Stallion Semen Cooling Using Native Phosphocaseinate-based Extender and Sodium Caseinate Cholesterol-loaded Cyclodextrin-based Extender

The objective of this study was to compare semen parameters and embryo recovery rates of cooled stallion semen extended with INRA 96 or BotuSemen Gold. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspended at 100 million sperm/mL with their respective extender. Semen was then cooled and stored as in experiment 1. In both experiments, sperm motility parameters, plasma membrane integrity, and high mitochondrial membrane potential were assessed at 0, 24, and 48 hours post cooling. For experiment 3, 12 mares (n = 24 cycles) were bred with 48 hour–cooled semen from one stallion. Semen was processed as described in experiment 1. Mares had embryo flushing performed by 8-day post-ovulation. In experiment 1, BotuSemen Gold displayed superior total and progressive motility relative to INRA 96 (P < .05). There were no significant differences between the types of containers in any experiment. In experiment 2, INRA 96 and BotuSemen Gold extenders had similar total and progressive motility, but BotuSemen Gold had superior sperm velocity parameters at all timepoints. Embryo recovery was identical for both extenders (50%). Finally, the results obtained herein suggest that BotuSemen Gold is a suitable alternative to be included in semen cooling tests against INRA 96 in clinical practice.     

Cryopreservation of Cooled Semen and Evaluation of Sperm Holding Media for Potential Use in Equine-Assisted Reproduction Procedures

Processing stallion semen for assisted reproductive procedures, such as intracytoplasmic sperm injection (ICSI), requires special considerations regarding cooling, concentrating, and handling of sperm. The aim of experiment 1 was to determine whether cooled semen could be frozen without removal of seminal plasma and at a low sperm concentration while maintaining motile sperm for ICSI selection procedures. In experiment 2, five media for holding stallion sperm were compared to evaluate sperm motility for an interval of time sufficient for ICSI sperm selection procedures. In experiment 1, semen samples from eight stallions were cooled for 24 hours in two extenders, CST (E-Z Mixin-CST “Cool-Store/Transport” Animal Reproduction Systems) and INRA96 (Institut National de la Recherche Agronomique, IMV International Corporation), before being frozen in four freezing diluents, and were evaluated at 0, 45, and 75 minutes after thawing. The cooling extender did not significantly affect sperm motility, but modified French and glycerol egg yolk diluents provided the best sperm motility for frozen–thawed groups. In experiment 2, semen samples from seven stallions were used to test five media for holding sperm. Samples were analyzed for total and progressive motility at hourly intervals. Mean total and progressive motility were not different (P > .05) among groups from 1 through 4 hours. At 5 hours, groups differed (P = .004), with sperm held in Tyrode’s with albumin, lactate, and pyruvate having higher (P < .05) total and progressive motility than all other samples. In conclusion, motile stallion sperm can be obtained after the sperm are cooled for 24 hours, frozen, and thawed; various media are available to maintain sperm motility during equine ICSI selection procedures.     

Quality of Raw and of Cold-Stored Semen in Icelandic Stallions

The aim of the present study was to evaluate the quality of raw and cooled semen in Icelandic stallions. Experiments were performed using seven stallions aged between 3 and 19 years. From each stallion, six ejaculates were collected, and semen quality was determined. Thereafter, the semen was split into eight equal parts and processed with and without centrifugation using the extenders INRA 82-egg yolk, INRA 96, GENT, and Equi-Pro to a final concentration of 30 × 10⁶ sperm/mL. The extended semen was then cooled in an Equitainer, where it was stored for 24 hours, and subsequently refrigerated for another 24 hours at 5°C. Immediately after dilution as well as after 24 and 48 hours storage, sperm motility was analyzed using computer-assisted sperm analyzer, and viability was assessed after dual DNA staining with SYBR-14 in combination with propidium iodide. The results show that the stallion had a significant (P < .05) influence on all variables evaluated in raw semen, and mean (±SEM) values of 43.4 ± 4.3 mL for the volume, 193.0 ± 17.0 × 10⁶ sperm/mL for the concentration, 6.7 ± 0.5 × 10⁹ for total sperm and 73.5 ± 2.1% for total sperm motility, 48.7 ± 2.0% for progressive motility, and 65.3 ± 2.0% for rapid cells were measured. In the cold-stored semen, all variables were significantly (P < .05) influenced by the stallion, extender, and storage time (48 hours). Except for Equi-Pro, all extenders examined were suitable for cooled semen preservation. For storage of more than 24 hours, centrifugation and removal of the seminal plasma were advantageous for all extenders with the exception of Equi-Pro.     

Studies on Motility and Fertility of Cooled Stallion Spermatozoa

This study on extended, cooled stallion spermatozoa aimed to compare the ability of three extenders to maintain sperm motility during 24 h of preservation, and to describe pregnancy and foaling rates after artificial insemination (AI) of stallion spermatozoa stored and transported in the extender chosen from the in vitro study. After 6 and 24 h of preservation, motility, both subjective and evaluated by the motility analyzer (total, progressive and rapid), was lower in non-fat, dried skim milk-glucose than in both other extenders: dried skim milk-glucose added to 2% centrifuged egg yolk, and ultra high temperature treated skim milk-sugar-saline solution added to 2% centrifuged egg yolk (INRA82-Y). Rapid spermatozoa and sperm velocity parameters, after 24 h, were significantly higher in INRA82-Y. In the fertility trial, semen collected from three Maremmano stallions, diluted in INRA82-Y, and transported in a refrigerated Styrofoam box, was used to inseminate 56 mares of the same breed. Pregnancy rates after the first cycle and per breeding season were significantly higher for the 31 mares inseminated in three AI centres (54.8 and 80.6%, respectively) than for the 25 mares inseminated at the breeder's facilities (28.0 and 52.0%). Foaling rates were not significantly different between the AI centres mares (54.8%) and the other mares (44.0%). In conclusion, INRA82-Y yielded satisfactory pregnancy and foaling rates, especially when employed in the more controlled situation of an AI centre, and can therefore be included among those available for cooled stallion semen preservation.     

Influence of Semen Storage and Cryoprotectant on Post-thaw Viability and Fertility of Stallion Spermatozoa

The aim of the current study was to verify that stallion spermatozoa could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryopreservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio^® extender containing one of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5°C for 20 minutes, then frozen in nitrogen vapor. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty-three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine semen for 24 hours before freezing, while maintaining sperm viability and fertility, is possible.     

红腹锦鸡精液冷冻稀释液配方的筛选

为加强濒危珍稀动物种质资源的保护,开展了濒危珍稀禽类—红腹锦鸡的人工授精研究。2005年5月26日~6月8日,对8只红腹锦鸡用手按摩其背、尾部采精12次。初测其精液品质,结果表明,采精量平均(0.114±0.016)mL(0.01~0.2 mL),每毫升精液中精子平均3.2亿个(3.0~3.3亿),活力9级以上,pH值6.5,偏酸性,淡乳白色(半透明似冲熟的藕粉),微腥。鲜精分别加11%蔗糖—卵黄稀释液(3号液);11%蔗糖—0.1%柠檬酸三钠—卵黄稀释液(5号液);5%葡萄糖—卵黄稀释液(8号液)。精子活力达8~9级。用3%柠檬酸三钠—卵黄稀释液(2号液)稀释,精子活力2级。冷冻时,用11%蔗糖溶液100 mL中加16 mL鲜卵黄,加5 mL甘油,配制的3号冷冻稀释液稀释,解冻后,精子活力达4级。优于试验中选拟的其它配方(加入5 mL甘油的5号冷冻液,解冻后精子活力为3级;加入5 mL甘油的8号冷冻液,解冻后未见活的精子)。如果冷冻稀释液中甘油改为6 mL,则解冻后精子全部死亡。     

Cryopreservation of Stallion Spermatozoa with INRA96 and Glycerol

The aim of the present study was to improve success of cryopreservation of stallion spermatozoa. Semen from eleven stallions was collected and frozen in INRA 96 with two different concentrations of glycerol (3.5% and 6.0%) and compared with a control freezing process. The mean post-thaw motility for the eleven stallions of 57.93% (3.5% glycerol) and 66.50% (6.0% glycerol), which was statistically higher (P < 0.05) when compared with the mean post-thaw motility (39.7%) for semen in a control egg-yolk extender (Equipro^® CryoGuard™ Complete, Minitube). The Equipro^® CryoGuard™ Complete is a commercial semen freezing protocol that has been one of the standard processes used in our laboratory for freezing equine spermatozoa. INRA 96 with 6% added glycerol was used in the fertility trial as it provided the highest spermatozoa survival. To evaluate fertility of the frozen semen, eight mares were bred over two cycles with both fresh and frozen semen. The pregnancy rate of mares bred with frozen semen (55.6%) was not statistically different (P > 0.05) from the pregnancy rate of mares bred with fresh semen (55.6%). INRA 96 with 6.0% glycerol improved the survivability of stallion spermatozoa through the cryopreservation process, and subsequent fertility was not different (P > 0.05) from fresh, extended semen.     

The Effects of Centrifuged Egg Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to Freeze Miniature Caspian Horse Semen

The aim of this study was to determine the synergistic effects of centrifuged egg yolk (EY) and soybean lecithin on post-thaw Caspian horse sperm motility, morphological abnormalities, and assessment of membrane integrity. The centrifuged EY (CEY) was added at concentrations of 2% and 4% to a defined INRA plus 1.25% soybean lecithin extender used to freeze Caspian horse semen. In this experiment, ejaculates collected from each Caspian horse (n = 4) were divided into three equal aliquots and diluted in CEY 2% (INRA2), 4% (INRA4) supplemented, and without any CEY (INRA0) in INRA plus 1.25% soybean lecithin extender, respectively. Thereafter, samples were frozen and thawed following a standard protocol. Sperm cryosurvival was evaluated in vitro by microscopy assessments of post-thaw sperm motility (by means of computer-assisted semen motility analysis [CASA]), acrosomal and other abnormalities (head, mid-pieces, and tail) and plasma membrane integrity (evaluated by HOST). In Caspian stallion, semen extended with INRA2 had significantly higher CASA motility and CASA progressive motility than those extended with the rest of extenders after freezing and thawing (P < .001). There was no significant difference in path velocity (VAP), VCL, and ALH among three groups (P > .05). For straight line velocity (P < .01) and LIN (P < .001), the highest values were obtained from the INRA4 group. The highest percentages of acrosomal and other abnormalities were found in semen diluted in INRA4 (P < .001). In the group frozen INRA2, the percentage of membrane integrity was significantly higher than that of the other groups (P < .001). The use of CEY 2% in combination with soybean lecithin significantly improved Caspian horse semen freezability.     

Pregnancy rates of mares inseminated with semen cooled for 18 hours and then frozen

The ability to ship cooled stallion sperm for subsequent freezing at a facility specializing in cryopreservation would be beneficial to the equine industry. Stallion sperm has been centrifuged, cooled to 5 degrees C for 12 h, and frozen without a detrimental effect on motility in a previous study; however, no fertility data were available. Experiment 1 compared the post-thaw motility of sperm cooled for 18 h at 15 or 5 degrees C at either 400 or 200 x 10(6) sperm/mL and then frozen. Storage temperature, sperm concentration, or the interaction of temperature and concentration had no effect on total (TM) and progressive motility (PM) after cooling. Post-thaw TM and PM were higher for control than (P < 0.05) for treated samples. There was no difference in post-thaw TM and PM due to temperature or concentration. Experiment 2 further evaluated procedures for cooling before freezing. Ejaculates were either cooled to 5 degrees C for 18 h and centrifuged, centrifuged at room temperature and then cooled to 5 degrees C for 18 h before freezing, or centrifuged and frozen immediately (control). There was no difference among treatments on post-thaw TM or PM. In Exp. 3, mares were inseminated with semen that had been extended in skim milk-egg yolk without glycerol, centrifuged, resuspended at 200 x 10(6) sperm/mL, cooled to 5 degrees C for 18 h, and then frozen or not cooled for 18 h before freezing (control). Pregnancy rates did not differ for mares receiving semen cooled and then frozen (21 of 30, 70%) or semen frozen directly without prior cooling (16 of 30, 53%). In summary, a procedure was developed for cooling stallion sperm for 18 h before freezing without a resultant decrease in fertility.     

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

Melatonin can improve viability and functional integrity of cooled and frozen/thawed rabbit spermatozoa

Melatonin is known to protect sperm against freezing-inflicted damage in different domestic species. The aim of the study was to evaluate the effect of supplementation of semen extender with melatonin on the quality and DNA integrity of cooled and frozen/thawed rabbit spermatozoa. We also investigated whether the addition of melatonin to the semen extender could improve the fertility of rabbit does artificially inseminated with frozen/thawed semen. Semen samples collected from eight rabbit bucks were pooled and then diluted in INRA-82 supplemented either with (0.5, 1.0 or 1.5 mM) or without (0.0 mM) melatonin. Diluted semen was cooled at 5°C for 24 hr. For cryopreservation and based on the first experiment's best result, semen samples were diluted in INRA-82 in the presence or absence of 1.0 mM melatonin and then frozen in 0.25 ml straws. Following cooling or thawing, sperm quality and DNA integrity were evaluated. Furthermore, the fertility of frozen/thawed semen was investigated after artificial insemination. Supplementation of semen extender with 1.0 mM melatonin improved (p < .05) motility, viability, membrane and acrosome integrities in cooled semen compared with other groups. Sperm quality and DNA integrity were higher (p < .05) in frozen/thawed semen diluted in 1.0 mM melatonin-supplemented extender than in the control group. Conception and birth rates were higher in does inseminated with 1.0 mM melatonin treated semen compared with the controls. In conclusion, supplementation of semen extender with 1.0 mM melatonin improved the quality of cooled and frozen/thawed rabbit spermatozoa. Melatonin can preserve DNA integrity and enhance the fertility of frozen/thawed rabbit spermatozoa.     

Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility

This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk–based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one “bad cooler” and one “good cooler” stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours.     

Effect of cryoprotectants and thawing temperatures on chicken sperm quality

There is need for standardization of freezing–thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post‐thaw motility and to analyse combined effect of the best permeating cryoprotectant (P‐CPA) with one of four non‐permeating cryoprotectants (N‐CPA) on post‐thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N‐methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N‐CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing–thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen‐thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post‐thaw motility. Moreover, ficoll addition to EG‐based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N‐CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank.     

Effect of feeding pomegranate seed oil as a source of conjugated linolenic acid on Arabian stallion semen quality in cooled and postthawed condition

The objective was to assess the influence of pomegranate seed oil supplementation on the quality of fresh, cooled and frozen–thawed Arabian breed stallion semen. Eight stallions (n = 4 per group) received their normal diet (control group) or normal diet top dressed with 200 ml of pomegranate seed oil (PSO group). Semen was collected every fifteen days for 90 days. Stallions were reversed across the treatments after a sixty‐day interval. In cooled and stored condition (2, 12 and 24 hr), spermatozoa motion characteristics, membrane integrity, viability, morphology and lipid peroxidation were analysed. In frozen–thawed semen, sperm dynamic characteristics were analysed by CASA, acrosome status and mitochondrial activity (evaluated by Flow cytometry) determined. The effects of treatment, time, semen type and their interactions were submitted to PROCMIX (SAS^®), and means compared by the Tukey test. Also, collected semen samples were artificially inseminated to evaluate fertility and pregnancy rate after day 60 of the experiment. The results from fresh condition showed that semen volume, sperm concentration, abnormality and live sperm were not affected by dietary treatment (p > 0.05). In cooled condition, the higher value for sperm plasma membrane integrity and viability was observed in PSO group compared to control after 24 hr cooled and stored in 5°C. In postthawed condition, the higher value for CASA total motility and acrosome status was observed in PSO group compared to control group (p < 0.05). One hundred and twenty‐six mares were artificially inseminated for fertility trial using control and PSO groups’ fresh semen. The average pregnancy rates were not significantly different between control and treated group (62.88% and 65.90%, respectively) (p > 0.05). We concluded that under the conditions of this study, dietary supplementation of 200 ml pomegranate seed oil seems to relatively improved Arabian horse sperm quality during storage in cooled and frozen condition via increasing plasma membrane integrity, viability and acrosome status, but did not improve the pregnancy rates.     

Intracellular calcium chelating agent (BAPTA‐AM) aids stallion semen cooling and freezing–thawing

This study aimed to investigate the effects of different concentrations of 1,2‐bis‐(o‐aminophenoxy)‐ethane‐N,N,N0 N0‐tetraacetic acid, tetra‐acetoxymethyl ester (BAPTA‐AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing–thawing. After collection, semen was extended (1:1 v/v) on a skim milk‐based extender, centrifuged and resuspended at 400 million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200 μΜ BAPTA‐AM. Motility parameters were assessed after cooling in Equitainer at 5°C for 12, 24, 48, 72 and 120 hr and after freezing–thawing. In addition, mitochondrial membrane potential, intracellular ATP, reactive oxygen species and malondialdehyde concentrations were measured in cryopreserved‐thawed semen. Cooled stored (48 hr) semen containing 50 μΜ BAPTA‐AM and control extender (0 μΜ BAPTA‐AM) was used to assess fertility. Inclusion of 50 μΜ BAPTA‐AM resulted in superior sperm motility parameters during cooled storage when compared to other groups (p < 0.05). Furthermore, semen cryopreserved in extender containing 50 μΜ BAPTA‐AM showed increased intracellular ATP and mitochondrial membrane potential, whereas reactive oxygen species and malondialdehyde were increased after thawing for all groups (p < 0.05). Addition of 50 μΜ BAPTA‐AM to cooling extender resulted in similar pregnancy rates to the control group (75% vs. 73.6%, respectively; p > 0.05). In conclusion, the addition of BAPTA‐AM to semen extenders aided stallion semen cryopreservation in a dose‐dependent manner. Furthermore, the cooling extender supplemented with 50 μΜ BAPTA‐AM could be used to prolong the sperm motility during cooling without apparently compromising fertility. Field trials should be conducted to assess fertility of cryopreserved stallion semen with BAPTA‐AM.     

Influence of partial or total replacement of glycerol by alternative cryoprotectants in Ghent freezing extender on post‐thaw sperm quality in stallions

Although glycerol is the cryoprotectant most commonly used in stallions, it has also a considerable toxicity for equine sperm. It was the aim of this study to analyse the quality of frozen‐thawed stallion semen after complete or partial replacement of glycerol in the freezing extender by alternative cryoprotectants. We hypothesized that partial or total replacement of glycerol by cryoprotectants occurring in cold‐resistant frog, insect or plant species results in similar or better semen quality after freezing–thawing. As basic medium, the commercial Ghent basic extender was used and either supplemented with glucose and urea, trehalose and proline, or trehalose and betaine. Based on a series of preliminary experiments, semen was frozen in either commercial Ghent cryopreservation extender (Ghent control), Ghent glucose–urea extender or a Ghent combined extender (glucose–urea, trehalose‐betaine and trehalose‐proline; volume ratio of 2:1:2) in a computer‐controlled rate freezer. After freezing–thawing, semen was analysed for motility, membrane integrity, phosphatidylserine translocation, mitochondrial membrane potential and chromatin condensation. No differences between Ghent control and Ghent glucose–urea extender were seen, while all endpoints except DNA integrity were negatively affected in Ghent combined extender (e.g., progressive motility: Ghent 49.2 ± 3.7, Ghent glucose–urea 46.5 ± 4.6, Ghent combined 24.4 ± 2.8%; p < .001). In conclusion, glycerol concentration in a commercial freezing extender for equine spermatozoa can be successfully reduced when urea as an additive cryoprotectant is added and the glucose concentration is elevated. However, total glycerol replacement with urea, betaine, proline and trehalose was less successful.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Study on Technology of Black Silkies Chicken Semen Freezing by DMA Pellet

The aim was to explore the effects of different kinds of dilution and thawing devices on the N,N-dimethylformamide (DMA) pellet frozen semen of Black Silkies. Firstly,the motility and fertility of the frozen semen thawed by different dilutions were compared;Then,the motility of the frozen semen was compared when the pellets were thawed using different tube and different number.Finally,the motility and fertility of the sperm thawed by three kinds of thawing devices (thermostat water bath,hotfunnel and hotplate ) were tested. The results showed that:①There was similar order of the sperm motility and fertility in the different dilution groups (LR > F > B > L),and there was significant difference among those groups (P < 0.05).②The motility was the best when the frozen semen was thawed with large thin-wall glass tube at 60℃.③The best temperature range of the 3 devices was different. The highest motility for thermostat water bath was 50 to 60℃ (0.51 to 0.59),and thermostat hotfunnel was 40 to 45℃ (0.42 to 0.46),while thermostat hotplate was 50 to 55℃ (0.61 to 0.63).There was no significant difference of the motility in the optimum temperature range for each device (P > 0.05).④The fertility of the different devices in their best thawing temperature was 26.91% (55℃,thermostat hotplate),23.08% (60℃, thermostat water bath), 20.93% (40℃, thermostat funnel),respectively,and there was no significant differences among those groups (P > 0.05).Therefore,the efficiency thawing condition for the Black Silkies frozen semen was the LR diluent,DMA cryoprotectant,pellet freezing,thawed in the thermostat hotplate at 54.9℃.     

Comparison of Various Extenders for Storage of Cooled Stallion Spermatozoa for 72 Hours

Multiple extenders have been developed to preserve cooled stallion semen. Comparisons of some extenders have been made but there is need for further research in this area. Extenders tested included EZ Mixin (Animal Reproduction Systems, Chino, CA), Kenney's, Universal (NASCO, Fort Atkinson, WI), EquiPro, EquiPro CellGuard (Minitube of America, Verona, WI), and INRA 96 (IMV, Maple Grove, MN). Semen was collected and each ejaculate was divided and extended in each of the aforementioned extenders and stored at 4°C. Motility measures were determined using computer-assisted sperm analysis at 0, 24, 48, and 72 hours after collection. Samples were evaluated for total motility, progressive motility (PM), straight-line velocity, curvilinear velocity, straight-line distance, and curvilinear distance. Total motility and PM decreased over time in storage (P < .05). Sperm stored in INRA 96, EquiPro, and EquiPro Cell Guard retained the most total motility and PM over the 72 hour period (P < .05). Universal, EquiPro, and EquiPro Cell Guard had the highest measurements for curvilinear velocity, straight-line velocity, and curvilinear distance (P < .05). There were no significant differences among the extenders for straight-line distance.     

黑丝羽乌骨鸡精液DMA颗粒冷冻技术研究

为研究不同稀释液与解冻装置对黑丝乌骨鸡N,N-二甲基甲酰胺（N,N-dimethylformamide,DMA）颗粒冻精解冻后精子质量的影响,试验首先采用不同稀释液对精液进行稀释并比较其解冻后精子活力与人工输精的受精率;其次,选取不同解冻管及冻精颗粒数进行恒温水浴解冻,比较其活力;最后,依据解冻后的精子活力,筛选恒温水浴、恒温漏斗、恒温板3种解冻装置各自的最佳解冻温度,并利用各自最佳解冻温度解冻后的精液进行人工输精,检测受精率。结果显示：①不同稀释液组的精子活力与受精率高低趋势一致,为LR > F > B > L组,且各组之间差异显著（P < 0.05）。②在60℃恒温水浴中,用薄壁大玻璃管解冻的精子活力最好。③不同解冻装置有各自最佳解冻活力的温度范围,恒温水浴为50~60℃（0.51~0.59）、恒温漏斗为40~45℃（0.42~0.46）、恒温板为50~55℃（0.61~0.63）,每种装置最佳温度段内的精子活力差异不显著（P > 0.05）。④3种解冻装置最佳解冻状态相比：在精子活力上,60℃恒温水浴与55℃恒温板分别显著高于40℃恒温漏斗（P < 0.05）,但60℃恒温水浴与55℃恒温板之间差异不显著（P > 0.05）;在受精率上,55℃恒温板最高（26.91%）,60℃恒温水浴次之（23.08%）、40℃恒温漏斗最低（20.93%）,三者之间差异不显著（P > 0.05）。因此,黑丝羽乌骨鸡精液应采用LR稀释液、DMA冷冻保护剂及颗粒冷冻技术,在54.9℃恒温板解冻可获得较高的受精率。