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The process of freezing and thawing induces certain cellular damage in rainbow trout (Oncorhynchusmykiss) spermatozoa. We have previously demonstrated that after freezing and thawing decreased fertility in rainbow trout (Oncorhynchusmykiss) spermatozoa, is related to sublethal damage to the plasma membrane. External cryoprotectants are known to stabilize the sperm cell membrane against such damage. In the current study, we used a basic freezing extender containing #6 Erdahl and Graham and 7% DMSO and added egg yolk, BSA, and a soybean-protein complex (DanPro S760) singly and in various combinations. To assess the effect of these cryoprotectants we evaluated the percentage of cells with progressive motility, permeability of cells to propidium iodide (viability) after exposure for 30 sec, 2, 5, 10 and 15 min. to hypo-and isoosmotic solutions of 10 and 300mOsm, and the in vitro fertility rate. Fertility trials were performed using 1.87 × 107spermatozoa/egg. Some of the tested stabilizers increased motility, increased viability, or reduced cell fragility after freezing and thawing. Nevertheless these quality improvements demonstrated by the “in vitro” tests do not always correlate with high fertility. The best membrane protection in terms of resistance to hypoosmotic shock was achieved when BSA and egg yolk were added to the extender. The highest fertility rates were obtained with DanPro S760®alone or in combination with BSA; the use of BSA with egg yolk did not improve this parameter. Our results demonstrated that some external cryoprotectants effectively increased membrane resistance during freezing and thawing, but some of the tested mixtures interfered with fertilization. Soybean protein concentrate provided good protection and increased fertility rates in cryopreserved trout spermatozoa.

Although cellular damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this damage and the fertility rates of eggs using postthawed milt. The apparent lack of such correlation may be due to other undetected sublethal cryodamage, which could affect the cell functionality and viability. This may be extremely important for freshwater fish spermatozoa whose ability to fertilize the egg requires dilution in water or hypoosmotic solutions, an hazardous environment for the cells. This study tested the change in cell permeability during cryopreservation, using Hoechst 33258 to assess cell permeability. The permeability of spermatozoa at different times after dilution in several hypoosmotic media were investigated. In the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied. Three CPT were tested (Me2SO, DMA, and methanol). In the second trial, the addition of egg yolk as a membrane stabilizer was investigated. Samples were frozen at −20°C/min in a programmable cooler and thawed in a 25°C water bath. Dilution in the CPTs slightly increased the susceptibility of cells to damage but freezing/thawing caused a dramatic increase in the fragility of cells, which were killed in a few seconds after their contact with the hypoosmotic solutions. Egg yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization media. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated sublethal cryodamage in the function of the sperm membrane that was not detected by standard procedures.

A pygmy sperm whale (Kogia breviceps; adult male; 350 kg) was stranded and died on a beach near Cabo Bustos (Asturias, North of Spain) on March 12th, 2005. Finding specimens of this species is a rare event on Spanish shores, although this whale is not considered endangered. Postmortem examination was performed 24 h later. Genitalia (testicles and epididymides) were extracted. The postmortem report indicated that vas deferens and seminal glands seemed to contain an important amount of semen, which was not recovered. Refrigerated genitalia were send to our laboratory, arriving around 40 h postmortem. The refrigerated testicles were in poor physical condition upon arrival, indicating advanced tissue detoriation. The epididymides (very long) were not closely attached to the testicles, but were connected by a loose conjunctive membrane. We divided the epididymides into four regions that approximated the (1) caput, (2) mid-region, (3) corpus, and (4) cauda. Physical characteristics of the genitalia are described in Table 1. The left testicle was larger, and possibly more active, than the right one. A sperm sample was obtained from the cauda region after incising the tissue. Osmolality and pH of the sample were 428 mOsm/kg and 6.62, respectively (maybe due to tissue breakdown) and the sperm concentration was 1194 106/mL. Spermatozoa were immotile, even after diluting in buffered medium; it is possible that postmortem damage occurred quickly. However, using flow cytometry we determined that 57? of cauda spermatozoa had intact plasma membranes and acrosomes (determined by staining with 37 mmol/mL propidium iodide and 1 ?g/mL PNA-FITC; Sigma, Madrid, Spain). Examination by phase contrast microscopy (600) showed many spermatozoa with abnormal heads and bent midpieces and flagella, even in the cauda (13? and 21?, respectively). Sperm head morphometry was studied using DiffQuick staining and an automated analysis system (SCA2000; Microptic, Barcelona, Spain). Mean sperm head size was 3.71 0.19 2.61 0.12 ?m in width and length, respectively. Computer analysis (AnalySiS-GmbH, Cologne, Germany) of phase contrast images revealed that the mean size of the sperm midpiece and flagellum were 3.44 0.19 and 40.95 2.02 ?m, respectively. The information obtained after postmortem recovery of the testes and epididymis should be useful to future conservation efforts of the pygmy sperm whale and similar species. The rapid deterioration of the testicular tissue by 40 h postmortem was not expected since good quality sperm samples have been obtained at similar postmortem intervals in other species. Therefore, we recommend that postmortem sperm recovery should be accomplished as rapidly as possible in this species.

Sperm morphology is an useful characteristic for estimating potential fertility. Currently, we are obtaining baseline information on various aspects of reproduction in the brown bear (Ursus arctos) with the intention of using the knowledge to establish a germplasm bank for the species. In the present report, we describe the results obtained using assisted sperm morphology analysis (ASMA, Sperm Class Analyzer; Microptic S.L, Barcelona, Spain) to analyze the morphological differences in epidydimal (caput, corpus, and cauda) and ejaculated brown bear spermatozoa. A post-mortem epididymal sperm sample was obtained from an adult brown bear after accidental death. The epididymides were excised, washed, and dissected into the three major segments; caput, corpus and cauda. Then multiple incisions were made in the tissue to allow migration of spermatozoa into the surrounding medium. Semen was collected by electroejaculation from five adult brown bears living in a semi-free ranging environment in the Cabarceno Park (Cantabria, Spain). Anesthesia was induced using tiletamine ? zolazepan (Zoletil 100; Virbac, Carras, France; 7 mg/kg), and ketamine (Imalgene 1000; Rhone Merieux, Lyon, France; 2 mg/kg). The electroejaculation unit (PT Electronics; Boring, Oregon) was connected to a 3-lateral electrode transrectal probe (26 mm in diameter, 320 mm in length). Ejaculation occurred at 6?10 V/250?300 mA. For head morphometry assessment, sperm samples were fixed in glutaraldehyde and slides were smeared and air-dried for 2 h. The samples were then stained with Diff-Quik staining (37C; 10 min in the red component and 15 min in the blue component). The area, perimeter, length and width, and ellipticity (length/width) of heads were measured from at least 100 spermatozoa/slide. As shown in Table 1, values obtained for each measure were similar in both epididymal and ejaculated spermatozoa. These results provide normal morphometry values for brown bear spermatozoa, a potentially useful characteristic for predicting fertility.  This work was supported in part by CANTUR S.A. and CICYT (CGL 2004?0278/BOS).

Brown bear is a highly endangered species in Spain and could benefit from biological resource banking. Currently, we are studying several reproductive aspects in order to aquire the knowledge for establishment of a germplasm bank for this species. One of our objectives is to develop adequate protocols for the evaluation of bear sperm before and after cryopreservation. We have used the fluorescent probe JC-1 protocol, which differentially stains mitochondria, according to its activity (Garner DL et al. 1997 Biol. Reprod. 57, 1401?1406). Here we describe one problem that arose using this staining for evaluation of extended bear semen. We electroejaculated 13 adult brown bears (Ursus arctos) (206?311 kg) housed in a half-freedom regime in the Cabarceno Park (Cantabria, Spain). Anesthesia was performed with tiletamine + zolazepan (Zoletil 100, 7 mg/kg; Virbac, Spain), and ketamine (Imalgene 1000, 2 mg/kg; Mericl, Sain). We used an electroejaculator (PT Electronics; Boring, OR, USA) with a 3-electrode transrectal probe (26 mm in diameter, 320 mm long). Ejaculation occurred at 10 V/250 mA. Samples were extended (prepared in our laboratory, Anel L et al. 2003 Theriogenology 60, 1293?1308; M3 modified) and cooled to 5C for 70 min (pre-freezing protocol). We analyzed individual (MI) and progressive (MP) motility by means of an automated motility analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA), using a phase contrast microscope (Nikon, 10). Mitochondrial status was analyzed after diluting the sample 1:100 with buffered medium (20 mM HEPES, 153 mM NaCl, 2.5 mM KOH, 10 mM glucose; Sigma, Madrid, Spain) and adding JC-1 (6.8 ?M final; Molecular Probes, The Netherlands). After 30 min at 37C, 100 cells were counted with an epifluorescence microscope (Nikon, 400), determining the percentage of sperm with orange-stained (active) mitochondria. We analyzed a total of 55 samples in three different models: fresh, pre-freezing, and thawed. We divided the samples into successful JC-1 staining (valids: V) or failed JC-1 staining (not valid: NV) (depending on the aspect of the stained cells). In not-valid samples we observed a greenish background, with almost no fluorescent spermatozoa. These observations were consistent in a given sample, giving the same V or NV result when we repeated the staining. In fresh and thawed groups there were no NV samples, but in the pre-freezing group there were 40 NV samples (73%). We calculated Pearson correlations (SAS; SAS Institute, Inc., Cary, NC, USA) between percent JC-1 orange population and MI and MP in fresh (r = 0.40 and 0.33; P < 0.001), thawed (r = 0.61 and 0.43; P < 0.001) and pre-freezing samples (r = ?0.11 and ?0.24; P > 0.05), all respectively. When pre-freezing samples were split between V and NV, the former had good correlations (r = 0.74 and 0.49; P < 0.05), and NV still did not (r = ?0.17, ?0.27; P > 0.05). We conclude that JC-1 staining is not reliable for the pre-freezing analysis of bear sperm, at least under the conditions described here. This could be due to the interaction of the extender or the refrigeration treatment with the sperm. However, this problem did not occur in the analysis of fresh and thawed samples. Nevertheless, it may be advisable to test other mitochondrial probes for analyzing this kind of samples.

The Somalia wild ass (Equus africanus somaliensis) is a critically endangered taxon (IUCN 2004 red list) which could benefit from biological resource banking. In this work, we studied the effect of different extenders applied to the cryopreservation of epididymal sperm obtained from one male of this subspecies. This animal (13 years old; housed in Cabarceno Park, Cantabria, Spain) was castrated because of very aggressive behavior with other mature males. Genitalia were dissected and weighed (testicles: right, 166 g, and left, 179 g; cauda epididymis: right, 9.3 g, and left, 11.8 g). Sperm were flushed from the cauda epididymis, yielding 15 mL of sample. Sperm concentration was 15 109 spermatozoa/mL, totaling 225 109 (allowing 4500 doses at 50 106 sperm/dose). Sperm motility (TM ? ? total motile; PM ? ? progressive; VAP ? average path velocity) was assessed by CASA (Microptic, Barcelona, Spain). Viability (VIAB ? ? viable sperm) and acrosomal status (ACR ? ? viable spermatozoa with intact acrosomes) were assessed using propidium iodide (37 ?mol/L) and PNA-FITC (1 ng/L) and flow cytometry. Chemicals were purchased from Sigma (Madrid, Spain). Part of the sample was divided into six aliquots and diluted 1:1 with different extenders: UL4: Tes-Tris-Fructose (TTF), 10? egg yolk (EG), and 4? glycerol (G); UL8: TTF, 20? EG, and 8? G; AND4: Andromed (Minitb, Tiefenbach, Germany) and 4? G; AND7: Andromed and 7? G; GENT: Gent 1045; and INRA: INRA96 and 4? G. Andromed, Gent, and INRA are commercial extenders. Samples were cooled to 5C (-0.2C/min) and then diluted to 200 106 sperm/mL. Samples were packed (0.5-mL straws) and frozen using a biofreezer (from 5C to -15C at -15C/min, and from -15C to -100C at -25C/min). Samples were thawed at 65C for 6 s, and assessed as for pre-freezing (Table 1). Post-thawing motility recovery using AND7 was excellent. The highest viability recovery was achieved by UL4, although that in AND7 was similar. The poor results of equine commercial extender Gent 1045 in this species are remarkable. Our results highlight the importance of species differences in the field of sperm cryopreservation. It is necessary to carry out continuous research for optimizing cryopreservation protocols in order to create germplasm banks for wild species.

Brown bear is a highly endangered species in Spain and could benefit from biological resource banking. Currently, we are studying several reproductive aspects in order to aquire the knowledge for establishment of a germplasm bank for this species. One of our objectives is to develop adequate protocols for the evaluation of bear sperm before and after cryopreservation. We have used the fluorescent probe JC-1 protocol, which differentially stains mitochondria, according to its activity (Garner DL et al. 1997 Biol. Reprod. 57, 1401?1406). Here we describe one problem that arose using this staining for evaluation of extended bear semen. We electroejaculated 13 adult brown bears (Ursus arctos) (206?311 kg) housed in a half-freedom regime in the Cabarceno Park (Cantabria, Spain). Anesthesia was performed with tiletamine + zolazepan (Zoletil 100, 7 mg/kg; Virbac, Spain), and ketamine (Imalgene 1000, 2 mg/kg; Mericl, Sain). We used an electroejaculator (PT Electronics; Boring, OR, USA) with a 3-electrode transrectal probe (26 mm in diameter, 320 mm long). Ejaculation occurred at 10 V/250 mA. Samples were extended (prepared in our laboratory, Anel L et al. 2003 Theriogenology 60, 1293?1308; M3 modified) and cooled to 5C for 70 min (pre-freezing protocol). We analyzed individual (MI) and progressive (MP) motility by means of an automated motility analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA), using a phase contrast microscope (Nikon, 10). Mitochondrial status was analyzed after diluting the sample 1:100 with buffered medium (20 mM HEPES, 153 mM NaCl, 2.5 mM KOH, 10 mM glucose; Sigma, Madrid, Spain) and adding JC-1 (6.8 ?M final; Molecular Probes, The Netherlands). After 30 min at 37C, 100 cells were counted with an epifluorescence microscope (Nikon, 400), determining the percentage of sperm with orange-stained (active) mitochondria. We analyzed a total of 55 samples in three different models: fresh, pre-freezing, and thawed. We divided the samples into successful JC-1 staining (valids: V) or failed JC-1 staining (not valid: NV) (depending on the aspect of the stained cells). In not-valid samples we observed a greenish background, with almost no fluorescent spermatozoa. These observations were consistent in a given sample, giving the same V or NV result when we repeated the staining. In fresh and thawed groups there were no NV samples, but in the pre-freezing group there were 40 NV samples (73%). We calculated Pearson correlations (SAS; SAS Institute, Inc., Cary, NC, USA) between percent JC-1 orange population and MI and MP in fresh (r = 0.40 and 0.33; P < 0.001), thawed (r = 0.61 and 0.43; P < 0.001) and pre-freezing samples (r = ?0.11 and ?0.24; P > 0.05), all respectively. When pre-freezing samples were split between V and NV, the former had good correlations (r = 0.74 and 0.49; P < 0.05), and NV still did not (r = ?0.17, ?0.27; P > 0.05). We conclude that JC-1 staining is not reliable for the pre-freezing analysis of bear sperm, at least under the conditions described here. This could be due to the interaction of the extender or the refrigeration treatment with the sperm. However, this problem did not occur in the analysis of fresh and thawed samples. Nevertheless, it may be advisable to test other mitochondrial probes for analyzing this kind of samples.