Arcuate nucleus kisspeptin response to increased nutrition in rams.

Rams respond to acute nutritional supplementation by increasing the frequency of gonadotrophin-releasing hormone (GnRH) pulses. Kisspeptin neurons may mediate the effect of environmental cues on GnRH secretion, so we tested whether the ram response to nutrition involves activation of kisspeptin neurons in the arcuate nucleus (ARC), namely kisspeptin, neurokin B, dynorphin (KNDy) neurons. Rams were given extra lupin grain with their normal ration. Blood was sampled before feeding, and continued until animals were killed for collection of brain tissue at 2 or 11h after supplementation. In supplemented rams, LH pulse frequency increased after feeding, whereas control animals showed no change. Within the caudal ARC, there were more kisspeptin neurons in supplemented rams than in controls and a higher proportion of kisspeptin cells coexpressed Fos, regardless of the time the rams were killed. There were more Fos cells in the mid-ARC and mid-dorsomedial hypothalamus of the supplemented compared with control rams. No effect of nutrition was found on kisspeptin expression in the rostral or mid-ARC, or on GnRH expression in the preoptic area. Kisspeptin neurons in the caudal ARC appear to mediate the increase in GnRH and LH production due to acute nutritional supplementation, supporting the hypothesised role of the KNDy neurons as the pulse generator for GnRH.

Volatiles from Merino fleece evoke antennal and behavioural responses in the Australian sheep blow fly Lucilia cuprina.

To identify flystrike-related volatile compounds in wool from Merino sheep, the attractiveness of wool to Lucilia cuprina Wiedmann (Diptera: Calliphoridae) was examined. First, a selection of wool samples guided by previous knowledge of sheep lines, predicted to be more susceptible or more resistant to flystrike, was tested. The attractiveness of the 10 samples selected was not associated with field susceptibility: two samples from the more resistant line were identified as most attractive and two samples from the more susceptible line were identified as least attractive, based on the behavioural assays with gravid flies. Comparison of the headspace volatiles of these samples, using solid phase microextraction and gas chromatography-mass spectrometry-electroantennographic detection, revealed octanal and nonanal to be present in the attractive wool samples that elicited responses from the fly antenna. Furthermore, the two compounds were not present in wool that was least attractive to L. cuprina. In laboratory bioassays, octanal and nonanal evoked antennal and behavioural responses in gravid L. cuprina, thus confirming their potential role as semiochemicals responsible for attracting L. cuprina to Merino sheep.

Appraisal and standardization of curvilinear velocity (VCL) cut-off values for CASA analysis of Japanese quail (Coturnix japonica) sperm.

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut-off values of "velocity curvilinear" (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA® -CASA. The median and mean VCL values were 85 and 97 µm/s. To define the VCL cut-off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut-off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut-off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the "25%" setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two-step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 µm/s, respectively. Together, the mean from two methods suggested that, for SCA® -CASA categorization of quail sperm, sperm should be classed as "rapid" at VCL ≥160 µm/s and "slow" at VCL ≤45 µm/s.

Monitoring stress in captive and free-ranging African wild dogs (Lycaon pictus) using faecal glucocorticoid metabolites.

An understanding of stress physiology is important for species management because high levels of stress can hamper reproduction and affect an individual's ability to cope with threats to their survival, such as disease and human-wildlife conflict. A commonly used indicator of stress, faecal concentrations of cortisol metabolites (FCM), can be used to assess the impact of social, biological and environmental factors. Measurements of FCM are particularly valuable for endangered species that are logistically challenging to study and where non-invasive techniques are preferred. As the second most endangered canid in Africa, the African wild dog (Lycaon pictus) has been the focus of considerable conservation research, yet there is still little understanding of factors associated with stress, in either captive or free-ranging populations. The present study therefore aimed to determine whether stress levels differ between captive and free-ranging populations, and to detect social, biological and environmental factors that are stressful in these populations. Faecal samples were collected from 20 captive and 62 free-ranging animals. Within free-ranging populations, the sexes differed significantly, but there was no effect of social status, age or breeding period for either sex. Captive females had higher FCM concentrations than free-ranging females. In captive populations, FCM concentrations differed among zoos and with reproductive status in females, but were not related to age class or group-housing structure. In conclusion, FCM is a useful indicator of stress and should be considered an integrative aspect of management, for both in situ and ex situ African wild dog populations.

Relationships among Puberty, Muscle and Fat, and Liveweight Gain during Mating in Young Female Sheep.

Greater depths of muscle are associated with better reproductive performance in ewe lambs, but, in adult ewes, reproductive performance also seems to vary with liveweight gain during the mating period. Therefore, in a large field study with Merino ewe lambs, we tested whether the relationships among eye muscle depth (EMD), fat depth (FAT) and reproductive performance depend on liveweight gain during the mating period. We selected lambs with a wide range in phenotypic values for depths of eye muscle (EMD) and fat (FAT) and assigned them to dietary treatments designed to achieve low (LOW, n = 244) or high (HIGH, n = 237) rates of liveweight gain during a 28-day mating period. The LOW treatment maintained live weight, whereas the HIGH treatment gained 179 ± 3.8 g/day (p < 0.001). From those ewe lambs that attained puberty, first oestrus was detected at live weight 37.8 ± 0.2 kg and age 232 days. The proportion of ewes that attained puberty increased with EMD (p < 0.01). Ewes from the HIGH treatment were more fertile (pregnant ewes per 100 ewes exposed to rams) and had a higher reproductive rate (foetuses in utero per 100 ewes exposed to rams; p < 0.001) than those from the LOW treatment. Fertility and reproductive rate were positively correlated with weight gain during mating as well as live weight at the start of mating, FAT and EMD (p < 0.05 to <0.001). We conclude that faster growth, due to either extra nutrition during mating or higher phenotypic potential for fat and muscle, will increase reproductive performance in ewe lambs mated at 8 months of age.

Follicle development, endocrine profiles and ovulation rate in adult Merino ewes: effects of early nutrition (pre- and post-natal) and supplementation with lupin grain.

In adult ewes, we tested whether ovarian function, including the response to short-term supplementation, was affected by the nutrition of their mothers during the pre-/post-natal period. A 2×2 factorial design was used with nutrition in early life (low or high) and a 6-day supplement (with or without) as factors. All ewes received three prostaglandin (PG) injections 7 days apart, and the supplement (lupin grain) was fed for 6 days from 2 days after the second until the third PG injection. We measured reproductive and metabolic hormones, studied follicle dynamics (ultrasonography), and evaluated granulosa cell numbers, aromatase activity and oestradiol (E2) concentrations in follicular fluid in healthy follicles at days 3 and 7 of supplementation. Ovulation rate was increased by 25% by exposure to high pre-/post-natal nutrition (1.5 vs 1.2; P<0.05), in association with a small decrease in FSH concentrations (P=0.06) and a small increase in insulin concentrations (P=0.07). The number of healthy antral follicles was not affected. Acute supplementation increased the number of granulosa cells (3.7±0.2 vs 3.0±0.2 million; P<0.05) in the largest follicle, and the circulating concentrations of E2 (4.6±0.3 vs 3.9±0.3 pmol/l; P<0.05) and glucose (3.4±0.03 vs 3.3±0.03 mmol/l; P<0.01). Both early life nutrition and acute supplementation appear to affect ovulation rate through changes in glucose-insulin homoeostasis that alter follicular responsiveness to FSH and therefore E2-FSH balance.

Genetic selection for temperament affects behaviour and the secretion of adrenal and reproductive hormones in sheep subjected to stress.

We investigated the effect of genetic selection for temperament on the way that stressors affect the behaviour and the adrenal and reproductive axes of sheep. We tested three hypotheses: (i) isolation would increase cortisol secretion and decrease luteinising hormone (LH) secretion more in nervous sheep than in calm sheep; (ii) isolation combined with simulated human presence would increase cortisol secretion and decrease LH secretion more in nervous sheep than in calm sheep and (iii) isolation combined with stressors that were not specific to the selection process (i.e. non-selection stressors) would increase cortisol secretion and decrease LH secretion equally in calm and nervous sheep. Isolation alone increased cortisol secretion and decreased LH secretion in nervous sheep but not in calm sheep. Compared to calm sheep, nervous sheep were more agitated during the first 2 h of isolation but not during the second 2 h of isolation. Exposure to non-selection stressors increased cortisol secretion, decreased LH pulse amplitude and the mean plasma concentrations of LH in both calm and nervous sheep. We conclude that genetic selection for temperament affects the behavioural expression of the stress response and the secretion of adrenal and reproductive hormones during isolation, but has less impact on their reactivity to non-selection stressors.

Apoptosis-related protein expression during pre- and post-natal testicular development after administration of glucocorticoid in utero in the sheep.

Pre-natal glucocorticoids are used in women at risk of preterm delivery to induce foetal lung maturation. However, glucocorticoids can produce negative outcomes for other tissues such as the reproductive system. We therefore tested the effects of pre-natal betamethasone on testicular morphology and apoptotic protein immune expression during pre- and post-natal development. Pregnant ewes (n = 42) bearing singleton male foetuses were randomly allocated to receive intramuscular injections of saline or betamethasone (0. 5 mg/kg) at 104, 111 and 118 days of gestation (DG). Testes were collected at 121 and 132 DG, and at 45 and 90 post-natal days (PD) and subjected to morphometric analysis (volume densities of sex cords and interstitial tissues; sex cord diameter). Immunohistochemistry (% stained area) was used to assess active caspase-3, Bax, Bcl-2 and cell-cycle proteins (PCNA). Compared with control values, betamethasone treatment decreased sex cord diameter at 121 DG, 45 and 90 PD, and sex cord volume at 90 PD. Active caspase-3 was decreased by betamethasone at 121 DG and 90 PD, but Bax was increased in all betamethasone groups. Bcl-2 and PCNA decreased in the betamethasone groups at 121 DG and 45 PD, but increased at 132 DG and 90 PD. We conclude that high levels of pre-natally administered glucocorticoid reduce foetal testicular development, perhaps via changes in the balance between pro- and anti-apoptotic proteins and cell-cycle proteins. These outcomes could compromise the future spermatogenic potential of male offspring.

In vitro initiation of the acrosome reaction in the emu (Dromaius novaehollandiae).

1. An assessment of the efficiency of the acrosome reaction (AR) provides an important predictor of the fertilizing potential of semen and for diagnosis of the causes of infertility. A standardized protocol was therefore developed for initiation of the acrosome reaction in emu spermatozoa in vitro, and the role of CaCl2 or perivitelline membrane (PVM) proteins in determining the outcome of the reaction was investigated. 2. The acrosome reaction (assessed by FITC-PNA) was successfully induced in live spermatozoa by incubation for 2 min in NaCl-TES medium supplemented with 5 mM CaCl2. The maximum response was 32% live acrosome-reacted spermatozoa (LAR) achieved after 10 min incubation. 3. Compared to the outcome with 5 mM CaCl2 or PVM protein alone, the response was significantly better with a combination of PVM protein and CaCl2. 4. A significant variation in the percentage of LAR spermatozoa among individual males was observed. No treatment affected the percentage of dead acrosome-reacted spermatozoa. 5. The results emphasize the important role played by both PVM proteins and Ca(2+) in the in vitro initiation of the acrosome reaction.

Role of male novelty and familiarity in male-induced LH secretion in female sheep.

Ewes supposedly need to be separated from rams before male stimuli can increase gonadotrophin secretion and induce ovulation. In the present study, we investigated the LH response of ewes to 'novel' and 'familiar' rams after varying periods of separation. In Experiment 1, ewes (n = 8 per treatment) were separated from familiar rams for 15 min or 1 month and then exposed to either familiar rams, novel rams or novel wethers. After 15 min or 1 month of separation, exposure to novel rams increased pulsatile LH secretion (P < 0.05) and induced an LH surge in all ewes whereas exposure to familiar rams or novel wethers had no effect on LH secretion (P > 0.1). After 1 month of separation, re-exposure to the same familiar rams increased pulsatile LH secretion (P < 0.05) in six of eight ewes, but only induced an LH surge in two of eight ewes. In Experiment 2, familiar rams were removed and returned after 15 min, 1 day or 17 days (n = 5 per treatment). None of these treatments affected LH secretion. We conclude that separation of ewes from rams is a prerequisite for familiar rams to increase LH secretion, but is not necessary if the rams are novel.

Embryo losses in sheep during short-term nutritional supplementation.

To test whether a nutritional supplement fed from 6 days before until 15 days after insemination reduces progesterone concentrations and increases embryo losses, Merino ewes were artificially inseminated (Day 0). Control ewes (n = 116) were not supplemented whereas Lupin6 ewes (n = 112) were supplemented with 500 g lupin grain daily for 6 days before insemination, and Lupin6+15 ewes (n = 122) from 6 days before until 15 days after insemination. There were no major differences between treatment groups in progesterone concentrations over the first 17 days of pregnancy. Embryo losses over Days 10-17 were lower in the Lupin6+15 than in the Control and Lupin6 groups, but the opposite occurred from Day 17-30. The concentrations of insulin and IGF-I were higher in Lupin6+15 ewes on Days 5, 12 and 17, compared with Lupin6 and Control ewes, while leptin concentrations decreased by Day 17 in the Lupin6+15 group. We conclude that feeding ewes for 15 days after mating improved embryo survival, which was associated with an increase in the concentrations of metabolic hormones and lower progesterone concentrations. However, the decrease in leptin concentrations promoted by the interruption of supplementation seems be linked to increased embryo mortality up to Day 30.

Extending the viability of emu spermatozoa during in vitro storage by manipulation of temperature and diluent potassium concentration.

1. Survival of emu spermatozoa during in vitro storage is not affected by increasing the extracellular [K(+)] to the point where it does not adversely affect spermatozoa function. 2. In three experiments, the effects were studied of [K(+)] in a diluent in the range 12·5-80 mM/l on emu spermatozoa survival for up to 48 h at 5, 10 or 20°C. 3. At the end of the storage period, spermatozoa viability, motility, fertilising ability and morphology were measured. 4. In Experiment 1, spermatozoa viability and morphology were adversely affected after storage (P < 0·001) only in the diluent containing 80 mM/l [K(+)] whereas spermatozoa motility decreased as [K(+)] increased from 12·5 to 80 mM/l. 5. In Experiment 2, during storage at 5°C, the spermatozoa viability was comparable among any of the diluents (standard or modified) but morphology was better (P < 0·001) in all of the modified diluents than in the standard E3 diluent. 6. In Experiment 3, after 48 h of storage in a diluent containing 40 mM/l of [K(+)], the spermatozoa functions were better preserved at 10°C than at 5 or 20°C. 7. It is concluded that a higher than physiological level of potassium can be used in a diluent without detrimental effect on emu spermatozoa survival during 48 h storage and that the best outcome was with storage at 10°C rather than 5 or 20°C.

Modification of spermatozoa quality in mature small ruminants.

This review is based largely, but not entirely, on the assumption that gamete quality is directly linked to sperm output and thus testicular mass, an approach made necessary by the absence of a large body of data on factors that affect gamete quality in ruminants. On the other hand, there is a change in the efficiency of sperm production per gram of testicular tissue when the testis is growing or shrinking, a clear indicator of changes in the rates of cell loss during the process of spermatogenesis, probably through apoptosis. We therefore postulate that the spermatozoa that do survive when the testis is shrinking are of a lower quality than those that are produced when the testis is growing and the rate of sperm survival is increasing. In adult small ruminants in particular, testicular mass and sperm production are highly labile and can be manipulated by management of photoperiod (melatonin), nutrition, genetics and behaviour ('mating pressure'). Importantly, these factors do not act independently of each other - rather, the outcomes in terms of sperm production are dictated by interactions. It therefore seems likely that spermatozoa quality will be affected by these same factors, but definitive answers await detailed studies.

Regulation of folliculogenesis and the determination of ovulation rate in ruminants.

The paper presents an update of our 1993 model of ovarian follicular development in ruminants, based on knowledge gained from the past 15 years of research. The model addresses the sequence of events from follicular formation in fetal life, through the successive waves of follicular growth and atresia, culminating with the emergence of ovulatory follicles during reproductive cycles. The original concept of five developmental classes of follicles, defined primarily by their responses to gonadotrophins, is retained: primordial, committed, gonadotrophin-responsive, gonadotrophin-dependent and ovulatory follicles. The updated model has more extensive integration of the morphological, molecular and cellular events during folliculogenesis with systemic events in the whole animal. It also incorporates knowledge on factors that influence oocyte quality and the critical roles of the oocyte in regulating follicular development and ovulation rate. The original hypothetical mechanisms determining ovulation rate are retained but with some refinements; the enhanced viability of gonadotrophin-dependent follicles and increases in the number of gonadotrophin-responsive follicles by increases in the throughput of follicles to this stage of growth. Finally, we reexamine how these two mechanisms, which are thought not to be mutually exclusive, appear to account for most of the known genetic and environmental effects on ovulation rate.

Ovulation and ovulation rate in ewes under grazing conditions: factors affecting the response to short-term supplementation.

The relationships between ovulation rate and nutrition remain confused, probably because of uncontrolled variation in experimental conditions. To help resolve the problem, we analyzed data from 20 experiments conducted between 2002 and 2016, in Uruguay with grazing ewes. All experiments were carried out by a single laboratory under comparable conditions of experimental design and measured variables. The studies used a total of 3 720 ewes, of purebred Corriedale, Polwarth, or East Friesian x Polwarth genotypes. In all experiments, a control group grazed native pastures and extra nutrition was provided to the treatment groups using either improved pastures or supplements. Ovulation rate was measured by counting corpora lutea using laparoscopy or rectal ultrasound or by counting foetuses at ultrasound on day 45 of gestation. For statistical analysis, data were grouped according to nutritional treatment (control or supplemented) and, within these groups, type of supplement to provide energy or protein (protected or not from rumen degradation). Across all experiments, 92-99% of the ewes ovulated and the effects of diet, length of supplementation, and initial live weight and genotype are reported. Within diets, ovulation was most affected by overall energy intake during supplementation (P < 0.01). Ewes that grazed native pastures supplemented with protein supplements had higher ovulation rates (P < 0.05) than control ewes grazing only native pastures. The addition of tannins to the protein supplement, to protect it from degradation in the rumen, did not further increase the ovulation rate. In unsupplemented ewes that had access to legume pastures, ovulation rates did not increase when the legume pasture was rich in tannins although only ewes that grazed tanniniferous legumes had marginally higher ovulation rates than the control ewes (P < 0.05). When ewes grazing native pastures were supplemented with energy, their ovulation rate did not increase above those of nonsupplemented ewes. Live weight at the start of supplementation also affected ovulation rate. We conclude that ovulation was most affected by overall energy intake, whereas the factors that affected ovulation rate during short-tern nutritional supplementation were intake of protein from highly digested supplements or dietary protein protected from ruminal degradation.

Feasibility of two screen media reduction interventions: Results from the SCREENS pilot trial.

BACKGROUND: Advancements in screen media devices has transformed the way families engage with screen media. Although these modern devices offer many opportunities, e.g. communication and research online, an in-depth understanding of how these devices affect our health, is lacking. Before a definite randomized controlled trial, the SCREENS pilot study was conducted to assess compliance to and feasibility of two interventions, a measurement protocol, and a survey-based recruitment strategy. Also, the potential of the interventions to impact leisure time spent non-sedentary in children six-to-ten years of age was explored. METHODS: Families (N = 12) were recruited through a population-based survey sent out in October of 2018 to adults (N = 1,675) in the Municipality of Middelfart, Denmark. Families were randomized to one of two two-week interventions; an Evening Restriction intervention (no screen media use after six pm) and a General Restrict intervention (limit entertainment-based screen media to three hours/week/person). Intervention compliance was assessed objectively by measuring household TV usage, smartphone and tablet activity via an application, and via screen media diaries. During baseline and follow-up, as part of larger protocol, family members wore two triaxial accelerometers for seven consecutive days. The potential of the interventions to impact non-sedentary time was explored based on means and standard errors (SEs). RESULTS: Despite almost 85% and 75% reductions in leisure screen media use 0% and 50% of families were compliant in the Evening Restrict group and General Restrict group, respectively, based on strict a priori criteria. Participant feedback indicated that the General Restrict intervention generally was feasibly. Compliance to the accelerometry wear protocol was high (median non-wear was <1 hour/week). Moreover, the recruitment strategy was implemented and was feasible. The General restrict intervention might increase children's non-sedentary time (mean (SE): 36.6 (23) min/day, N = 6). CONCLUSIONS: The General Restriction intervention, the accelerometer wear protocol and recruitment strategy, appeared feasible. TRIAL REGISTRATION: NCT03788525 at https://clinicaltrials.gov [Retrospectively registered; 27th of December, 2018].

The use of a 'first-wave' model to study the effect of nutrition on ovarian follicular dynamics and ovulation rate in the sheep.

We have developed an experimental model in which groups of ewes are simultaneously experiencing the first ovarian follicular wave of their oestrous cycle. We used this 'first-wave model' in a 2 × 2 factorial experiment (ten ewes per group) to study the effect of body condition (BC) and a short-term supplement on follicular dynamics and ovulation rate. The 'first-wave' was established by giving ewes three injections of prostaglandin (PG), 7 days apart. The 6-day supplement (lupin grain) began 2 days after the second PG injection and continued until the third. Follicles were studied by ultrasound, and blood was sampled to measure glucose and hormones. The supplement increased (P<0.01) the concentrations of glucose, insulin and leptin, decreased FSH concentrations (P<0.01) and tended to increase oestradiol concentrations (P=0.06). The supplement tended to increase the number of 3 mm follicles (P=0.06). Compared with low-BC ewes, high-BC ewes had more follicular waves (P<0.05), higher concentrations of insulin, leptin and IGF1 (P<0.05) and tended to have higher FSH concentrations (P=0.09). Leptin and insulin concentrations remained high until the end of supplementation in high-BC ewes, whereas they decreased after the third day of supplementation in low-BC ewes. In conclusion, high concentrations of metabolic hormones in fat ewes are associated with the development of more follicular waves. When a supplement is superimposed on this situation, changes in glucose and metabolic hormones allow more follicles to be selected to ovulate.

Dietary protein during gestation affects circulating indicators of placental function and fetal development in heifers.

The influences of nutritional protein during the first and second trimesters of pregnancy on placental hormones and fetal growth were determined in composite beef heifers. At artificial insemination, heifers were stratified by weight within each composite genotype into 4 treatment groups: High High (HH=1.4kg crude protein (CP)/day for first and second trimesters of gestation; n=16), High Low (HL=1.4kg CP/day for first trimester and 0.4kg CP/day for second trimester; n=19), Low High (LH=0.4kg CP/day for first trimester and 1.4kg CP/day for second trimester; n=17) or Low Low (LL=0.4kg CP/day for first and second trimesters; n=19). Maternal plasma bovine pregnancy associated glycoprotein (bPAG) and progesterone (P4) were determined at gestation day (gd) 28, 82, 179 and 271 (mean gestation length 286 days) in addition to P4 at term. Estrone sulphate (ES) and bovine placental lactogen (bPL) concentrations were measured at gd 124, 179, 236 and 271 and at term in addition to ES at gd 82. Low dietary protein increased placental function as indicated by increased bPAG (P<0.001) and ES (P=0.02) concentrations in first trimester and increased bPL concentrations (P=0.01) in the second trimester of gestation. In the third trimester, when dietary treatment had ceased, placental function was no longer associated with previous dietary treatments. Dam genotype affected placental function as measured by bPL (P<0.001) and ES concentrations (P=0.02). Calf gender, heifer age and maternal insulin-like growth factor (IGF)-I, -II and leptin did not affect hormonal indicators or circulating markers of placental function. Enhanced placental function during the third trimester, as measured by ES, was associated with increased calf birth weight (P=0.003).

Map-based cloning of a protein kinase gene conferring disease resistance in tomato.

The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.

Morphological study of the effects of the GnRH superagonist deslorelin on the canine testis and prostate gland.

The present study is part of a programme of research designed to evaluate the efficacy of the GnRH superagonist,deslorelin (D-Trp6-Pro9-des-Gly10-LHRH ethylamide), as a contraceptive for male dogs. Adult dogs were assigned to a completely randomized design comprising six groups of four animals. Each dog in the control group received a blank implant (placebo) and each dog in the other five groups received a 6 mg deslorelin implant. One group of deslorelin treated dogs was sacrificed on each of days 16, 26, 41, 101 and 620, and testicular and prostate tissues were collected for study by light and electron microscopy. On days 16 and 26 after implantation, we observed partial disruption of the seminiferous tubules, with early spermatids shed into the lumen. On days 41 and 101 after implantation, 90­100% of the seminiferous tubules were atrophic and aspermatogenic.On day 101 after implantation, 99% of all sections showed atrophy of the epithelium and shrinkage of epithelial height in the ductus epididymides. On days 41 and 101 after implantation, prostate tissue showed complete atrophy of the glandular epithelium (100% of sections) and an apparent increase in the relative proportion of connective tissue. At the electron microscopic level, in dogs treated with deslorelin for 41 and 101 days, the Sertoli cells were smaller and their nucleoli appeared smaller than in the control dogs. The nucleoli of the Leydig cells were atrophied and prostate glandular epithelium showed reduced epithelial height, a trophy of the nucleolus and an absence of secretory granules.Tissues collected during the recovery phase revealed a complete recovery of spermatogenesis. In conclusion, slow release implants containing deslorelin induce a striking a trophy of the testes and prostate gland by 26 days after implantation, explaining the previously reported loss of ejaculate and arrest of sperm output. At histological level,the entire process appears to be completely reversible, in accordance with data on endocrine variables and semen production.