In vitro Inhibitory Activity of IgY Antibodies Against Salmonella Ser. Newport Isolated from Horses.

Equine salmonellosis is caused by several Salmonella serotypes, including Salmonella Newport, which cause enterocolitis and diarrhea. Treatment usually includes the administration of antibiotics. However, since multidrug-resistant Salmonella is commonly detected, alternative options to control the pathogen are needed. One of these options is the use of specific egg yolk antibodies (IgY) for passive immunotherapy. Thus, the aim of our work was to produce IgY antibodies against an equine S. Newport strain and assess their in vitro inhibitory activity. To this end, laying hens were immunized with an inactivated S. Newport strain by using either Freund's or Montanide adjuvant and egg yolk extracts were obtained. The levels of specific IgY antibodies against Salmonella in sera and egg extracts were determined by dot-blot and microagglutination. Besides, the IgY extracts were characterized by total protein analysis, SDS-PAGE, Western Blot, and inhibition of bacterial motility. IgY extracts showed high purity (87.7 to 91.8 %), high microagglutination titers, and the ability to inhibit the motility of the bacterium. The results using Montanide were similar to those using the traditional Freund's adjuvant. Thus, Montanide may also be a good adjuvant to produce IgY. IgY-technology represents a potential tool for the control of salmonellosis in horses.

Uterine and Corpus Luteum Blood Flow Evaluation Prior to Uterine Flushing in Llama Embryo Donors.

The aim of this study was to assess the uterine blood flow (UBF) and corpus luteum blood flow (CLBF) in llamas 8 days post-mating, using color-Doppler ultrasonography (CDU), to determine the possible relationship between vascularization and the presence of an embryo. Adult females (n = 25) were used to monitor ovarian dynamics by palpation and transrectal ultrasonography until detection of a ≥6 mm growing follicle. Females were randomly assigned to one of two groups: Group I (n = 19), were mated and ovulation was induced by a single dose of buserelin (GnRH analog) that same day (Day 0); and Group II (n = 6), only ovulation was induced (control). On Day 8, UBF and CLBF were evaluated transrectally in both groups. The color-flow images obtained were analyzed with Image J1.52a software to determine the vascularization area and the percentage of corpus luteum with blood flow emission (CLBF%) together with the percentage for each uterine horn (UBF%). Statistical analysis was performed using an ANOVA test. In Group I, uterine flushing was performed to obtain the embryos, thus dividing the females into Group I+ (n = 10), when an embryo was recovered and Group I- (n = 9), when no embryo was recovered. Embryo recovery rate was 52.63% (10/19). In Group I+, UBF% was significantly higher compared to Group I- and Group II (P <0.05). UBF appears to be a good predictor for embryo presence, with an area under the curve (AUC) of 0.9 and an optimal cut-off value of 9.37% (with a sensitivity of 90% and specificity of 88.9%). The CLBF% did not differ between groups (P > 0.05). In conclusion, it is possible to detect a local increase of UBF in the presence of an embryo on day 8 post-mating in llamas. This could be useful to achieve an early pregnancy diagnosis or to decide whether to carry out the uterine flushing in a llama embryo transfer program.

Air-Drying Llama Sperm Affects DNA Integrity.

The objective of this study was to evaluate the effects of air-drying preservation on llama sperm DNA. Semen collections were carried out using electroejaculation under general anesthesia. A total of 16 ejaculates were processed from 4 males (n = 4, r = 4). Each sample was diluted 4:1 in a collagenase solution in TALP media, then incubated and centrifuged at 800 g for 8 min. The pellet was re-suspended to a concentration of 20 million sperm/ml in TALP. Then the samples were placed onto sterile slides forming lines and were left to dry under laminar flow for 15 min. After this, the slides were placed into Falcon centrifuge tubes and kept at 5°C. Sperm characteristics (motility, membrane function, viability and morphology) were evaluated in raw semen and in the air-dried samples kept at 5°C for 30 min. DNA evaluation (integrity and degree of chromatin condensation) was carried out in raw semen and in the air-dried samples after 30 min, 7, 14, 21, 30, and 60 days after preservation. To compare raw semen to the air-dried samples, a Wilcoxon test was used for all sperm characteristics except for DNA, where a paired Student t-test was applied. A split plot design was used to compare chromatin condensation between the different periods of preservation and a Kruskal Wallis test was used to compare DNA integrity. Motility, membrane function, viability and sperm with intact DNA decreased in the air-dried samples (p < 0.05), while morphology and chromatin condensation were not affected (p > 0.05). No significant differences were observed in the percentage of sperm with condensed chromatin between the different periods of preservation (p > 0.05). On the other hand, a significant decrease in the percentage of sperm with intact DNA was observed as from day 7 of preservation (p < 0.05). In conclusion the air-drying process has a negative effect on llama sperm DNA, hence the media used will need to be improved to protect DNA and be able to implement this technique in this species.

Apoptosis in porcine cumulus-oocyte complexes: Relationship with their morphology and the developmental competence.

The aim of this study was to assess the presence and distribution of apoptosis in porcine cumulus-oocyte complexes (COCs) and its relations with COC morphology and developmental competence. The COCs were obtained from slaughterhouse ovaries, classified into A1 (top category), A2, B1, B2, C, and D based on their morphology. A1, A2, and B1 were matured and fertilized in vitro, and blastocyst rate was compared among them. Before and after in vitro maturation (IVM), annexin-V staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to assess early and late apoptosis, respectively. There was a significant increase in both annexin-V (+) oocytes and TUNEL (+) cumulus cells as morphology further deteriorated. There were no statistical differences regarding annexin-V (+) oocytes within immature and post-IVM COCs, but TUNEL (+) oocytes were only observed in post-IVM COCs. Early and late apoptosis was detected in cumulus cells of all categories of immature and post-IVM COCs. However, the difference was only significant for annexin-V (+). There were no significant differences in embryo development. Therefore, apoptosis increases as the morphological features of the immature COCs decrease. In conclusion, the selection of COCs from Categories A1, A2, and B1 may be used as a selection criterion for in vitro development.

Corpus luteum vascularization during the maternal recognition of pregnancy in llamas (Lama glama).

The aim of this study was to characterize corpus luteum vascularization and its association with plasma progesterone concentration in early stages of pregnancy, when maternal recognition of pregnancy is expected to occur. In all animals, both plasma progesterone concentration and corpus luteum vascularization increased from Day 6 to Day 8 post-mating and afterwards in non-pregnant llamas they started to decrease to reach basal levels around Days 12 to 14 post-mating, while in pregnant animals, both variables remained elevated until the end of the study. A lineal positive relationship between corpus luteum vascularization and plasma progesterone concentration was observed in pregnant (r2  = .46, p < .0001) and non-pregnant llamas (r2  = .66, p < .0001). Pregnant animals showed higher plasma progesterone concentration and corpus luteum vascularization than the non-pregnant ones from Day 12 post-mating until the end of the study (p Ë‚ .05 and p Ë‚ .01, respectively). These results suggest that maternal recognition of pregnancy should occur before Day 12 post-mating in order to expand luteal lifespan, maintaining corpus luteum vascularization and progesterone production. Also, the assessment of CL vascularization area could be a useful and non-invasive method for early pregnancy diagnosis due to its association with plasma progesterone concentration.

Induction of apoptosis in the bursa of Fabricius by vaccination against Gumboro disease.

Infectious bursal disease is a severe acute viral disease of young chickens, affecting mainly the B-lymphocytes in the bursa of Fabricius, leading to severe immunosuppression as a result of the death of lymphoid cells. In the bursa infected with infectious bursal disease virus, viral replication is associated with apoptosis of lymphoid cells, inflammatory change and atrophy. Vaccination has appeared to be a crucial factor for control, with live attenuated vaccines being the most used. However, the apoptotic effect of these vaccines on the bursa has not been tested. We determined the apoptotic effect caused by the most used vaccines in local production on the bursa of Fabricius cells and the correlation with histological changes. In this study, it was demonstrated that apoptosis levels in the vaccinated groups were higher than those observed in the non-vaccinated birds leading to the conclusion that the action of the live virus vaccine strains modifies the boundary of the bursa and shapes processes of cell death by apoptosis. In contrast to other studies, the vaccine strains used did not show the phenomenon of bursal atrophy during the experimental period.

Embryo transfer technique: Factors affecting the viability of the corpus luteum in llamas.

The aim of the present study was to evaluate the effect of the embryo transfer (ET) maneuvers on plasma progesterone concentrations in recipient Lama glama females and the relationship between the site the embryo was transferred to and corpus luteum (CL) localization. Experiment I (effect of transcervical threading): adult non-pregnant, non-lactating llama females were randomly assigned into two groups: control group (without cervical threading, n=10) and group A (with cervical threading, n=10). In both groups, CL activity was evaluated through measurement of progesterone plasma concentrations. In group A, on Day 6 after inducing ovulation with buserelin, the cervix was threaded to evaluate the effect of the maneuver on CL viability. No significant differences were observed in mean progesterone concentrations between groups (P>0.05). Experiment II (effect of depositing PBS): females (n=66) were randomly assigned into six groups (n=10 per group and control group: n=6) to evaluate the effect of depositing PBS in different sites in the uterus in relation to the localization of the CL: group 'Left-Ipsilateral': transcervical placing of PBS in the left uterine horn (CL in left ovary); group 'Left-Contralateral': transcervical placing of PBS in the left uterine horn (CL in right ovary); group 'Right-Ipsilateral': transcervical placing of PBS in the right uterine horn (CL in right ovary); group 'Body-Left': transcervical placing of PBS in the uterine body (CL in left ovary); group 'Body-Right': transcervical placing of PBS in the uterine body (CL in right ovary) and control group. Corpus luteum activity was evaluated in all groups by measuring plasma progesterone concentrations. On Day 6 post-buserelin, the corresponding maneuver was carried out according to the group. No significant differences were found for the mean plasma progesterone concentrations between groups (P>0.05). Experiment III (effect of ET on CL viability): females (n=22) were used as embryo donors and 50 females as recipients, in order to evaluate if placing the embryo in different areas of the uterus influences CL viability. Recipients were randomly divided into five groups, according to the place in the uterus where the ET was conducted with respect to the ovary where ovulation occurred: group 'Left-Ipsilateral': ET in the left uterine horn (CL in left ovary); group 'Left-Contralateral': ET in the left uterine horn (CL in right ovary); group 'Right-Ipsilateral': ET in the right uterine horn (CL in right ovary); group 'Body-Left': ET in the uterine body (CL in left ovary) and group 'Body-Right': ET in the uterine body (CL in right ovary). Corpus luteum activity was evaluated in all groups by measuring plasma progesterone concentrations. Embryos were recovered by flushing the uterus on Day 8 after the first mating of the donor and transcervical ET was carried out in recipients 6 days after buserelin administration. Pregnancy rates were: group 'Left-Ipsilateral': 50%; group 'Left-Contralateral': 20%; group 'Right-Ipsilateral': 30%; group 'Body-Left' and 'Body-Right': 10%. No significant differences (P=0.4728) were detected between the pregnancy rates in the five groups. Threading the cervix and transcervical placing of PBS either in the uterine horns or the body did not affect plasma progesterone concentrations in the llama, indicating that the different embryo transfer maneuvers do not interfere with CL viability. To improve pregnancy rates it could be suggested that ET in the left uterine horn with an ipsilateral CL, is the most desirable option.