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(6)  sperm ml(-1) ) (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(6)  sperm ml(-1) , 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.

Effect of colloid (Androcoll-Bear, Percoll, and PureSperm) selection on the freezability of brown bear (Ursus arctos) sperm.

The development of a species-specific conservation protocol that involves artificial insemination with frozen semen needs to validate an effective methodology for freezing semen. Colloid centrifugation has been suggested and widely applied as an effective tool for selecting animal spermatozoa for artificial breeding. The objective of the present study was to compare different methods of centrifugation, single layer using Androcoll-Bear and Percoll and double layer using PureSperm 100 (in two different discontinuous gradients 40%-80% and 45%-90%), for the selection of fresh brown bear sperm samples. In the before freezing group, all selected samples showed a higher progressive motility and viability (except Percoll for motility 43.0 ± 5.3 [P < 0.05]); all colloids except PureSperm 45/90% rendered samples with fewer damaged acrosomes. In the after thawing group, all tested centrifugation colloids showed a good capacity to decrease the number of damaged acrosomes. Furthermore, PureSperm treatment (45/90%) resulted in an increase in apoptotic-like changes not only immediately after thawing but also after the incubation test, leading us to suggest that this gradient could induce some kind of deleterious effects on the sperm samples. On the other hand, PureSperm treatment (40/80%) yielded a quality preservation capacity similar to Androcoll-Bear in number of damaged acrosomes, different relative to the control (control, 5.3 ± 0.6; PureSperm 80, 2.0 ± 0.3; Androcoll, 2.1 ± 0.9 [P < 0.05]) but a decrease in the number of viable spermatozoa recovered after thawing relative to the control (control, 21.2 ± 3.1; PureSperm 80, 13.7 ± 2.7 [P < 0.05]). In conclusion, Androcoll-Bear constitutes a useful tool for handling of brown bear ejaculates owing to its simple handling and procedure with a reliable sperm selection and freezability. This colloid yielded an improvement in several sperm parameters in brown bear frozen-thawed semen; the selected spermatozoa of fresh samples with this colloid showed a better resistance to freezing compared with the control sample not only for motility but also for viability.

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 < 0.05); for 48 hours, the LS-T5 sample reached higher total and progressive motility (25.9% and 9%, respectively) and nonapoptotic values (36.5%). Recovery rates revealed susceptibility to freezing at LS-15 or LS-RT samples at 24 hours (viability) or 48 hours (viability and motility). In experiment 2, samples were stored at 5 °C up to 48 hours and three glycerol concentrations were evaluated: 0% (0Gly), 3% (3Gly), and 6% (6Gly). Postthawing viability and motility increased progressively with the percentage of glycerol for 24 hours at 5 °C; 6% glycerol during 48-hour storage had beneficial effects on sperm cryopreservation. Besides, 6% glycerol had a clearly superior freezability for viability (42.7% and 40.8% for 24 hours and 48 hours, respectively) and motility (24 hours: total, 44.1%; progressive, 17.1%; 48 hours: total, 38.4%; progressive, 16%). In experiment 3, samples were stored up to 48 hours at 5 °C with 6% of glycerol and two dilution methods were evaluated: dilution 1:1 (average: 1782 × 10(6) sperm/mL; low) or final dilution (100 × 10(6) 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 < 0.05). In conclusion, our results suggested that the best conditions for long-term prefreezing storage (up to 48 hours) of brown bear electroejaculates are at 5 °C, at a concentration of 100 × 10(6) sperm/mL, and with 6% glycerol.

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 100×10(6) sperm/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 11-12 h) at 5°C from final dilution to freezing. After thawing, non-equilibrated samples (0 h) 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 72 h) at 5°C before freezing using storage for 1h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48-72 h 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.

Use of commercial extenders and alternatives to prevent sperm agglutination for cryopreservation of brown bear semen.

The objective of this study was to evaluate different bovine and canine commercial semen extenders for cryopreservation of brown bear ejaculates and the effect of semen collection directly into extender on sperm agglutination. Semen samples were obtained by electroejaculation from 13 adult males. In experiment 1, eleven ejaculates from eight bears were used to evaluate Bioxcell and Andromed as extenders, whereas in experiment 2, nine ejaculates from six bears were used to evaluate Triladyl canine, CaniPro, and Extender 2 as extenders. An extender specifically developed for brown bears (Test-Tris-fructose-egg yolk-glycerol, TTF-ULE/bear) served as a control extender in both experiments. After thawing, total and progressive sperm motility and sperm viability were greater (P < 0.05) for TTF-ULE/bear and Andromed extenders than for Bioxcell in experiment 1 and greater (P < 0.05) for TTF-ULE/bear extender than for Triladyl Canine, CaniPro, and Extender 2 in experiment 2. In experiment 3, addition of handling extender (TTF-H) to the semen collection tube for eight ejaculates from seven bears resulted in less (P < 0.05) sperm agglutination in fresh samples (score 0.5 ± 0.2 vs. 1.8 ± 0.4 in diluted and control samples, respectively) with no effect on pre-freeze and post-thawing semen quality. In conclusion, TTF-ULE/bear is the most suitable extender for brown bear semen cryopreservation, but comparable results can be obtained with the commercial extender Andromed. In addition, collection of ejaculates directly in TTF-H extender decreases sperm agglutination in fresh samples.

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 × 10(6) 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.

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

Cryopreservation 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.

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

The 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).

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

The 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 72h 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 24h post-mortem, was higher compared to the Ex-EPID samples, although differences decreased at 48 and 72h. 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 72h. At 24h, 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 24h, 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 48h. The D320 extender preserve motility of epididymal spermatozoa but does not protect the status of the acrosome.