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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. 相似文献
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Hori T Odaka S Oba H Mizutani T Kawakami E Tsutsui T 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2006,68(10):1055-1061
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. 相似文献
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Ishikawa A Matsu M Sakamoto H Katagiri S Takahashi Y 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2002,64(4):373-376
Four adult Hokkaido brown bears were used as semen donors, and semen characteristics were examined before freezing and after thawing. A total of 10 electroejaculates were diluted with Tris-egg yolk extender and cooled to 4 degrees C over 90 min. Spermatozoa were equilibrated with 4.7% glycerol for 80 min. Semen packed in 0.25 ml plastic straws were frozen with liquid nitrogen vapor. Percentages (mean +/- SD) of motile and live sperm were 96+/-2 and 86.5+/-7.2% before freezing, and 43+/-5 and 67.4+/-3.9% after thawing, respectively. Although the number of progressively motile sperm after thawing varied among samples (1.8+/-1.2 x 10(8) cells/ejaculate), frozen semen in the present study might serve for artificial insemination. 相似文献
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A Córdova JF Pérez B Lleó C García Artiga & S Martín Rillo 《Reproduction in domestic animals》2001,36(3-4):199-202
The aim of this study was to evaluate the straw size effect used for freezing on the in vitro fertilizing capacity. Twenty-one ejaculates from seven fertile boars were frozen under controlled conditions in 0.5 and 5 ml straws. Thawed semen was compared to fresh semen. For fresh and thawed semen in 0.5 and 5 ml straws, the results were: 92.18, 77.38 and 79.04% sperm penetration; 80.68, 66.89 and 69.33% monospermy; 11.51, 10.49 and 9.74% polyspermy; 86.19, 47.14 and 47.02% motility and 75.52, 48.19 and 46.81% normal apical ridge (NAR), respectively. Analysis of variance and test of multiple comparisons showed that under the conditions employed, penetration, monospermy, motility and NAR were significantly reduced by freezing–thawing, but polyspermy was much less affected. The results obtained suggest that frozen boar semen is adequate for in vitro fertilization. In addition freezing in 5 ml straws did not have any detrimental effect on either penetration, monospermy, polyspermy, motility and NAR, in comparison with freezing in 0.5 ml straws. 相似文献
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WMC Maxwell I Parrilla I Caballero E Garcia J Roca EA Martinez JM Vazquez D Rath 《Reproduction in domestic animals》2007,42(5):489-494
The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing. 相似文献
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Eriksson BM Rodriguez-Martinez H 《Journal of veterinary medicine. A, Physiology, pathology, clinical medicine》2000,47(2):89-97
The motility and membrane integrity of spermatozoa from nine boars frozen with a programmable freezing machine in plastic bags, 'cochettes', and in 'maxi-straws', in total doses of 5 x 10(9) spermatozoa/5 ml with glycerol (3%) used as cryoprotectant, were assessed after thawing. A computer-based cell motion analyser was used to evaluate sperm motility, while the integrity of the plasmalemma was assessed with fluorescent supravital dyes (C-FDA/PI). The fertilizing capacity of the semen frozen in the two containers was investigated by inseminating (AI) gilts. Pregnancy was monitored by Doppler-ultrasound, and the numbers of corpora lutea and viable embryos counted at slaughter, between days 30 and 38 after AI. The cochettes sustained the overall procedure of freezing/thawing (FT), with 30 min post-thaw (PT) sperm motility being significantly higher than for straws, 46.9 vs. 39.5%. The only significant difference in motility patterns detected when comparing the packages was a higher sperm velocity (VCL) in cochettes at 30 min PT. However, percentages of FT-spermatozoa with intact membranes, detected with the supravital probes, were higher in maxi-straws than in cochettes, 46.8 vs. 43.0% (P < 0.05). There were no significant differences found in fertilizing capacity between spermatozoa frozen in maxi-straws and those frozen in cochettes. The results indicate that although the deep-freezing of AI-doses of boar semen in large plastic bags is feasible, problems such as their inconvenient size for storage and inconsistent thawing must be solved before this type of container can be used for the commercial cryopreservation of boar semen. 相似文献
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试验对绵羊精液分别采用液氮熏蒸冷冻法和冷冻仪冷冻法进行冷冻,通过测定精液解冻后精子活率、顶体完整率、质膜完整率和谷草转氨酶活性及乳酸脱氢酶活性来比较不同的冷冻方法对解冻后精液品质的影响;通过测定精液稀释后、平衡后、冷冻仪法冷冻后的酶活性来比较谷草转氨酶与乳酸脱氢酶在不同阶段的释放量。结果表明,采用冷冻仪法冷冻后的精子活率和质膜完整率极显著高于液氮熏蒸冷冻法（P<0.01）;冷冻仪法冷冻后的谷草转氨酶和乳酸脱氢酶活性极显著低于液氮熏蒸冷冻法（P<0.01）;精液稀释后的谷草转氨酶和乳酸脱氢酶活性极显著低于精液冷冻后的活性（P<0.01）。表明采用冷冻仪冷冻法的绵羊精液品质好于液氮熏蒸冷冻法。精子中谷草转氨酶主要在平衡阶段释放,而乳酸脱氢酶主要在冷冻阶段释放。 相似文献
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Transfer of frozen-thawed embryos in sheep 总被引：3，自引：0，他引：3
Embryos collected from ewes six days after oestrus were frozen in straws using ethylene-glycol as a cryoprotectant. The efficiency of the simplified freezing and thawing procedure was assessed after transfer, which resulted in an overall survival rate of 58.3 per cent. Forty-two lambs were born from 72 frozen embryos which had been transferred without any attempt to evaluate them after the thawing and sucrose dilution process. 相似文献
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利用细管冻精进行布尔山羊人工授精，其受胎率总是徘徊在40％左右，与牛人工授精90％的受胎率相比差异甚大，从而严重制约了布尔山羊的快速发展。为解决这一难题，现从理论上重点阐述进行细管冻精人工授精的综合操作技术以提高布尔山羊受胎率。 相似文献
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The objective of this study was to determine the effects of thawing groups of 2, 5, 10, 15, or 20 .5-ml French straws on post-thaw spermatozoal viability. Thermostatically controlled and nonthermostatically controlled thawing baths were compared. Using a split-plot design, semen from 10 bulls was extended in egg yolk citrate, frozen, and then thawed (in the respective groups) at 36 degrees C in two types of thawing baths. Motility and percentage of intact acrosomes were determined immediately after thawing (0 h) and again after 4 h of incubation at the respective temperature of each thawing bath. Neither percentage of intact acrosomes nor motility was influenced by the number of straws thawed at 0 h (P greater than .05). Thawing bath had no effect (P greater than .05) on motility or percentage of intact acrosomes at 0 h. Bull variation was significant in both the 0- and 4-h evaluations. After 4 h of incubation, there was a significant (P less than .05) straw number x thawing bath interaction. When 15 or 20 straws were thawed in the thermostatically controlled bath there was a reduction (P less than .05) in motility and percentage of intact acrosomes. However, in the nonthermostatically controlled bath there was no reduction in motility and percentage of intact acrosomes as the size of straw group increased. Our results indicate that, when using a nonthermostatically controlled thawing bath, semen packaged in .5-ml straws can be thawed in groups of 20 without an effect on post-thaw sperm viability. 相似文献
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Control of sperm quality after commercial freezing/thawing of bull semen is still restricted to the subjective assessment of sperm motility, despite its low correlation to fertility (Söderquist et al. 1991, Kjaestad et al. 1993). Although no single in vitro method has yet been designed to predict the fertilizing ability of a given semen sample, the quantitation of viable spermatozoa (with intact plasma and acrosome membranes, and metabolically active) seems to be most promising (Woelders et al. 1991). The present report describes the use of a bioluminiscence technique to determine ATP-levels and a novel supravital stain (using fluorescent dyes) to assess the amount of viable spermatozoa in frozen/thawed semen from 3 A.I. dairy bulls with significantly different motility after thawing. 相似文献
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为了建立重庆板角山羊精液的细管冷冻保存方法,实验进行了不同冷冻稀释液(配方Ⅰ、Ⅱ、Ⅲ)、不同冷冻保存剂(甘油、EG)及不同离心速度(1000、1200、1400r/min)对重庆板角山羊细管精液冷冻保存效果的研究,结果表明:配方Ⅱ对重庆板角山羊精液的冻后活率显著优于配方Ⅰ和Ⅲ(P<0.05)。在配方Ⅱ中添加相同剂量(5%)的EG和甘油,精液冻后活率差异不显著(P>0.05)。以1200r/min的速度对山羊鲜精作离心处理后,冻后活率相对于对照组有所提高,但差异不显著(P>0.05)。 相似文献
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Initial cooling time before freezing affects post‐thaw quality and reproductive performance of rabbit semen 
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下载免费PDF全文 Michele Di Iorio Maria Antonietta Colonna Marsia Miranda Paola Principe Michele Schiavitto Silvia Cerolini Angelo Manchisi Nicolaia Iaffaldano 《Animal Science Journal》2018,89(9):1240-1244
The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post‐thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris‐citrate‐glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post‐thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms. 相似文献
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Contents: At the Bundesanstalt Wels tests with frozen-thawed boar semen in plastic straws were conducted. The influences of straw volume, method of freezing and thawing extender were investigated. The straw volumes of 1, 0 and 0, 5 ml showed significantly better thawing results in a preliminary trial than the 5, 0 ml Macrotub. When semen was frozen in a computerized freezer with automatic seeding, all tested straw. volumes gave significantly better results than the straws frozen in static N2-vapor. A ready to use commercial extender was used for thawing with good results to simplify the handling of deep frozen semen for on farm insemination. In the main trial 60 ejaculates from 10 boars were frozen in 1, 0 ml straws in the computerized freezer. Three in vitro parameters for fresh semen (motility, osmotic resistance and keeping quality under standard conditions) and two parameters for thawed semen (motility and percentage of sperm with normal apical ridge) were recorded and correlated. The osmotic resistance test proved to be well suited as means of predicting the fitness of an ejaculate for deep freezing, but the other two fresh semen parameters showed poor correlation with the parameters for thawed semen. From the parameters for thawed semen a freezing score was derived as a measure of the freezability of single ejaculates and boars. A preliminary insemination trial gave satisfying farrowing rates. Inhalt: Zur Tiefgefrierung von Ebersamen: Untersuchungen zum Einfluß von verschiedenen “Straw”-Volumina, Einfriewerfahren und Auftaubedingungen Ander Bundesanstalt Wels wurden Tiefgefrierversuche mit Eberspermain Kunststoffpailletten durchgeführt. Die jeweiligen Einflüsse von Paillettenvolumen, Gefrierverfahren und Auftauverdünner wurden untersucht. In Vorversuchen erwiesen sich die Paillettenvolumina 1, 00 und 0, 5 ml gegenüber den 5,0 ml Makrotüb hinsichtlich Kopfkappenintegrität und Auftaumotilität signifkant überlegen. Die Auftauergebnisse der im computergesteuerten Freezer mit automatischem Seeding eingefrorenen Proben waren für alle untersuchten Paillettengröβen signifikant bis hoch signifikant besser, als die Werte der im statischen Stickstoffdampf eingefrorenen Proben. Als Auftauverdünner wurde ein handelsüblicher Fertigverdünner gewählt um eine besonders für den Eigenbestandsbesamer wichtige einfache Handhabung der TG-Besamung zu gewährleisten. Im Hauptversuch wurden 60 Ejakulate von 10 Ebern verwendet und in 1, 0 ml Pailletten im programmierbaren Freezer eingefroren. Drei Frischsamenparameter (Motilität, osmotische Resistenz und Haltbarkeit unter Laborbedingungen) und zwei Auftausamenparameter (Motilität und Kopfkappenintegrität) wurden erhoben und korreliert. Dabei enwies sichder osmotische Resistemtest als für die Beurteilung von Frischsperma hinsichtlich TG-Eignung gut geeignet, während die beiden anderen Frischsamenparameter keine bzw. nur schwache Korrelation zu den Auftauergebnissen aufwiesen. Aus den Auftausamenparametern wurde eine “Einfrierbarkeitszahl” als Maβ für die Eignung des Spermas zur Tiefgefrierkonservierung der einzelnen Eber bzw. Ejakulate erstellt. Dabei enwies sich der osmotische Resistenztest als für die Beurteilung von Frischsperma hinsichtlich TG-Eignung gut geeignet. Bei einem orientierenden Besamungsversuch wurden zufriedenstellende Abferkelraten erzielt. 相似文献
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Tsutsui T Tanaka A Takagi Y Nakagawa K Fujimoto Y Murai M Anzai M Hori T 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2000,62(12):1247-1251
Frozen feline semen was prepared using two types of extenders, egg yolk Tris-fructose citric acid (EYT-FC) and egg yolk sodium citrate solution (EYC), and the semen qualities after thawing and the conception rates obtained by unilateral intrauterine horn insemination (UIUI) were investigated. Cats used in the experiment were six males and 11 females aged 2-12 years (the number of experimental cases was 17). For preparation of frozen semen, semen collected by the artificial vagina method was adjusted to I x 10(8) sperm/m/ and 7% glycerol, put in 250 microl straws, and then frozen using a cell freezer. The mean sperm motility after thawing was 30.0+/-9.7 (SE) % in the semen prepared with EYT-FC and 30.0+/-3.3% in the semen prepared with EYC. Four of seven animals were fertilized by UIUI using two straws in both extenders, and the conception rate was 57.1%. The mean ratios of number of kits to the number of ovulations in the inseminated side were 61.1+/-24.5% and 30.5+/-3.4% for EYT-FC and EYC, respectively, showing that the ratio tended to be higher in the semen prepared with EYT-FC. The above findings, comparing the two extenders for preparation of frozen feline semen, showed that EYT-FC is slightly superior to EYC. To increase conception and fertility rates, it may be important to increase the sperm count for insemination and to inseminate both uterine horns. 相似文献