Fertility prediction in dairy goats from Murciano-Granadina breed: The role of sperm evaluation and female traits.

Fertility is one of the most economically important traits in farm animals, due to the direct and indirect costs associated to low pregnancy rates. Thus, one of the priority goals in animal reproduction is to predict the performance that the semen doses will have in vivo based on the quality values obtained in laboratory assays. Attempts have been made for getting a predictive model of fertility of frozen-thawed sperm in dairy goats, but similar studies have not been conducted for chilled goat buck sperm doses that are mostly used for artificial insemination in many countries including Spain. We study how parameters of in vitro sperm quality and characteristics of Murciano-Granadina dairy goats may affect the in vivo fertility obtained after artificial insemination with semen doses chilled at 4 °C. Moreover, this information was used for obtaining predictive models of the fertility. Sixty-three ejaculates from 13 males were used to prepare chilled doses for the insemination of 495 goats over 13 sessions. Fresh and chilled sperm were evaluated for motility and plasma membrane integrity with a computer-assisted sperm analysis system and flow cytometry, respectively. Fertility was determined at parturition, according to the kidding goats. Overall fertility was 59.6%. Pearson's correlation coefficients between in vivo fertility and quality variables of fresh sperm were not significant and were low (below 0.34 in absolute value) for chilled sperm. Females' characteristics had a low negative impact on fertility (correlation coefficients of -0.19 with age, -0.20 with parturitions and -0.11 with total milk yield obtained in the best lactation). Fixed and mixed logistic regression procedures were used trying to explain the fertility results. None of the models accurately predicted fertility, but the best models included the percentage of total motile sperm or average path velocity from fresh semen, age of the females and the session effect (uncontrolled environmental effects). These analyses showed that primiparous goats were 2.42 times more likely to get pregnant than goats that had kidded four or more times. Our field assay data on fertility in Murciano-Granadina dairy goats highlighted the importance of making quality controls of sperm, of choosing the doses presenting high percentages of motile sperm exhibiting regular trajectories and of selecting the youngest goats for AI, after their first kidding. Efforts should continue to obtain better predictive models for improving fertility in goat dairy herds.

Factors affecting milking speed in Murciano-Granadina breed goats.

Milk flow during the first minute of milking was analyzed using data from 1,132 Murciano-Granadina breed goats belonging to 17 herds. During the individual lactations, 2 test days were scheduled for recording several milk flow traits, total milk, milk composition (fat and protein percentages), and somatic cell count. Average lag time from teatcup attachment to arrival of milk at the milk claw (T0) was 4.9 s and at the milk meter (T1) was 15.8 s. Average milk flow after 30 s (MF0.5) was 0.29kg/30 s (0 to 1.1kg/30 s) and milk flow at 60 s or milking speed (MF1) was 0.67kg/min (0.1 to 2.1kg/min). Repeatabilities of T0, T1, MF0.5, and MF1 were 0.45, 0.58, 0.62, and 0.68, respectively. The MF1 showed high phenotypic correlation withT1(-0.63) and MF0.5 (0.90), medium values withT0(-0.42) and total milk (0.22), and very low values (-0.04 to -0.12) with fat, protein, and somatic cell count. We found no differences between flows during the first 3 lactations, with a reduction as the lactation number increased. Months in milk since parturition affected MF1, being highest in the first 3mo (0.67-0.71kg/min) and decreasing until the end of lactation (0.58kg/min). The effect of herd-test day was significant for all traits. Inclusion of all these effects for the analysis of milk flow traits is considered necessary.

The impact of a specific blend of essential oil components and sodium butyrate in feed on growth performance and Salmonella counts in experimentally challenged broilers.

Essential oils (EO) and short-chain fatty acids have potential antimicrobial activity in broilers. This study aimed to investigate the effect of a specific blend of EO and a combination of this blend of EO with sodium-butyrate on growth performance and Salmonella colonization in broilers. A total of 480 one-day-old male broilers were distributed into 5 treatments (8 pens per treatment and 12 birds per pen) and reared during 42 d in experimental conditions. Dietary treatments consisted of the addition of different doses of EO (0 mg/kg, control; 50 mg/kg, EO50 and 100 mg/kg, EO100) or a combination of EO with 1 g/kg of sodium-butyrate (B; EO50 + B, EOB50 and EO100 + B, EOB100) to a basal diet. All birds were orally infected with 10(8) cfu of Salmonella Enteritidis on d 7 of study. Individual BW and feed intake per pen were measured at arrival and on a weekly basis. The prevalence and enumeration of Salmonella in feces was determined per treatment at 72 h postinfection and on d 23 and 37 of study. At slaughter, cecal content and liver samples from 16 birds per treatment were cultured for Salmonella and cecal pH was measured. No differences were observed on growth performance among treatments. All fecal samples analyzed were positive for Salmonella from d 10 to the end of the rearing period. At slaughter, Salmonella contamination (positive samples) in cecum was lower in birds fed EOB50 compared with the other treatments (P < 0.05), whereas birds fed the control diet showed the highest colonization rates. The pH of the cecal content was not different among treatments. Thus, EO or its combination with sodium-butyrate did not affect growth performance. However, a clear effectiveness of these products was observed in Salmonella control, especially when low doses of EO were combined with sodium-butyrate (EOB50).

Development of methods for cryopreservation of rooster sperm from the endangered breed "Gallina Valenciana de Chulilla" using low glycerol concentrations.

Glycerol (11%; v:v) is the cryoprotectant most often used for the cryopreservation of rooster sperm. However, chicken breeds differ in the resistance of their sperm to the cryopreservation process and endangered or local breeds usually present low fertilizing ability when conventional sperm cryopreservation protocols are used. The objective of this study was to optimize the protocol for the cryopreservation of the sperm from the endangered breed "Gallina Valenciana de Chulilla". For this purpose, 10 pools of semen from 43 roosters of this breed were cryopreserved using 8%, 7%, 6%, or 4% glycerol, and the sperm quality was determined immediately after thawing and in the insemination doses. Lohmann Brown Classic laying hens (n = 40) were used for the insemination trials. The sperm quality after cryopreservation progressively decreased as the glycerol concentration was reduced (P < 0.01); samples frozen using 4% glycerol exhibited the lowest quality (38% total motile sperm and 49% live sperm), and samples frozen using 8% glycerol exhibited the highest quality (67% total motile sperm and 66% live sperm). These differences were also observed after the glycerol was removed (P < 0.01). However, the sperm fertilizing ability was similar for all the treatments (23%-30% fertilized eggs), and increased as the glycerol concentration decreased. In conclusion, semen from roosters frozen using 4% glycerol exhibited lower sperm quality but similar fertilizing ability compared with samples processed using higher glycerol concentrations. These results may provide useful information for developing cryopreservation protocols for other breeds.

Optimizing conditions for treating goat semen with cholesterol-loaded cyclodextrins prior to freezing to improve cryosurvival.

The fertility of goat sperm is highly variable and new methods for improving sperm cryosurvival are needed. Cholesterol plays important roles in membrane fluidity, cold shock sensitivity and cryodamage, and treating sperm from cold-shock sensitive species with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances sperm cryosurvival. The aim of this study was to develop a CLC-treatment to optimize goat sperm cryopreservation. A total of 45 ejaculates coming from eleven adult Murciano-Granadina bucks were used and three experiments were conducted to determine: (1) the optimal CLC concentration to treat goat sperm; (2) the optimal time to treat the sperm (before or after seminal plasma removal); and (3) optimal freezing diluent (either of two Tris-citrate diluents containing 2% or 20% egg yolk and 4% glycerol or a skim milk diluent with 7% glycerol) to cryopreserve goat sperm. Goat sperm cryosurvival rates were greatest when they were treated with 1mg CLC/120 × 10(6)sperm prior to freezing. The benefit was also greatest if the sperm were treated with CLC after seminal plasma removal. Finally, CLC treatment improved sperm cryosurvival rates for sperm frozen in all three diluents, however, CLC treatment was most effective for sperm frozen in egg-yolk diluents. In conclusion, treating goat sperm, with CLC prior to cryopreservation, improved sperm cryosurvival rates. In addition, CLC treatment was effective for all freezing diluents tested, making this technology practical for the industry using current cryopreservation techniques. Nevertheless, additional studies should be conducted to determine how CLC might affect sperm functionality and fertilizing ability.

Semiclassical propagation of Wigner functions.

We present a comprehensive study of semiclassical phase-space propagation in the Wigner representation, emphasizing numerical applications, in particular as an initial-value representation. Two semiclassical approximation schemes are discussed. The propagator of the Wigner function based on van Vleck's approximation replaces the Liouville propagator by a quantum spot with an oscillatory pattern reflecting the interference between pairs of classical trajectories. Employing phase-space path integration instead, caustics in the quantum spot are resolved in terms of Airy functions. We apply both to two benchmark models of nonlinear molecular potentials, the Morse oscillator and the quartic double well, to test them in standard tasks such as computing autocorrelation functions and propagating coherent states. The performance of semiclassical Wigner propagation is very good even in the presence of marked quantum effects, e.g., in coherent tunneling and in propagating Schrodinger cat states, and of classical chaos in four-dimensional phase space. We suggest options for an effective numerical implementation of our method and for integrating it in Monte-Carlo-Metropolis algorithms suitable for high-dimensional systems.

Crossbreeding parameters for growth and feed consumption traits from a five diallel mating scheme in rabbits.

Twenty-five genetic groups of young rabbits originated from a diallel-crossbreeding scheme among 5 selected lines: 3 maternal and 2 terminal sire lines belonging to 2 Spanish research institutions. A sample of 2,773 young rabbits from 525 litters was controlled during the fattening period lasting from 5 to 9 wk. Growth and feed consumption traits were evaluated throughout different biweekly batches. A Bayesian approach was used for inference from an animal model with common litter effects. On average, genetic groups coming from lines selected for growth rate were heavier (+58.9 g at 32 d and +315.5 g at 60 d), had greater growth rate (9.24 and 8.15 g/d from individual or cage analysis, respectively) and feed intake (+13.24 g/d), and showed better feed conversion ratio (-0.21 g of intake/g of gain), than the genetic groups originated from crosses among lines selected for litter size. Crossbreeding parameters were estimated from the samples of the marginal posterior distribution of the genetic group effect according to the Dickerson model. Maternal genetic and individual heterosis effects were null or very low. Direct genetic effects mainly regulated the expression of growth traits.

Effect of oxytocin treatment on artificial insemination with frozen-thawed semen in Murciano-Granadina goats.

The site where the semen is deposited appears to be one of the most important factors affecting pregnancy of inseminated goats. In Murciano-Granadina (MG) goats, post-cervical insemination is achieved in a limited number of females. An effective way to increase fertility rate could be by increasing post-cervical inseminations. Effect of exogenous oxytocin application to facilitate the cervical penetration and its effect on kidding rate and prolificacy in MG goats were investigated. Oestrus was synchronized using progesterone-impregnated sponges for 11 days. Females were randomly divided into three groups (n = 190) and received either an i.v. injection of 100 or 200 IU of oxytocin or saline solution 15 min before being inseminated. Data on semen deposition depth were recorded for each animal using a catheter scaled in centimetres (up to 4 cm). Depth of semen deposition was affected by the oxytocin treatment (p < 0.05). Oxytocin enhanced cervical passage only with the dose of 200 IU compared with the control group, increasing the deposition depth (2.9 cm vs 1.9 cm). No significant effect of oxytocin treatment on kidding rate and prolificacy was detected. Depth of semen deposition affected kidding rate (p < 0.01). In conclusion, oxytocin treatment improved the depth of semen deposition in AI of MG goats, but kidding rate and prolificacy was not affected. More studies must be conducted to assess the minimal effective dose required for sufficient cervical dilation, and to determine the effects of such doses of oxytocin on uterine motility, sperm transport and fertility in goats.

Effect of solid storage on caprine semen conservation at 5 degrees C.

In this work, we investigated the effect of storage in solid-phase extender on buck semen conserved at 5 degrees C. Furthermore, we studied the effect of addition of cysteine to the extender and the washing of seminal plasma on sperm survival. In Experiment 1, milk-based extender (M) was used as a control to study the effect of solid media storage (G) and cysteine supplementation (C), and the combination of both (GC), on in vitro sperm survival of washed and non-washed semen, conserved up to 72 h at 5 degrees C. Motility, acrosome integrity (NAR) and hypo-osmotic swelling tests (HOST) were evaluated to assess in vitro sperm survival. In Experiment 2, an artificial insemination (AI) field trial was performed to compare G versus M. Solid media (G) maintained motility of spermatozoa during storage higher than any other extender (67% G versus 62% GC; 61% M and 59% C; P<0.05), but there was no difference in NAR or HOST between extenders (P>0.05). No improvement in sperm viability was obtained by addition of cysteine to the media. Washing of semen improved motility (65% versus 60%; P<0.05), NAR (70% versus 64%; P<0.05) and HOST (37% versus 28%; P<0.05). No significant differences in fertility were obtained between G and M extenders (47% versus 41%; P>0.05). In conclusion, washing of semen and dilution in gelatin-supplemented milk extender (solid storage) appears to be a successful method for goat semen storage at 5 degrees C.

Morphometric changes in goat sperm heads induced by cryopreservation.

Two experiments were designed to evaluate the effect of cryopreservation on morphometric characteristics of the goat sperm head. To address this question, we evaluated the size of the sperm head in fresh control cells, post-cooling cells after equilibration with the glycerol preservation solution, and post-thawing cells. Assessment was by automated morphometric sperm head analysis (ASMA) using phase-contrast microscopy without staining. In the first experiment, ASMA was performed on heterospermic pooled samples (fresh, post-cooling after equilibration with the glycerol preservation solution and post-thawing): length, width, area and perimeter were measured. In the second experiment, sperm viability was assessed by Hoechst staining and head morphometry was carried out as before, simultaneously during the cryopreservation process, and the head size was identified for both live and dead spermatozoa. The data were analysed by principal component analysis (PCA). The purpose of PCA is to derive a small number of linear combinations (principal components) from a set of variables (length, width, area and perimeter), that retain as much of the information in the original variables as possible. The main findings that have emerged from this study are that (i) a simple procedure has been developed for measuring spermatozoa heads without staining, which minimises the possibility that sperm head dimensions were influenced by procedural artefacts; (ii) the dimensions of goat sperm heads after cryopreservation in skimmed milk-glucose medium were smaller than in fresh sperm, but this was due to the equilibration phase with the cryoprotectant and not to the cryopreservation process itself; and (iii) dead spermatozoa showed smaller heads than live sperm, consequent upon the loss of membrane function. No differences were observed between post-cooling cells after equilibration with the glycerol preservation solution and post-thawing spermatozoa and only minor osmotic differences between them and fresh sperm were observed.

Factors affecting pregnancy rate in artificial insemination with frozen semen during non-breeding season in Murciano-Granadina goats: a field assay.

An artificial insemination programme was carried out to study the effect of factors such as depth of semen deposition, inseminator skill, farm, sire and expression of oestrus on pregnancy rate in Murciano-Granadina (MG) goats during non-breeding season and using frozen semen. Frozen-thawed semen from six males was applied by three technicians to inseminate a total of 551 goats in 17 farms distributed throughout the Mediterranean area of Spain. Pregnancy rate was determined at 6 weeks after insemination by transabdominal ecography. Overall pregnancy rate was 57%. Farm and depth of semen deposition affected pregnancy rate, whereas the sire and the technician had no effect. The deeper the semen was deposited in the genital tract, the higher was the rate of pregnancy obtained, being greater when the catheter reached the uterus. In spite of the relevant difference observed (48.2% vs 59.0%), pregnancy rate of females not coming into oestrus until 30 h after sponge removal was not significantly different, compared with those showing oestrus during the OD procedure. In conclusion, our field assay data on AI in MG goat with frozen-thawed semen showed that post-cervical insemination presented significantly greater pregnancy rate in comparison to when semen is deposited in the vagina or in the caudal part of the cervix.

Effect of changing female stimulus on intensive semen collection in young Murciano-granadina male goats.

The aim of this research was to study the effect of changing female stimulus on libido and semen characteristics from young Murciano-granadina male goats submitted to intensive semen collection using females not in estrus as teasers. Males were submitted to two different sexual stimulation procedures. In the first procedure, the same doe was used as the female stimulus for three consecutive presentations. In the second, the doe was replaced after the second presentation by a new female. Semen volume, concentration, forward progressive motility, and live spermatozoa were scored. To analyze reaction time (RT), three types of analysis were performed. In the first one, RT was analyzed by multifactor ANOVA, taking as a missing value 300 s when a buck did not ejaculate. In the second, RT also was analyzed by multifactor ANOVA, but data from males that did not ejaculate were removed. In the third, a Cox Survival analysis was carried out by censoring data when a buck did not ejaculate within 5 min of entering the test arena. A decrease in semen volume and sperm concentration in the successive ejaculations was observed, being highly marked in the third ejaculation independent of the stimulation procedure (0.62 vs. 0.38 and 0.43 mL, and 2,828 vs. 2,183 and 2,223 million spermatozoa/mL to the first and third ejaculation respectively; P < 0.05). No significant differences were observed either in forward progressive motility or live sperm rate. Changing the female stimulus in the third presentation had no significant effect on any seminal characteristic. Regarding libido and mounting behavior variables, there was a substantial decrease in RT in the third service when the female was changed (with both types of ANOVA). When censored data were taken into account, the relative risk showed that the probability of a male ejaculating in the third presentation increased almost fourfold when the female was replaced than when the female was the same in all services (P < 0.05). In conclusion, young Murciano-granadina bucks can be used as semen donors because none of the most important semen variables used to reject or accept an ejaculate before freezing process decreased after intensive semen collection. We also recommend changing the female stimulus to make the semen collection procedure more efficient and using survival analysis methodology to analyze time data, mainly when a high rate of censored data are scored.

Elliptical selection experiment for the estimation of genetic parameters of the growth rate and feed conversion ratio in rabbits.

Two elliptical selection experiments were performed in two contemporary sire lines of rabbits (C and R) in order to optimize the experimental design for estimating the genetic parameters of the growth rate (GR) and feed conversion ratio (FCR). Twelve males and 19 females from line C, and 13 males and 23 females from line R, were selected from an ellipse defined by a quadratic index based on these traits. Data from 160 rabbits of each of the parental generations of lines C and R and their offspring (275 and 266 animals, respectively) were used for the analysis. A Bayesian framework was adopted for inference. Marginal posterior distributions of the genetic parameters were obtained by Gibbs sampling. An animal model including batch, parity order, litter size, and common environmental litter effects was assumed. Posterior means (posterior standard deviations) for heritabilities of GR and FCR were estimated to be 0.31 (0.10) and 0.31 (0.10), respectively, in line C and 0.21 (0.08) and 0.25 (0.12) in line R. Posterior means of the proportion of the variance due to common litter environmental effects were 0.14 (0.06) and 0.21 (0.06) for GR and FCR, respectively, in line C and 0.17 (0.06) and 0.22 (0.06) in line R. Posterior means of genetic correlation between both traits were -0.49 (0.25) in line C and -0.47 (0.32) in line R, indicating that selection for GR was expected to result in a similar correlated response in FCR in both lines.

Randomised efficacy and safety study of two 3-day artesunate rectal capsule/mefloquine regimens versus artesunate alone for uncomplicated malaria in Ecuadorian children.

The combination of artesunate and mefloquine is one of the most effective treatments against multidrug-resistant falciparum malaria. Experience in children is however limited. The objective of this study was to compare the efficacy and safety of two artesunate/mefloquine combinations with artesunate monotherapy in Ecuadorian children. A total of 150 children with an age between 2 and 12 years, confirmed to have uncomplicated falciparum malaria, were randomly selected and divided in three treatment groups of 50 patients each. Group 1 received 50 mg rectal capsules alone (40 mg/kg total dose) administered over 6 days. Group 2 received 50 mg rectal capsules (30 mg/kg total dose) for 3 days combined with mefloquine (20 mg/kg total dose) on day 1. Group 3 was treated with 50 mg rectal capsules (30 mg/kg total dose) for 3 days, combined with mefloquine on days 1 and 3 (15-17 mg/kg total dose). Patients were continuously followed up and controlled by clinical and laboratory examinations for 7 days as well as on days 14, 21 and 28. An additional parasite examination was performed at 2 months following therapy. Clearance of parasitaemia was comparable between treatment groups. These were 9.2, 9.2 and 8.3 h for Groups 1, 2 and 3, respectively. Cure rates at day 28 were 76, 96 and 94% and after 2 months 60, 88 and 80%, respectively. There were no adverse events (AEs) reported during the study. Vital signs and laboratory examinations revealed no changes of clinical relevance. It can be concluded that the combination of artesunate rectal capsules with mefloquine is effective and safe. Starting concomitant administration already on day 1 is well tolerated. This combination significantly reduces the incidence of recrudescence compared to artesunate monotherapy. Comparing the two tested artesunate/mefloquine regimens, a total mefloquine dose of 20 mg/kg seems to be more effective compared to a total dose of 15-17 mg/kg. Further studies seem to be warranted.

Usefulness of sampling with cotton swab for PCR-diagnosis of cutaneous leishmaniasis in the New World.

In this study, we tested the polymerase chain reaction (PCR)-method to diagnose cutaneous leishmaniasis (CL) by taking exudate materials from lesions with cotton swabs, using our previously tested (PCR) panel comprised of Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) mexicana and L. (L.) amazonensis. The objectives of the present study were to improve the sampling method convenient for the patients and to test the usefulness of samples taken with cotton swabs. Sixteen patients were clinically diagnosed to have CL including one case of diffuse cutaneous leishmaniasis (DCL) in Ecuador and the causative Leishmania parasites were identified by PCR. All the 12 samples from CL patients of La Mana, positive for Leishmania DNA, were identified as L. (V.) panamensis, while two from CL of Huigra and one from DCL of San Ignacio were L. (L.) mexicana. In the field condition, taking biopsy material is not only painful but sometimes causes iatrogenic bacterial infections. Considering the sensitivity of the test, and convenient sampling procedure, it may be suggested that collection of exudates using cotton swabs may be a better alternative to biopsy sample for PCR-diagnosis of CL.

Detection of species of the subgenus Leishmania parasites using polymerase chain reaction and southern blotting.

In this study, an attempt was made to identify different Leishmania species by polymerase chain reaction (PCR). Fourteen Leishmania strains from stock were tested by PCR and Southern blotting. A pair of primers were employed that anneal to the kinetoplast DNA sequence conserved among subgenus Leishmania. Of the 14 Leishmania strains used in this study, six showed strong bands of approximately 170 bp, and all the positive strains belonged to the species of the subgenus Leishmania viz., Leishmania (Leishmania) garnhami, L. (L.) amazonensis, L. (L.) pifanoi, L. (L.) mexicana, L. (L.) chagasi, and L. (L.) major All the species belonging to the subgenus Viannia used in this study were negative by PCR. These results suggest that the primer pair may be useful for identification of the species belonging to the subgenus Leishmania of the New World as well as to distinguish subgenus Leishmania from subgenus Viannia.

A preliminary study aimed at the detection of Leishmania parasites in subjects with cutaneous leishmaniasis using polymerase chain reaction.

As a basic study for future diagnosis of cutaneous leishmaniasis, we tried to detect Leishmania parasites representing different species in the subgenera Leishmania and Viannia from subject patients with cutaneous leishmaniasis by using the polymerase chain reaction (PCR) with the subgenus Viannia specific primer. Four out of the 14 specimens revealed an amplified DNA of 70 bp specific for the subgenus Viannia (L. braziliensis complex). No bands were detected in the rest of the specimens belonging to the subgenus Leishmania and unclassified groups. The base sequences of the amplified DNA corresponded with those of the L. (V). braziliensis kinetoplast minicircle. We concluded that PCR using the present primer specific for the subgenus Viannia would be useful in detecting Leishmania parasites in lesions of cutaneous leishmaniasis caused by the L. braziliensis complex.

Rapid identification of Leishmania species from formalin-fixed biopsy samples by polymorphism-specific polymerase chain reaction.

The precise identification and classification of Leishmania species is important for public health surveillance since different species cause different clinical features of the disease. A highly specific polymerase chain reaction (PCR) panel was developed to enable the identification of the five major Leishmania species that cause New World cutaneous leishmaniases. The primers used for this panel were designed to distinguish the polymorphism in sequences of commonly amplified DNA bands of the parasites produced by arbitrarily primed PCR. These polymorphism-specific PCR diagnoses were performed with formalin-fixed biopsy specimens of the leishmanial lesions from four patients in Ecuador and one hamster skin lesion, and these lesions were determined to be caused by Leishmania (Viannia) panamensis, L. (Leishmania) mexicana, and L. (L.) amazonensis. The PCR panel may offer an important and practical approach to the standardized identification of Leishmania species in field examinations.