Fertility of Deep Frozen Boar Spermatozoa

In the present investigation the results of two insemination trials with deep frozen boar spermatozoa are presented. The aim of the trials was to study the effect of different thawing diluents and to compare the fertility of deep frozen spermatozoa from four boars. The trials utilized a total of 139 gilts. The thawing diluents used were boar seminal plasma, protein free seminal plasma, the thawing diluent OLEP and isotonic glucose solution. The composition of OLEP was based on physical and biochemical analyses of boar seminal plasma. The electrolyte levels, pH and osmotic pressure of OLEP are similar to those of boar seminal plasma. From the results it is evident that thawing in boar seminal plasma, protein free seminal plasma and OLEP yielded equal results. Thawing in isotonic glucose solution yielded significantly poorer results concerning percentage of fertilized ova 24–48 hrs. after insemination and almost significantly poorer fertility results four weeks after insemination. The possible effects of the thawing diluents are discussed. With the freezing procedure applied, electrolyte levels, pH and osmotic pressure seem to be factors of importance for the survival of the frozen and thawed spermatozoa and for the maintenance of their fertilizing capacity. Almost significant differences were found in fertility of spermatozoa from different boars. These differences were reflected in pregnancy rates as well as ratio of foetuses to c. 1. in pregnant gilts. The differences were found to be independent of thawing diluent. The variation seems to be caused by differences in resistance of the spermatozoa to the freezing and thawing procedure. The need for laboratory methods for selection of boars with spermatozoa of good freezability is stressed.     

Fertility of Deep Frozen Boar Spermatozoa at Various Intervals between Insemination and Induced Ovulation

This study was performed to investigate the influence of boars and thawing diluents on the fertilizing capacity of deep frozen spermatozoa at various intervals between inseminations and ovulation. Forty-four Swedish crossbred gilts were inseminated following injection of HCG late in the prooestrus. Inseminations were performed 22, 28, 34 and 38 hrs. after injection of HCG. Ovulation was expected to occur 40 hrs. after injection of HCG. Two boars, previously tested for fertility with frozen semen, supplied the spermatozoa. Roar seminal plasma and OLEP were utilized as thawing diluents. The gilts were slaughtered 32–48 hrs. after estimated ovulation. The genital tracts were removed immediately after stunning and bleeding and the numbers of recent ovulations, recovered ova and fertilized ova were recorded. Additionally recovered ova were classified according to estimated numbers of spermatozoa attached to the zona pellucida. Similar fertilization rates were obtained when inseminations were performed 2 and 6 hrs. before estimated ovulation. A clear decline in fertility appeared when inseminations were performed earlier than 6 hrs. before expected ovulation. The results were influenced by the boars as well as by the thawing diluents. Seminal plasma yielded a higher fertilization rate than OLEP in inseminations performed 2 hrs. before estimated ovulation. The boars yielded similar fertility in inseminations performed 2 hrs. before estimated ovulation. With increasing intervals between inseminations and ovulation the difference between the boars increased. The single gilt in which fertilized ova were found after insemination 18 hrs. before ovulation was inseminated with spermatozoa from the superior boar, thawed in seminal plasma. The present results indicate that spermatozoa with low resistance to freezing-thawing have a short fertile life in the female genital tract after insemination.     

On the Fertility and Survival of Deep Frozen Boar Spermatozoa Thawed in Skim Milk

Satisfactory conception rates of deep frozen boar spermatozoa were obtained, with insemination by way of the cervix, after thawing the deep frozen spermatozoa in boar seminal plasma, both in preliminary trials (Crabo & Einarsson 1971, Crabo et al. 1972 b) and in a large field trial (Einarsson et al. 1972). Fertility with pellet frozen boar spermatozoa, thawed without dilution, was reported by Graham et al. (1971 a, b) and Pursel & Johnson (1971).     

Influence of Thawing Diluents on Vitality,Acrosome Morphology,Ultrastructure and Enzyme Release of Deep Frozen Roar Spermatozoa*

The present investigation was performed to study the effect of freezing and thawing on boar spermatozoa. Thirty-one ejaculates from four boars were investigated after thawing in three different thawing diluents (seminal plasma, OLEP, isotonic glucose solution). From each ejaculate one sample of 1 × 109 spermatozoa was thawed in each of the thawing diluents. Each sample was examined in a thermoresistance test in which motility was stimulated with caffeine 30 min. and 3 hrs. after thawing. Furthermore, acrosome morphology and ASAT release from the spermatozoa were investigated for each sample. One ejaculate from the two most frequently used boars was examined by electron microscopy after thawing in each of the thawing diluents. Differences in the aspects studied appeared between isotonic glucose solution and the other two thawing diluents in the thermoresistance test, in the response to caffeine stimulation 3 hrs. after thawing and in the amount of ASAT released from the spermatozoa. The influence on the acrosome morphology varied between the thawing diluents, but the acrosomal alterations did not seem to be connected with the damage reflected by the thermoresistance test and by the measurement of extracellular ASAT activity. The ultrastructural investigation showed that all spermatozoa examined had some degree of ultrastructural alteration as compared with freshly ejaculated boar spermatozoa treated in the same way. This alteration could not be related to any of the thawing diluents. Of the various laboratory tests the thermoresistance test and the measurement of ASAT release are suggested to be sensitive indicators of sperm damage during freezing and thawing. These tests might be useful indicators of variations in sensitivity of spermatozoa to the freezing-thawing procedure.     

III: Ultrastructure of Boar Spermatozoa Frozen Ultra-Rapidly at Various Stages of Conventional Freezing and Thawing

Ejaculated boar spermatozoa subjected to a conventional freezing and thawing process, were ultra-rapidly fixed, freeze-substituted and examined by electron microscopy to monitor the presence of real or potential intracellular ice and the degree of cell protection attained with the different extenders used during the process. Numerous ice crystal marks representing the degree of hydration of the cells were located in the perinuclear space of those spermatozoa not in proper contact with the extender containing glycerol (i.e. prior to freezing). The spermatozoa which were in proper contact with the extenders presented a high degree of preservation of the acrosomes, plasma membranes as well as the nuclear envelopes. No ice marks were detected in acrosomes before thawing, indicating that the conventional assayed cryopreservation method provided a good protection against cryoinjury. The presence of acrosomal changes (internal vesiculization, hydration and swelling) in thawed samples however, raises serious questions about the thawing procedure employed.     

猪精子冷冻损伤研究进展

综述了猪冻存精子顶体、质膜和线粒体的形态学变化及其对精子受精能力的影响,同时分析了冻融精子中所发生的蛋白质、DNA等生物大分子的变化及其可能对冻存精子质量及受精能力的影响,指出冷冻保存过程对精子蛋白质、DNA等生物大分子质和量的影响是精子冷冻损伤的实质,应用先进的蛋白组学进行猪精子冷冻损伤机理研究,有助于深刻揭示冷冻损伤的分子机理,为推动猪精液冷冻保存技术研究取得突破性进展提供理论依据。     

Abaxial Implantation of the Middle Piece in Spermatozoa and Spermatids in Related Sterile Boars

Spermatozoa and developing spermatids showing neck region abnormalities have been studied in material from 2 genetically related boars. In both boars the defects were abaxial implantation of the tails and lack of substance in the neck region. In many spermatozoa, a too wide space between the capitulum and the basal plate was more pronounced in epididymal spermatozoa compaired to testicular material. This implies that the defect aggravated, and might be connected with the migration of the cytoplasmic droplet in the epididymis. Since the defects were observed in spermatids, it its concluded that the defects were heriditary. This conclusion was further supported by the observation of similar defects in 6 other related boars, examined by light microscopy only.     

大熊猫冷冻精液解冻速度和解冻液中添加Pentyoxyfilline对其解冻后活力的影响

建立大熊猫的精子库,进行远距离圈养大熊猫种群间的人工授精和遗传物质的转运,维持遗传多样性,是目前大熊猫遗传管理的优先方法。要成为最有效的工具,精子库保存的精子解冻后的活力必须很好。本文对大熊猫冷冻精液的解冻速度和解冻液中添加化学激活剂Pentyoxyfilline(PF)后的精子活力进行了试验。试验用的精液采自11只成年大熊猫,精液冷冻速度为每分钟-40℃～-100℃。试验Ⅰ:将冷冻精液放入3种不同温度的水浴中解冻:(1)22℃(慢速解冻);(2)37℃(中速解冻)(3)50℃(快速解冻)。将冷冻前精子活力(78 1±2 9%)和解冻后的平均精子活力进行比较,快速解冻后的精子活力(57 5±5 4%)显著地降低(P<0 05),而中速解冻的精子活力(67 5±3 1%)和慢速解冻的精子活力(73 33±2 1%)与冷冻前的活力接近。试验Ⅱ:使用中速解冻方法解冻精液后,分别加入最终浓度为0mM、1mM、5mM和10mM的PF,然后分别保温15min和24h。在PF(0mM、1mM、5mM和10mM)中分别孵育15min的解冻精子活力,运动状态,活率和顶体正常率在试验期的90min内都很相似(P>0 05)。在1mMPF中孵育24h的精子活力没有变化(P>0 05)。在5mM和10mMPF中孵育过的精子活力(5mM:24 0±4 7%;10mM19 5±3 6%)比没有加PF的对照组的精子活力(38 3±5 2%)显著地低(P<0 05)。而且,在10mM     

猪精子凝集素在精卵结合中的作用机制

猪精子凝集素（ＢＳＬ）位于精子头部,既与精子蛋白结合,亦与透明带糖蛋白ＺＰ３结合。并且ＢＳＬ自身分子间亦会聚合。与ＺＰ３结合的片段亦结合于岩藻素－Ｓｅｐｈａｒｏｓｅ亲和柱,但不与胎球蛋白－Ｓｅｐｈａｒｏｓｅ柱结合。该片段具有较强的抑制精卵结合的活性。ＢＳＬ经ＮＢＳ修饰后,血凝活力大大下降,同时推动与精子结合的活性,但不影响它与ＺＰ３的结合。修饰后的ＢＳＬ亦显著抑制精卵结合。表明ＢＳＬ以两个不同的部     

Effects of Cholesterol-Loaded Cyclodextrins on the Rate and the Quality of Motility in Frozen and Thawed Rabbit Sperm

The motility of sperm after freezing and thawing is critical for effective cryopreservation. It is known that supplementation with cholesterol-loaded cyclodextrin (CLC) improves cryosurvival of sperm in various animals. To clarify the effects of supplementation with CLC on rabbit sperm motility after freezing and thawing, rabbit sperm motility was analyzed using a computer-assisted sperm analysis system. Sperm motility with CLC supplementation was 29.4 ± 9.6% (mean ± SD), which was significantly higher than that of controls (20.8 ± 7.1%, P<0.05). The curvilinear velocity of sperm with CLC exceeded that of controls, whereas the values for linearity and wobble were significantly lower in sperm with CLC compared with controls. After artificial insemination, 44.3% of recovered ova were fertilized in the CLC-supplemented group, which was higher than the percentage in the control group (36.4%). The results indicate that supplementation with CLC improves the rate and quality of motility in rabbit sperm after freezing and thawing, and would be advantageous for successful cryopreservation.     

冻存液添加维生素B12,解冻液添加BSA及肝素对大熊猫精子的影响

大熊猫冻精的解冻活力一直徘徊在35%左右,顶体完整率低于40%,较大地影响着人工授精的成功率。本文通过在冻存液中添加维生素B12和在解冻液中添加BSA及肝素,以提高冻精活力和顶体完整率进行了实验研究,结果表明:(1)冻存液中添加维生素B121mg/ml,使精子顶体完整率从76.66%提高到83.34%(P<0.05);(2)选用美国Hyclone公司的M199、Ham's F-10液和Irving Scientific公司的两种Ham's F-10液(代号168、175)共4种解冻液,均添加5%的FBS进行比较实验,其中以Irving Scientific公司的Ham's F-10(175)对提高解冻精子质量效果最好,精子活力达到74.7%±2.706%,运动速度(SOP)为3±0.82(P<0.05);(3)解冻液中添加高浓度BSA对精子的运动速度有明显提高(P<0.05),而添加低浓度BSA对精子的影响不显著;(4)解冻液中添加肝素对精子活力及运动速度的影响均不明显(P>0.05)。     

低剂量三聚氰胺对小鼠精子质量影响的研究

采用灌胃法,研究了三聚氰胺在1.0 mg/kg、2.0 mg/kg、4.0 mg/kg浓度下染毒35 d时,小鼠体重、精子运动参数和精子形态的变化.结果显示,4.0 mg/kg 处理组小鼠部分精子运动参数显著性降低(P＜0.05),1.0 mg/kg 和2.0 mg/kg 浓度组各项精子运动参数与对照比较变化不显著(P＞0.05);在染毒一周后,2.0 mg/kg 浓度组小鼠体重增长显著性下降(P＜0.05),4.0 mg/kg 浓度组小鼠体重极显著降低(P＜0.01);3个处理浓度组对小鼠精子形态影响均不显著.总之,食物中低浓度三聚氰胺对小鼠精子活性和形态无明显的影响.     

Influence of Boar and Semen Parameters on Motility and Acrosome Integrity in Liquid Boar Semen Stored for Five Days

Ninety ejaculates from a total of 76 AI boars were extended in Beltsville Thawing Solution (BTS). Boar identity, breed, weight of the ejaculate and sperm concentration were registered. Motility and acrosome integrity were assessed after storage at 16–18°C for 6, 30, 54, 78, and 102 h. Storage time had a significant influence on both motility (p < 0.01) and acrosome integrity (p < 0.001). The Least Square Means for percentage of motility showed a small decline from 79.8% after 6 h of storage to 78.4% at 102 h. Motility at 78 and 102 h was significantly different from motility at 6 h (p < 0.05). The percentage of sperm cells with normal acrosomes declined throughout the experiment. The Least Square Means for 6, 30, 54, 78, and 102 h of storage were 93.9%, 90.6%, 88.0%, 84.8%, and 78.2%, respectively. The decrease in acrosome integrity from one storage time to the next was highly significant throughout the trial (p < 0.001). There was a significant influence of boar (p < 0.001) and sperm concentration (p < 0.01) on motility, while acrosome integrity was affected only by boar (p < 0.001). Breed of the boars and weight of the ejaculate did not influence the dependent variables.     

女性受教育程度与其生育水平关系的数学模型分析

本文对我国女性受教育程度与其活产子女数、总和孩次递比等重要的生育水平指标间的关系进行了全面而深入的统计分析，并得到反映其定量关系的回归方程数学模型，从而揭示出女性受教育程度对其生育水平所具有的重大作用。     

Normal Fertility Requires the Expression of Carbonic Anhydrases II and IV in Sperm

HCO₃⁻ is a key factor in the regulation of sperm motility. High concentrations of HCO₃⁻ in the female genital tract induce an increase in sperm beat frequency, which speeds progress of the sperm through the female reproductive tract. Carbonic anhydrases (CA), which catalyze the reversible hydration of CO₂ to HCO₃⁻, represent potential candidates in the regulation of the HCO₃⁻ homeostasis in sperm and the composition of the male and female genital tract fluids. We show that two CA isoforms, CAII and CAIV, are distributed along the epididymal epithelium and appear with the onset of puberty. Expression analyses reveal an up-regulation of CAII and CAIV in the different epididymal sections of the knockout lines. In sperm, we find that CAII is located in the principal piece, whereas CAIV is present in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an imbalanced HCO₃⁻ homeostasis, resulting in substantially reduced sperm motility, swimming speed, and HCO₃⁻-enhanced beat frequency. The CA activity remaining in the sperm of CAII- and CAIV-null mutants is 35% and 68% of that found in WT mice. Sperm of the double knockout mutant mice show responses to stimulus by HCO₃⁻ or CO₂ that were delayed in onset and reduced in magnitude. In comparison with sperm from CAII and CAIV double knockout animals, pharmacological loss of CAIV in sperm from CAII knockout animals, show an even lower response to HCO₃⁻. These results suggest that CAII and CAIV are required for optimal fertilization.     

Influence of Various Factors on the Survival of Lactobacillus leichmannii during Freezing and Thawing

S ummary : The influence of the age and activity of bacteria, their concentration in the protective medium, the pH value of the medium, the temperature of thawing, and the time of storage on the survival of L. leichmannii during freezing and thawing has been investigated.     

Inactivation of Salmonellae on Chilled and Deep Frozen Broiler Carcasses by Irradiation

Chilled and deep-frozen broiler carcasses were examined for total counts of salmonellae using pooled samples taken from 76 batches each of five birds. By means of a most probable number technique (MPN) it was found that counts expressed/100 g of skin or /500 ml of thaw water varied between < 2 and 1400 with 90% being < 100. Irradiation of carcasses using a dose of 250 krad was found to be highly effective in destroying salmonellae whether the birds had been chilled or deep-frozen.