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1.
Flow cytometrically-sorted sperm has been involved in the production of sex preselected offspring. More than 30,000 bovine offspring have been produced using AI and other means using spermatozoa separated by flow cytometer. Flow cytometric sperm sorting based on differences in their DNA content is the best method for separation of X- and Y-chromosome bearing spermatozoa. At first, flow cytometers were modified for DNA confirmation and sorting of sperm with high resolution. The beveled insertion needle can regulate orientation of flat-shaped bull sperm heads. The forward fluorescence detector is essential for measuring the DNA content of sperm. Recently, high-speed sperm sorting with orienting nozzles has resulted in production of 90% pure X- and Y-sperm at rate of 15-20 million sperm per hour. Application of this new technique will enable conduct of more conventional technologies for both artificial insemination and cryopreservation in the bovine and in other farm animals using X- or Y-sperm.  相似文献   

2.
Application and Commercialization of Flow Cytometrically Sex-Sorted Semen   总被引：1，自引：0，他引：1  
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.  相似文献   

3.
Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples.  相似文献   

4.
Flow cytometric sorting technology was used to measure the difference in DNA content between X- and Y-chromosome-bearing spermatozoa in bucks. Spermatozoa were analysed by flow cytometry to characterize X- and Y-chromosome-bearing sperm populations and to quantify the DNA difference between them. Two symmetrical, overlapping and clearly separated peaks, corresponding to X- and Y-bearing spermatozoa, were detected. The difference in fluorescence intensity between the peaks was 4.4 +/- 0.03% without any significant inter- or intra-animal variations. Therefore, the identification and selection of high-purity samples of sperm populations for sex sorting is easier in bucks compared with other domestic species.  相似文献   

5.
The Beltsville sperm sexing technology is currently the only effective means of altering the sex ratio of offspring in livestock. The method is based on the flow-cytometric separation of X- and Y-chromosome-bearing sperm based on X/Y DNA content difference. It is an effective means of producing progeny of predetermined sex in cattle, swine, sheep, and laboratory animals. The method involves treating sperm with a DNA-binding fluorochrome, Hoechst 33342, and flow-cytometrically sorting them into separate X and Y populations that can subsequently be used for surgical intratubal or intrauterine insemination, deep-uterine insemination, regular artificial insemination in some cases, in vitro fertilization to produce sexed embryos for transfer, and intracytoplasmic sperm injection of ova. Skewed sex ratios of 85 to 95% of one sex or the other have been repeatably achieved in most species. The method has been used worldwide to produce several hundred morphologically normal animal offspring of the predicted sex. It has also been validated in the laboratory using DNA reanalysis of the sorted sperm populations and by fluorescence in situ hybridization and PCR of individual sperm. We developed a new orienting nozzle that we have fitted to both conventional and high-speed cell sorters that have been modified for sperm sorting. Recently we completed the adaptation of the new orienting nozzle to a Cytomation MoFlo high-speed cell sorter modified for sperm. This adaptation of the nozzle has increased the overall production rate of sorted X and Y sperm from about .35 million/h to 5 or 6 million sperm/h (each population). Calves have been born from cows artificially inseminated using conventional technique and sexed sperm. In addition, numerous litters of pigs have been born after transfer of embryos produced from X or Y sorted sperm.  相似文献   

6.
猪深部输精技术应用研究进展   总被引：2，自引：0，他引：2  
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。  相似文献   

7.
Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white ( Ceratotherium simum ), black ( Diceros bicornis ), Indian ( Rhinoceros unicornis )] and both elephant species, the Asian and the African elephant ( Elephas maximus, Loxodonta Africana ), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants.  相似文献   

8.
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.  相似文献   

9.
The differential proteins associated with plasma membrane of spermatozoa are less known, identification of which shall help overcome limitations of currently used methods of sperm sexing, considered as a high priority for livestock sector of many countries. This study has reported plasma membrane proteomics of unsorted spermatozoa and differential expression of plasma membrane-associated proteins between X- and Y-chromosome bearing spermatozoa of indicus cattle (Bos indicus). Isolation of plasma membrane fraction using percoll gradient, relatively a rapid method, from bovine spermatozoa has been reported to enrich isolation of plasma membrane proteins. Significant enrichment for plasma membrane-associated proteins was observed in plasma membrane fraction (p < .05) as compared to the total cell lysate using LC-MS/MS. Furthermore, these experiments were conducted in flow cytometry sorted, sexed-semen samples. Thirteen proteins were identified as differentially abundant between X- and Y-sorted spermatozoa. Among these, two proteins were downregulated in Y-sorted spermatozoa compared to the X-sorted spermatozoa (p < .05), while four and seven proteins could be noted in X- and Y-sorted spermatozoa, respectively. Proteins that are presumed to support sperm capacitation and sperm migration velocity were found to be abundant in Y-sorted spermatozoa while those associated with structural molecule activity were identified as abundant in X-sorted spermatozoa in the present study. Our study provides better insight into the plasma membrane proteomics of spermatozoa of indicus cattle and furnishes data that might aid in design and development of alternate and open technology for sex-sorting of semen.  相似文献   

10.
利用流式细胞仪分析水牛分离和未分离精液中精子的DNA含量,所得的直方图用高斯曲线拟合,分析计算出样本中X和Y精子的比率。结果表明:未分离的水牛精液中X精子的比率为50%,与正常水牛后代性别比率没有显著差异;而水牛的分离X精液样本中X精子占93%,分离Y精液样本中Y精子占89%。实验结果显示了流式细胞仪DNA分析法鉴定水牛精液中X和Y精子比率的可靠性,而流式细胞仪分离精子程序和方法在水牛上的应用是可靠而有效的。  相似文献   

11.
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.  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
This study was designed to apply the method of discontinuous Percoll gradients for sex preselection in bovine semen by using a current developed molecular technique, fluorescence in situ hybridization (FISH). In addition, we attempted to amplify the level of enrichment of X- or Y-bearing spermatozoa by treating for activating sperm motility performance with 10 mM caffeine. Bovine spermatozoa were fractionated on Percoll gradients into two major subpopulations of motile spermatozoa (bottom fraction) and weak motile spermatozoa (top fraction). The percentage of Y-bearing spermatozoa in the top fraction (52.9%) slightly exceeded and that in the bottom fraction (44.3%) decreased significantly (P<0.001) compared with the theoretical ratio (50:50). Washing sperm with BO medium affected a deviation between the two sex populations, whereas semen activated with caffeine showed no difference in the percentage of X- and Y-bearing spermatozoa in both fractions compared with the theoretical ratio (50:50). These results show that the proportion of X- and Y-bearing bovine spermatozoa can deviate after discontinuous Percoll gradients, although the proportion of X- and Y-bearing bovine spermatozoa was affected by sperm motility of the sample applied.  相似文献   

15.
To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa – including sex sorting – as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry‐based assays. After sorting, oxidative stress increased from 26% to 33% in pre‐ and post‐incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post‐sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.  相似文献   

16.
使用流式细胞仪分离精子进行仔猪性别控制的研究   总被引：2，自引：2，他引：0  
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。  相似文献   

17.
Background: The application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction. However, a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation, rendering the frozen-thawed semen impractical for routine use.Cryopreservation is known to reduce sperm lifespan and fertility. Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination(AI) centers. Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process.Results: Our strategy of sperm selection based on rheotaxis and thermotaxis(SSRT) on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity, viability and biological function related to fertilization competence. This strategy has a positive effect on the cryosurvival and fertilizing abilities of spermatozoa as supported by the improvement on pregnancy rate of Japanese Black heifers and Holstein repeat breeders. This technique protected further sublethal damage to bovine spermatozoa(higher %cryosurvival than the control) and resulted in the improvement of DNA integrity. Prefreeze selected spermatozoa demonstrated slower and controlled capacitation than unprocessed control which is thought to be related to sperm longevity and consequently to appropriate timing during in vivo fertilization.Conclusions: These results provide solid evidence that improvement of post-thawed semen quality by SSRT method is beneficial in terms of cryosurvival, longevity of post-thawed sperm, and optimization of in vivo fertilization, embryo development and calving as supported by the favorable results of field fertility study.  相似文献   

18.
The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.  相似文献   

19.
Successful sex‐sorting of goat spermatozoa and subsequent birth of pre‐sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm‐sorting (using a modified flow cytometer, MoFlo SX®) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post‐sorting and (ii) frozen in Tris–citrate–glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled‐rate freezer. Post‐sort and post‐thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC‐PNA). Sex‐sorted goat spermatozoa frozen in pellets displayed significantly higher post‐thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex‐sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex‐sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non‐sorted goat spermatozoa, non‐sorted ram spermatozoa and sex‐sorted ram spermatozoa. Following intrauterine artificial insemination with sex‐sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non‐sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex‐sorted by flow cytometry, successfully frozen and used to produce pre‐sexed kids.  相似文献   

20.
Finding a laboratory test reliable enough to predict the potential fertility of a given semen sample or a given sire for artificial insemination (AI) is still considered utopian, as indicated by the modest correlations seen between results obtained in vitro and field fertility. Male fertility is complex, and depends upon a heterogeneous population of spermatozoa interacting at various levels of the female genital tract, the vestments of the oocyte, and the oocyte itself. For this reason, laboratory assessment of semen must include the testing of most sperm attributes relevant for fertilization and embryo development, not only in individual spermatozoa but within a large sperm population as well. Strategies for the discovery of in vitro predictors of semen fertility require evaluations of low sperm doses for AI, so that differences in innate in vivo fertility can be accurately detected.  相似文献   

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