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Background: Assisted reproduction techniques in birds have contributed to many species' conservation and sustainable use. One of these techniques is semen cryopreservation, which is possible following the discovery of suitable cryoprotectants. Aims: This study aimed to characterize the fresh and post-thaw ejaculates of different species of birds of prey. Methods: The following species were included in the study: red-tailed hawk (Buteo jamaicensis) n=3, golden eagle (Aquila chrysaetos) n=3, and Harris's hawk (Parabuteo unicinctus) n=3. Twenty-five ejaculates were obtained for each species. The percentage of spermatozoa motility, viability, and morphology were evaluated. Results: Evident differences were observed among the ejaculates of the three species, particularly in sperm length and between the fresh and post-thaw parameters of the same species in which the motility reduced to approximately 40% after thawing. It was demonstrated that sperm cryopreservation of the studied species was possible using the same freezing protocol. Conclusion: This study showed that sperm characteristics could influence the parameters obtained during their in vitro conservation, both in the fresh and post-thaw states.

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RESUMEN La anestesia aviar constituye un área de estudio controvertida debido a la morfofisiología diferente entre aves y mamíferos. Lo anterior hace necesario desarrollar protocolos confiables que contribuyan al bienestar de las aves en cautiverio. La anestesiología en aves representa una actividad clínica que demanda especial cuidado de los pacientes que requieren procedimientos quirúrgicos. Este estudio describe los parámetros de SpO2, frecuencias cardiaca y respiratoria durante la anestesia con isoflurano de Melopsittacus undulatus. Se monitorearon 12 machos y 7 hembras durante las etapas anestésicas. La inducción anestésica duró 1:30 ± 0:31 min en machos y 2:19 ± 0:16 min en hembras, con promedio de mantenimiento de 7:00 ± 1:39 min. No se encontraron diferencias significativas en los tiempos anestésicos entre hembras y machos (p>0,05). Se presentó una variación estadísticamente significativa (p<0,05) de la SpO2 en el periodo de recuperación, las hembras presentaron mayor saturación de oxígeno (71±4 %) en comparación con los machos (89±2 %). En la valoración de la función cardiaca durante la anestesia, no se detectaron diferencias significativas entre machos y hembras (p>0,05). Se concluye como un protocolo anestésico seguro para procedimientos clínicos de corta duración para aves pequeñas como M. undulatus.

ABSTRACT Avian anesthesia is a controversial area of study due to the differences between birds and mammals morpho physiology. This makes necessary to develop reliable protocols for birds in captivity, which contributes to their welfare under human care. Bird anesthesiology today represents a veterinary clinical activity that demands special care for patients requiring surgical procedures. This study describes the parameters of SpO2, cardiac activity, heart and respiratory rate during anesthesia with isoflurane for Melopsittacus undulatus. 12 males and 7 females were monitored during the anesthetic stages. Anesthetic induction lasted 1:30 ± 0:31 min in males and 2:19 ± 0:16 min in females, with an average maintenance time of 7:00 ± 1:39 min. No significant differences were found in anesthetic times between females and males (p>0.05). There was a statistically significant variation (p<0.05) of SpO2 in the recovery period, females had higher oxygen saturation (71±4%) compared to males (89±2%). In the assessment of cardiac function during anesthesia, no significant differences were detected between males and females (p>0.05). It is concluded as a safe anesthetic protocol for clinical procedures of short duration for small birds such as M. undulates.

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The apoptosis of ß cells induced by hyperglycemia has been associated with p53 mobilization to mitochondria and p53 phosphorylation. Murine double minute 2 (Mdm2) induces the degradation of p53 and thereby protects cells from apoptosis. We studied the effect of glucose at high concentration on the ability of Mdm2 to ubiquitinate p53 and promote its degradation. RINm5F cells were grown in RPMI-1640 medium with 5 or 30 mM glucose for varying periods of time. After this treatment, the expression of Mdm2 was measured using real-time PCR. The phosphorylation of Mdm2 at Ser166, p53 at Ser15, and the kinases Akt and ATM were measured by Western blotting. The formation of the p53-Mdm2 complex and p53 ubiquitination was assessed by p53 immunoprecipitation and immunofluorescence. Our results showed that high glucose reduced Mdm2 mRNA expression and protein concentration and increased Mdm2 and Akt phosphorylation, albeit with slower kinetics for Akt. It also promoted p53-Mdm2 complex formation, whereas p53 ubiquitination was suppressed. Furthermore, phosphorylation of both p53 Ser15 and ATM was increased in the presence of 30 mM glucose. These data indicate that high concentration glucose decrease the mRNA expression and cytosolic concentration of Mdm2. However, although the increase in glucose promoted the phosphorylation of Mdm2, it also decreased p53 ubiquitination, thus avoiding p53 degradation. In hyperglycemic conditions, such as diabetes mellitus, the reduction of pancreatic ß cells mass is favored by stabilization of p53 in association with low p53 ubiquitination and reduced expression of Mdm2.

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In the spermatozoa of some species, the ubiquitin-proteasome system detects altered proteins and tags them for elimination by the proteasome. In some species' ejaculates, a high proportion of ubiquitinated spermatozoa (i.e. those having ubiquitin bound to the altered or damaged membrane proteins) has been related to infertility. The aim of this study was to assess whether the percentage of ubiquitinated spermatozoa relates to fertility of dairy bulls and whether ubiquitination increases during protein remodelling that occurs during in vitro spermatic capacitation. Thirty-two frozen semen straws from four high-fertility (ReproMax(®) ) and four normal-fertility (Normal) Holstein-Friesian sires were evaluated. Ubiquitinated and capacitated spermatozoa were quantified by sperm ubiquitin tag immunoassay and chlortetracycline stain, respectively. Fertilizing capacity of sires was assessed by in vitro fertilization. No differences were found between Normal and ReproMax(®) sires with regard to the observed percentage of ubiquitinated spermatozoa (42.97 ± 3.69% and 49.68 ± 9.27%, respectively; p > 0.05). Additionally, no differences were found in the percentage of ubiquitinated spermatozoa as a consequence of spermatic capacitation in either Normal (42.97 ± 3.69% before capacitation vs 44.67 ± 7.5% after; p > 0.05) or ReproMax(®) sires (49.68 ± 9.27% before vs 45.05 ± 7.51% after; p > 0.05). The percentage of ubiquitinated spermatozoa in a thawed sperm samples did not correlate with its in vitro fertilizing capacity; thus, this assay does not prove useful to detect in vivo fertility differences between sires. Additionally, protein degradation occurring during remodelling of the spermatozoon plasma membrane during the capacitation process does not seem to involve the ubiquitin-proteasome system.

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This study was conducted to evaluate phosphatidylserine translocation in head plasma membrane of Percoll-gradient purified of rabbit cauda epididymal sperm during capacitation and acrosome reaction (AR) using Annexin-V. Propidium iodide was used as control to reject dead or dying cells. The presence and distribution of Annexin-V binding sites were analyzed using flow fluorocytometry and confocal microscopy. After 6 h of incubation of sperm in capacitation medium, the number of cells positively stained with Annexin-V showed a small but significant increment. The Annexin-V binding sites produced during capacitation were found mainly in the post-acrosomal region of the sperm head plasma membrane. After AR induction with progesterone, the localization of phosphatidylserine was changed and the Annexin-V binding sites were found almost only in the acrosomal region, but with higher number of binding sites in the equatorial area. On the contrary, after AR induction with A23187, phosphatidylserine translocation, although predominant over the acrosomal region, was also observed in the post-acrosomal region. Plasma membrane destabilization during capacitation and AR may be important for sperm-oocyte fusion.

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Cell death can occur through apoptotic or necrotic death pathways. Membrane disruption leads to inflammation, a typical feature of necrosis. Apoptosis constitutes a genetically controlled physiologic process of cell removal. It is characterized by cell shrinkage, chromatin condensation, and DNA cleavage. Apoptotic cells are rapidly recognized and engulfed by phagocytes thus inhibiting an inflammatory response following necrosis. Apoptosis has been proposed as a basic event to protect tissue homeostasis. This paper analyzes the genetic, biochemical, and morphologic characteristics related to apoptosis, as well as its relationship to certain illnesses.

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The differential quantitative participation of apoptosis and necrosis in ewe antral follicles of two different sizes, separated in four stages of atresia using macroscopic, histologic, and esteroid quantification methods was assessed. Annexin V binding and propidium iodide (PI) uptake was used to detect healthy live cells (Annexin V negative/PI negative), early apoptotic cells (Annexin V+/PI-), and necrotic or late apoptotic cells (PI+). Additionally we used internucleosomal DNA fragmentation as a quantitative estimate of apoptosis. Presence and distribution of lysosomal enzymes in follicular fluid and granulosa cells was used as a measure of necrotic cell death. DNA flow cytometry and gel electrophoresis were positively correlated with the progression of atresia, small atretic follicles tend to have higher percentages of internucleosomal cleaved DNA than follicles >6 mm. Annexin/PI binding also indicates that apoptosis and necrosis increase with atresia progression, generally apoptosis outweighs necrosis in small follicles. Acid phosphatase and glucosaminidase in follicular fluid of 3-6 mm follicles showed no significant modifications between healthy and initially atretic follicles, and only a small, but significant increase in activity in advancedly atretic follicles. On the contrary, lysosomal enzyme activity in follicles >6 mm showed positive correlation between atresia stages and the activities of acid phosphatase and glucosaminidase in follicular fluid. A similar size-differential behavior was found in free or membrane-bound lysosomal enzyme activity of granulosa cells. Necrosis, but principally apoptosis, were present during all stages of follicular maturation indicating that growth and maturation of ovarian follicles involves a continuous renewal of granulosa cells, regulated by apoptosis. Mechanisms regulating this equilibrium may participate in the final destiny, whether ovulation or atresia of ovarian follicles.

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