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.

In vitro production of llama (Lama glama) embryos by IVF and ICSI with fresh semen.

The interest for South American camelids has increased in the last years. The aim of the present research was to compare the in vitro production of Lama glama embryos using two techniques: in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). For IVF technique, we compared the effect of adding or not, heparin, penicillamine and hypotaurine as sperm capacitating agents. In the oocyte group subjected to ICSI, activation with or without, ionomycin and 6-dimethylaminopurine (6-DMAP) was assessed. Semen samples were obtained by electroejaculation and incubated at 38 degrees C in a 25% (v/v) collagenase solution. The cleavage and embryo development rates were compared between the different experimental groups. Only the number of cleaved oocytes was less when ICSI with no activation was used (p<0.05).

Membrane changes during different stages of a freeze-thaw protocol for equine semen cryopreservation.

Many theories have been postulated concerning the possible effects of cryopreservation on spermatozoa, including suggestions the freeze-thawing process produces membranes that have greater fluidity and are more fusogenic, thus inducing changes similar to those of capacitation. The main objectives of this study were to determine at what stage of the freeze-thaw process membrane changes occur and whether evaluation with chlortetracycline (CTC) stain could predict the freezability of stallion sperm. Sperm viability and state of capacitation were simultaneously evaluated using CTC and Hoechst 33258 (H258) techniques. Membrane function was evaluated using the hypo-osmotic swelling test (HOS) and progressive motility (PM) was evaluated under light microscopy at each stage of a freeze-thaw protocol. Evaluated were raw semen; after dilution and centrifugation; after redilution and equilibration at room temperature; after cooling to 5 degrees C; after super cooling to -15 degrees C; and after thawing. The most pronounced functional damage to membranes and the greatest decrease in PM occurred in samples of all stallions after thawing (P<0.05). Cryopreservation, as evaluated by CTC/H258 staining, significantly (P<0.05) affected sperm membrane integrity after centrifugation, after redilution and equilibration at room temperature and after cooling to 5 degrees C. The HOS and H258 tests gave similar results (R values of approximately 0.75) and correlated inversely with the number of live noncapacitated sperm cells (R values of approximately -0.75). Remarkably, the subpopulation of capacitated live cells was unaffected in all freeze-thawing steps and the number of live acrosome reacted cells increased by a factor of 4. However, it was not possible to determine whether the changing CTC patterns reflect a true capacitation phenomenon or an intermediate destabilized state of the sperm cell membrane. This increase may indicate that the subpopulation of functional sperm cells capable of binding to the zona pellucida increases after freeze-thawing despite the deteriorative effect of this procedure for the entire live sperm population.

Metabolic and histological pancreatic changes induced by ovariectomy in the female dog

En perras se estudiaron las acciones de la ovariectomía llevada a cabo con 4 meses de anticipación sobre la citología pancreatica y tambíen sobre la glucemia, insulina sérica inmunorreactiva y ácidos grasos libres circulantes durante las pruebas de glucosa e insulina. Concluimos que la ovariectomía en esas condiciones no afecta la glucemia- basalmente o durante las pruebas-, el espacio de glucosa y la desaparición de la glucosa circulante. Por el contrario, la respuesta insulinémica integrada durante la prueba de glucosa fue muy aumentada (965%) en dichos animales por la ovariectomía; el aumento ocorrió a pesar de que ellos presentan un espacio de insulina agrandado (59%), es apenas medido por una reducción (132%) en la velocidad de aclaramiento de la insulina circulante y parece ser principalmente mediado por un enorme aumento de la secreción de insulina. La inmunocitolocalización de la insulina en el tejido pancreático de las perras así ovariectomizadas demostró la existencia de hipertrofia de islotes de Langerhans, desgranulación beta pero no vacuolización. Sin embargo, el amontonamiento de los gránulos-beta, como así también la aparición de un sinnúmero de pequeños islotes, y microislotes dispersos entre los acinos, ausentes en los páncreas de las perras controles no tratados quirúrgicamente, que estaban en anestro, indican que la potencia secretoria, de insulina del páncreas de las perras ovariectomizadas debería ser elevada. En la perra, la ovariectomía afectó los niveles de ácidos grasos libres séricos a través de dos mecanismos, a saber: a) estimulación del almacenamiento de lípidos durante las pruebas de glucosa, y b) reducción en la lipomovilización cuando predomina el antagonismo insulínico

Metabolic and histological pancreatic changes induced by ovariectomy in the female dog

En perras se estudiaron las acciones de la ovariectomía llevada a cabo con 4 meses de anticipación sobre la citología pancreatica y tambíen sobre la glucemia, insulina sérica inmunorreactiva y ácidos grasos libres circulantes durante las pruebas de glucosa e insulina. Concluimos que la ovariectomía en esas condiciones no afecta la glucemia- basalmente o durante las pruebas-, el espacio de glucosa y la desaparición de la glucosa circulante. Por el contrario, la respuesta insulinémica integrada durante la prueba de glucosa fue muy aumentada (965%) en dichos animales por la ovariectomía; el aumento ocorrió a pesar de que ellos presentan un espacio de insulina agrandado (59%), es apenas medido por una reducción (132%) en la velocidad de aclaramiento de la insulina circulante y parece ser principalmente mediado por un enorme aumento de la secreción de insulina. La inmunocitolocalización de la insulina en el tejido pancreático de las perras así ovariectomizadas demostró la existencia de hipertrofia de islotes de Langerhans, desgranulación beta pero no vacuolización. Sin embargo, el amontonamiento de los gránulos-beta, como así también la aparición de un sinnúmero de pequeños islotes, y microislotes dispersos entre los acinos, ausentes en los páncreas de las perras controles no tratados quirúrgicamente, que estaban en anestro, indican que la potencia secretoria, de insulina del páncreas de las perras ovariectomizadas debería ser elevada. En la perra, la ovariectomía afectó los niveles de ácidos grasos libres séricos a través de dos mecanismos, a saber: a) estimulación del almacenamiento de lípidos durante las pruebas de glucosa, y b) reducción en la lipomovilización cuando predomina el antagonismo insulínico (AU)