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

López-Urueña, E.; Álvarez, M.; Gomes-Alves, S.; Manrique, P Luis Phanor; Anel-López, Luis; Chamorro, C.A.; Borragán, S.; Paz, P. de; Anel, L.

doi:10.1016/j.cryobiol.2014.10.001

Cited by 5 publications

(3 citation statements)

References 46 publications

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“…To prevent disease transmission in most domestic species, the addition of antibiotics to sperm extenders is mandatory in the European Union and so common in other countries outside ( 9 ). In this context, the antibiotic supplementation as an additive in most of the handling and preservative sperm extenders has been widely used in many species; in domestic ones such as ram ( 10 ), bull ( 11 ), stallion ( 12 ), or boar ( 13 ) and wild species: red deer ( 14 ) or brown bear ( 15 ). Many studies have demonstrated the beneficial effect of antibiotics inhibiting the bacterial proliferation in the sperm samples during their storage ( 1 , 16 – 18 ).…”

Section: Introductionmentioning

confidence: 99%

Comparing the Effect of Different Antibiotics in Frozen-Thawed Ram Sperm: Is It Possible to Avoid Their Addition?

et al. 2021

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It is crucial to perform a deep study about the most extensively used antibiotics in sperm extenders. Most of the protocols and concentrations used in ram are direct extrapolations from other species. It is important to establish species-specific antibiotic treatments to optimize their use and if it is possible to reduce the quantity. Previews studies have assessed some aspects of sperm quality in vitro, but this study aimed to go further and assess the effect of three different antibiotic treatments, which are the most extensively used, not only in sperm quality or assessing the inhibitory effect on bacterial growth but also assessing these important parameters of productivity such as fertility, prolificacy, fecundity, and sex-ratio during a freeze-thaw process. Gentamicyn (G) treatment showed the worst results, not only concerning sperm quality but also in the reproductive trials exhibiting a toxical effect at the experiment concentration, and being the most powerful inhibiting bacterial growth. For its part, Lincomicyn-spectinomycin (LS) showed similar results inhibiting bacterial growth but it did not show a detrimental effect either in sperm quality or in reproductive parameters. Penicillin-streptomycin (PS) showed good results in the sperm quality and in the reproductive in vivo trials, but it showed a very poor effect inhibiting bacterial growth probably due to some kind of antibiotic resistance. According to our results, there is not a significant positive relationship between the higher bacterial inhibitory activity of LS and PS samples, and the sperm quality respect Control samples (without antibiotics). In the case of G, which exhibited the most effective as antibacterial, we observed a toxic effect on sperm quality that could be translated on productivity parameters. Our results suggest that the bacterial contamination control in frozen-thawed semen may be possible without the use of antibiotics, although the effects of longer periods of cooling storage and different temperatures of storage need to be further investigated for animal semen. At this point, a reflection about a drastic reduction in the use of antibiotic treatments in sperm cryopreservation is mandatory, since freezing conditions could keep sperm doses contamination within the levels recommended by regulatory health agencies.

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Section: Introductionmentioning

confidence: 99%

Comparing the Effect of Different Antibiotics in Frozen-Thawed Ram Sperm: Is It Possible to Avoid Their Addition?

et al. 2021

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“…This is possible because this period allows water to exit and facilitates the entrance of permeable cryoprotectants (e.g., glycerol), which exert their cryoprotective effect on the sperm [ 35 , 51 , 52 , 53 ]. In several studies of other species, an association has previously been found between an average equilibration time (2–4 h) and the preservation of the motility and integrity of sperm membranes [ 52 , 53 , 54 , 55 , 56 ]. However, there are no published studies regarding different durations of this stage and their effect on the thawing of Iberian red deer epididymal spermatozoa, with the most frequently used equilibrium time being 120 min [ 6 , 26 ].…”

Section: Discussionmentioning

confidence: 99%

Freezing Protocol Optimization for Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm under Field Conditions

Medina-Chávez¹,

Soler²,

Martín-Maestro³

et al. 2022

Animals

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Creating germplasm banks of wild species, such as the Iberian red Deer (Cervus elaphus hispanicus) can be challenging. One of the main difficulties is the obtention and cryopreservation of good-quality reproductive cells when the spermatozoa are obtained from epididymides after death. To avoid a loss of seminal quality during transport, developing alternative methods for cooling and freezing sperm samples under field conditions is necessary. The objective of this study was to evaluate the effects of different durations of equilibrium and different techniques of cooling and freezing on Iberian red deer epididymal sperm quality after thawing to optimize the processing conditions in this species. Three experiments were carried out: (I) evaluation of refrigeration in straws or tubes of 15 mL; (II) study of equilibration period (0, 30, 60, or 120 min); and (III) comparison of four freezing techniques (liquid nitrogen vapor in a tank (C), liquid nitrogen vapor in a polystyrene box (B), dry ice (DY), and placing straws on a solid metallic plate floating on the surface of liquid nitrogen (MP)). For all experiments, sperm motility and kinematic parameters, acrosomal integrity, sperm viability, mitochondrial membrane potential, and DNA integrity were evaluated after thawing. All statistical analyses were performed by GLM-ANOVA analysis. Samples refrigerated in straws showed higher values (p ≤ 0.05) for mitochondrial activity and lower values (p ≤ 0.05) for apoptotic cells. Moreover, the acrosome integrity showed significant differences (p ≤ 0.05) between 0 and 120 min, but not between 30 and 60 min, of equilibration. Finally, no significant differences were found between freezing in liquid nitrogen vapors in a tank or in a box, although there was a low quality after thawing when the samples were cryopreserved in dry ice or by placing straws on a solid metallic plate floating on the surface of liquid nitrogen. In conclusion, under field conditions, it would be possible to refrigerate the sperm samples by storing them in straws with a 120 min equilibration period and freezing them in liquid nitrogen vapors in a tank or box.

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“…The cryopreservation protocol for brown bear sperm developed by our group (Anel et al., , ; Lopez‐Urueña, Alvarez, Gomes‐Alves, Manrique et al., ; Lopez‐Urueña, Alvarez, Gomes‐Alves, Martinez‐Rodriguez et al, ; de Paz et al., ), involves the addition of glycerol in two steps (an initial 1:1 dilution at room temperature [RT]) and up to final concentration at 5°C using a specific extender (TTF‐ULE‐Bear). Gelatine was added at RT because seminal samples with gelatine become solid at below 20°C and for this reason the final dilution (100 × 10 6 sperm ml −1 , glycerol 6%) was made and packaged into plastic straws at RT.…”

Section: Introductionmentioning

confidence: 99%

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

López-Urueña

Anel-López

Borragán³

et al. 2016

Reprod Domestic Animals

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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.

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

Cited by 5 publications

References 46 publications

Comparing the Effect of Different Antibiotics in Frozen-Thawed Ram Sperm: Is It Possible to Avoid Their Addition?

Comparing the Effect of Different Antibiotics in Frozen-Thawed Ram Sperm: Is It Possible to Avoid Their Addition?

Freezing Protocol Optimization for Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm under Field Conditions

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

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