猪深部输精技术应用研究进展

常规子宫颈人工授精(cervical artificial insemination,CAI)技术输精量大,存在浪费和不经济的问题,尤其在使用高附加值的精液产品时更为突出。近年来,一种高效利用猪精液的深部输精技术得到了研究和应用,这种技术与定时输精技术相结合,直接将精液输送至子宫体、子宫角和输卵管内,减少了每头母猪受孕所需精子数目,大幅度提高了良种公猪的利用价值,有助于实现冷冻保存精液和性控精液在养猪生产上的商业化应用。作者概述了猪人工授精中3种深部输精技术在液态保存、冷冻保存和性控精液上的研究和应用情况。无论实施哪种输精技术,公猪繁殖力、精子活力、母猪的饲养管理、输精时间和人工授精技术操作的规范性都是提高受胎率必须要考虑的因素。     

奶牛性控冻精人工授精影响因素研究

用分离X和Y精子的性控精液进行人工授精是控制家畜性别之最简单可行的方法.然而,低密度性控精液输精效果还不如常规人工授精,许多技术环节都有待改进.以常规冻精和稀释常规冻精为对照,研究解冻方法、输精时间和部位、不同精液来源和输精员以及育成和经产牛等因素对性控冻精人工授精妊娠率的影响.结果显示,精液解冻水浴温度和持续时间对人工授精效果有显著影响,性控精液对解冻水浴温度更敏感;性控冻精和稀释常规冻精比常规冻精对输精时间要求更严格;3种精液输精到排卵卵泡同侧子宫角基部受胎率都显著高于输精于子宫体和同侧子宫角前端;3种精液育成牛受胎率(80%)都显著高于经产牛(50%);于输精同时注射促排卵素3号明显提高性控冻精受胎率;经严格挑选、能够从事胚胎移植操作的技术熟练输精员之间性控冻精受胎率差异不显著;在所设计的不同条件下,性控冻精与稀释同样倍数的常规冻精行为相似,说明精子分离过程没有对精子造成特殊损伤.研究结果说明,精确控制人工授精各个技术环节可以实现消除性控与非性控、低密度与高密度精演之间的差别,获得高妊娠率.     

Application and Commercialization of Flow Cytometrically Sex-Sorted Semen

The current technology to sort X and Y chromosome bearing sperm population requires individual identification and selection of spermatozoa in a modified high-speed flow cytometer. For farm animal species, the technology is capable of producing sexed sperm at greater than 90% purity. However, only in the bovine, the technology has reached a developmental level that allows its commercial application. Meanwhile, the demand for female calves has grown rapidly, which encourages the demand for sex-sorted semen from high genetic value bulls. The success of the technology will depend mainly on the fertilizing capacity of the sorted spermatozoa, as this is the most affecting and economically relevant factor. To date, fertility is still variable and is quite dependant on post-sort processing. New processing techniques are under investigation and will likely be able to improve the fertility rates after AI with sex-sorted semen. It is of great importance to select the right bulls and to test the sorted samples on a routine basis. In addition to the demand for sex-sorted semen by the cattle industry, there is also a significant demand expressed by pig farmers. However, it is still unknown if the use of sex-sorted semen through commercial pig AI will be economically feasible. For the pig, the combination of in vitro fertilization with sexed semen and non-surgical embryo transfer is an alternative that merits further scientific attention. Recent developments in ovine AI and ET will make it very likely that commercial sheep industry will adopt the sexing technology in their breeding concepts.     

Pregnancy Rates in Lactating Holstein–Friesian Cows after Artificial Insemination with Sexed Sperm

The effects of artificial insemination (AI) using sexed sperm on pregnancy rates have seldom been studied in lactating dairy cows on commercial dairy farms. We evaluated pregnancy results after AI of 306 lactating dairy cows, of which 157 were inseminated with 2x10(6) frozen/thawed sexed sperm and 149 with 15x10(6) frozen/thawed unsexed sperm. The average pregnancy and calving rates were 21.0% and 20% for the sexed-sperm AIs and 46% and 45% for the unseparated control-sperm AIs respectively (p<0.001). The proportion of female calves derived from sexed-sperm AI was 82% compared with 49% for control AI (p<0.01). The proportion of live and healthy calves in single births was 100% for sexed-sperm AI and 97% for control AI (p>0.05). Our results indicate that AI with low-dose sexed sperm under field conditions in commercial dairy herds without oestrus synchronization results in significantly reduced pregnancy rates compared with normal-dose AI. Improved insemination strategies combined with increased sperm doses are needed before the use of sexed sperm can be of any significant benefit for the dairy and beef industry.     

In vitro fertilization using non-sexed and sexed bovine sperm: sperm concentration, sorter pressure, and bull effects

The objective of these experiments was to study bovine in vitro fertilization (IVF) conditions for blastocyst production using non-sexed sperm (Experiment 1) and sexed sperm (Experiment 2). For Experiment 1, in vitro-matured oocytes (N=707) were allocated to a 2 × 3 × 4 factorial design: time of co-incubation of gametes for fertilization (4 and 18 h), sperm dose (1, 0.33, and 0.11 × 10(6) frozen-thawed sperm/ml, and sperm source (four bulls). Pronuclear status was evaluated for a subset. Experiment 2 (N=2155 oocytes) was a 2 × 3 × 2 × 6 factorial design: sex of sperm (X and Y), sperm dose (1, 0.33, and 0.11 × 10(6) frozen-thawed sperm/ml), and sperm-sorting pressures (40 and 50 psi), replicated with sperm of six bulls. Presumptive zygotes were cultured 60 h in chemically defined medium-1 (CDM-1), and for 114 h in CDM-2. For Experiment 1, pronuclear formation, cleavage and blastocysts rates were greater for 1, and 0.33 × 10(6) than 0.11 × 10(6) sperm/ml (72 and 62 vs 42%; 89 and 81 vs 58%; and 21 and 17 vs 9%, respectively; all p<0.01); polyspermy was greater for 1, than 0.33 and 0.11 × 10(6) sperm concentrations (24 vs 2 and 0%; p<0.01). There were greater main effects (p<0.01) of pronuclear formation (69 vs 48%), polyspermy (13 vs 4%), and cleavage (63 vs 54%), at 18 than at 4 h of co-incubation of gametes (all p<0.01). For Experiment 2, cleavage and blastocyst rates were greater for 1 × 10(6) sperm/ml vs 0.33 and 0.11 (69%, 47%, and 30% cleavage and 30%, 14%, and 8% blastocysts) and 40 vs 50 psi (54% and 44% cleavage and 18% and 15% blastocysts) (p<0.01). A marked bull by fertilization sperm dose interaction was found for cleavage (p<0.05). The main conclusion was that the optimal sperm concentration for cleavage and producing blastocysts via IVF with sexed sperm was considerably higher and more variable among bulls than for unsexed sperm.     

First Established Pregnancies in Mediterranean Italian Buffaloes (Bubalus bubalis) Following Deposition of Sexed Spermatozoa near the Utero Tubal Junction

At the time of AI following Ovsynch protocol, a total of 51 buffaloes were randomly divided in a first group (n = 30) subjected to conventional AI into the uterine body with 20 million non-sex sorted frozen-thawed spermatozoa, while a second group (n = 21) was inseminated near the utero-tubal junction (UTJ) ipsilateral to the ovary carrying the preovulatory follicle with 2.5 million live (4 million total) sex-sorted frozen-thawed spermatozoa. The semen used for flowcytometric sorting was collected and processed on a farm in Italy, and then shipped to a laboratory in Germany. Eleven buffaloes were inseminated with X-chromosome bearing spermatozoa and 10 with Y-chromosome bearing spermatozoa. Conception rates after conventional and UTJ inseminations were 43.3% (n = 13) and 42.8% (n = 9) respectively (p = 0.97). Eight of the nine foetuses obtained after insemination with sexed spermatozoa corresponded to the sex as predicted by the cell sorting procedure (five male and four female foetuses by ultrasound vs six male and three female foetuses by cell sorting). In conclusion, for the first time buffalo semen has been successfully subjected to procedures for flowcytometric sperm sorting and freezing. Low doses of sexed spermatozoa have been deposited near the UTJ giving conception rates similar to those of conventional AI with full dose.     

谷氨酰胺替代部分甘油对冻融猪精子获能和体外受精能力的影响

本试验旨在探索用谷氨酰胺（Gln）替代部分甘油对冻融猪精子体外获能和受精能力的影响，试验分为6组：3%甘油对照组和5个处理组（Ⅰ~Ⅴ组：2%甘油＋谷氨酰胺（0、20、40、80和100 mmol/L））。对冻融松辽黑猪精子的精子活力、质膜完整性、顶体完整性、线粒体膜电位、鱼精蛋白水平、获能及体外受精等指标进行了检测。结果显示，用谷氨酰胺替代部分甘油均对冻融精子质量有一定的改善作用，改善的程度受谷氨酰胺浓度的影响。与对照组相比，Ⅰ组精子的质量参数均显著下降（P<0.05）；与Ⅰ组相比，Ⅱ组精子活力、顶体完整性和活率显著提高（P<0.05），Ⅲ组精子线粒体膜电位显著提高（P<0.05），Ⅴ组精子活力、质膜完整性、顶体完整性、活率和线粒体膜电位均显著提高（P<0.05）。说明用谷氨酰胺替代部分甘油对精子质量具有很大的影响，且当谷氨酰胺为100 mmol/L时可得到更高质量的精子，因此，后续试验使用浓度为100 mmol/L的谷氨酰胺进行研究。与对照组相比，2%甘油＋100 mmol/L谷氨酰胺处理组精子鱼精蛋白缺失率显著下降（P<0.05），精子获能无显著差异（P>0.05），但胚胎卵裂率显著提高（P<0.05）。综上所述，谷氨酰胺可作为一种新型冷冻保护剂替代部分甘油来提高猪精液的质量，并降低甘油对猪精液的毒性作用，为猪精液的冷冻保存及商业化生产提供技术支撑。     

Sexing sperm of domestic animals

The ability to preselect or predetermine the sex of offspring prior to conception is a highly desired technological tool for assisted female breeding programs specifically for milk production, and in males, for meat production and increasing livestock numbers. The current technology is based on the well-known differences in X- and Y-sperm in the amount of DNA. The technology uses modified flow cytometric instrumentation for sorting X- and Y-bearing sperm. The method can be validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content, and embryo biopsy for sex determination. Currently, the sex of animals has been predetermined with 90 % accuracy by sexing spermatozoa. In the bovine breeding industry, flow cytometric sperm sexing has not fulfilled its original promise. Sexed sperm doses are too expensive for widespread application while the fertility of sexed sperm doses is lower than unsexed ones. Essentially all bovine sexed semen is frozen and then applied through artificial insemination (AI) or in vitro fertilization. There is still a need in the animal breeding industry to develop a technique for sperm sexing that provides sufficient spermatozoa for AI doses, does not compromise sperm fertility, and is widely applicable to a range of species. In this review, we will summarize the current state-of-the-art in sex preselection in domestic animals and some wildlife species using flow cytometric sperm-sorting of X from Y sperm based on DNA differences.     

Validating bovine sexed semen samples using quantitative PCR

In cattle, separation of X‐ and Y‐bearing sperm cells by flow‐sorting technology makes it possible to predetermine the sex of calves. Due to high costs and decrease in fertilization, the extensive use of sexed semen in livestock depends heavily on sorting purity of sperm cells. Validating the accuracy of sperm sexing requires reliable procedures, therefore a real‐time polymerase chain reaction (PCR) assay was established to calculate the male cell proportion in the sexed semen based on the relationship between the amplification of a SRY fragment and an autosomal gene (MSHR) fragment. Our results showed stable amplification of SRY for 100–1 ng of genomic DNA, which allows detection of 1% of male cells if 100 ng of target DNA is used. To account for the discrepancy in the efficiency of the MSHR and the SRY amplification correction of the difference of the mean values was performed. The ratio of male to female sperm cells in unsexed semen cells was very accurately determined. The fractions of the sexed samples, however, were different from the expected range appearing lower than estimated. Thus, the study reveals that real‐time PCR provides a good basis for the examination of sexed sperm cells, but needs to be optimized for the samples.     

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.     

Effects of sperm pretreatment on efficiency of ICSI-mediated gene transfer in pigs

Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has recently been shown to be an effective technique for producing transgenic pigs; however, the types of sperm pretreatment having the most beneficial effects on post-ICSI embryogenesis or transgenic efficiency have not been clarified. In the present study, we performed ICSI-mediated gene transfer using pig sperm subjected to various pretreatments and determined the developmental potential of sperm-injected oocytes and introduction efficiency of exogenous DNA. Embryos were then transferred to recipient pigs to confirm gene transfer efficiency during the fetal period. When ICSI was performed using unfrozen sperm heads with tails removed by piezo-pulse, the rates of blastocyst formation (14.2%, 17/120) and transgene (EGFP) expression (11.8%, 2/17) were both low. When unfrozen sperm heads were used that were removed by sonication, EGFP expression efficiency (11/21, 52.4%) improved significantly (P<0.05). Pretreatment of unfrozen sperm with a surfactant or acrosomal reaction did not further improve the rates of blastocyst formation and EGFP expression. However, use of the heads of sperm frozen-thawed with or without a cryoprotective agent resulted in rates of blastocyst formation and EGFP expression that tended to be generally high (23.0%, 14/61-33.8%, 26/77 and 42.9%, 6/14-66.7%, 10/15). A total of 219 in vitro matured oocytes were fertilized by ICSI-mediated gene transfer using the heads of frozen-thawed sperm and then transferred into two recipient pigs. Seven fetuses were obtained, and EGFP expression and integration of the transgene (10-30 copies) were confirmed in two of the seven fetuses. Use of unfrozen sperm thus confers no advantages on ICSI-mediated gene transfer, and although further investigations are needed, frozen-thawed sperm heads appear to be useful in ICSI-mediated gene transfer.     

Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry

The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P < 0.05) ROS formation, decreased (P < 0.05) JC-1-aggregate fluorescence intensity, and decreased (P < 0.05) motion variables by 25 to 60%. The mechanism of inhibition of motility by ROS formation may be related to a decrease in mitochondrial charge potential below a critical threshold. Catalase and superoxide dismutase treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not superoxide dismutase, was capable of attenuating ROS-induced inhibition of motility. Whereas basal intracellular hydrogen peroxide formation was low in viable fresh and frozen-thawed boar sperm, both were quite susceptible to external sources of hydrogen peroxide.     

Effects of sex-sorted spermatozoa on the efficiency of in vitro fertilization and ultrastructure of in vitro produced bovine blastocysts

Frozen-thawed sexed semen from six bulls (Holstein) was used for studying their efficiency in an in vitro fertilization (IVF)-programme and to compare their ultrastructure with in vitro produced bovine blastocysts produced with non-sorted sperm. Progressive motility of sorted spermatozoa, their IVF rate, development of produced blastocysts and the ultrastructure of the blastocysts were analysed. The cleavage rates of sexed sperm of bulls (groups S1, S2 and S4) were significantly lower than that of unsorted control sperm (P < 0.01). Blastocyst development at day 7 of the sexed semen groups varied between 3.5% and 28.8% versus 33.6% for non-sexed semen. The individual blastocyst yield with sexed semen of group S5 (28.8%) was similar to the mean blastocyst production of the non-sexed control spermatozoa (C, 33.6%; P > 0.05). The remaining five sexed sperm groups resulted in significantly lower developmental rates of blastocysts on day 7 (S1, 4.9%; S2, 0%; S3, 0%, S4, 3.5%; S6, 25.8%, P < 0.01). Group S2 showed microbiological contamination in 50% (four of eight) and S3 in 100% of the experiments (eight of eight). Progressive motility of sexed sperm was significantly lower than that of unsorted sperm (S1, 48 +/- 12.0%; S2, 41 +/- 11.9%; S3, 39.0 +/- 9.9%; S4, 42 +/- 4.6%; P < 0.01; S5, 72 +/- 7.1% and S6, 64 +/- 9.3; P < 0.05 versus C 82 +/- 4.6%). The percentage of progressive motile spermatozoa showed a good correlation with the developmental capacity of blastocysts (r(2): >0.70), the regression parameter was significant (P < 0.01). Furthermore, with a straw containing 10 x 10(6) sexed spermatozoa significantly lower number oocytes was fertilized than with the same concentration of non-sexed sperm (P < 0.01). Our results demonstrate that the suitability of sperm sorting for in vitro fertilization (IVF) is lower than no sexed sperm. Our ultrastructural studies showed that blastocysts produced with flow-cytometrically sex-sorted spermatozoa possessed deviations in the number and structure of organelles like mitochondria, rough endoplasmic reticulum (ER) and nuclear envelope. These morphological alterations may be responsible for compromised development that observed in embryos produced with sex-sorted spermatozoa. Thus, we conclude that sperm sex sorting can markedly affect the efficiency of an IVF-programme.     

海藻糖替代部分甘油对延边黄牛冻融精子质量的影响

[目的] 探讨是否可以通过补充海藻糖来降低冷冻保护剂中甘油的浓度,从而提高冻融精子的质量。[方法] 分别用6%甘油（Ⅰ组）及3%甘油+0、50、100和150 mmol/L海藻糖（Ⅱ、Ⅲ、Ⅳ和Ⅴ组）处理精子,用精子-微生物动（静）态图像检测分析系统（CASA）检测冻融精子的活力、质膜完整性和顶体完整性及动力学相关参数,筛选出最佳海藻糖处理浓度用于后续试验。通过Western blotting方法检测精子蛋白酪氨酸磷酸化水平评价精子获能状态,Hoechst 33342/PI/JC-1联合染色法检测精子的活率及线粒体膜电位,色霉素A₃（CMA₃）染色法检测精子DNA的完整性,部花青540（M540）和Yo-Pro-1染色检测精子膜脂质紊乱水平。[结果] 与Ⅰ组相比,Ⅱ组精子的活力、质膜完整性、顶体完整性以及曲线速度（VCL）和直线率（STR）均显著下降（P<0.05）,Ⅲ、Ⅳ和Ⅴ组组精子的活力、VCL、直线速度（VSL）、平均路径速度（VAP）、直线性运动（LIN）和STR均显著升高（P<0.05）。因此,后续试验选用100 mmol/L海藻糖处理精子。与新鲜精子相比,冻融精子获能后其蛋白酪氨酸磷酸化的条带显著减少（P<0.05）,冻融精子的活率和线粒体膜电位均显著降低（P<0.05）;冻融活精子和死精子的高度膜脂质紊乱水平均显著升高（P<0.05）,100 mmol/L海藻糖可显著降低高度膜脂质紊乱水平（P<0.05）;冻融精子的鱼精蛋白缺失率显著增加（P<0.05）,且Ⅱ组和100 mmol/L海藻糖组精子的鱼精蛋白缺失率显著低于Ⅰ组（P<0.05）。[结论] 海藻糖可作为一种新型冷冻保护剂降低甘油毒性,用100 mmol/L海藻糖替代部分甘油可显著改善延边黄牛冻融精子的质量。     

使用流式细胞仪分离精子进行仔猪性别控制的研究

本研究旨在探索流式细胞仪分离精子在猪性别控制中的应用。使用流式细胞仪分离猪XY精子,而后通过母猪输卵管授精生产"预知"性别的仔猪,并使用吖啶橙染色法检测粗分离对精子核酸含量的影响。结果,成功利用分离获得的猪X和Y精子对母猪进行输卵管授精,母猪怀孕率、产仔率均为100%;输Y精子母猪产仔雄性率100%(♂6/6),对照母猪产仔雄性率57.14%(♂8/14);3头输X精子母猪产母仔率91.67%(♀11/12),对照母猪产雌性仔猪40%(♀2/5);使用性别分离精子不影响母猪的怀孕率、产仔率,但窝产仔数较低;吖啶橙染色法检测结果表明,流式细胞仪粗分离对猪精子核酸含量没有显著影响(P>0.05)。本研究结果提示,使用流式细胞仪分离精子授精可以有效改变仔猪的性别比例。本研究结果为猪分离精子性别控制技术的推广应用奠定了基础。     

用去透明带仓鼠卵穿透试验检测牛冷冻精子的受精能力

研究了用去透明带仓鼠卵穿透试验检测牛冷冻精子的受精能力。对36头西门塔尔、23头夏洛来和12头利木赞牛冷冻精子的实验结果表明：三个品种的牛冻精均能穿入去透明带仓鼠卵，并形成发育良好的雄原核。穿透率在70％－80％左右，有原核卵百分率在30％左右。去透明带 鼠卵穿透试验是检测牛冻精受精能力的有效方法。     

Characteristics of calves produced with sperm sexed by flow cytometry/cell sorting

The objectives of this study were to determine whether calves produced by sexed sperm differed from controls and to what extent the sex ratio of calves was altered by the sexing procedure. Data were collected from 1,169 calves produced from sperm sexed by flow cytometry/cell sorting after staining with Hoechst 33342, and 793 calves produced from control sperm during breeding trials between 1997 and 2001. Least squares ANOVA were completed using factors of treatment (sexed vs. control sperm), 19 management groups from 13 field trials, and calf sex. Responses analyzed include gestation length, birth weight, calving ease, calf vigor, weaning weight, abortion rate, and death rates (neonatal and through weaning). No significant difference was observed for any response due to treatment or treatment interactions (P > 0.10). Therefore, calves produced from sexed sperm grew and developed normally both pre- and postnatally. A neurological disorder was observed in four control calves and one sexed calf from one farm. No gross anatomical abnormalities were reported for any calves in the study. Differences were observed for all responses among management groups (P < 0.03 for abortions and P < 0.01 for all other responses). Heifer and bull calves differed (P < 0.001) in gestation length (278.4 and 279.6 d), birth weight (32.8 and 35.2 kg), calving ease (1.15 and 1.30), and weaning weight (233 and 247 kg). Gestation length did not affect characteristics of calves. The sex ratio at birth of calves from unsexed control sperm was 49.2% male. Sexing accuracy of X-sorted sperm was 87.8% female calves, and Y-sorted sperm produced 92.1% male calves. Flow cytometry/cell sorting can be used to preselect sex of calves safely with approximately 90% accuracy.     

不同添加剂及剂型对猪冷冻精液品质的影响

试验旨在研究咖啡因、维生素E、超氧化物歧化酶(SOD)及剂型对猪冷冻精液品质的影响。采用添加了不同浓度咖啡因、维生素E、超氧化物歧化酶(SOD)的冷冻稀释液和3种剂型的冻精管进行精液冷冻。3种添加剂对冻精解冻后的精液品质都有一定的促进作用,试验中其添加最适浓度分别为:咖啡因为0.2 mg/mL,维生素E为0.4 mg/mL,SOD为300 IU/mL;0.25mL剂型的冻精解冻后精子品质最好。咖啡因、抗氧化剂及冻精管剂型对冷冻精液的效果有一定的影响。     

肉碱对冻融猪精子总抗氧化能力、抗氧化酶基因及凋亡相关基因表达的影响

试验旨在探究肉碱对冻融猪精子质量、抗氧化、抗凋亡及精卵结合能力的影响。试验分鲜精组和冻融组,冻融组的肉碱浓度分别为0、0.025、0.05、0.075 mg/mL,对冻融后的精子质量、总抗氧化能力、抗氧化酶相关基因及凋亡相关基因mRNA表达、精卵结合能力进行检测。结果显示,在冻融组中,经过肉碱处理的精子存活率、质膜完整率和顶体膜完整率均显著高于0 mg/mL处理组（P < 0.05）,其中肉碱浓度为0.05 mg/mL时改善效果最好;经过冻融处理的精子中丙二醛（MDA）浓度与鲜精组相比显著升高（P < 0.05）,其中肉碱浓度为0.05 mg/mL时显著低于其他肉碱处理组（P < 0.05）;与鲜精组相比,各冻融组精子总抗氧化能力均显著降低（P < 0.05）,肉碱浓度为0.05 mg/mL时总抗氧化能力最高。此外,与鲜精组相比,0 mg/mL肉碱处理组中凋亡相关基因Caspase-3与Bax的相对表达量显著升高（P < 0.05）,抗凋亡基因Bcl-2的相对表达量显著降低（P < 0.05）,抗氧化酶相关基因SOD2、CAT与GPx的相对表达量显著降低（P < 0.05）;冻融组精卵结合能力均下降,但肉碱浓度为0.05 mg/mL时可得到显著改善（P < 0.05）。结果表明,添加肉碱可以改善冻融猪精子的质量、抗氧化能力、抗凋亡能力及精卵结合能力。     

精子活率与精子完整率和质膜完整率的相关性分析

采用考马斯亮蓝（R250）染色法和低渗膨胀试验（HOST试验）检测精子顶体反应和质膜完整性,以检测和评价这2种方法在猪精液质量检测中的实际应用价值。结果表明,冷冻复苏后,精子活率为37.97%,精子平均速度为55.72μm/s,顶体完整率和质膜完整率分别为51.01%和39.95%。经相关性分析得出,猪精子顶体反应率和质膜完整性与精子活率相关系数为0.904 0和0.817 6,呈显著相关（P〈0.01）,而精子的平均游动速度与顶体反应率和质膜完整性相关系数为-0.056 2和-0.151 8,无明显相关性（P〉0.05）,精子活率与精子平均游动速度间也无明显相关性（P〉0.05）。