Banking North American buffalo semen

The purpose of this study was to develop a procedure to collect and preserve semen from wood bison (Bison bison athabascae) and plains bison (Bison bison bison). Semen samples from three wood and three plains bison bulls were collected by electroejaculation from June through October. In addition, sperm was collected from the cauda epididymis of seven plains bison. Semen was cryopreserved using two commercially available cryopreservation media, an egg yolk-based medium (Triladyl), and a medium free of products of animal origin (Andromed). Sperm morphology and motility were recorded on fresh and post-thawed semen samples. Total sperm motility was not different between plains and wood bison for the months of June (50%), July (69%) and October (54%). However, total sperm motility for wood bison was higher (P < 0.05) than plains bison for the months of August and September (August: 80% vs 55%; September: 73% vs 40%). Plains and wood bison did not differ in mean total and mean progressive motility (35 and 15%, respectively) of frozen-thawed sperm samples. The post-thaw motility of Triladyl-treated sperm was higher (P < 0.05) than Andromed-treated sperm (35% vs 13%, respectively). Interestingly, post-thawed epididymal spermatozoa had higher total motility (P < 0.05) than post-thawed electroejaculated sperm when cryopreserved with a medium free of products of animal origin (Andromed; 35% vs 9%, respectively). In conclusion, we used electroejaculation to collect high quality bison semen, and cryopreserved it for future needs.     

Sperm characteristics in plains (Bison bison bison) versus wood (Bison bison athabascae) bison

The objective was to compare sperm characteristics between the two subspecies of North American bison, plains bison (Bison bison bison) and wood bison (Bison bison athabascae). Frozen-thawed ejaculated sperm from age-matched plains (n = 3) and wood (n = 2) bison were evaluated for morphometry, motility, viability, protein profile, and in vitro fertilization characteristics. Sperm morphometry and motility were assessed with computer-based systems, viability was assessed with SYBR-14 and propidium iodide, and fertilizing ability was determined using a heterologous in vitro fertilization system (using bovine oocytes). For plains versus wood bison, there were significant differences for head width (4.76 ± 0.22 vs 4.71 ± 0.19 μm; mean ± SD), head area (35.64 ± 1.91 vs 34.72 ± 2.64 μm²), head perimeter (23.61 ± 0.68 vs 23.31 ± 0.98 μm), midpiece length (14.58 ± 0.4 vs 14.36 ± 0.51 μm), midpiece width (0.81 ± 0.06 vs 0.79 ± 0.07 μm), and tail length (46.61 ± 2.15 vs 45.98 ± 2.08 μm). However, there was no significant difference in head length (overall, 9.04 ± 0.37 μm), progressive motility (41.16 ± 8.39%), or viability (41.58 ± 5.58%). Based on two-dimensional gel electrophoresis, 93 out of 113 protein spots were similar in their expression patterns. Furthermore, we inferred that differences in sperm biometry between these subspecies did not affect in vitro fertilization percentage (overall, 82.62 ± 12.13%). Based on these findings, we concluded that plains bison were an appropriate research model for developing reproductive technologies for wood bison.     

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.     

Quantification of damage at different stages of cryopreservation of endangered North American bison (Bison bison) semen and the effects of extender and freeze rate on post-thaw sperm quality

Semen cryopreservation is an important technique for the banking of animal germplasm from endangered species and exploitation of genetically superior sires through artificial insemination. Being a member of bovidae family, bison semen has poor freezing ability as compared to dairy and beef bulls' semen. This study was designed to quantify the damage to bison sperm at different stages of cryopreservation, and to determine the effects of extender (commercial Triladyl(?) vs. custom made tris-citric acid [TCA]) and freeze rate (-10, -25 and -40°C/min) on post-thaw quality of bison semen. Semen was collected from five bison bulls (three woods and two plains) via electroejaculation. In Experiment 1, semen was diluted in Triladyl? extender and frozen with freeze rate -10°C/min. Sperm motility characteristics were recorded in fresh, diluted, cooled (4°C) and freeze-thawed semen using computer-assisted sperm analyzer (CASA). In Experiment 2, semen was diluted in Triladyl? or TCA extender, and frozen with three different freeze rates, i.e. -10, -25 or -40°C/min. Thawing was performed at 37°C for 60s. Post-thaw sperm motility characteristics were assessed using CASA, and sperm structural characteristics (plasma membrane, mitochondrial membrane potential and acrosomes) were evaluated using flow cytometer, at 0 and 3h while incubating semen at 37°C. In Experiment 1, total and progressive motilities did not differ among pre-freeze stages of cryopreservation (P>0.05). However, sperm total and progressive motilities declined (P<0.001) in freeze-thawed semen by 35% and 42%, respectively, compared to after cooling (pre-freeze) semen. In Experiment 2, Triladyl?, as compared to TCA, yielded greater (P<0.05) post-thaw sperm total motility (41% compared to 36%) and progressive motility (34% compared to 29%) at 0h, respectively. The percent change in post-thaw sperm total and progressive motilities, VAP, VCL, VSL, IPM-high ΔΨm and IPM-IACR during 3h incubation at 37°C, was less (P<0.05) in TCA than in Triladyl?. There was an effect of freeze rate on post-thaw sperm average path velocity at 0h, and total motility, progressive motility, VCL, IPM and IPM-IACR at 3h were the greatest (P<0.05) when bison semen was frozen at -40°C/min. Likewise, the percent change in post-thaw sperm total and progressive motilities, during 3h incubation at 37°C, was less (P<0.05) in bison semen frozen at -40°C/min. All post-thaw bison sperm characteristics decreased (P<0.05) from 0h to 3h, during incubation at 37°C. In conclusion, the maximum damage to bison sperm occurred during freeze-thaw processes. Post-thaw total and progressive motilities of bison sperm were greater in Triladyl? at 0h whereas sperm survival was greater in TCA extender during 3h post-thaw incubation. Bison sperm had greater survival (P<0.05) when frozen at -40°C/min freeze rate.     

Genetic variation within and relatedness among wood and plains bison populations.

There are two recognized subspecies of bison, wood (Bison bison athabascae) and plains (Bison bison bison) bison. The establishment of most bison populations from a small number of individuals has raised concerns about their genetic variation. To this end, 11 bison populations were surveyed with 11 microsatellite loci in order to calculate genetic variation and genetic distances. Mean number of alleles ranged between 3.18 at Antelope Island State Park (Utah) and 6.55 at Wood Buffalo National Park (Alberta and Northwest Territories). Mean heterozygosity ranged from 0.295 at Antelope Island State Park to 0.669 at Custer State Park (South Dakota). The amount of genetic variability present in the bison populations as measured by mean number of alleles and overall probability of identity was found to correlate with the number of founders for all sampled populations. The G-test for heterogeneity revealed some evidence for the existence of subpopulations at Wood Buffalo National Park, however very small genetic distances between these subpopulations suggest that nuclear material from the plains bison introduced into Wood Buffalo National Park has diffused throughout the park. Genetic distances between the sampled populations were generally larger between than within the two bison subspecies.     

Recovery and cryopreservation of epididymal sperm from agouti (Dasiprocta aguti) using powdered coconut water (ACP-109c) and Tris extenders

The objective was to compare the use of powdered coconut water (ACP-109c; ACP Biotecnologia, Fortaleza, CE, Brazil) and Tris extenders for recovery and cryopreservation of epididymal sperm from agouti. The caudae epididymus and proximal ductus deferens from 10 sexually mature agoutis were subjected to retrograde washing using ACP-109c (ACP Biotecnologia) or Tris. Epididymal sperm were evaluated for motility, vigor, sperm viability, membrane integrity, and morphology. Samples were centrifuged, and extended in the same diluents plus egg yolk (20%) and glycerol (6%), frozen in liquid nitrogen, and subsequently thawed at 37°C for 1 min, followed by re-evaluation of sperm characteristics. The two extenders were similarly efficient for epididymal recovery, with regard to the number and quality of sperm recovered. However, for both extenders, sperm quality decreased (P < 0.05) after centrifugation and dilution. After sperm cryopreservation and thawing, there were (mean ± SEM) 26.5 ± 2.6% motile sperm with 2.6 ± 0.2 vigor in the ACP-109c (ACP Biotecnologia) group, which was significantly better than 9.7 ± 2.6% motile sperm with 1.2 ± 0.3 vigor in Tris. In conclusion, agouti epididymal sperm were successfully recovered using either ACP-109c (ACP Biotecnologia) or Tris extenders; however, ACP-109c (ACP Biotecnologia) was a significantly better extender for processing and cryopreserving these sperm.     

几种冷冻稀释液与单胺类防冻剂对中缅树鼩精子冷冻存活率的影响

该文实验精子采自昆明地区经笼养驯化的树鼩(Tupaia belangeri),检测其冷冻前后运动度、顶体完整率以及检测部分冷冻精子的受精能力。实验一:选用8种已报道的冷冻稀释液TTE、TCG、TCF、TTG、BWW、BTS、DM、SR稀释鲜精,并添加0.4mol/LDMSO,4℃预冷平衡2h后,TTE、DM和SR稀释液的精子的运动度与鲜精无差别(P>0.05),其余处理组均显著下降(P<0.05)。冷冻复苏后,各组的运动度显著低于预冷平衡处理后的运动度(P<0.05);DM组的复苏运动度显著高于其他稀释液组(P<0.05),BWW组最低(P<0.05)。对于顶体完整率,与鲜精相比,4℃平衡2h后,TTE和DM组精子的顶体完整率显著高于BWW、BTS和SR组(P<0.05)。冷冻复苏后,DM组精子的顶体完整率显著高于其它(除了TTE)冷冻组(P<0.05)。实验二:在DM稀释液基础上分别添加4种浓度0.2、0.4、0.8和1.2mol/L的二甲基甲酰胺(DF)、甲酰胺(F)、二甲乙酰胺(DA)和乙酰胺(A)以及0.4mol/LDMSO,经过预冷平衡处理后,与鲜精相比,各防冻剂组的精子运动度没有下降(P>0.05);冷冻解冻后,各冷冻组精子的运动度显著低于预冷平衡处理后的精子运动度(P<0.05);0.8mol/LDF和0.4mol/LDMSO组精子的运动度显著高于其它冷冻组(P<0.05)。对于顶体完整率,预冷平衡处理后各高浓度组的比率显著下降;冷冻复苏后,0.4mol/LF和0.4mol/LDF组精子的顶体完整率相对较高。实验三,人工授精实验中,DM+0.8mol/LDF冷冻精子的受精率为16.7%,DM+0.4mol/LDMSO的受精率为50.0%。以上实验结果提示,含卵黄的非离子冷冻稀释液对树鼩精子冷冻保护效果好,但单胺类防冻剂的防冻效果还需进一步深入研究。     

Phylogenetic relationships among the European and American bison and seven cattle breeds reconstructed using the BovineSNP50 Illumina Genotyping BeadChip

Here we present the first attempt to use the BovineSNP50 Illumina Genotyping BeadChip for genome-wide screening of European bison Bison bonasus bonasus (EB), two subspecies of American bison: the plains bison Bison bison bison (PB), the wood bison Bison bison athabascae (WB) and seven cattle Bos taurus breeds. Our aims were to (1) reconstruct their evolutionary relationships, (2) detect any genetic signature of past bottlenecks and to quantify the consequences of bottlenecks on the genetic distances amongst bison subspecies and cattle, and (3) detect loci under positive or stabilizing selection. A Bayesian clustering procedure (STRUCTURE) detected ten genetically distinct clusters, with separation among all seven cattle breeds and European and American bison, but no separation between plain and wood bison. A linkage disequilibrium based program (LDNE) was used to estimate the effective population size (N _e) for the cattle breeds; N _e was generally low, relative to the census size of the breeds (cattle breeds: mean N _e = 299.5, min N _e = 18.1, max N _e = 755.0). BOTTLENECK 1.2 detected signs of population bottlenecks in EB, PB and WB populations (sign test and standardized sign test: p = 0.0001). Evidence for loci under selection was found in cattle but not in bison. All extant wild populations of bison have shown to have survived severe bottlenecks, which has likely had large effects on genetic diversity within and differentiation among groups.     

The seasonal fertility of North American bison (Bison bison) bulls

The objective of this study was to determine the presence and magnitude of seasonal fluctuations in semen quality and other reproductive indices in bison bulls. Testicles from 288 commercially slaughtered bison bulls were collected monthly over a 1-year period. Carcass and testicle weight were determined and measurements of seminiferous tubule lumen, diameter, and epithelial thickness were made. Sperm cell morphology and defects were described and quantified using epididymal semen from each testicle. Twenty-one Plains (Bison bison bison) and Wood bison (Bison bison athabascae) breeding bulls, averaging 6.0 years of age (range 2.5-8.0), from three farms were selected for semen collection and evaluation on the basis of producer co-operation. Semen was collected by electro-ejaculation on four seasonal occasions during a 12-month period. Ejaculate quality was judged on the basis of volume, density, gross and individual motility, morphology, live/dead ratio, and concentration. Sperm cell morphologies were evaluated microscopically and classified according to criteria used for bovine semen. Fecal testosterone was measured at each semen collection using a commercial competitive binding radioimmunoassay. There was an increase in carcass weights over the study period and testis weights were moderately correlated (r=0.44) with carcass weights. However, mean testes weights were heavier (P<0.05) in the summer than in winter, spring, or fall periods. There were no differences in the proportion of normal and abnormal epididymal sperm between seasons but there were seasonal changes in the testicular parenchyma. Seminiferous tubule and lumen diameter, and epithelial thickness were greatest (P<0.05) in summer. Live bulls gained weight between April and November, but lost weight over the winter. Normal sperm cell percentages as well as individual sperm cell motility in electro-ejaculated sperm samples were higher (P<0.05) at the pre-breeding collection relative to other collections, but no change in sperm cell concentrations occurred over the study period. Fecal testosterone concentrations were highest at the pre-breeding period (June) but decreased (P<0.05) in each subsequent collection to reach their lowest levels in the April. While many changes in seen characteristics were not significant, overall results indicate the presence of some reproductive seasonality and increased testicular capacity in the summer breeding season. Bulls showing marginal semen quality in the winter but otherwise carrying desirable genetic traits may warrant another evaluation in late spring prior to being culled from a breeding program.     

Effect of semen collection method (artificial vagina vs. electroejaculation), extender and centrifugation on post-thaw sperm quality of Blanca-Celtibérica buck ejaculates

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl(?), Andromed(?) and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1-/PI- (intact spermatozoa), and Mitotracker+/YO-PRO-1- (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl(?) and Andromed(?) resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl(?) or Andromed(?) are used as freezing extenders.     

Cryopreservation of goat spermatozoa: comparison of two freezing extenders based on post-thaw sperm quality and fertility rates after artificial insemination

TRIS-glucose or skim milk extenders are most commonly used for cryopreserving goat sperm. The aim of this study was to compare the ability of two extenders based on TRIS and skimmed milk buffer to maintain sperm viability after cryopreservation. Goat semen samples (n=110) were frozen with TRIS and with milk extender and thaw. Sperm motion parameters, morphology and acrosomal integrity were assessed in fresh and frozen-thawed samples by Sperm Class Analyzer (SCA) and Diff-Quik and Spermac staining techniques. Pregnancy rates were obtained after cervical insemination with frozen semen doses. The cryopreservation process had a significant effect on acrosome and kinematic parameters. TRIS extender provided more effective preservation of total motility, velocity parameters and amplitude of lateral head displacement after freezing. The percentage of acrosome intact spermatozoa was significantly higher in samples diluted with milk extender. In the insemination doses, mean values of velocity parameters and lateral head displacement were higher in doses processed in TRIS. Spermatozoa frozen in milk extender was mathematically greater than for those frozen with TRIS extenders, though no significant difference exists. We conclude that post-thaw kinematic parameters and acrosome integrity assessed after 1h of incubation was acceptable in both extenders which indicated the feasibility of cryopreserving goat spermatozoa. TRIS extender results in better in vitro performance compared to milk, though these improvements were not reflected in fertility results. Semen doses cryopreserved in milk extender provided greater pregnancy rates after intra-cervical insemination compared to those in TRIS extender (52.4% versus 42.9%).     

Effect of glycerol concentration, Equex STM® supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (Vicugna pacos) sperm

Two experiments were conducted to determine the effects of glycerol concentration and Equex STM® paste on the post-thaw motility and acrosome integrity of epididymal alpaca sperm. In Experiment 1, epididymal sperm were harvested from male alpacas, diluted, and cooled to 4 °C in a Lactose cooling extender, and pellet-frozen in a Lactose cryodiluent containing final glycerol concentrations of 2, 3, or 4%. In Experiment 2, epididymal sperm were diluted in Biladyl®, cooled to 4 °C, stored at that temperature for 18-24 h, and further diluted with Biladyl® without or with Equex STM® paste (final concentration 1% v:v) before pellet freezing. In Experiment 1, sperm motility was not affected by glycerol concentration immediately (2%: 16.1 ± 4.6%; 3%: 20.5 ± 5.9% and 4%: 18.5 ± 6.6%; P > 0.05) or 3h post thaw (< 5% for all groups; P > 0.05). Post-thaw acrosome integrity was similar for sperm frozen in 2% (83.6 ± 1.6%), 3% (81.3 ± 2.0%) and 4% glycerol (84.8 ± 2.0%; P > 0.05) but was higher 3h post-thaw for sperm frozen in 3% (75.7 ± 3.8%) and 4% (77.2 ± 4.1%) than 2% glycerol (66.9 ± 2.7%; P < 0.05). In Experiment 2, sperm motility was higher immediately after thawing for sperm frozen in the presence of Equex STM® (Equex®: 21.5 ± 3.5%; control: 14.4 ± 2.1%; P < 0.05) but was similar at 3h post-thaw (P > 0.05). Acrosome integrity was similar for sperm frozen with or without Equex STM® paste immediately (control: 89.6 ± 1.2%; Equex®: 91.1 ± 1.4%; P > 0.05) and 3 h post-thaw (control: 69.3 ± 3.7%; Equex®: 59.9 ± 5.8%; P > 0.05). Sperm cryopreserved in medium containing 3-4% glycerol and 1% Equex STM® retained the best motility and acrosome integrity, even after liquid storage for 18-24 h at 4 °C prior to cryopreservation.     

Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: Comparison between cysteine and rosemary (Rosmarinus officinalis)

Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10 mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3 h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10 mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3 h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system.     

Long-term preservation of chilled canine semen: effect of commercial and laboratory prepared extenders

The present study was conducted to evaluate chilled semen conservation over time in 3 commercial and 4 laboratory prepared extenders, including a new Tris-glucose extender. The beneficial effect of adding egg yolk to these media was also analyzed. The effects of these extenders on motility and acrosome reaction were characterized objectively using a computer-aided semen analyzer and the chlortetracycline staining, respectively. No significant differences were observed when comparing the different commercial extenders without egg yolk, but addition of egg yolk improved all motility parameters significantly (preservation of 50% of motility was observed at 3.2+/-1, 2.9+/-0.5, 2.3+/-0.5, 8.5+/-0.2, 5.4+/-1.1, 5.2+/-0.4 d, for Biladyl, green extender and fresh-phos extenders without and with egg yolk, respectively). Motility parameters were best preserved in egg yolk supplemented Biladyl extender with a mean percentage of 86.3+/-10.5 motile spermatozoa after 7 d at 4 degrees C. Efficacy of egg yolk-supplemented commercial extenders on sperm motility at 4 degrees C was (in decreasing order) as follows: Biladyl > green extender > fresh-phos. However, high quality motility and the percentage of motile spermatozoa were highest with some of the laboratory prepared extenders: a 50% conservation rate of motile spermatozoa was observed following the use of supplemented egg yolk extenders. These are classified in decreasing order as follows: Tris-glucose (13+/-1 d) > Tris-fructose (9.7+/-0.6) > EDTA (4.+/-0.6 d) > Tris-bes (3.6+/-1.1 d). A low concentration of motile spermatozoa was still observed in the Tris-glucose egg yolk extender 16 d after collection, clearly demonstrating the importance of the medium and the beneficial effect of egg yolk on sperm motility of 4 degrees C chilled semen. Similar effects of extender were observed for acrosome reactions. Egg yolk clearly had a protective effect reducing acrosome reactions significantly in all media tested as follows: the highest acrosome losses were observed in the fresh-phos and EDTA extenders without egg yolk; the lowest rate was observed with Tris-glucose supplemented egg yolk extender. In conclusion, at 4 degrees C, egg yolk extender best-protected sperm motility parameters. Differences in osmolarity between the extenders in terms of substrate related to sperm metabolic activity may explain the optimal results obtained using egg yolk-supplemented Tris-glucose extender, which preserved motility and acrosome integrity in chilled dog semen. These results indicated that good quality dog spermatozoa could be preserved for up to 10 d.     

Trehalose improves rabbit sperm quality during cryopreservation

High levels of reactive oxygen species are associated with spermatozoa cryopreservation, which bring damage to functional spermatozoa. The aim of the present study was to investigate whether and how the freezing extenders supplemented with trehalose was beneficial for the survival of rabbit spermatozoa. semen was diluted with Tris-citrate-glucose extender addition of different concentrations of trehalose. Addition of 100 mM trehaose significantly improved post-thaw rabbit sperm parameters, such as motility, acrosome integriy, membrane integrity and mitochondrial membrane potential. Moreover, when freezing extenders supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced, meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA) content were decreased. The results suggest that freezing extenders supplemented with 100 mM trehalose resulted in less ROS level and MDA content, higher motility and mitochondrial membrane potential as well as the integrity of acrosome and plasma membrane. Supplementation of trehalose with freezing extenders is beneficial to the rabbit breeding industry.     

Cryopreservation of canine epididymal sperm using ACP-106c and TRIS

The objective was to cryopreserve sperm recovered from the canine epididymal cauda immediately after an orchiectomy. The sperm was stored for 12 h at 4 °C using ACP-106c and TRIS as extenders. Sixty adult male dogs were used. The testis–epididymis complex (TEC) was removed, immersed in 0.9% saline and transported to the laboratory. The 60 TEC were divided into groups according to the 4 °C cooling time (0 h or 12 h) and according to the extender used for sperm recovery (ACP-106c or TRIS), forming 4 experimental groups: G0h-ACP, G12h-ACP, G0h-TRIS and G12h-TRIS. The sperm were recovered from the epididymal cauda using the retrograde flow technique. Next, 1.0 mL of ACP-106c or 1.0 mL of TRIS (preheated to 37 °C for 5 min) was added to the sperm of each epididymis. One week later, the sperm was thawed at 37 °C for 1 min, and its morphology, functionality and total and progressive sperm motilities were analyzed. Other parameters were obtained by Computer Assisted Semen Analysis (CASA). The data were submitted to multivariate analysis of variance (MANOVA) (P < 0.05). The total motility values were 52.17 ± 1.78 and 49.8 ± 1.93 for groups G0h-ACP and G12h-ACP and 50.7 ± 2.06 and 43.90 ± 2.51 for groups G0h-TRIS and G12h-TRIS, respectively. A decrease in total sperm motility was observed after 12 h of cooling for both extenders (P < 0.05). ACP-106c can be used as an extender for freezing canine epididymal sperm, and the freezing procedure must be performed immediately after sperm recovery.     

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing–thawing caused a significant decrease (P < 0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P < 0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P < 0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.     

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3–5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.     

Incorporation of ascorbic acid and α-tocopherol to the extender media to enhance antioxidant system of cryopreserved sea bass sperm

Despite the overwhelming application of sperm cryopreservation in aquaculture and broodstock management, its detrimental effects on sperm quality must be taken into account. Imbalance of reactive oxygen species is considered one of the main triggers of cell damage after cryopreservation, because the spermatozoa antioxidant system is decimated during this process, mainly because the natural antioxidants present in seminal plasma diminish when sperm is diluted in extenders. It has been demonstrated that the addition of antioxidants to the extender improves the quality of thawed sperm. Thus, the aim of the present work was to evaluate the status of the antioxidant system in cryopreserved sea bass sperm, and the possibility of enhancing this system to reduce oxidation of the membrane compounds by extender supplementation with vitamins. To do this, sperm from European sea bass (Dicentrarchus labrax) was cryopreserved using an extender control (NAM), supplemented with 0.1 mm α-tocopherol or 0.1 mm ascorbic acid. Sperm motility (computer assisted sperm analysis (CASA) parameters), viability (SYBR Green/propidium iodide (PI)), lipid peroxidation (malondialdehyde (MDA) levels) and protein oxidation (DNPH levels) were analyzed, as well as the status of the sperm antioxidant system by determining glutathione peroxidase, glutathione reductase and superoxide dismutase (GPX, GSR and SOD) activity. The results demonstrated that extenders containing vitamins significantly increased sperm motility. Total motility, velocity and linearity increased from 31.2 ± 3.0 μm/sec, 18.3 ± 1.7 μm/sec and 46.9 ± 2.0% in extender containing 0.1 mm α-tocopherol or 30.6 ± 3.9 μm/sec, 19.5 ± 1.6 μm/sec and 47.9 ± 2.2% in extender containing 1 mm ascorbic acid respect to the extender control (20.7 ± 3.3 μm/sec, 13.8 ± 1.7 μm/sec and 37.3 ± 4.1%). However, viability and levels of lipid peroxidation and protein oxidation were not affected by the presence of these antioxidants, suggesting that membrane impairment could be more associated to osmotic shock or membrane destabilization than oxidative damage. The increased activity of both GPX and GSR after cryopreservation showed that the antioxidant system of sea bass sperm must play an important role in preventing oxidation of the membrane compounds. In conclusion, the addition of α-tocopherol and ascorbic acid to the extender media, together with the antioxidant system of the spermatozoa improved sea bass sperm motility, which is one of the impairment parameters most affected by cryopreservation.     

Evaluation of Tris–citric acid,skim milk and sodium citrate extenders for liquid storage of Punjab Urial (Ovis vignei punjabiensis) spermatozoa

The Punjab Urial (Ovis vignei punjabiensis) is an endangered subspecie of ovidae, distributed as small scattered populations in the forest belt of the Himalayan foothills of Pakistan and in the areas enclosed by the Indus and the Jhelum rivers. The present study was conducted to evaluate the liquid storage of Punjab Urial spermatozoa in different extenders for use in future in situ conservation activities. Semen was collected by electro-ejaculation from three captive Punjab Urial rams. Suitable ejaculates of individual animals were pooled and divided into three aliquots for dilution with the experimental extenders (Tris–citric acid, skim milk and sodium citrate) at 37 °C. Extended semen was cooled from 37 °C to 5 °C in 2 h, and stored for three days at 5 °C. Sperm motility (%), viability (%; live/dead), acrosome integrity (%) and plasma membrane integrity (%) were assessed on days 1, 2 and 3 of storage. On day 1, sperm motility, viability as well as acrosome and plasma membrane integrity were similar (p > 0.05) in all three experimental extenders. On day 2, sperm motility, viability, acrosome and plasma membrane integrity were higher (p < 0.05) in Tris–citric acid extender compared to sodium citrate based extender. On day 3 of storage, the values of motility, viability and acrosome integrity were higher (p < 0.05) in Tris–citric acid extender than in skim milk and sodium citrate based extenders. In conclusion, Tris–citric acid extender appears to be a better option compared with skim milk and sodium citrate extenders for liquid storage of Punjab Urial semen.