Cryopreservation of European Mouflon (Ovis Gmelini Musimon) Semen During the non-Breeding Season is Enhanced by the Use of Trehalose

The influence of trehalose on European mouflon spermatozoa cryopreservation during the non-breeding season was tested. Semen was frozen in two different extenders: (a) recommended Tris-based ram extender (CTR); (b) CTR extender supplemented with trehalose 0.147 mm (TRH). Sperm viability and acrosome integrity were assessed using propidium iodide and fluorescein isothiocynate labelled Pisum Sativum agglutinin. Trehalose significantly enhanced sperm viability after thawing compared with CTR extender (62.7% vs 51.8%; p < 0.05), whereas no differences were observed on acrosome integrity (42.9% vs 42.1%). Trehalose influence was also evidenced in the in vitro fertility test performed with sheep oocytes matured in vitro. Both fertilization rates (60.9% TRH vs 43.6% CTR; p < 0.05) and cleavage rates (58% TRH vs 39.8% CTR; p < 0.001) were higher for trehalose frozen semen compared with control extender frozen semen. A higher percentage of zygotes resulting from fertilization with trehalose cryopreserved semen presented the first cleavage earlier if compared with the group fertilized with control semen (48.7% vs 31.5%, respectively; p < 0.01). This result was confirmed by embryo kinetic development. Fertilization with trehalose cryopreserved semen leaded to an higher percentage of blastocysts (40.2% vs 27.8% CTR; p < 0.05), and enhanced in particular the number of blastocysts that developed on the day 6th of culture (28.6% vs 17% CTR; p < 0.05). Our data demonstrated that, during mouflon non-breeding season, trehalose extender enhances spermatozoa viability and its in vitro fertilizing capacity, allowing the production of an higher number of blastocysts.     

Fertility after different artificial insemination methods using a synthetic semen extender in sheep

The present study aimed to investigate the fertility of ewes artificially inseminated with three different methods using a synthetic semen extender, AndroMed. The three methods of artificial insemination (AI) were cervical AI with fresh-diluted or frozen-diluted semen at observed estrus, and an intrauterine AI with frozen-thawed semen. A total of 80 ewes were treated with a controlled internal drug release (CIDR) containing 0.3 g progesterone per device for 12 days. In Experiment 1 (26 Suffolk ewes), superovulation was induced with 20 mg follicle-stimulating hormone and 250 IU equine chorionic gonadotropin (eCG) two days and one day before CIDR removal, respectively, during the non-breeding season. In Experiment 2 (54 Suffolk and Suffolk crossbred ewes), an intramuscular injection of 500 IU eCG was administered one day before CIDR removal to synchronize estrus and ovulation during the breeding season. In Experiment 1, fresh-diluted or frozen-thawed semen was deposited into the cervical orifice after estrus detection, and an intrauterine AI with frozen-thawed semen was performed by laparoscopy at a fixed-time basis without estrus detection. Embryos were recovered by uterine flushing 6 days after AI, and the rates of recovered, fertilized (cleaved) ova and embryos at the morula or blastocyst stage were compared among the three AI methods. In Experiment 2, the pregnancy rates after the three AI methods were compared. In Experiment 1, the rates of recovered ova were not significantly different among the three AI methods (52.5-56.7%). The rate of fertilized ova (81.0%) by laparoscopic AI with frozen-thawed semen was significantly higher compared with cervical AI of fresh-diluted (25.5%) or frozen-thawed (3.5%) semen, but the rate of embryos at the morula or blastocyst stage (17.6%) was significantly lower than that of the cervical AI with fresh-diluted semen (69.2%). The rates of ewes yielding fertilized ova were not significantly different among the three groups (44.4, 11.1 and 62.5% for cervical AI with fresh-diluted and frozen-thawed semen and intrauterine AI with frozen-thawed semen). In Experiment 2, the pregnancy rate of ewes intrauterinally inseminated with frozen-thawed semen (72.2%) was significantly higher than those of ewes inseminated cervically with fresh-diluted (5.5%) or frozen-thawed (0.0%) semen. The present results showed that acceptable fertilization and pregnancy rates could be obtained by an intrauterine AI with frozen-thawed semen using a synthetic semen extender (AndroMed), but not sufficient by the cervical AI with either fresh or frozen semen.     

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。     

Insemination with Stallion Semen Frozen in 0,5 ml Straws

Contents: An insemination trial using frozen semen is described. The freezing procedure was slightly modified from the Hannover method. The insemination dose consisted of 7 medium straws containing approx 1 10⁹ spermatozoa. A total of 28 mares of the Norwegian Trotter breed were inseminated during the 1991 season. During oestrus the mares were examined at 12 hour intervals, and the insemination was carried out after detection of ovulation. The pregnancy rate was 43% after the first insemination, increased to 68% after second and further to 75% after the third and last insemination. The foaling rate was 61.5%.     

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℃.     

Fertility of ewes inseminated intrauterinally with frozen semen using extender containing bovine serum albumin

The present study was conducted to examine the fertility of ewes inseminated intrauterinally with frozen semen using semen extender containing either egg yolk or bovine serum albumin (BSA). Sixty Suffolk and cross-bred ewes were treated with controlled internal drug release (CIDR) devices during the non-breeding season (July 2006). A CIDR was inserted into the vagina for 12 days and an intramuscular injection of 500 IU equine chorionic gonadotropin was administered one day before its removal. Ejaculates from a suffolk ram were diluted with a Tris-based extender containing either 15% (v/v) egg yolk or 10% (w/v) BSA, and the diluted semen was frozen in 0.25 ml straws. A fixed-time intrauterine artificial insemination (AI) was performed 43-47 h after CIDR removal, regardless of incidence of estrus. There was no significant difference in pregnancy rates at 60 days after AI between the extenders containing egg yolk (66.7%, 20/30 animals) or BSA (65.5%, 19/29 animals). Furthermore, there were no significant difference in the lambing rates (66.7% and 62.1%) and prolificacy (1.25 and 1.56) between the two semen extenders. The present study indicates that a semi-defined semen extender containing 10% BSA produces fertility after intrauterine AI that is similar to that achieved with semen extender containing egg yolk.     

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。     

A retrospective study of artificial insemination of 251 mares using chilled and fixed time frozen-thawed semen

REASONS FOR PERFORMING STUDY: Historically, artificial insemination (AI) using frozen semen has been perceived to have poorer success rates and be more labour intensive than using chilled semen. A retrospective study was therefore conducted to compare the conception rate achieved by AI between chilled and frozen semen, using fixed time insemination protocols over 2 breeding seasons. HYPOTHESIS: Artificial insemination using chilled semen produces a higher conception rate than that achieved with frozen semen. METHOD: Mares (n = 251) were inseminated with either chilled (n = 112) or frozen (n = 139) semen in the 2006 and 2007 northern hemisphere breeding season. Per rectum ultrasonography of the mare's reproductive tract determined the timing of insemination, and deslorelin acetate was used to induce ovulation. Chilled semen insemination was performed using a single preovulatory dose delivered into the uterine body. Frozen semen was administered as 2 doses (pre- and post ovulation) using a deep uterine insemination technique. Pregnancy was detected ultrasonographically at 15 days post insemination. Conception rates were compared using a Chi-squared test. RESULTS: Insemination with frozen semen produced a significantly (P = 0.022) higher seasonal conception rate (82.0%) than that achieved with chilled semen (69.6%). CONCLUSIONS AND POTENTIAL RELEVANCE: Insemination with frozen semen can achieve conception rates equal to those with chilled semen, enabling the mare owner a greater selection of stallions.     

BMY牛冷冻精液质量控制及影响因素研究

[目的]为了加大BMY牛的推广应用力度和提高人工授精受胎率,开展了BMY牛冷冻精液质量控制研究,优选出冷冻稀释液及得出冷冻精液质量控制技术程序。[方法]采用不同的初始冷冻温度、不同种类的稀释液、不同的冷冻方法对BMY牛精液冷冻及解冻效果进行试验。[结果]不同的初始冷冻温度、不同种类的稀释液、不同的冷冻方法,BMY牛精液冷冻解冻效果各异。初始冷冻温度为-121--140℃时,TRIS液为基础稀释液添加糖类和氨基酸一步法稀释BMY牛精液冷冻6-10 min BMY牛冷冻精液解冻效果较好,用程控冷冻仪冷冻BMY牛精液,同样获得良好的解冻效果,解冻活力达到0.372±0.026。BMY牛冷冻精液平均解冻活力可达0.357±0.029以上,精子解冻复苏率达50%以上。精子畸形率平均为16.8%±4.26%,BMY牛采精成功率为75.3%。[结论]全放牧条件下BMY牛电刺激采精法获得很好效果,精液质量好,更有利于种质资源的保存和利用。     

The Effect of Removal of Seminal Plasma, Egg Yolk Level and Season on Sperm Freezability of Canary Buck (Capra hircus)

Goat semen is different from that of other domestic species in its limited tolerance to the inclusion of egg yolk in the freezing medium, and this tolerance depends on the presence of enzymes in the seminal plasma that react with egg yolk, producing toxic compounds to the spermatozoa. Moreover, the goat is a seasonal breeder that shows variations in semen quality throughout the year, and those variations may affect semen freezability; hence in freezing protocols, for instance, removal of seminal plasma (washing) yields varying results. This work was designed to study this problem in Canary goats: semen from six males was collected in spring, autumn or winter, washed or non-washed, diluted in a freezing extender with 1.5, 6 or 12% egg yolk, frozen, and thawed after 2 days, 2 or 6 months of cryopreservation. The effect of egg yolk concentration in the freezing extender was far more important than the effect of washing or season on sperm cryosurvival. The quality of frozen-thawed semen tended to improve as egg yolk concentration increased regardless of the effects of season, washing or period of cryopreservation. Washing produced a positive effect on frozen-thawed semen collected during spring or autumn, but the difference decreased as the concentration of yolk increased. However, washing produced a negative effect on frozen-thawed semen collected during winter, diluted with either 6 or 12% egg yolk. There was no apparent seasonal effect on gross measures of sperm production but the seasonal effect was ever present and was reinforced by freezing.     

海藻糖、二甲乙酰胺对公猪精液冷冻保存效果的研究

采用5% 二甲乙酰胺(DMA)(V/V)完全替代甘油,比较乳糖、海藻糖对精液冷冻保存效果的影响。结果表明,海藻糖显著提高了冷冻——解冻后精子成活力(49.32%±1.52%)与顶体完整性 (47.33%±1.16%)(P<0.05)。然后利用海藻糖替代乳糖,评价不同浓度的DMA对公猪精液冷冻保存的影响。结果表明,当DMA添加量为4%时,解冻后精子活率、成活力、顶体完整率分别为(45.17±0.56)%、(50.33±0.67)%、(48.30±1.44)%,均显著高于3% DMA、6% DMA添加组(P<0.05),精子活率显著高于5% DMA添加组(P<0.05),但精子成活力、顶体完整性与其差异不显著(P>0.05)。因此,当利用海藻糖作为冷冻保存基础稀释液,DMA最适添加量为4%。     

Cryopreserving turkey semen in straws and nitrogen vapour using DMSO or DMA: effects of cryoprotectant concentration,freezing rate and thawing rate on post-thaw semen quality

1. This study was designed to identify a suitable protocol for freezing turkey semen in straws exposed to nitrogen vapour by examining the effects of dimethylacetamide (DMA) or dimethylsulfoxide (DMSO) as cryoprotectant (CPA), CPA concentration, freezing rate and thawing rate on in vitro post-thaw semen quality.

2. Pooled semen samples were diluted 1:1 (v:v) with a freezing extender composed of Tselutin diluent containing DMA or DMSO to give final concentrations of 8% or 18% DMA and 4% or 10% DMSO. The semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen (LN₂) surface (1, 5 and 10 cm) for 10 min. Semen samples were thawed at 4°C for 5 min or at 50°C for 10 s. After thawing, sperm motility, viability and osmotic tolerance were determined.

3. Cryosurvival of turkey sperm was affected by DMSO concentration. Freezing rate affected the motility of sperm cryopreserved using both CPAs, while thawing rates showed an effect on the motility of sperm cryopreserved using DMA and on the viability of sperm cryopreserved using DMSO. Significant interactions between freezing rate × thawing rate on sperm viability in the DMA protocol were found.

4. The most effective freezing protocol was the use of 18% DMA or 10% DMSO with freezing 10 cm above the LN₂ surface and a thawing temperature of 50°C. An efficient protocol for turkey semen would improve prospects for sperm cryobanks and the commercial use of frozen turkey semen.     

Factors Affecting Pregnancy Rate in Artificial Insemination with Frozen Semen During Non-Breeding Season in Murciano–Granadina Goats: a Field Assay

An artificial insemination programme was carried out to study the effect of factors such as depth of semen deposition, inseminator skill, farm, sire and expression of oestrus on pregnancy rate in Murciano-Granadina (MG) goats during non-breeding season and using frozen semen. Frozen-thawed semen from six males was applied by three technicians to inseminate a total of 551 goats in 17 farms distributed throughout the Mediterranean area of Spain. Pregnancy rate was determined at 6 weeks after insemination by transabdominal ecography. Overall pregnancy rate was 57%. Farm and depth of semen deposition affected pregnancy rate, whereas the sire and the technician had no effect. The deeper the semen was deposited in the genital tract, the higher was the rate of pregnancy obtained, being greater when the catheter reached the uterus. In spite of the relevant difference observed (48.2% vs 59.0%), pregnancy rate of females not coming into oestrus until 30 h after sponge removal was not significantly different, compared with those showing oestrus during the OD procedure. In conclusion, our field assay data on AI in MG goat with frozen-thawed semen showed that post-cervical insemination presented significantly greater pregnancy rate in comparison to when semen is deposited in the vagina or in the caudal part of the cervix.     

Influence of season on the freezability of free-range poultry semen

The purpose of the present study was to examine the seasonal variation in freezing damage in free-range rooster sperm. Over a period of 1 year, heterospermic semen samples were collected weekly by massage from the roosters of 14 Spanish chicken breeds, all housed under natural photoperiod and climatic conditions. All samples were frozen in straws using DMA as a cryoprotectant, placing them first in nitrogen vapour and then plunging them into liquid nitrogen. No seasonal effects on fresh sperm quality were found. Neither did season affect the percentage of viable frozen-thawed spermatozoa nor the percentage with an intact acrosome. However, the collection season influenced (p < 0.05) most frozen-thawed sperm motility values. The percentage of immotile frozen-thawed spermatozoa was lower (p < 0.05) in spring-collected sperm than in summer- or autumn-collected samples. The percentage of spermatozoa showing progressive motility was higher in spring-collected sperm compared with winter-, summer- or autumn-collected samples (p < 0.05). The curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) values of spring-collected sperm were also higher (p < 0.05). In conclusion, spring would appear to be the best season for collecting and freezing the semen of free-range Mediterranean chicken breeds.     

The Effect of Season on Spermatozoa Motility,Plasma Membrane and Acrosome Integrity in Fresh and Frozen–Thawed Semen from Xinong Saanen Bucks

The aim of this study was to evaluate whether the season of ejaculate collection influences seminal quality parameters of pre‐ and post‐freeze–thawing in Xinong Saanen bucks. Ejaculates were collected from eight bucks throughout the four seasons (spring, summer, autumn and winter) in a 12 months’ time period, identified in the Northern Hemisphere. Semen samples were evaluated by the combinations of conventional and Computer‐Assisted Sperm Analysis (CASA) when fresh and after frozen–thawed, respectively. The results clearly demonstrated that season of ejaculate collection influenced (p < 0.05) fresh semen quality. Highest semen quality was observed during autumn. On the contrary, undesirable indices (significantly lower, p < 0.05) were observed in winter as compared with the other remaining seasons. CASA has clearly shown the influences of seasonal variations on semen motility parameters. Furthermore, season of ejaculate collection was also found to influence sperm freezability. Semen characteristics after frozen–thawed followed a similar pattern with that of fresh ejaculate except in spring. The results revealed that sperm quality was higher (p < 0.01) in summer and autumn than in spring and winter. In conclusion, seasonal variation influences semen quality in Xinong Saanen bucks. In addition to summer and autumn, fresh ejaculates in spring can also be successfully used for AI. Sperm from ejaculates collected during summer and autumn are more suitable for cryopreservation. Hence, it is possible to increase the efficiency of goat breeding by manipulating the seasonal variations of semen quality for immediate AI and/or cryopreservation.     

水牛性控精液微生物分析

研究针对目前分离水牛X和Y精子性别控制过程中,容易导致微生物快速繁殖进而污染性控精液的问题,对水牛新鲜及冷冻性控精液中的微生物进行分析研究,以期改进水牛分离精子保存方法。结果表明:新鲜采集的水牛精液、常规冷冻精液及分离后的冷冻精液中,平均菌落密度分别为10 619个/mL、293个/mL和18935个/mL,三者差异显著(P0.05)。新鲜精液检测到的菌落中,革兰氏阳性球菌占48%,阴性杆菌占41%,阳性杆菌占9%,霉菌占2%;性控冷冻精液中的菌落100%为革兰氏阴性杆菌。对性控冻精菌落药敏检测发现,菌落对妥布霉素和庆大霉素均呈现耐药性,而对丁胺卡那霉素以及药物环丙氟哌酸药物敏感性较强。本研究结果,为改进水牛分离精子稀释液以及冷冻保存液中的抗生素使用提供了有价值的参考。     

Effects of liquid nitrogen vapor sensitization conditions on the quality of frozen-thawed dog spermatozoa

The freezing conditions for preparation of frozen canine semen by the plunging method were investigated with regard to the period of sensitization in liquid nitrogen (LN2) vapor and the height from LN2, and the semen qualities after thawing were compared with those of canine semen prepared by the simple freezer method previously reported by us. In the plunging method, 9 semen straws were prepared under the same conditions, horizontally kept at 5, 7, and 10 cm above the LN2 surface in a styrene foam box for 5, 10, and 15 min, and then plunged into LN2. The semen qualities immediately after thawing were high in the 7 cm/10 min (cooling rate: -4 to -22 degrees C/min) and 10 cm/15 min groups (cooling rate: -6 to -10 degrees C/min). On comparison of frozen semen prepared by the plunging method (7 cm/10 min) with frozen semen prepared by the simple freezer method, sperm motility and viability were significantly higher for the frozen semen prepared by the plunging method. The cooling rate in freezing was higher for the simple freezer method (cooling rate: -6 to -50.9 degrees C/min) than the plunging method. Based on these findings, horizontal placement of canine semen straws above LN2 to reduce the temperature at a slow cooling rate of about -10 degrees C/min, followed by plunging into LN2 after sensitization for 10-15 min, provides good semen qualities after thawing.     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

Post-thaw Survival and Longevity of Bull Spermatozoa Frozen with an Egg Yolk-based or Two Egg Yolk-free Extenders after an Equilibration Period of 18 h

The aim of the present study was to determine the suitability of using two egg yolk-free commercial extenders, Andromed and Biociphos Plus as compared with the Tris-egg yolk based diluent Biladyl, for the cryopreservation of bull spermatozoa when the freezing protocol involved holding the extended semen at 4 degrees C for 18 h before the freezing. Six ejaculates from each of 10 Holstein bulls were collected by using artificial vagina. The ejaculates were evaluated for volume, sperm concentration and motility, divided in to three equal volumes, and diluted, respectively, with the three extenders as specified above. Extended semen was equilibrated for 18 h at 4 degrees C and frozen in 0.25-ml straws. After thawing, 100-mul aliquots of semen were labelled with SYBR-14, PI and PE-PNA (Phycoerythrin-conjugated Peanut agglutinin) and analysed by flow cytometry at 0, 3, 6 and 9 h after incubation at 37 degrees C. A General Lineal Model procedure for repeated measures was used to determine the effects of extender, bull, replicate and the interaction between them, on sperm viability and acrosomal integrity. Semen samples frozen with Biladyl showed higher (p < 0.001) sperm survival after 0 h (47.9%) and 9 h (30.3%) of incubation than those frozen with Andromed (38.5% and 17.3%, after 0 and 9 h respectively) or Biociphos Plus (34.9% and 21.6%, after 0 and 9 h respectively). The bull and replicate had significant effects (p < 0.001) on both sperm viability and acrosomal integrity, but the interactions between bull and extender and between replicate and extender were not significant. It was concluded that, when holding the semen overnight before freezing, the use of Biladyl results in higher sperm survival and longevity than the use of Andromed or Biociphos Plus.     

聚乙烯吡咯烷酮对公猪精液冷冻的影响

试验旨在评价聚乙烯吡咯烷酮（polyvinylpyrrolidone,PVP）对公猪精液冷冻的影响。试验分为5组,分别为对照组（不添加PVP）和PVP处理组（在冷冻基础液中分别加入0.25%、0.50%、1.00%、2.00% PVP）。采用手握法采集松辽黑猪精液,用5种冷冻基础液稀释,在25 ℃平衡1 h,17 ℃平衡2 h,4 ℃平衡3 h后灌装于0.5 mL细管中,在液氮上方3 cm处熏蒸10 min,保存在液氮罐中30 d后进行检测。样本解冻后分别检测精子活力、质膜完整性、顶体完整性、线粒体活性、DNA完整性、过氧化氢酶（catalase,CAT）活性、超氧化物歧化酶（superoxide dismutase,SOD）活性、谷胱甘肽过氧化物酶（glutathioneperoxidase,GSH-Px）活性、活性氧簇（reactive oxygen species,ROS）水平及丙二醛（malondialdehyde,MDA）水平。结果显示,与对照组相比,冷冻基础液中添加0.50% PVP显著提高冻融后精子的活力、质膜完整性、顶体完整性、线粒体活性、CAT活性、SOD活性、GSH-Px活性（P<0.05）,显著降低精子ROS和MDA水平（P<0.05）;与对照组相比,添加PVP有利于提高DNA完整性,但差异不显著（P>0.05）。因此,猪精液冷冻基础液中添加PVP可改善冻融后精子质量,添加0.50%效果最佳。