Boar sperm motility is modulated by CCK at a low concentration of bicarbonate under capacitation conditions.

In a previous study, our group detected the cholecystokinin (CCK) protein in the porcine oviduct. This fact, together with the involvement of CCK in the regulation of sperm protein tyrosine phosphorylation by the modulation of HCO3 - uptake (in mice and humans) suggests a role for CCK during sperm capacitation. Therefore, on the one hand, the expression of CCK receptors (CCK1R and CCK2R) on boar testes has been investigated and probed; on the other hand, boar spermatozoa (from seminal doses of 1-day and 5-day storage) were exposed to different concentrations of CCK (0-control, 25 or 50 µM) in a medium supporting capacitation supplemented with 0, 5 or 25 mmol/L of HCO3 - for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 µM of CCK) were observed when HCO3 - was absent in the media (p > .05). However, the results showed that when the media was supplemented with 5 mmol/L HCO3 - in 1-day seminal dose storage, the linearity index (LIN, %), straightness index (STR, %) and oscillation index (WOB, %) (sperm kinetics parameters) increased in the presence of CCK regardless the concentration (p < .05). Nevertheless, CCK in sperm from 5-day storage only increased the WOB parameter in comparison to the control (p < .05). Furthermore, the average amplitude of the lateral displacement of the sperm head (ALH, µm) and curvilinear velocity (VCL, µm/s) decreased when CCK was present, depending on its concentration and sperm aging (1-day vs. 5-days) (p < .05). In the case of the media supporting capacitation supplemented with 25 mmol/L HCO3 - , any differences were observed except for sperm viability in the 5-day seminal doses, which increased in the 50 µM-CCK group compared to the control (p < .05). In conclusion, these data suggest an implication of CCK protein during sperm capacitation under low bicarbonate concentration increasing the sperm linear trajectory.

In vitro supplementation of testosterone or prolactin affects spermatozoa freezability in small ruminants.

In small ruminants, testosterone and prolactin plasma concentrations show circannual fluctuations as an adaptation mechanism to their seasonal breeding behavior. Sperm resistance to the freezing-thawing process shows seasonal fluctuation throughout the year, with lower sperm freezability at the beginning of the breeding season when prolactin and testosterone levels reach their maximum concentration. Nevertheless, whether these hormones directly affect post-thaw sperm quality parameters is still unclear. The objective was to study the effect of testosterone or prolactin added in vitro on sperm freezability in domestic ram (Ovis aries) and buck (Capra hircus). Sperm samples were incubated for 1 h with a range of testosterone (0, 2, 4, or 6 ng/mL; Exp. 1) or prolactin (0, 20, 100, 200, or 400 ng/mL; Exp. 2) concentrations. Samples were cryopreserved by slow freezing in straws at 0 h and after 1 h incubation. Sperm viability, acrosome integrity, motility, and kinetic parameters were assessed at 0 and 1 h in fresh and frozen-thawed samples. Results showed no hormone effect in fresh sperm, whereas these hormones affected post-thaw sperm parameters. In Exp. 1, in vitro incubation with testosterone decreased the post-thaw acrosome integrity of ram sperm (from 68.1 ± 6.3% to 49.6 ± 3.9%; P < 0.05). In Exp. 2, in vitro incubation with prolactin decreased the post-thaw acrosome integrity of ram (from 78.2 ± 3.4% to 66.3 ± 3.5%; P < 0.05) and buck sperm (from 81.7 ± 2.5% to 67.6 ± 3.5%; P < 0.05). Moreover, prolactin increased the post-thaw amplitude of lateral head displacement in ram sperm (from 3.3 ± 0.1 µm to 3.8 ± 0.2 µm; P < 0.05). In conclusion, either testosterone or prolactin added in vitro decreased the post-thaw acrosome integrity of ram and buck sperm. This suggests a destabilization process that could be decreasing sperm freezability when physiological levels of these hormones are high in vivo.

Effect of boar seminal dose type (cervical compared with post-cervical insemination) on cooling curve, sperm quality and storage time.

Seminal doses used for cervical and post-cervical artificial insemination (CAI and PCAI, respectively) vary in volume, the number of spermatozoa and packaging. The aim was to evaluate the outcomes when there was use of routine processing procedures for CAI- and PCAI-doses. Two different types of seminal doses were processed: 1) CAI: 2.7 × 109 sperm/80 ml; 2) PCAI: 1.5 × 109 sperm/45 ml. In Experiment 1, the cooling curve of seminal doses during processing occurred in two phases: 1st) At room temperature (23.4 ±â€¯0.5 °C) from 0 (just after packaging) to 120 min; 2nd) At refrigeration (15.7 ±â€¯0.8 °C) from 121-240 min. For the PCAI-doses, the time required to reach room temperature was 47 min compared to 107 min for CAI-doses (decreasing velocity of 0.093 °C/min and 0.048 °C/min, respectively). During refrigeration, for the PCAI-doses the time required to reach the desired preservation temperature was 20 min less than for CAI-doses (PCAI: 90 min, 0.074 °C/min; CAI: 110 min, 0.066 °C/min). In Experiment 2, sperm motility, kinetic parameters and acrosome damage for both types of doses were evaluated at 0, 24, 48 and 72 h of refrigeration. Also, morphology, pH, and osmolality were assessed at 0 and 72 h. Values for all these did not differ between CAI- and PCAI-doses. In conclusion, PCAI-doses took less time than CAI-doses to reach the desired temperature, but sperm quality was similar for CAI- and PCAI-doses during storage. Nevertheless, the different cooling curves should be taken into consideration for further investigation.

Effect of temperature and cell concentration on dolphin (Tursiops truncatus) spermatozoa quality evaluated at different days of refrigeration.

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Sperm cryopreservation in the bottlenose dolphin (Tursiops truncatus) has been developed for the exchange of gametes within the ex situ population. The aim of this study was to develop an effective method for refrigeration of bottlenose dolphin spermatozoa diluted in a commercial extender (BTS). In Experiment 1, the effect of temperature (5 compared with 15 °C) on sperm quality was evaluated during 7 days of storage at 100 × 106 spermatozoa/ml. In Experiment 2, the effect of the storage concentration (100 × 106 compared with 20 × 106 spermatozoa/ml) on sperm quality was assessed during 7 days of storage at 5 °C. In Experiment 1, total motility (including % of rapid sperm) was greater at 5 than 15 °C. When the effect of storage concentration was evaluated (Experiment 2), total motility and ALH were greater at the higher storage concentration (100 × 106 spermatozoa/ml). For both experiments, values for viability, acrosome integrity, and normal morphology variables were consistent throughout the 7 days of refrigeration. In Experiment 3, a microbiological study was performed to evaluate the effect of the refrigeration temperature and days of storage on bacterial growth. The results of microbiological analysis indicated there was Staphylococcus aureus in some samples, however, there was no effect of temperature or days of refrigeration. In conclusion, bottlenose dolphin semen can be refrigerated for a short to medium period of storage and there is maintenance of functionality of sperm when stored at 100 × 106 spermatozoa/ml at 5 °C.

Effect of Porcine circovirus 2 (PCV-2) maternally derived antibodies on performance and PCV-2 viremia in vaccinated piglets under field conditions.

BACKGROUND: Nowadays, the most common presentation of PCV-2 is the subclinical infection in piglets after weaning. The success of PCV-2 vaccination is associated with the control of the clinical disease as well as the improvement of production parameters. In consequence, the objective of the present study was to analyse the effect of PCV-2 maternally derived antibody (MDA) levels on vaccine efficacy in piglets vaccinated at three weeks of age with a commercial PCV-2 subunit vaccine. The study was performed analysing a database with 6112 wean-to-slaughter piglets from 4 different European regions. RESULTS: Results showed that the use of the vaccine was able to decrease the PCV-2 viremia calculated as area under the curve (AUC = 60.29 ± 3.73), increase average daily weight gain (ADWG = 0.65 ± 0.01 kg/day) and reduce mortality (7%) in vaccinated piglets compared to non-vaccinated ones (AUC of 198.27 ± 6.14, 0.62 ± 0.01 kg/day and 11% respectively). The overall difference of ADWG between both groups was close to 30 g per day (p < 0.05), also when they were split for low and high levels of MDA titres. Moreover, the animals with the highest ADWG were observed in the group of piglets vaccinated with high or extremely high antibody titres (0.66 and 0.65 kg/day respectively). Considering only animals with extremely high antibody titres, both study groups performed similar, however there was a numerical difference of 10 g/day in favour of vaccinated piglets. Likewise, lack of correlation between ADWG and MDA was observed suggesting that no maternal antibody interference was present with the tested vaccine because the vaccinated animals grew faster compared to unvaccinated control animals, regardless of the level of maternal antibodies present at the time of vaccination. CONCLUSIONS: The results of the present study demonstrated that the MDA against PCV-2 transferred through the colostrum intake has a protective effect against this viral infection. The vaccine used in the present study (Ingelvac CircoFLEX®) was effective when applied at three weeks of age and was not affected by the level of MDA at the time of vaccination.

Seminal plasma mitigates the adverse effect of uterine fluid on boar spermatozoa.

After natural or artificial insemination, spermatozoa start their journey within the uterus to reach the site of fertilization, but only few of them attain this goal. Part of this spermatozoa loss happens in the uterus, in which uterine fluid (UF) seems to be involved. It is known from other species that UF provokes damage to spermatozoa, which is avoided when seminal plasma (SP) is present. Therefore, the aim of the present study was to evaluate the effect of UF on the quality of ejaculated (previously contacted with SP) and epididymal (without previous contact with SP) boar spermatozoa analyzing motility, kinetic parameters, viability and acrosome integrity in the presence or absence of SP over time. Three experimental groups were evaluated in each source of spermatozoa (ejaculated and epididymal): 1) Control: spermatozoa with 20% of SP; 2) UF: spermatozoa with 20% of UF; and 3) UF-SP: spermatozoa with 20% of SP and 20% of UF. Total and progressive motility, kinetic parameters (VCL, VSL, VAP, LIN, STR, WOB, and BCF), viability and acrosome damage were analyzed at 15, 60, 120 and 180 min after incubation. Total and progressive motility decreased when ejaculated spermatozoa were incubated in UF in contrast to control and UF-SP groups (p < 0.0007), with no differences between control and UF-SP. The VCL decreased in the UF group compared to the control and UF-SP groups in ejaculated spermatozoa (p = 0.0002). The VSL, VAP, LIN and STR kinetic parameters were greater when ejaculated spermatozoa were incubated in the UF-SP group than in the UF group (all: p ≤ 0.02). Acrosome damage increased in ejaculated and epididymal spermatozoa incubated in the UF group compared to the control and UF-SP groups (both: p < 0.0001). Also, the viability of epididymal spermatozoa decreased in the UF group, while it did not change in the control and UF-SP groups (p = 0.0004). The rest of the parameters in either ejaculated or epididymal spermatozoa did not differ between experimental groups, except for WOB when epididymal spermatozoa were used (UF-SP higher than the control group, with UF being similar for both; p = 0.03). In conclusion, both ejaculated and epididymal spermatozoa are affected by UF, suggesting a negative effect on their quality. This negative effect is reduced by the presence of SP, improving the spermatozoa functionality, preserving motility, viability and acrosome integrity.

Post-cervical artificial insemination in porcine: The technique that came to stay.

The porcine industry is of great importance worldwide, and so any technological innovation in one or more of the associated production areas is of interest for meat production. Among such innovations in the reproduction area, post-cervical or intrauterine artificial insemination (PCAI) has emerged as a new approach in artificial insemination (AI). PCAI is gradually replacing traditional cervical insemination (CAI), particularly in countries with intensive pig production industries. This type of insemination, which deposits the semen in the body of the uterus (as opposed to traditional cervical deposition), is increasingly used in the field due to its simplicity and the numerous advantages that it provides at production level (e.g. reduced number of sperm, less time required to perform insemination and faster genetic improvement) and, consequently, from an economic point of view. In addition, since its inception, PCAI has been combined with other reproductive biotechnologies, such as the use of frozen-thawed sperm, fixed-time AI or sperm-mediated gene transfer. However, despite its wide acceptance and application, new approaches for increasing the efficiency of PCAI are constantly being sought, such as the adjustment and standardization in sperm numbers, the conservation of the PCAI semen dose, its association with other biotechnologies (sex-sorted sperm) or its efficacy in young (nulliparous and primiparous) females.

Morphological changes in the porcine cervix: A comparison between nulliparous and multiparous sows with regard to post-cervical artificial insemination.

In recent decades, new artificial insemination (AI) methods, such as post-cervical AI (PCAI), have been developed in pig. PCAI involves crossing the cervix to deposit the sperm in the uterine body. Although PCAI application in sows is frequent, its application in nulliparous (gilts) females it is still limited due to the difficulty of passing through the cranial part of the cervical lumen. We hypothesized that ageing and parity would modify the cervical canal, facilitating the introduction of AI devices through the cervix. The aim was to compare the morphology of the uterus at different levels between multiparous and nulliparous females. Morphological analysis of the uterus pointed to a longer cervix (25.9 ±â€¯4.6 vs. 21.6 ±â€¯3.3 cm, p < 0.001) and greater length of the part of the reproductive tract involved in PCAI (from rima vulvae to the last cervical cushion) (56.2 ±â€¯6.0 vs. 50.3 ±â€¯5.2 cm, p < 0.001) in multiparous sows compared with nulliparous animals. As regards the structure of the vaginal and uterine parts of the cervix (the part in contact with the vagina and uterine body, respectively), the cross-sectional area, perimeter and total thickness were greater in the uterine part of multiparous than of nulliparous animals (area: 4.07 ±â€¯1.46 vs. 2.46 ±â€¯0.56 cm2, p < 0.01; perimeter: 8.50 ±â€¯1.44 cm vs. 6.28 ±â€¯0.92 cm, p < 0.001; thickness: 10.79 ±â€¯0.96 vs. 8.35 ±â€¯0.62 mm, p < 0.05), but not in the vaginal part. The tissue content analysed in histological cross-sections also showed differences between female groups, a greater content of connective tissue (58.86 ±â€¯10.78 vs. 67.60 ±â€¯13.38%, p < 0.001) and a lower amount of muscle fibres (39.79 ±â€¯10.24 vs. 30.66 ±â€¯13.69%, p < 0.001) being observed in multiparous sows. Finally, silicone casts of the cervical lumen revealed differences between the two groups in the size and shape of the ridges in the lumen trajectory. Parity, which is also influenced by ageing, determines important changes in the size, structure and tissue content of the cervix wall, as well as in the morphology of the cervical canal, which may be responsible for the different levels of performance of PCAI in the female populations. Therefore, the future design of AI strategies and catheters should take into consideration the morphological variations of the cervix lumen, which will depend on age and parity of the females.

Re-emergence of rabies virus maintained by canid populations in Paraguay.

Paraguay has registered no human cases of rabies since 2004, and the last case in dogs, reported in 2009, was due to a variant maintained in the common vampire bat "Desmodus rotundus". In 2014, a dog was diagnosed as positive for rabies with aggression towards a boy and all required measures of control were successfully adopted. Epidemiological investigation revealed that the dog was not vaccinated and had been attacked by a crab-eating fox, "zorro" (Cerdocyon thous). The sample was diagnosed by the Official Veterinary Service of the Country and sent to the Center on Rabies Research from the University of São Paulo, Brazil, for antigenic and genetic characterization. A second sample from a dog positive for rabies in the same region in 2015 and 11 samples from a rabies outbreak from Asuncion in 1996 were also characterized. The antigenic profile of the samples, AgV2, was compatible with one of the variants maintained by dogs in Latin America. In genetic characterization, the samples segregated in the canine (domestic and wild species)-related group in an independent subgroup that also included samples from Argentina. These results and the epidemiology of the case indicate that even with the control of rabies in domestic animals, the virus can still circulate in wildlife and may be transmitted to domestic animals and humans, demonstrating the importance of continuous and improved surveillance and control of rabies, including in wild species, to prevent outbreaks in controlled areas.

Implementation of an extended ZINB model in the study of low levels of natural gastrointestinal nematode infections in adult sheep.

BACKGROUND: In this study, two traits related with resistance to gastrointestinal nematodes (GIN) were measured in 529 adult sheep: faecal egg count (FEC) and activity of immunoglobulin A in plasma (IgA). In dry years, FEC can be very low in semi-extensive systems, such as the one studied here, which makes identifying animals that are resistant or susceptible to infection a difficult task. A zero inflated negative binomial model (ZINB) model was used to calculate the extent of zero inflation for FEC; the model was extended to include information from the IgA responses. RESULTS: In this dataset, 64% of animals had zero FEC while the ZINB model suggested that 38% of sheep had not been recently infected with GIN. Therefore 26% of sheep were predicted to be infected animals with egg counts that were zero or below the detection limit and likely to be relatively resistant to nematode infection. IgA activities of all animals were then used to decide which of the sheep with zero egg counts had been exposed and which sheep had not been recently exposed. Animals with zero FEC and high IgA activity were considered resistant while animals with zero FEC and low IgA activity were considered as not recently infected. For the animals considered as exposed to the infection, the correlations among the studied traits were estimated, and the influence of these traits on the discrimination between unexposed and infected animals was assessed. CONCLUSIONS: The model presented here improved the detection of infected animals with zero FEC. The correlations calculated here will be useful in the development of a reliable index of GIN resistance that could be of assistance for the study of host resistance in studies based on natural infection, especially in adult sheep, and also the design of breeding programs aimed at increasing resistance to parasites.

Resistance of gastrointestinal nematodes to the most commonly used anthelmintics in sheep, cattle and horses in Spain.

The objective of this study was to evaluate the status of anthelmintic resistance (AR) in ruminants and horses in Spain. The efficacy of commonly used macrocyclic lactones (MLs) - ivermectin (IVM) and moxidectin (MOX) - was measured in sheep, cattle and horses. In addition, albendazole (ABZ) and levamisole (LEV) were evaluated in sheep and oxibendazole (OXI) and pyrantel (PYR) in horses. Efficacy was evaluated based on the difference between the arithmetic mean pre- and post-treatment faecal egg count (in cattle and horses), or compared to an untreated control group (in sheep). AR was present when the percentage reduction in egg count was <95% and the lower 95% confidence interval (CI) was <90%; if only one of these two criteria was met, the finding was recorded as suspected AR (SAR). In horses, AR-PYR and OXI was considered when the percentage reduction in egg count was ≤ 90% and the lower 95% CI was ≤ 80%. For each animal species, at least 10 study sites were selected. AR to at least one of the drugs was detected in all 10 sheep flocks; the main parasite identified after treatment was Teladorsagia circumcincta. Moreover, in 5 flocks multidrug resistance was identified, on 4 farms to drugs from different families, on one farm to both MOX and IVM and on another farm to all drugs tested. In cattle, the efficacy of both MOX and IVM was 100% on 4 and 3 farms, respectively, and therefore 60% of these farms were considered to have AR or SAR to both MLs. The most frequent parasite identified after treatment was Trichostrongylus spp., although Ostertagia ostertagi was also identified after treatment on one farm. In contrast to ruminants, the 4 drugs evaluated in horses were highly efficacious against strongyles, with efficacies for the MLs and OXI between 95 and 100% and between 94 and 100% for PYR, although 3 herds were SAR against PYR. In conclusion, AR to at least one of the commonly used drugs was identified on all sheep flocks investigated in the northwest of Spain. The occurrence of AR to MLs in cattle was higher than expected but consistent with what was observed in sheep. In horses, all currently used drugs were confirmed as effective against strongyles.

Four glycoproteins are expressed in the cat zona pellucida.

The mammalian oocyte is surrounded by a matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding and may be involved in speciation. In cat (Felis catus), this matrix is composed of at least three glycoproteins called ZP2, ZP3, and ZP4. However, recent studies have pointed to the presence of a fourth protein in several mammals (rat, human, hamster or rabbit), meaning that a reevaluation of cat ZP is needed. For this reason, the objective of this research was to analyze the protein composition of cat ZP by means of proteomic analysis. Using ZP from ovaries and oocytes, several peptides corresponding to four proteins were detected, yielding a coverage of 33.17%, 71.50%, 50.23%, and 49.64% for ZP1, ZP2, ZP3, and ZP4, respectively. Moreover, the expression of four genes was confirmed by molecular analysis. Using total RNA isolated from cat ovaries, the complementary deoxyribonucleic acids encoding cat ZP were partially amplified by reverse-transcribed polymerase chain reaction. Furthermore, ZP1 was totally amplified for the first time in this species. As far as we are aware, this is the first study that confirms the presence of four proteins in cat ZP.

Boar sperm with defective motility are discriminated in the backflow moments after insemination.

During insemination, a large number of spermatozoa are deposited in the female genital tract, but a very low percentage is able to colonize the site of fertilization. The influx of neutrophils into the uterine lumen and semen reflux (backflow) are known mechanisms that decrease the number of spermatozoa within the uterus. No report has attempted to ascertain whether the backflow is a random or selective process of the spermatozoa. In this work, sows were inseminated using two populations of spermatozoa in the same proportion: (1) unstained spermatozoa with high motility and (2) stained spermatozoa with low, medium, or high motility. Volume, number, and percentage of stained spermatozoa were evaluated in the backflow (collected at 0-15, 16-30, and 31-60 minutes after insemination). This article provides evidence that (1) the motility characteristics of the spermatozoa do not influence the percentage of sows with backflow, the volume and number of spermatozoa in the backflow; (2) the discarding of spermatozoa in the backflow is not specific during the first moments after insemination (0-15 minutes), whereas later (16-60 minutes), spermatozoa with defective motility (low and medium groups) are discarded in a higher proportion than high group in the backflow ([16-30 minutes: low, 85.13 ± 4.32%; medium, 72.99 ± 5.05%; and high, 54.91 ± 2.38%; P < 0.0001; 31-60 minutes: low, 87.16 ± 6.01%; medium, 87.02 ± 4.01%; and high, 59.35 ± 2.86%; P = 0.001]). Spermatozoa with poor motility are discarded in the backflow probably as a selective process, on the part of the female genital tract or as a result of the intrinsic low spermatozoa motility.

Analysis of genetic variability of Fasciola hepatica populations from different geographical locations by ISSR-PCR.

Inter-simple sequence repeats markers were used to determinate the genetic variability of Fasciola hepatica populations recovered from sheep and cattle from Spain (Sp1, Sp2, Sp3 and Sp4), UK (Eng), Ireland (Ir) and Mexico (Mex). Twenty five primers were tested but only five produced 39 reproducible bands, being 71.79% polymorphic bands. This percentage ranged from 10.26% in Sp4 to 48.72% in Sp1, and per host between 28.21 and 48.72% in sheep and between 10.26 and 38.46% in cattle. This relatively low range of genetic diversity within populations, with a mean of 34.40%, implies that a large proportion of variation resided among populations. The population differentiation (Gst = 0.547) indicated that 54.7% of variation is due to differences between populations and 45.3% due to differences within population. The Nei's distance ranged between 0.091 and 0.230 in sheep and between 0.150 and 0.337 in cattle. The genetic relationships between populations and individuals were shown by a UPGMA dendrogram and a principal coordinate analysis; both grouped all populations separately from Sp4, a population of from the Midwest of Spain with the lowest level of diversity. Small genetic distances were observed between Eng and Ir, on the one hand, and Sp1, Sp2, Sp3, from the Northwest of Spain, together with Mex, on the other.

Immunological features of LPS from Ochrobactrum intermedium on sheep experimentally infected with Fasciola hepatica.

The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium in sheep with fasciolosis was reported previously, resulting in lower fecal egg counts and fluke burden. In the current study, we analyzed its immunological effects in two groups of sheep, treated (T) and controls (C). Fasciolosis induces a T helper (Th) type-2 response, characterized by IL-4 and IL-10 production; however, at the beginning of the infection, the IFN-γ production predominates (Th type-1 response). Although we did not find differences in IL-4 production or in the expression level of this gene in the hepatic lymph nodes, the expression level of IL-10 was higher (P < 0.05) in the T group at 4 wpi. The IFN-γ production was higher (P < 0.01) at 12 wpi as well as its level of expression at 4 wpi (P < 0.05) in the T group. We found a higher expression level of TGF-ß at 4 wpi in the T group (P < 0.05), associated with the previous report of thicker fibrous tracks in a treated group. Immunoglobulin G1, related with a Th type-2 response, was higher (P < 0.01) in the T group at 4 and 12 wpi. In conclusion, the effects of LPS from O. intermedium could have resulted from a predominant Th type-2 immune response.

Effect of dietary supplementation with flaxseed oil or vitamin E on sheep experimentally infected with Fasciola hepatica.

The effects of two diets supplemented with flaxseed oil (FO) or vitamin E (VE) were studied in sheep infected with Fasciola hepatica. The control group (CO), not supplemented, had higher weight gain and faecal egg count (P < 0.05) at the end of the study. At necropsy, the number (71.2 ± 26.5) and size of flukes were lower in the FO group (P < 0.01), probably due to the higher levels of white blood cells and lymphocytes (P < 0.01). This group also had higher red blood cell and haematocrit values (P < 0.01). The CO group had more severe hypoproteinaemia and hypoalbuminaemia, due to having the highest fluke burden (123.0 ± 35.2), and also the highest IgG1 titre (P < 0.01). The diet did not affect production or gene expression levels of IFN-γ and IL-4. On the other hand, the supplementation with VE led to a reduction in adult fluke burden (97.7 ± 39.9) and lower lipid oxidation in the liver (P < 0.001).

Development of an egg hatch assay for the detection of anthelmintic resistance to albendazole in Fasciola hepatica isolated from sheep.

The aim of this study was to develop an egg hatch assay (EHA) to detect the resistance of Fasciola hepatica to albendazole (ABZ). With this purpose, two different F. hepatica isolates were tested: (i) susceptible (Shrewsbury/South Gloucester strain) to ABZ (SA); (ii) resistant to ABZ (RA). A commercial formulation of ABZ (Sinvermin) diluted in dimethyl sulfoxide (DMSO) was used at concentrations of 0.0002, 0.002, 0.02, 0.2, and 2 µg/ml. In the SA isolates, eggs from feces exposed to ABZ at the three highest concentrations showed significantly lower hatching percentages than the control well (P<0.01); also for the two highest concentrations, the percentage of development was significantly lower than the control (P<0.01). However, in the RA isolate, no differences among treatments and control were found in the percentage of hatched or developed eggs. On the other hand, the EHA was conducted over a 15 day period following treatment with ABZ for sheep infected by the RA isolate. On day 7 post-treatment (pt), no differences among treatments and controls were observed. On day 15 pt, the development and hatching rates exposed to the highest ABZ concentration were lower than at lower concentrations, suggesting susceptibility. The ovicidal effect of ABZ and the potential use of EHA to differentiate between ABZ susceptible and resistant isolates are confirmed in the current study. However, the use of this technique should be confirmed with a higher number of isolates.

Efficacy of an anthelmintic combination in sheep infected with Fasciola hepatica resistant to albendazole and clorsulon.

In the current study, we identified five sheep flocks with fasciolosis in the province of León (northwestern Spain) in order to determine the anthelmintic resistance status to three commonly used anthelmintics, namely albendazole (ABZ), triclabendazole (TCBZ) and clorsulon (CLOR). The identification of one flock resistant to ABZ and CLOR was shown after the faecal egg count reduction test (FECRT). The reductions in eggs per gram values were -17.6% and -68% against immature and adult flukes, respectively, after ABZ treatment; 85.15% and 44.91% against immature and adult flukes, respectively, after CLOR treatment; and 97.06% against both stages, after the administration of TCBZ. As an alternative to control the infection, two combinations of ABZ and CLOR were tested. In the first, both drugs were administered at the recommended dose of each; in this case, the efficiency reached values above 95% against both immature and adult flukes. However, when the combined drugs were administered at half the recommended dose of each, the efficiency of the combination was very low, i.e. 16.67% and -11.11% against mature and immature flukes, respectively. In conclusion, this preliminary report suggests a possible interaction between ABZ and CLOR after their joint administration. However, these results should be confirmed in other flocks.

The diagnosis of fasciolosis in feces of sheep by means of a PCR and its application in the detection of anthelmintic resistance in sheep flocks naturally infected.

The aim of this study was to develop a PCR for the diagnosis of Fasciola hepatica infection in feces of sheep based on the ribosomal internal transcribed spacer. Detection of infection was possible from the second week post-infection in experimentally infected sheep by amplification of a 292bp fragment. This PCR was employed for the detection of anthelmintic resistance (AR) in naturally infected sheep flocks, and results were compared with techniques such as the fecal egg count reduction test (FECRT) and the copro-antigen reduction test (CRT). The FECRT was carried out in two flocks, Santillan de la Vega (SV) and Corullón (CR), with sheep treated with albendazole (ABZ), clorsulon (CL), or triclabendazole (TCBZ). Feces were collected from individuals on days 0, 7, 15, and 30 post-treatment (pt). The FECRT showed adult F. hepatica to be resistant to ABZ and CL in both flocks. All parasite stages in the SV flock were susceptible to TCBZ, while in the CR flock, adult flukes showed resistance and immature forms were susceptible to the treatment. To compare FECRT and the PCR results, we calculated the percent of positive sheep on day 1 pt. In both flocks, the percent positive sheep was consistently higher by PCR than by sedimentation, confirming that the PCR is a more sensitive method of diagnosing infection and therefore to detect the resistance in infected animals. The CRT was carried out in the SV flock using a sandwich ELISA kit. The percent of sheep found positive by PCR was higher than with ELISA. Comparison of FECRT, CRT, and PCR for the detection of AR showed PCR to be the most sensitive.

Effect of lipopolysaccharide (LPS) from Ochrobactrum intermedium on sheep experimentally infected with Fasciola hepatica.

The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium was evaluated in sheep experimentally infected with Fasciola hepatica. Animals were divided into four groups: two treated with the LPS (T1/T2) and two controls (C1/C2). T1/C1 were slaughtered at 30 days postinfection (dpi) and T2/C2 at 85 dpi. Body weight and body condition were found higher in T1 and T2 than in controls, although differences were not significant. Treated sheep showed lower cumulative fecal egg count than controls (p < 0.01). Levels of red blood cells (RBC), white blood cells, hemoglobin, and hematocrit (HCT) were higher in T1 and T2. Significant differences (p < 0.05) in RBC and HCT were found between groups at 84 dpi. More severe macrocytic and hypochromic anemia was observed in C1 and C2 than in treated groups. Total protein and albumin values were higher in T1 and T2 (p < 0.01) until 29 dpi. At the end of the trial, no significant differences were observed in hepatic enzymes, although gamma-glutamyl transferase and aspartate aminotransferase values were higher in C2, and alanine aminotransferase was higher in T2. At necropsy, the mean weight of liver, fibrosis in portal triads, and ganglion size were similar in all groups. The number and size of flukes was greater in C2 than in T2 (p < 0.05). The histological examinations revealed a higher degree of parenchymatous fibrosis in T2 compared to C2 (p < 0.05). The administration of LPS from O. intermedium increased the nonspecific resistance against F. hepatica in experimentally infected sheep.