeptin in the arcuate nucleus of the hypothalamus than during the non-breeding season in males and females whereas there was no seasonal difference in the expression of GnIH. There were more cells producing the appetite stimulating peptide, NPY, in the arcuate/median eminence regions of the hypothalamus of females during the non-breeding season whereas the levels of an appetite suppressing peptide, γ-MSH, were highest in the breeding season. Male deer brains exhibited the converse, with NPY cell numbers highest in the breeding season and γ-MSH levels highest in the non-breeding season. These results support a role for kisspeptin as an important stimulatory regulator of seasonal breeding in deer, as in other species, but suggest a lack of involvement of GnIH in the seasonality of reproduction in deer. In the case of appetite regulation, the pattern exhibited by females for NPY and γ-MSH was as expected for the breeding and non-breeding seasons, based on previous studies of these peptides in sheep and the seasonal cycle of appetite reported for various species of deer. An inverse result in male deer most probably reflects the response of appetite regulating cells to negative energy balance during the mating season. Differences between the sexes in the seasonal changes in appetite regulating peptide cells of the hypothalamus present an interesting model for future studies.

Comparison of analytical and predictive methods for water, protein, fat, sugar, and gross energy in marine mammal milk.

Mammalian milks may differ greatly in composition from cow milk, and these differences may affect the performance of analytical methods. High-fat, high-protein milks with a preponderance of oligosaccharides, such as those produced by many marine mammals, present a particular challenge. We compared the performance of several methods against reference procedures using Weddell seal (Leptonychotes weddellii) milk of highly varied composition (by reference methods: 27-63% water, 24-62% fat, 8-12% crude protein, 0.5-1.8% sugar). A microdrying step preparatory to carbon-hydrogen-nitrogen (CHN) gas analysis slightly underestimated water content and had a higher repeatability relative standard deviation (RSDr) than did reference oven drying at 100°C. Compared with a reference macro-Kjeldahl protein procedure, the CHN (or Dumas) combustion method had a somewhat higher RSDr (1.56 vs. 0.60%) but correlation between methods was high (0.992), means were not different (CHN: 17.2±0.46% dry matter basis; Kjeldahl 17.3±0.49% dry matter basis), there were no significant proportional or constant errors, and predictive performance was high. A carbon stoichiometric procedure based on CHN analysis failed to adequately predict fat (reference: Röse-Gottlieb method) or total sugar (reference: phenol-sulfuric acid method). Gross energy content, calculated from energetic factors and results from reference methods for fat, protein, and total sugar, accurately predicted gross energy as measured by bomb calorimetry. We conclude that the CHN (Dumas) combustion method and calculation of gross energy are acceptable analytical approaches for marine mammal milk, but fat and sugar require separate analysis by appropriate analytic methods and cannot be adequately estimated by carbon stoichiometry. Some other alternative methods-low-temperature drying for water determination; Bradford, Lowry, and biuret methods for protein; the Folch and the Bligh and Dyer methods for fat; and enzymatic and reducing sugar methods for total sugar-appear likely to produce substantial error in marine mammal milks. It is important that alternative analytical methods be properly validated against a reference method before being used, especially for mammalian milks that differ greatly from cow milk in analyte characteristics and concentrations.

Caloric restriction, but not caloric loading, affects circulating fetal and maternal C-type natriuretic peptide concentrations in late ovine gestation.

The factors regulating the greatly elevated concentrations of maternal plasma C-type natriuretic peptide (CNP) forms in ruminant pregnancy are largely unknown, but nutrient status is likely to be important. Previous work has shown that increases in maternal plasma CNP, sourced from the placenta, occur in response to caloric restriction in late gestation. Whether oversupply of nutrients also regulates CNP secretion in pregnancy has not been studied. Hypothesising that CNP in fetal and maternal tissues will be responsive to both deficiency and excess, we studied changes in CNP and a cosecreted fragment, namely N-terminal pro-CNP (NTproCNP), during short-term periods of caloric restriction (CR) and loading (CL). Twin-bearing ewes received CR (fasted Days 121-124), CL (Days 110-124) or control maintenance diets. During CR, fetal plasma CNP forms, insulin-like growth factor (IGF)-1 and liveweight all fell, and maternal plasma NTproCNP increased. During CL, fetal IGF-1 increased, whereas CNP forms and liveweight were unchanged, as were maternal concentrations of CNP forms. The high abundance of CNP peptides in placental tissues was unaffected by these short-term changes in nutrient supply. We conclude that CNP in the fetal-maternal unit is acutely responsive to undernutrition, but is unaffected by oversupply in late gestation.

The trophoblast binucleate cell is the source of maternal circulating C-type natriuretic peptide during ovine pregnancy.

Maternal plasma concentrations of C-type natriuretic peptide (CNP) and a co-secreted bioinactive amino-terminal fragment (NTproCNP) are elevated during ovine pregnancy. Although the uteroplacental unit has been implicated as a likely source of CNP, the relative contributions of specific uterine and placental tissues, and identity of the cellular site/s of production remain unknown. Therefore, we measured CNP and NTproCNP in intercaruncular uterine tissue and maternal (caruncle) and fetal (cotyledon) placental tissues throughout gestation. Concentrations of CNP forms in placental tissues greatly exceeded those in intercaruncular uterine tissue throughout pregnancy (P < 0.05). Mean caruncular concentrations (CNP 32 ± 4, NTproCNP 56 ± 6 pmol g(-1)) peaked at day 60 whereas in the cotyledon there was a progressive increase in CNP forms to peak values (CNP 66 ± 6, NTproCNP 134 ± 9 pmol g(-1)) at day 100-135 followed by a sharp decline just prior to term (day 143). At term CNP gene expression was 6-fold greater in placental tissue compared with intercaruncular uterine tissue. Changes in maternal plasma concentration of CNP forms closely followed those in cotyledonary tissue whereas fetal plasma levels fell progressively throughout gestation. Immunohistochemistry revealed staining in binucleate cells (BNC) and around placental blood vessels. CNP's localization to the BNC suggests a novel endocrine role during pregnancy, in addition to its paracrine actions within the placental vasculature. The function of CNP in maternal circulation remains to be determined, but as proposed for other BNC products, may involve manipulation of maternal physiology and placental function to favour fetal growth.

Assessment of the production of analgesia induced by application of a rubber ring or local anaesthetic to the antler pedicle of yearling stags.

AIMS: To compare two methods of applying rubber rings to the pedicles of yearling stags to induce analgesia in the antler prior to removal. To compare the application of a rubber ring with that of a lignocaine ring block of the antler pedicle on the efficacy and time course of the analgesia produced in yearling stags. METHODS: Rubber rings were applied to the pedicles of 36 yearling stags that required velvet antler removal. The standard method, a doubled-over ring expanded and lowered down from the distal end of the antler and released midway down the pedicle, was compared with a cable-tie method on the other pedicle, where a ring was pulled around the pedicle by an electrical cable tie threaded through the ring. Brief electrical stimulation (train-of-four mode) was applied proximal and distal to the ring before, and at regular intervals for 1 hour after, application of the ring to a level that produced an auriculopalpebral reflex response. In a second experiment, each pair of antlers per yearling stag (n=36) was allocated to one of three pairs of treatments, viz no treatment (control) and the cable-tie method as described above, control and local anaesthesia (a ring block of 2 ml 2% lignocaine per cm pedicle circumference), or the cable-tie method and local anaesthesia. Electrical stimulation (tetanic mode) was applied to each antler approximately 25 mm distal to the pedicle/antler junction before, and at intervals up to 1 hour after, application of treatments at a level required to produce a head/neck avoidance behavioural response. In a third experiment, the two electrical stimulation protocols used above were directly compared by measuring the response of stags (n=8) to one protocol on each pedicle/antler prior to, and at intervals for 1 hour after, application of a rubber ring. At the end of each treatment in all three experiments, analgesia of the antler was established as a nil behavioural response of the stag to a saw cut to the antler (the 'nick test'). RESULTS: For both methods of application of a ring the minimum electrical stimulation required distal to the ring to elicit a reflex response increased from around 16 to 55 mA by 60 minutes. In contrast, the electrical stimulation required proximal to the ring remained low (approximately 17.0 mA) throughout. No stag subjected to either of the methods of application responded to the nick test 60 minutes after application of the ring. The electrical stimulation required to produce a behavioural response increased very rapidly in stags treated with local anaesthetic and at a slower rate in those treated with the cable-tie method but showed no significant increase in control stags. After 4 and 30 minutes, for local anaesthesia and the cable-tie method, respectively, 95% of stags were not responding to 80 mA. A significantly greater proportion of stags with antlers treated with local anaesthetic and the cable-tie method did not respond to the nick test than controls, and there was no significant difference in the frequency of the response between stags with treated antlers. The minimum current required to produce a response proximal to a rubber ring was slightly higher on average for train-of-four electrical stimulus (mean 18.1 (SD 2.6) mA) than for the tetanic mode (mean 11.9 (SD 2.5) mA). The increase in minimum current required to produce the respective response to stimulation distal to the ring was similar for both methods, although the maximum predicted value (67.4 mA) was lower for train-of-four than for the tetanic mode (84.5 mA). No stag responded to the nick test > or = 60 minutes after application of the ring. CONCLUSIONS: The cable-tie method was no different from the standard method as a procedure for producing analgesia in the antlers of yearling stags and should be accepted as an appropriate procedure for applying analgesic rings to yearling stags. The analgesia produced in the antlers of yearling stags by rubber rings applied by the cable-tie method to the pedicle was similar to that of a lignocaine ring block, but the time course for the development of analgesia was markedly different. Given that a lignocaine ring block is accepted as an adequate method of pain relief for antler removal, the application of rubber rings followed by a period of > or = 30 minutes after application can be advocated as a viable alternative for pain relief.

Immunological influences on reproductive neuroendocrinology.

One of the consequences of activation of the immune system, with its associated inflammatory responses and operation of the stress axes, is a generalised inhibition of reproductive function. This can be considered as part of the all-encompassing effects of an activated immune system, included in which is the 'immunological cost' arising from the nutritional demand required to maintain a competent, responsive immune system, and the pathological effects produced by severe immune responses. Elucidation of specific immune-neuroendocrine linkages has largely involved examination of corticosteroid-based mechanisms or use of bacterial endotoxin as a model stimulus and examination of effects on GnRH and LH pulsatility, on GnRH and LH surge processes and on pituitary responsiveness to GnRH, using various sheep models. Although there is good evidence for prostaglandins as common mediators for endotoxin-induced and stress axis-induced impairment of neuroendocrine reproductive processes, both mechanisms appear to have prostaglandin-independent pathways as well. At the anterior pituitary gland level, the type II glucocorticoid receptor appears to mediate corticosteroid effects. Otherwise, the identity of specific cytokines, their sites of action and the cell level mechanisms underlying the inhibition of the reproductive axis at hypothalamic and anterior pituitary levels, especially in sheep, remains largely unresolved.

Glycine betaine excretion is not directly linked to plasma glucose concentrations in hyperglycaemia.

Diabetes mellitus subjects, type 1 and type 2, have increased glycine betaine excretion compared to normal subjects that correlated with plasma glucose and HbA(1C) concentrations. The current study was undertaken to determine whether elevated glucose concentration directly increases glycine betaine excretion in an animal model. Non-pregnant female Coopworth sheep received an intravenous glucose load (12.5,25 and 50% w/v; rate 200 ml/h) for 6 h followed by a 12 h physiological saline washout (0.9% w/v). Plasma and urine samples were analyzed for glycine betaine and glucose. Urine volumes and osmolality were also measured. Using the non-parametric Kruskal Wallis analysis of variance test we found no difference in glycine betaine excretion between glucose loaded and saline infused control animals (P=0.861). However, a significant negative correlation (r=-0.28, P<0.001) was observed between urine osmolality and glycine betaine excretion independent of treatment. We conclude that acute elevations of plasma glucose concentrations did not result in increased glycine betaine excretion and is therefore unlikely to be directly responsible for elevated glycine betaine excretion observed in diabetes mellitus subjects.

Importance of photoperiodic signal quality to entrainment of the circannual reproductive rhythm of the ewe.

An endogenous circannual rhythm drives the seasonal reproductive cycle of a broad spectrum of species. This rhythm is synchronized to the seasons (i.e., entrained) by photoperiod, which acts by regulating the circadian pattern of melatonin secretion from the pineal gland. Prior work has revealed that melatonin patterns secreted in spring/summer entrain the circannual rhythm of reproductive neuroendocrine activity in sheep, whereas secretions in winter do not. The goal of this study was to determine if inability of the winter-melatonin pattern to entrain the rhythm is due to the specific melatonin pattern secreted in winter or to the stage of the circannual rhythm at that time of year. Either a summer- or a winter-melatonin pattern was infused for 70 days into pinealectomized ewes, centered around the summer solstice, when an effective stimulus readily entrains the rhythm. The ewes were ovariectomized and treated with constant-release estradiol implants, and circannual cycles of reproductive neuroendocrine activity were monitored by serum LH concentrations. Only the summer-melatonin pattern entrained the circannual reproductive rhythm. The inability of the winter pattern to do so indicates that the mere presence of a circadian melatonin pattern, in itself, is insufficient for entrainment. Rather, the characteristics of the melatonin pattern, in particular a pattern that mimics the photoperiodic signals of summer, determines entrainment of the circannual rhythm of reproductive neuroendocrine activity in the ewe.

Bone turnover associated with antler growth in red deer (Cervus elaphus).

Although it is known that skeletal bone depletion occurs during antler growth in deer, it is not clear whether repletion of the skeleton takes place before or after completion of antler development. This study attempted to correlate repeated scanning electron microscopic measures of ilium and rib bone porosity from six approximately 2-monthly biopsy samples (using back-scattered imaging) and biochemical markers of bone turnover (serum hydroxyproline and osteocalcin concentrations) taken for 11 months with antler growth in six red deer stags. No changes were detected in ilium samples but changes in porosity of rib bones and an elevation of the biochemical markers indicated that skeletal depletion occurred during the antler growth period. However, the decrease in rib bone porosity and decline in markers of bone turnover took place before completion of antler growth, indicating that a considerable amount of skeletal repletion could have occurred whilst antlers were also undergoing bone accretion. This latter finding extends the current view of antler growth being accompanied by a form of reversible osteoporosis in the skeleton by showing that there is a period when the antlers and skeleton are both undergoing net bone formation.

Immunocytochemical localization of oestrogen receptors in perichondrium of antlers in red deer (Cervus elaphus).

Remodelling of cancellous bone of antlers to compact bone can be stimulated by administration of oestrogens and previous work has demonstrated the presence of specific oestrogen binding in this tissue. In this study the presence of oestrogen receptors (ER) in antler tissue from red deer males was examined by immunocytochemistry using a monoclonal mouse anti-human ER serum. Strong positive staining was detected in the tip regions of immature antlers and was confined primarily to the cells forming a fibrous layer of the perichondrium. This finding indicates that the effects of oestrogens on remodelling of cancellous bone of the antler may be mediated indirectly by the surrounding connective tissue layers.

Involvement of serotoninergic pathways in the control of luteinizing hormone secretion in red deer hinds.

Two experiments were conducted to determine whether serotoninergic pathways, which are implicated in the neuroendocrine regulation of luteininzing hormone (LH) secretion in domestic animals, have a similar action in red deer hinds. In the non-breeding season (August), ovariectomized (n = 5) and ovariectomized-thyroidectomized (n = 5) hinds received a vehicle solution followed 4 h later by either serotonin (66 microg kg(-1) i.v.) every 10 min for a further 4 h or the serotonin antagonist, cyproheptadine (3 mg kg(-1) i.v.) as a single injection. This procedure was repeated in the breeding season (June). In the non-breeding season serotonin was without effect, but cyproheptadine reduced LH pulse frequency and amplitude in ovariectomized-thyroidectomized hinds (P<0.01). During the breeding season, serotonin reduced LH pulse amplitude in ovariectomized hinds (P<0.05) and cyproheptadine reduced LH pulse frequency in both ovariectomized and ovariectomized-thyroidectomized hinds (P<0.05 and P<0.01, respectively). On each occasion, cyproheptadine increased (P<0.01) plasma prolactin concentration, whereas serotonin had no effect. These results indicate a stimulatory role for serotoninergic neurons on the hypothalamic GnRH pulse generator mechanism of red deer hinds during the breeding season. In a second experiment, the LH response to GnRH (5 microg i.v.) was examined in ovariectomized hinds (n = 5) following administration of a serotonin infusion (6.6 microg kg(-1) min(-1) i.v. for 15 min), cyproheptadine (3 mg kg(-1) i.v. as a single dose) or vehicle, in the breeding season (July) after induction of halothane anaesthesia and in the non-breeding season (December) without anaesthesia. Halothane anaesthesia eliminated endogenous pulses of LH. In comparison with the vehicle-treated controls, the response of plasma LH to exogenous GnRH was not altered by serotonin or cyproheptadine in either season, which shows that serotonin has no effect on LH release at the pituitary gland level in these animals. It was concluded that in the regulation of LH release in red deer hinds, serotoninergic pathways have a stimulatory role operating at the hypothalamic level.

Effects of thyroidectomy and thyroxine replacement on seasonal reproduction in the red deer hind.

Two experiments were conducted to test whether thyroid hormones are required for the cessation of the breeding season in female red deer. In Expt 1, 16 mature hinds were allocated early in the breeding season (May) to the following groups: thyroidectomized (THX), thyroidectomized and treated with subcutaneous thyroxine implants which increased plasma tri-iodothyronine to physiological concentrations (THX + T4), or euthyroid controls. Plasma progesterone concentrations consistent with oestrous cyclicity were evident in all hinds during the breeding season (May-September) but after 23 September plasma progesterone concentrations became generally low (< 2.0 nmol l-1) in control and THX + T4 hinds. In contrast, thyroidectomized hinds not receiving thyroxine continued to exhibit circulating progesterone comparable with breeding season concentrations until the end of the experiment (late December). Nine hinds were ovariectomized or ovariectomized and thyroidectomized in May and treated with subcutaneous oestradiol-impregnated capsules, which were removed for periods of about 1 months during August, November and January, to test whether thyroid hormones are involved in the seasonal decline of LH secretion (Expt 2). In the presence of oestradiol, basal LH concentrations and episodic LH secretion were low during the non-breeding season (September-March) (P < 0.05). During this season, basal and GnRH-induced LH concentrations and LH pulse frequency remained similar for both groups except in the absence of oestradiol, when basal LH (3.1 +/- 0.8 versus 1.3 +/- 0.5 ng ml-1, P < 0.05 and GnRH-induced LH (25.9 +/- 3.6 versus 4.7 +/- 0.4 ng ml-1, P < 0.001) concentrations were higher in thyroidectomized hinds than in euthyroid hinds, respectively. Twenty-five hinds were ovariectomized and thyroidectomized during the breeding season and treated with subcutaneous thyroxine implants at different times to identify when thyroid hormones are required to be present to suppress LH concentrations during the non-breeding season in the absence of oestradiol. Thyroxine treatment at the beginning of or during the non-breeding season was effective in suppressing plasma LH concentration, but this action of thyroid hormones did not occur during the breeding season. These results indicate that, in red deer hinds thyroxine is required for termination of the breeding season and that thyroid gland secretions specifically block steroid-independent inhibition of reproductive activity during the non-breeding season. This inhibitory mechanism requires thyroid hormones to be present only from around the time of the end of the breeding season for their normal expression, and they remain responsive to thyroid hormones after this period.

Pulsatile luteinizing hormone secretion in the ovariectomized, thyroidectomized red deer hind following treatment with dopaminergic and opioidergic agonists and antagonists.

Two experiments were conducted to determine whether dopaminergic or opioidergic pathways are modulated by thyroid gland secretions for seasonal suppression of LH secretion in red deer hinds. Ovariectomized (n = 5) or ovariectomized and thyroidectomized (n = 4) hinds, treated with estradiol implants, received the dopamine agonist bromocriptine or the antagonist sulpiride during pulse bleeds in July (breeding season) and October (nonbreeding season). Comparison of July and October mean plasma LH concentration (3.5 +/- 1.3, 0.7 +/- 0.1 ng/ml, respectively), pulse frequency (1.9 +/- 0.4, 0.7 +/- 0.2 pulses/4 h), and pulse amplitude (1.3 +/- 0.5, 0.7 +/- 0. 02 ng/ml) showed lower (p < 0.05) levels in October, and these levels were not significantly affected by thyroidectomy or drug treatment. In the absence of estradiol implants, the hinds received bromocriptine or morphine during the breeding season (July) and their antagonists, sulpiride or naloxone, respectively, in the nonbreeding season (November). In euthyroid hinds there was a seasonal decrease (p < 0.05) in mean plasma LH concentration, pulse frequency, and pulse amplitude, which did not occur in thyroidectomized hinds. There were no effects of drug treatment on LH concentration except for a small increase following sulpiride in November. Plasma prolactin concentration was significantly increased by antagonists and decreased by agonists on most occasions. We conclude that in red deer hinds, seasonal regulation of LH secretion does not involve dopamine or endogenous opioids and the thyroid gland is required specifically for LH suppression in the absence of estradiol.

Elevation of feed intake in parasite-infected lambs by central administration of a cholecystokinin receptor antagonist.

The role of cholecystokinin (CCK) in modulating feed intake depression in parasite-infected lambs was investigated using CCK receptor antagonists (L364-718 and loxiglumide). Four experiments were carried out using ewe lambs infected with 4000 Trichostrongylus colubriformis larvae/d or non-infected controls (n8, live weight 25 kg). Animals were fed daily on a nutritionally complete pelleted diet and had free access to water. In the first experiment, infected and non-infected animals were injected subcutaneously with CCK antagonist (100 micrograms L364-718) or carrier alone as a single dose. In the second experiment, CCK antagonist (loxiglumide: 0, 5, 10 or 20 mg/kg live weight) was injected into a jugular vein immediately before feeding. In the third experiment, animals were infused continuously with the CCK antagonist (loxiglumide; 10 mg/kg per h) for 10 min before feeding and for the first 2 h of feeding. In the final experiment, lambs were fitted with an indwelling cerebral ventricular cannula and infused with a CCK antagonist (loxiglumide, 162 micrograms/min), CCK agonist (CCK-8, 2.5 pmol/min), loxiglumide plus CCK-8 or sterile saline solution alone via the cannula for 30 min before feeding and for the first 60 min of feeding. In all the experiments short-term feed intake was recorded at 10 and 15 min intervals for the first and second hours of feeding respectively, then at hourly intervals for the remainder of the 8 h recording period. Peripheral injection with L364-718 or loxiglumide did not elevate feed intake in either the infected or non-infected animals. However, feed intake was increased (P < 0.05) in the short term by central infusion of loxiglumide, this effect being greater in the infected animals and apparently due to an elevation in intake during the second hour of feeding. CCK-8 depressed short term feed intake only in the infected animals (P < 0.05). Total daily feed consumption was not influenced by any of the pharmacological agents. The results indicate an involvement of central CCK receptors in regulation of feed intake depression following gastrointestinal parasitism of sheep and the possibility of a similar role in non-infected sheep. They do not support the singular importance of a peripheral action of CCK in determining satiety.

Targeting gene expression to the wool follicle in transgenic sheep.

To establish the feasibility of overexpressing foreign genes in the wool follicle, transgenic sheep were produced by pronuclear microinjection of a DNA construct consisting of a mouse ultrahigh-sulfur keratin promoter linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Four of 31 lambs born were transgenic. The overall efficiency of transgenesis was 1.1% of zygotes injected and transferred. Two transgenic rams were mated to nontransgenic ewes, and both transmitted the gene to their offspring in Mendelian fashion. CAT expression was found in the skin of one G0 ram and in 9 out of 26 transgenic G1 progeny. Two G1 lambs were sacrificed to study tissue specificity. Both had high levels of expression in skin but One had high expression in spleen and kidney with lower levels of expression in lung; the other had low expression in spleen, lung, and muscle. In situ hybridization demonstrated that transgene expression in the skin was confined to the keratogenous zone of the wool follicle cortex. Expression of CAT activity in skin was correlated with diet-induced or seasonal changes in the rate of wool growth. This keratin promoter appears useful for overexpressing factors in the wool follicle that might influence wool production or properties.

Anti-fertility effects of oral medroxyprogesterone acetate in rabbits.

Studies on the anti-fertility effects of medroxyprogesterone acetate (MPA) were conducted in rabbits. The bioavailability of MPA and plasma concentrations of progesterone and luteinizing hormone (LH) after mating were monitored following a single meal containing MPA (1000 mg) in entire does (n = 4); the response to gonadotrophin-releasing hormone (GnRH; 250 ng) was also observed in MPA-treated, ovariectomized does (n = 6). The reproductive tracts of rabbits mated following MPA treatment were examined 28-30 h after mating. Another group of rabbits (n = 4) received a single dose of MPA on Days 1, 10 or 19 after mating or daily for five days from Day 24. After dosage with 1000 mg MPA, plasma concentrations of MPA were detectable for eight days. However, following multiple dosing (10 mg, 5 days) MPA was detectable in the plasma for two days. MPA reduced the rate of ovulation and suppressed the increase in plasma concentrations of progesterone and LH observed after mating for four days, but had no effect on the response to GnRH. When administered late in gestation, MPA caused the death of fetuses. These results demonstrate an inhibitory effect of MPA on ovulation, probably at the hypothalamic level, and impairment of gestation or parturition.

Regional distribution of estradiol receptors in growing antlers.

This study of estrogen receptors (ER) was carried out to confirm their presence and to determine their localisation in antler bones. Partially grown antlers were amputated from red deer (Cervus elaphus) stags, the skin removed, and samples taken of periosteum, cartilaginous tissue including perichondrium, and bone. Capacity and binding of free ER in the samples were calculated by Scatchard analysis of data obtained from a radioreceptor assay which utilised [3H]estradiol as tracer. High affinity ER (ka 1.3-3.4 x 10(10)/M) were detected in all tissues sampled with the exception of bone. Receptor capacity ranged from 12-74 fmol/mg protein, ranking the tissues for capacity in the following descending order: periosteum, cartilage, calcified cartilage. These results demonstrate the presence of ER in growing antlers and indicate regional localization of the receptors within these structures. The absence of ER in bone tissue within the antler suggests that the effect of estradiol on stimulation of mineralization in this tissue is indirect and must occur via its binding to the non-calcified tissues of antlers, e.g., periosteum, perichondrium, and cartilage.

Thyroid hormones are required for the expression of seasonal changes in red deer (Cervus elaphus) stags.

Experiment 1, from 6 July 1990 (winter) to 8 November 1990 (spring), examined the effect of an early elevation of plasma thyroid hormone concentrations on the expression of seasonal changes in 20-month-old stags. Four stags were injected subcutaneously with 1 mg thyroxine (T4) daily, and four with vehicle only. Plasma T4 concentration was elevated by injection of T4; the mean concentration 24 h after injection was 192 +/- 31 nmol L-1 compared with 58 +/- 5 nmol L-1 in controls on 21 September. However, live weight, testicular diameter, plasma testosterone concentration and moulting underwent similar changes in both groups of animals. Experiment 2 tested the hypothesis that thyroid hormones are required for the expression of changes that occur during spring and early summer in red deer stags. Thyroidectomized (THX) stags (n = 4) of mixed age (> 22 months old) were given a replacement dosage of T4 or vehicle only (controls) during spring (from 7 September). T4-treated THX stags underwent testicular regression, antler regeneration and spring moult during spring and early summer (October-December), whereas control THX stags did not display these changes. During spring and summer, T4-treated stags gained more live weight (P < 0.05) than control stags. These results show that, in stags, thyroid hormones are required for the expression of seasonal changes and that these changes are not caused by the seasonal elevation of plasma concentrations of thyroid hormones.