| 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. 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 intrauterine insemination, deep-uterine insemination or producing sexed embryos for transfer. Insemination of lower sperm numbers in cattle has proven to be an effective means of utilizing the sexing technology. Offspring produced at bovine using the technology have been morphologically normal and reproductively capable in succeeding generations. Improvements in the technology will no doubt lead to much greater usage of sexed sperm for applications such as endangered species, laboratory animals, hobby or pet species, even human beings. Flow-sorted X- and Y-sperm are also the good research material to study other methods for sex control. Thus semen from wapiti, sika, goat and bovine were collected and separated into X- and Y- chromosome-bearing sperm after analysis and reanalysis using a Cytomation MoFlo high-speed cell sorter modified for sperm, and then inseminated or produced sexed-embryos for transfer. Further research in screening low abundances of differentially expressed genes in X or Y sperm by subtractive hybridization with high-purity bovine X and Y sperm sorted by flow cytometry were studied. The main results were showed as following:1. Sixteen wapiti stags were electroejaculated and their semen were cryopreservated. Semen evaluation and their fertility were assessed and recorded. TIANLU 3 diluent for stag was screened of the freezing method and final concentration of glycerol. The results of the experiment show that the best frequency of electroejaculation was 2 times each week for wapiti stag. The voltage had risen year by year; the electro ejaculation patience of wapiti stags had been proved. It was excellent for TIANLU 3 diluent contained 6% glycerol to crypreserve wapiti sperm. The post-thaw motility reached up to 48.14±13.15%. Acrosome integrity reached up to 56.34±14.84%. 126 hinds (estrus synchronization) and 107 hinds (normal estrus) had been applied insemination with this frozen thawed semen, and the calving rate reached up to 50.80% and 78.50%, respectively. 2. Semen was collected by electro-ejaculation or artificial vagina from wapiti stags, sika stags and goat, and transported to the laboratory and separated into X- and Y- chromosome-bearing sperm after analysis and reanalysis using a modified high-speed cell sorter for sperm. Wapiti hinds, sika hinds and nanny goats induced estrus synchronization with intra-vaginal progesterone-impregnated CIDR devices and administrations of PMSG were intra-uterine inseminated by rectum manipulation or laparoscopy with a low numbers (106 motile sperm) of sex-sorted cryopreserved sperm. (1) Wapiti: no significant differences in the post-thaw motility of control (43±6%), X-sorted (46±5%) and Y-sorted (43±5%) samples were recorded, sex ratio of sperm were 93.1% and 95.2%, respectively. Ultimately 7 out of the 7 calves produced by wapiti hinds inseminated with Y-sorted sperm were male (100%) and 8/8 calves or 100% of the calves from hinds inseminated with X-sorted sperm were female. The birth body weight, 85d body weight and gestation period for male calves were not significantly lower than those of the control, respectively. (2) Sika: no significant differences in the post-thaw motility of control (43±4%), X-sorted (45±4%) and Y-sorted (43±3%) samples were recorded, sex ratio of sperm were 91.2% and 94.1%, respectively. Ultimately 14 out of the 15 calves produced by Sika hinds inseminated with Y-sorted sperm were male (92.9%) and 5/5 calves or 100% of the calves from Sika hinds inseminated with X-sorted sperm were female. The birth body weight, 85d body weight and gestation period for male calves were not significantly lower than those of the control, respectively. (3) Goat: post thaw motility of sorted frozen X- and Y-chromosome-bearing sperm were 43±4% and 42±4%, sex ratio of sperm were 94.4% and 96.2%, respectively. The birthrate after insemination with sorted frozen thawed Y-sperm was significantly lower (25%, 4/16) than that of the control (90%, 9/10), and 4 kids were all male. The birth body weight, 70d body weight and gestation period for male kits were not significantly lower than those of the control, respectively. Normal calves or healthy kids of the predicted sex can be produced after intra-uterine insemination conducted by laparoscopy or rectum manipulation with low numbers of sex-sorted cryopreserved sperm in wapiti, sika and goat.3. Embryo was produced in superovulated wapiti hinds induced by eight declining doses of FSH starting on days 9-12 of the estrus cycle and inseminated with 1×10~7 unsorted (unsorted, n=6) or 1×10~7 Y-sorted frozen-thawed (Y, n=6) and 1×10~8non-sorted frozen-thawed (a commercial dose control, n=6) semen via a single time rectum manipulation. Twenty-five embryos of predicted sex were produced. Sex identification of samples from wapiti based PCR amplification using the bovine amelogenin gene primers was developed. The sex ratio of the embryos from hinds inseminated with Y sperm (4M:0F) was significantly (P<0.05) deviated for the 53.3% (male, 8/15) and 46.7% (female, 7/15) in the non-sexed group. No significant differences in the birthrate of control (non-sexed embryo) and sexed embryo groups were recorded.The sex ratio of the offspring from sexed embryo (8M:0F) was significantly (P<0.05) deviated for the 55% (male, 11/20) and 45% (female, 9/20) in the non-sexed embryo group.4. The laminar analysis column method and Trizol single-step method were combined for RNA extraction from swimming-up bovine frozen-thaw sperm. The bovine sperm swimming-up from frozen straws were not contaminated by somatic cells, and high-quality total RNA was obtained in about 2 hours. The value of OD260/OD280 was 1.8, and the≥200bp total RNA extracted from bovine sperm yielded 0.92μg/107sperm.Following RT-PCR, clear bands of SRY, LEP, and 23S rRNA of sperm cDNA were shown in a garose gel electrophoresis respectively, not a band of 18S rRNA.The purity and integrality of total RNA isolated from the bovine sperm of frozen straw with the method of combined the laminar analysis column with Trizol single-step were significantly satisfactory for the demands of molecular biological experiments.5. The low abundances of differentially expressed genes in X or Y sperm were screened by subtractive hybridization with high-purity bovine X and Y sperm sorted by flow cytometry. Twenty-seven clones isolated from Y-X subtracted cDNA library which had different insert fragments were sequenced. All sequences from 27 clones can be found matching the cow genome sequence following searching the Bos taruus genomic BLAST databases. There exist 4 bovine ESTs in Y-X subtracted cDNA library. One of them is known in bovine, which is the sequence of sry gene, and three are unknown in bovine.
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