Cryopreservation of testicular tissue from the dog (Canis familiaris) and wild boar (Sus scrofa) by slow freezing and vitrification: Differences in cryoresistance according to cell type

8 Pág.Sperm cryopreservation is the most common procedure used to establish germplasm banks for endangered species - but sometimes sperm cells cannot be obtained. In such cases, freezing testicular tissue may be the only option. The testes contains germ cells at different stages of differentiation, including spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa, among which differences in cryoresistance might be expected. The present work compares the viability and DNA integrity of 'rounded' cells, and of elongated spermatids and spermatozoa, from the dog and wild boar, following the cryopreservation of testicular tissue by slow freezing or vitrification. Cell viability was analyzed by PI/SYBR14 staining, and DNA integrity via the TUNEL technique. For wild boar, no significant differences were seen between the two methods with respect to the percentage of viable cells, nor in the percentage of cells with DNA damage. In the dog, the percentage of viable rounded germ cells (65.0 ± 2.4%) was higher (P < 0.05) after vitrification than after slow freezing (45.1 ± 6.7%). No difference was found between the two methods in terms of the viability of elongated cells. For rounded cells, the percentage of intact DNA was greater (P < 0.05) after vitrification (90.5 ± 2.1%) than after slow freezing (42.6 ± 11.0%), while for elongated spermatids and spermatozoa it was higher (P < 0.05) after slow freezing (66.9 ± 6.1%) than after vitrification (50.7 ± 4.5%). Thus, the response to cryopreservation is cell type-, cryopreservation type-, and species-dependent. Vitrification would appear to be the most appropriate method for preserving dog testicular tissue given the associated high cell viability and low degree of DNA fragmentation, while in wild boar, either method might be used.This study was funded by grant PID2020-113288RB-100/AEI/10.13039/501100011033.Peer reviewe

Slow and ultra-rapid freezing protocols for cryopreserving roe deer (Capreolus capreolus) epididymal sperm collected at different times of year

10 Pág. Centro de Investigación en Sanidad Animal (CISA) / Departamento de Reproducción animalThe roe deer is a monoestrous species with a very short rutting season. The present work reports the most suitable period for collecting epididymal sperm and describes the effect of two cooling rates on the post-thaw quality of sperm. Testes were collected 24–48 h after death. Samples of sperm flushed from the epididymis were subjected to either (1) dilution in a Tris-citric acid-glucose-egg yolk-based medium with glycerol, and slow freezing in straws, or (2) dilution in the same extender but replacing the glycerol with 100 mM of sucrose, and ultra-rapid freezing in pellets. Sperm motility, acrosome and membrane integrity, morphometry and morphological abnormalities were analysed before and after cryopreservation. Spermatogenic activity was investigated via histological examination of testis sections. Several testes collected between April, May and September showed no spermatogenic activity. All those collected in June–August showed spermatogenic activity. No significant difference was detected in the cryoresistance ratios associated with the conventional slow freezing, between sperm collected during the pre-rutting (April–May) and rutting (June–August) periods. No significant differences were seen between the slow-frozen-thawed and the ultra-rapid-frozen-thawed sperm in terms of percentage of viable sperm or the percentage of sperm with morphological abnormalities. Slow freezing returned significantly better (P<0.05) values for post-thaw acrosome integrity (43.3% vs. 25.0%) and straight-line velocity (19 μm/s vs. 4 μm/s). For both freezing methods, sperm heads were smaller post-thawing than pre-freezing (P<0.001). In conclusion, both the pre-rutting and rutting season are suitable periods for freezing roe deer sperm. Ultra-rapid freezing did not provide suitable results.This research was funded by MINECO/AEI/FEDER and EU grant AGL2017-85753-R. P. Bóveda was the recipient of a grant for pre-doctoral researchers from MINECO (AEI/FSE, UE). Octavio Mejía was the recipient of a research fellowship from the PASPA-DGAPA-UNAM (México). V.N. Flores-Gil was funded by FONDECYT-CONCYTEC (grant contract number 000245-2015-FONDECYT).Peer reviewe

2013. Documento Sevilla de Consenso sobre Alternativas a la Transfusión de Sangre Alogénica. Actualización del Documento Sevilla

La transfusión de sangre alogénica (TSA) no es inocua, y como consecuencia han surgido múltiples alternativas a la misma (ATSA). Existe variabilidad respecto a las indicaciones y buen uso de las ATSA. Dependiendo de la especialidad de los médicos que tratan a los pacientes, el grado de anemia, la política transfusional, la disponibilidad de las ATSA y el criterio personal, estas se usan de forma variable. Puesto que las ATSA tampoco son inocuas y pueden no cumplir criterios de coste-efectividad, la variabilidad en su uso es inaceptable. Las sociedades españolas de Anestesiología y Reanimación (SEDAR), Hematología y Hemoterapia (SEHH), Farmacia Hospitalaria (SEFH), Medicina Intensiva y Unidades Coronarias (SEMICYUC), Trombosis y Hemostasia (SETH) y Transfusiones Sanguíneas (SETS) han elaborado un documento de consenso para el buen uso de la ATSA. Un panel de expertos de las 6 sociedades ha llevado a cabo una revisión sistemática de la literatura médica y elaborado el 2013. Documento Sevilla de Consenso sobre Alternativas a la Transfusión de Sangre Alogénica. Solo se contempla las ATSA dirigidas a disminuir la transfusión de concentrado de hematíes. Se definen las ATSA como toda medida farmacológica y no farmacológica encaminada a disminuir la transfusión de concentrado de hematíes, preservando siempre la seguridad del paciente. La cuestión principal que se plantea en cada ítem se formula, en forma positiva o negativa, como: «La ATSA en cuestión reduce/no reduce la tasa transfusional». Para formular el grado de recomendación se ha usado la metodología Grades of Recommendation Assessment, Development and Evaluation (GRADE)

Terrestrial behavior in titi monkeys (Callicebus, Cheracebus, and Plecturocebus) : potential correlates, patterns, and differences between genera

For arboreal primates, ground use may increase dispersal opportunities, tolerance to habitat change, access to ground-based resources, and resilience to human disturbances, and so has conservation implications. We collated published and unpublished data from 86 studies across 65 localities to assess titi monkey (Callicebinae) terrestriality. We examined whether the frequency of terrestrial activity correlated with study duration (a proxy for sampling effort), rainfall level (a proxy for food availability seasonality), and forest height (a proxy for vertical niche dimension). Terrestrial activity was recorded frequently for Callicebus and Plecturocebus spp., but rarely for Cheracebus spp. Terrestrial resting, anti-predator behavior, geophagy, and playing frequencies in Callicebus and Plecturocebus spp., but feeding and moving differed. Callicebus spp. often ate or searched for new leaves terrestrially. Plecturocebus spp. descended primarily to ingest terrestrial invertebrates and soil. Study duration correlated positively and rainfall level negatively with terrestrial activity. Though differences in sampling effort and methods limited comparisons and interpretation, overall, titi monkeys commonly engaged in a variety of terrestrial activities. Terrestrial behavior in Callicebus and Plecturocebus capacities may bolster resistance to habitat fragmentation. However, it is uncertain if the low frequency of terrestriality recorded for Cheracebus spp. is a genus-specific trait associated with a more basal phylogenetic position, or because studies of this genus occurred in pristine habitats. Observations of terrestrial behavior increased with increasing sampling effort and decreasing food availability. Overall, we found a high frequency of terrestrial behavior in titi monkeys, unlike that observed in other pitheciids

Slow and ultra-rapid freezing protocols for cryopreserving mouflon (Ovis musimon) and fallow deer (Dama dama) epididymal sperm

This study examines the effectiveness of two methods for cryopreserving post-mortem epididymal sperm - conventional slow freezing employing a short equilibration time with glycerol, and ultra-rapid freezing - from the wild ruminant species Ovis musimon (mouflon) and Dama dama (fallow deer). A Tris-citric acid-glucose (TCG) + 12% egg yolk-based medium was used for the conventional slow freezing of the fallow deer sperm, whereas a Tes-Tris-glucose (TEST) + 6% egg yolk-based medium was used for the mouflon sperm. Glycerol was added to a final concentration of 5% to both media. The same diluents were used for ultra-rapid freezing but replacing the glycerol with 100 mM of sucrose. Sperm variables (motility, viability, acrosome integrity, membrane integrity, and morphological abnormalities) were analyzed before and after cryopreservation. Although values were generally better after the thawing of the conventionally cryopreserved sperm, total sperm motility (38.40 ± 4.44% in mouflon and 31.25 ± 3.37% in fallow deer) and total live sperm (47.19 ± 5.18% in mouflon and 43.13 ± 2.43% in fallow deer) were acceptable for the ultra-rapidly cooled sperm. Independent of the cryopreservation method, membrane integrity, acrosome integrity and the percentages of dead sperm and sperms with a damaged acrosome were better for the cryopreserved mouflon sperm than the fallow deer sperm (P < 0.05). Despite exerting a more harmful effect on sperm variables than conventional freezing, ultra-rapid freezing may be a useful alternative for the cryopreservation of these species' epididymal sperm in the field, as this simple technique does not require sophisticated equipment and expertise

Progesterone promotes foetal growth in a restricted interspecies gestation (Ovis canadensis × Ovis aries)

Gestations between bighorn (Ovis canadensis) and domestic sheep (O. aries) can be considered for ex situ conservation of bighorn. In the first exper­iment, domestic sheep were inseminated with bighorn or domestic semen. Bighorn inseminated sheep showed lower fertility than domestic inseminat­ed sheep (40% vs. 65%, p = 0.11). Bighorn inseminated sheep had lon­ger gestation periods (152.13 days vs. 146.54 days, p < 0.001) and lower progesterone levels during the last third. Hybrid lambs weighed less than domestic lambs (2.46 kg vs. 5.10 kg, p < 0.001). Their placentas were not as long (48.67 cm vs. 72.17 cm, p < 0.001), were less wide (17.83 cm vs. 23.83 cm, p < 0.001), and the weight of cotyledons was lower (1.50 g vs. 3.20 g, p < 0.001). In the second experiment, hybrid embryos (O. canadensis × O. aries) were transferred into domestic recipients, and preg­nant ewes were divided into the treated group, which had a progesterone daily dose of 25 mg from weeks 7 to 20, and the non treated group. Gesta­tion in domestic sheep that received one hybrid embryo and progesterone reached 152.60 days, which was similar to the 153.33 days (p = 0.51) in the non treated sheep. Hybrid offspring of the group treated with progesterone were heavier, 3.41 kg, than the control, 2.21 kg (p < 0.001), and their pla­centas were longer (71.20 vs. 50.83 cm, p = 0.002). Although progesterone levels were lower in domestic females inseminated with bighorn and in the recipients of hybrid embryos, it is possible to establish pregnancies between both species and the birth of viable offspring. The administration of proges­terone during gestation increases the length of the placenta and promotes higher birth weights of hybrids.Gestations between bighorn (Ovis canadensis) and domestic sheep (O. aries) can be considered for ex situ conservation of bighorn. In the first exper­iment, domestic sheep were inseminated with bighorn or domestic semen. Bighorn inseminated sheep showed lower fertility than domestic inseminat­ed sheep (40% vs. 65%, p = 0.11). Bighorn inseminated sheep had lon­ger gestation periods (152.13 days vs. 146.54 days, p < 0.001) and lower progesterone levels during the last third. Hybrid lambs weighed less than domestic lambs (2.46 kg vs. 5.10 kg, p < 0.001). Their placentas were not as long (48.67 cm vs. 72.17 cm, p < 0.001), were less wide (17.83 cm vs. 23.83 cm, p < 0.001), and the weight of cotyledons was lower (1.50 g vs. 3.20 g, p < 0.001). In the second experiment, hybrid embryos (O. canadensis × O. aries) were transferred into domestic recipients, and preg­nant ewes were divided into the treated group, which had a progesterone daily dose of 25 mg from weeks 7 to 20, and the non treated group. Gesta­tion in domestic sheep that received one hybrid embryo and progesterone reached 152.60 days, which was similar to the 153.33 days (p = 0.51) in the non treated sheep. Hybrid offspring of the group treated with progesterone were heavier, 3.41 kg, than the control, 2.21 kg (p < 0.001), and their pla­centas were longer (71.20 vs. 50.83 cm, p = 0.002). Although progesterone levels were lower in domestic females inseminated with bighorn and in the recipients of hybrid embryos, it is possible to establish pregnancies between both species and the birth of viable offspring. The administration of proges­terone during gestation increases the length of the placenta and promotes higher birth weights of hybrids.Gestations between bighorn (Ovis canadensis) and domestic sheep (O. aries) can be considered for ex situ conservation of bighorn. In the first exper­iment, domestic sheep were inseminated with bighorn or domestic semen. Bighorn inseminated sheep showed lower fertility than domestic inseminat­ed sheep (40% vs. 65%, p = 0.11). Bighorn inseminated sheep had lon­ger gestation periods (152.13 days vs. 146.54 days, p < 0.001) and lower progesterone levels during the last third. Hybrid lambs weighed less than domestic lambs (2.46 kg vs. 5.10 kg, p < 0.001). Their placentas were not as long (48.67 cm vs. 72.17 cm, p < 0.001), were less wide (17.83 cm vs. 23.83 cm, p < 0.001), and the weight of cotyledons was lower (1.50 g vs. 3.20 g, p < 0.001). In the second experiment, hybrid embryos (O. canadensis × O. aries) were transferred into domestic recipients, and preg­nant ewes were divided into the treated group, which had a progesterone daily dose of 25 mg from weeks 7 to 20, and the non treated group. Gesta­tion in domestic sheep that received one hybrid embryo and progesterone reached 152.60 days, which was similar to the 153.33 days (p = 0.51) in the non treated sheep. Hybrid offspring of the group treated with progesterone were heavier, 3.41 kg, than the control, 2.21 kg (p < 0.001), and their pla­centas were longer (71.20 vs. 50.83 cm, p = 0.002). Although progesterone levels were lower in domestic females inseminated with bighorn and in the recipients of hybrid embryos, it is possible to establish pregnancies between both species and the birth of viable offspring. The administration of proges­terone during gestation increases the length of the placenta and promotes higher birth weights of hybrids