Estudio del factor térmico en los protocolos de crioconservación del semen de oso pardo ("Ursus arctos") = Evaluation of the temperature handling during cryopreservation of brown bear (Ursus arctos) semen

150 p.En esta tesis doctoral se han evaluado diferentes modelos de manejo durante la precongelación de eyaculados de oso pardo, con el fin de obtener un Banco de Recursos Genético eficaz y que sea de aplicación a la población de osos de las montañas cantábricas. Los modelos objeto de estudio han sido: 1)la aplicación de diferentes rampas de refrigeración y la simplificación de los protocolos (eliminación del tiempo de equilibrado sólo o con el periodo de refrigeración); 2) el almacenamiento a corto o largo plazo a 5 °C de las muestras espermáticas; 3) el control de la temperatura, el porcentaje de glicerol o la tasa de dilución, en el almacenamiento a largo plazo; 4) el uso de medio sólido para mejorar la calidad durante el almacenamiento a largo plazo

The percentage of spermatozoa lost during the centrifugation of brown bear (Ursus arctos) ejaculates is associated with some spermatozoa quality and seminal plasma characteristics

P. 113-121Cryopreservation of brown bear (Ursus arctos) semen requires centrifugation to increase concentration and/or remove urine contamination. However, a percentage of the spermatozoa are lost in the process. This percentage varies considerably between males and ejaculates, and we have studied the effect of sperm quality and seminal plasma characteristics on the spermatozoa recovery rate after centrifugation. One hundred and thirty one sperm samples obtained from fifteen brown bear males by electroejaculation under general anaesthesia were used. The ejaculates were centrifuged 600 × g for 6 min. Motility was assessed by CASA, and acrosomal status (PNA-FITC) and viability (SYBR-14/propidium iodide) were determined by flow cytometry. Seminal plasma characteristics (albumin, alkaline phosphatase, calcium, cholesterol, creatine, glucose, glutamic oxaloacetic transaminase (GOT), lactate, lipase, magnesium, phosphate and total protein) were determined by a biochemical and gas analysis. Total motility (r = 0.26; P = 0.005) and cell viability (r = 0.20; P = 0.033) were positively correlated with the percentage of recovered spermatozoa. Sperm recovery was correlated with the concentration of several components of seminal plasma: negatively with glucose concentration (r = −0.47; P = 0.005) and positively with the enzymes GOT (r = 0.36; P = 0.040) and lactate dehydrogenase (r = 0.36; P = 0.041). After sorting the data into classes according to sperm recovery (Low: 0–39, Medium: 40–69, High: 70–100), we observed that the samples with a lower recovery rate derived from ejaculates with lower values for TM, VAP and viability (P < 0.05). Multiple regression analysis rendered two models to define the post-centrifugation spermatozoa recovery which included total motility and damaged acrosome or glucose, GOT and lactate dehydrogenase. We discuss these relationships and their implications in the electroejaculation procedure and the handling of the sample during centrifugation.S

Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes

P. 76-82The effect of storage procedure at 5 °C on the quality of ram spermatozoa from the cauda epididymis was analyzed. Two strategies were tested at 0, 24, 48 and 72 h post-mortem: (1) spermatozoa held in the epididymal fluid and stored either in the cauda epididymis (In-EPID) or in vitro (Ex-EPID), (2) epididymal spermatozoa extended in three media at 320, 370 and 420 mOsm/kg (D320, D370, D420). Analyzed parameters were: osmolality, pH, motility, acrosomal status and viability. In experiment 1, osmolality of the In-EPID samples, but not in Ex-EPID, increased with post-mortem time. Motility of In-EPID spermatozoa in samples, after 24 h post-mortem, was higher compared to the Ex-EPID samples, although differences decreased at 48 and 72 h. In experiment 2, total (TM) and progressive motility (PM) were not significantly affected by storage time for D320 and In-EPID samples. However, the motility of D370 and D420 samples significantly decreased with time. TM and PM of D320 were significantly higher than D370 and D420 at 72 h. At 24 h, sperm viability was higher for In-EPID (80.7 ± 3.4%) than for the extended samples (44.8 ± 2.9%, 37.7 ± 3.9% and 48.6 ± 6.0% for D320, D370 and D420, respectively), which also decreased faster with time. At 24 h, the percentage of damaged acrosomes was low and similar for the four methods of storage, but damaged acrosomes increased with time for D320 and D370. Storing the spermatozoa in the epididymis is a good strategy for maintaining sperm quality in ram, at least for 48 h. The D320 extender preserve motility of epididymal spermatozoa but does not protect the status of the acrosome.S

Refrigerated storage of ram sperm in presence of Trolox and GSH antioxidants: Effect of temperature, extender and storage time

P. 137-147Antioxidants have a potential to improve the quality and fertility of refrigerated-stored ram semen. Reduced glutathione (GSH) and Trolox (0.2, 1 and 5 mM) were evaluated in ram semen preserved at 15 and 5 °C up to 48 and 96 h, respectively. Extenders were also evaluated (15 °C: Tris-citrate-fructose, TCF, without lipids, and TES-Tris-fructose 10% egg yolk, TTF-EY; 5 °C: TTF-EY and 3.5% soybean lecithin, TTF-SL; INRA96 at both temperatures). Storage at 5 °C resulted in poorer quality than 15 °C up to 48 h, while allowing acceptable quality at 96 h. Antioxidants had few effects on sperm quality, with use of Trolox resulting in reduced motility and viability in TCF. Storage at 15 °C in the TCF extender resulted in decreased motility, viability and mitochondrial activity compared with use of TTF-EY. Sperm quality when storage was at 5 °C was similar, but storage in TTF-SL resulted in decreased motility and mitochondrial activity. Acrosomal status was only slightly affected by extender and antioxidant. Mitochondrial activity was improved by antioxidants in TTF-SL, and GSH at 5 mM when semen was stored at 5 °C in TTF-EY. A preliminary artificial insemination trial indicated that supplementation with GSH has the potential for improving lambing (P < 0.1). In conclusion, use of antioxidants resulted in lesser effects than extender composition or storage time on quality of ram semen. Use of Trolox negatively impacted sperm quality and GSH had some positive impacts. The use of soybean lecithin requires further research to assess its impact on mitochondria.S

Specificity of the extender used for freezing ram sperm depends of the spermatozoa source (ejaculate, electroejaculate or epididymis)

P. 145-154The objective of this study was to identify possible specificity in the extender formulation for the cryopreservation of ram spermatozoa recovered from three origins (ejaculate, electroejaculate or epididymis), by evaluating post-thawing sperm quality and fertility. Ejaculated, electroejaculated or epididymal spermatozoa samples obtained from identical rams (8) were cryopreserved in four different extenders (TES-Tris–fructose with one of two egg yolk concentrations: 10% Y10 and 20% Y20, and with one of two glycerol rates: 4% G4 and 8% G8). Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability with SYBR-14/PI and acrosomal status with PNA/PI). Spermatozoa obtained by electroejaculation were of poorer quality after freezing/thawing, demonstrating that protocols for these samples need to be optimized. Egg yolk at 20% was more appropriate for freezing sperm from any of the sources. In general, 4% glycerol improved the quality of post-thawing samples recovered from ejaculate and electroejaculate, while 8% glycerol was more appropriate for samples recovered from the epididymis. Based on these results, an analysis of fertility was conducted. Fertility rates were similar between ewe groups inseminated with post-thawed sperm obtained from two sources: ejaculate (cryopreserved in Y20 + G4), and cauda epididymis (Y20 + G8), and this rate was less in the electroejaculated sample (Y20 + G4)S

Evolución de un programa de inseminación en la raza Assaf en la provincia de León

P. 675-677The Assaf breed has mostly been allocated in Castilla y León, particularly in the province of León. In 1998, a selection and genetic improvement programme was initiated for milk production in 38 flocks. Training of rams for semen collection using an artificial vagina reached a success rate of 74%, and the percentage of valid ejaculates has reached 90%. Fertility results of artificial insemination (lambing rates) have fluctuated between 35% y 40% since the programme was started. We found that fertility drops in ewes older than 4 years, and that it improved about 10% in the breeding season

The use of gelatine in long-term storage (up to 48 hr) at 5°C preserves the pre-freezing and post-thawing quality of brown bear sperm

Sedimentation of spermatozoa occurs during long-term liquid storage and this may produce deleterious changes. Our aim was to apply gelatine supplementation during long-term pre-freezing storage of bear sperm, applying final dilution and 6% glycerol at room temperature and cool in straws. We tested four models of sperm storage using a 1:1 dilution in TTF-ULE-Bear extender (TesT-fructose-egg yolk-glycerol 6%): (i) second 1:1 dilution at room temperature (RT), cooling at 5°C in a tube and final dilution (100 × 10 sperm ml) (Standard); (ii) final dilution at RT and cooling in a tube (FD-Tube); (iii) final dilution at RT and cooling in 0.25 ml plastic straw (FD-Straw); and (iv) final dilution at RT in extender supplemented with 1.5% gelatine (Gelatine) and cooling in a 0.25 ml plastic straw. A Standard sample was stored at 5°C for 1 hr (Control); the rest of the samples (Standard, FD-Tube, FD-Straw, Gelatine) were stored for 24 or 48 hrs before freezing (100 × 10 sperm ml, glycerol 6%). The quality of the samples was assessed for motility by CASA, and viability (SYBR-14/propidium iodide-PI-; VIAB), acrosomal status (PNA-FITC/PI; iACR) and apoptotic status (YO-PRO-1/PI; YOPRO-) by flow cytometry. At pre-freezing, after 48 hr, Gelatine showed significantly higher viability (for VIAB and YOPRO-) and progressiveness (PM, LIN and STR). At 48 hr, Gelatine showed similar YOPRO-, iACR, LIN, STR and ALH respect to Control. At both 24 and 48 h post-thawing, Gelatine sample had similar scores for YOPRO-, iACR, LIN, STR, WOB and VIAB (only 24 hr) when compared with Control, and lower for TM, PM, rapidPM, VAP and ALH. No differences were found among others experimental groups with respect to Control. In conclusion, gelatine could be a suitable alternative to preserve the viability and progressive motility of brown bear ejaculates during long-term pre-freezing storage at 5°C.This work was supported by MICINN (CGL2013-48255-R) and CANTUR S:A: Elena López-Urueña was supported by a PhD fellowship (ORDEN EDU/330/2008, co-finaced by Junta de Castilla y León and European Social Fund).Peer Reviewe

Alternative procedures for the cryopreservation of brown bear ejaculates depending on the flexibility of the >in cooling> period (5°C)

The adaptability of cryopreservation protocols for brown bear spermatozoa collected under field conditions and frozen in a nearby laboratory (transported for a few hours) or shipped to a reference laboratory for sex sorting (transported for a few days) was evaluated. Forty-nine electroejaculates from 15 mature brown bears were extended to 100×106sperm/mL in a TES-Tris-Fructose based extender and cryopreserved (-20°C/min to -100°C and stored at -196°C). After thawing, the quality of the seminal samples was assessed for total (TM), progressive (PM) motility and kinetic parameters - by CASA -, and viability (VIAB), viable and non-apoptotic status (YOPRO-), high membrane mitochondrial potential (MIT) and intact acrosomes (iACR) - by flow cytometry - In Experiment 1, we assessed different storage times (0, 0.5, 1 - control -, 4-5, 7-8 and 11-12h) at 5°C from final dilution to freezing. After thawing, non-equilibrated samples (0h) showed lower values of iACR, TM and PM. No significant differences were found for the different periods of equilibration tested. In Experiment 2, we evaluated three long-term storage times (24, 48 and 72h) at 5°C before freezing using storage for 1h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48-72h of pre-freezing. In conclusion, our findings suggest the possibility of extending the pre-freezing cooling period up to 24h post-collection without freezing. This knowledge should enable the adaptation of the freezing protocols for when a special handling conditions are required such as the shipment of seminal samples to technological centers for the pre-freezing application of enhancer spermatic biotechnologies.This work was supported in part by MICINN (CGL2010-19213/BOS) and CANTUR S.A.Peer Reviewe

Optimization of conditions for long-term prefreezing storage of brown bear sperm before cryopreservation

Brown bear ejaculates are usually collected in field conditions and may need to be shipped to a laboratory for the application of reproductive biotechnologies before cryopreservation. The aim of this study was to extend the prefreezing step to 48 hours (1 hour vs. long-term storage [LS] to 24 and 48 hours) to enable the sample to be transported. The effects of storage temperature (experiment 1), glycerol concentration (experiment 2), and dilution rate (experiment 3) on sperm were evaluated. Electroejaculates from brown bears were stored under different experimental conditions and cryopreserved. The sperm motility and viability, apoptotic status, and acrosomal status of sperm were assessed before freezing (prefreezing), after thawing, and after 2-hour incubation at 37 °C (thermal stress test). In all experiments, one control sample was frozen using a standard protocol (control). In experiment 1, three temperatures during LS with 6% glycerol were tested: 5 °C (T5), 15 °C (T15), and room temperature (RT). The LS-T5 sample yielded the highest postthawing results for viability (42.4%), progressive motility (15.6%), and intact acrosome (83.1%) after 24 hours in comparison with the other temperatures (P 6 sperm/mL; low) or final dilution (100 × 106 sperm/mL; high). Both dilution rates showed similar postthawing and postincubation results within 24 hours of long-term storage. After 48 hours, high dilution supported better postthawing quality. Both dilutions showed similar resistance to cryopreservation, except after 48 hours, when the high dilution reached a higher percent recovery rate of viability (38.8% vs. 21.6%, P 6 sperm/mL, and with 6% glycerol.This work was supported by Ministerio de Ciencia e Innovación (MICINN) (CGL2010-19213/BOS) and CANTUR S.A. E. López-Urueña was supported by a PhD fellowship (ORDEN EDU/330/2008, co-financed by Junta de Castilla y León and European Social Fund).Peer Reviewe

Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time

Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 106 spermatozoa/mL in a TES-Tris-Fructose-based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at -196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.This work was supported in part by MICINN (CGL2010–19213/BOS) and CANTUR S.A.Peer Reviewe