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 × 10⁶ 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 1 h 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 24 h 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.     

不同甘油浓度与平衡时间对食蟹猴精液冷冻效果的影响

探讨了不同甘油浓度(3%、5%、7%、11%)和不同平衡时间(30、60、90、120min)对食蟹猴(Macaca fascicularis)精液冷冻效果的影响,以建立和优化食蟹猴精液冷冻的程序。参照TTE稀释液成分组成改良型TTE,冷冻前和解冻后均检测精子的活力、畸形率、质膜完整性、顶体完整率。结果显示,平衡时间为30min时精子的冷冻解冻后活力、复苏率均高于平衡时间90min和120min组,差异显著(P<0.05),比60min组稍好;甘油浓度为3%、5%组的精子冷冻解冻后活力及复苏率均高于甘油浓度11%组,差异显著(P<0.05),比7%组好;不同甘油浓度各组间以及不同平衡时间各组间畸形率、质膜完整性、顶体完整率差异不显著(P<0.05)。由此得出如下结论,在食蟹猴精液冷冻中,在改良TTE中加入3%~5%的甘油且平衡30min可以获得较好效果,精子冻后活率和复苏率达到45%和62%。     

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.     

Effects of antioxidants and duration of pre-freezing equilibration on frozen-thawed ram semen

The objective was to evaluate the effects of various antioxidants and duration of pre-freezing equilibration on cryopreservation of ram semen. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control), or supplemented with reduced glutathione (GSH: 0.5, 1.0, and 2.0 mM), superoxide dismutase (SOD: 5, 10, and 20 U/mL), or catalase (CAT: 5, 10, and 20 U/mL), and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 12 or 24 h after equilibration. Total antioxidant capacity was determined in the in natura extenders and after addition of semen samples for various durations of processing (fresh/dilute, throughout refrigeration, and post-thaw). Plasma membrane (PI-CFDA), acrosome integrity (FITC-PNA), and mitochondrial membrane potential (JC-1) were determined in fresh/diluted, refrigerated, and post-thaw samples. Post-thaw sperm motility was assessed with a computerized analysis system (CASA). There were no significant differences in acrosome damage or mitochondrial membrane potential after refrigeration and freeze-thaw, regardless of antioxidant addition. Sperm plasma membrane integrity was worse (P < 0.05) with cryopreservation immediately after equilibration (average 20.1 ± 8.3; mean ± SD) than after 12 h of equilibration (average 42.5 ± 10.9); however, the addition of SOD and CAT (10 and 20 U/mL) resulted in no significant difference between post-equilibration intervals of 0 and 12 h. Total antioxidant activity was not different (P > 0.05) among treatments after sperm addition or throughout the refrigeration and post-thaw. In conclusion, adding GSH, SOD or CAT did not increase the total antioxidant capacity of semen, nor did it enhance the quality of the post-thaw sperm. However, maintenance of ram semen at 5 °C for 12 h prior to cryopreservation reduced membrane damage of frozen-thawed sperm.     

The effect of dichloroacetate and hydroxypyruvate on the entry of C from [1-C]alanine into urea in rat hepatocytes

Entry of metabolic ¹⁴CO₂ into urea is shown to be decreased by dichloroacetate although the production of ¹⁴CO₂ is stimulated 2-fold. Hydroxypyruvate, a product of dichloroacetate metabolism, increases the incorporation of metabolic ¹⁴CO₂ into urea. It is proposed that these effects result from changes in the cytoplasmic-mitochondrial pH gradient.     

Pre-freezing equilibration for 22 h improves post-thaw sperm functions in cryopreserved ram semen by reducing cholesterol efflux

Failure of cervical insemination with cryopreserved semen is hindering implementation of AI in sheep in field condition. Here the effect of equilibration time and catalase on post-thaw qualities of ram semen was investigated. Pooled semen was diluted (800 × 10⁶ sperm mL⁻¹) with a TES-Tris-fructose extender with 6% glycerol, 15% egg yolk and supplemented with 0, 50, 100 and 200 U mL⁻¹ catalase and packaged into 0.25 mL straws. In experiment 1, straws were equilibrated at 5 °C either for 3 h in a cold cabinet (E3) or for 10 (E10) and 22 h (E22) inside a refrigerator. In experiment 2, all straws were equilibrated for 22 h inside refrigerator. Straws were frozen at −25 °C min⁻¹ up to −125 °C using a cell freezer and finally plunged into liquid nitrogen. The post-thaw total and rapid motility were higher (P < 0.05) in E22 compared to E3 and E10. Sperm kinetics was comparable between E3 and E22, but lower in E10. Similarly, acrosome integrity, functional membrane integrity, percent high cholesterol (mCHO) and live-high mitochondrial membrane potential (MMP) were higher (P < 0.05) while live-high intracellular calcium and acrosome-reacted sperm were lower in E22 compared to E3 and E10. The percent rapid motile, high mCHO and live-high MMP were significantly (P < 0.05) lower in catalase-treated samples compared to the control, while the membrane integrity was comparable within the groups. In conclusion, pre-freezing equilibration for 22 h compared to 3 or 10 h resulted in higher post-thaw sperm functions while catalase had negative impact on cryopreservation of ram semen.     

Animal protein-free OptiXcell and shortened equilibration periods can replace egg yolk-based extender and slow cooling for rhinoceros semen cryopreservation

OptiXcell (OP) was tested as an animal protein-free alternative to an egg yolk-based extender for rhinoceros semen cryopreservation and shorter chilling/equilibration periods were evaluated. Semen was collected from three rhinoceros species: black (Diceros bicornis; n = 2), white (Ceratotherium simum; n = 2), and greater one-horned (GOH; Rhinoceros unicornis; n = 3). Controls were diluted with equine extender (EQ) or OP and equilibrated for 1 h. Treatments were diluted with extender and cooled for 15 min (fast: FEQ; FOP) or not cooled (immediate: IEQ; IOP), prior to cryopreservation. Motility decreased post-thaw (EQ: 50.7 ± 5.2%; OP: 52.9 ± 3.4%) from fresh (82.9 ± 2.9%), was higher in OP than IOP (38.6 ± 4.9%; P ≤ 0.05) and decreased over time (P ≤ 0.05). Post-thaw acrosomal integrity was lower in EQ, FEQ, and IEQ (56.9 ± 0.7; 56.6 ± 4.5; 54.9 ± 2.9%) than OP, FOP, IOP (71.8 ± 4.7; 71.9 ± 3.8; 69.9 ± 4.5%) and fresh (72.6 ± 1.4%; P ≤ 0.05). Progression and viability were lower in EQ (2.8 ± 0.2; 61.9 ± 7.4%) and OP (3.1 ± 0.2; 53.4 ± 6.9%) than fresh (3.7 ± 0.2; 87.2 ± 1.3%), decreased over time (P ≤ 0.05) but not different among treatments (P > 0.05). Morphology did not differ between fresh (75.0 ± 4.9% normal) and any treatment group (70.0–77.8%) or over time (P > 0.05). OptiXcell is comparable to egg yolk-based EQ when used for rhinoceros semen cryopreservation. Furthermore, chilling/equilibration can be reduced with little impact on sperm characteristics.     

Evaluations of reduction potential data in relation to coupling, kinetics and function

 This commentary addresses the effect and measurement of time-dependent contributions to reduction potentials. Reduction potentials form the basis for many quantitative or semi-quantitative judgements in biological redox chemistry. However, since data are obtained under an assumption of equilibrium being established, their relevance to biological functions requires consideration of the kinetics of the subprocesses that contribute to or influence the overall free energy change. Initial and final states effective in rapid and complex biological functions may differ considerably from those analysed after slow equilibration in a sample tube. A shortcoming of traditional potentiometric measurements is that the time domain is not probed. Voltammetry, a technique that has been much less widely applied in biological chemistry than in chemistry, examines redox transformations in both potential and time domains, and may enable a more realistic picture to be derived. Received, accepted: 26 November 1996     

Improved cryopreservation protocol for Blanca-Celtibérica buck semen collected by electroejaculation

The collection of sperm samples by electroejaculation (EE) leads to an increase of the production of seminal plasma which could modify the tolerance of spermatozoa to the cryopreservation procedure. This study aims to compare a standard sperm cryopreservation protocol for samples collected by artificial vagina (AV) with the same protocol and modifications to this for samples obtained by EE. Semen from six males of Blanca-Celtibérica goat breed was collected by AV (control) and EE, and three experiments were conducted. In Experiment 1, it was examined the effects of egg yolk concentration contained in freezing extender (0%, 1.5%, 10% and 20% of egg yolk); in Experiment 2, it was evaluated the cooling rate from 30 to 5 °C (fast: 10 min and slow: 90 min) and the temperature of glycerol addition (30 and 5 °C); and in Experiment 3, it was examined the time of equilibration at 5 °C (0, 1, 2 or 3 h). A heterologous in vitro fertilization test was carried out in order to compare the fertility of control samples with that resulting from the EE protocol which showed the highest sperm quality. Results showed greater sperm motility parameters after thawing for control samples cryopreserved in standard conditions in the three experiments. For samples collected by EE, extender with 20% egg yolk, a slow cooling rate and a longer equilibration time (3 h) provided higher sperm quality, and no differences were observed between temperatures of glycerol addition. Samples collected by EE and cryopreserved with the protocol which yielded the best sperm quality after thawing showed higher fertility compared to AV.     

Vitrification of porcine immature oocytes: Association of equilibration manners with warming procedures,and permeating cryoprotectants effects under two temperatures

The aim of this study was to evaluate the association of equilibration manners with warming procedures, and the different permeating cryoprotectants (pCPAs) effects under two temperatures, in terms of survival, maturation and subsequent parthenogenetic development of porcine immature oocytes after Cryotop vitrification. In Experiment 1, oocytes were equilibrated by exposure to 5% (v/v) ethylene glycol (EG) for 10 min (EM1) or stepwise to 7.5% (v/v) and 15% (v/v) EG for 2.5 min respectively (EM2). Warming procedures were performed in 1.0 M sucrose for 1 min, then in 0.5 and 0.25 M sucrose for 2.5 min respectively (WP1), or in 0.5, 0.25 and 0.125 M sucrose each step for 2 min (WP2), or in 0.25, 0.125 and 0.063 M sucrose each step for 2 min (WP3). After 2 h of warming, the survival rate of oocytes treated by EM1 and WP1 was significantly higher (P < 0.05) than that of the other groups. Moreover, a similar proportion of survival and nuclear maturation in all vitrified groups was obtained after completion of the IVM. No significant difference in blastocyst development was observed among vitrified groups except the group treated by EM2 and WP3. In Experiment 2, oocytes were vitrified by using EG alone, EG combined with dimethyl sulphoxide (EG + DMSO) or propylene glycol (EG + PROH) as pCPAs under 25 °C and 39 °C. The percentages of cryosurvival and nuclear maturation were similar in all vitrified groups. Under 25 °C, the embryo development and total cell numbers of blastocysts were not significantly different among EG, EG + DMSO and EG + PROH groups. However, the application of EG + PROH at 39 °C resulted in significantly decreased both cleavage and blastocyst formation rates. In conclusion, our data showed that equilibration manner and warming procedure affect the cryosurvival of porcine immature oocytes, and the combination of pCPAs cannot give a better cryopreservation outcome whether 25 °C or 39 °C. Notably, the Cryotop vitrification accompanied by our modified strategy for porcine immature oocytes could achieve high survival and respectable blastocyst production.