In vitro production of sexed embryos for gender preselection: high-speed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer

The objectives for the present experiments were to apply sperm sexing technology to an in vitro production system with porcine oocytes obtained from slaughterhouse material. On six experimental days, ovaries were obtained from an abattoir, and cumulus-oocyte-complexes were matured in vitro. Semen was collected from mature boars of proven fertility and was sorted for X-chromosome-bearing sperm, using the Beltsville Sperm Sexing Technology incorporating the use of high-speed sorting. A total of 5,378 oocytes were submitted for in vitro fertilization (IVF). Of these, 559 ova were stained for cytogenetic analysis 18 h after IVF. From the remaining 4,819 ova, 1,595 cleaved, and 1,300 of the cleaved embryos were transferred into 26 synchronized recipients (5 control gilts for unsorted sperm, 21 gilts for X-sorted sperm). In a test of two fertilization media (FERT-A vs FERT-B) higher cleavage rates (P<.05) were obtained when FERT-B was used as a fertilization medium for unsorted (43.4+/-5.1%) and sorted sperm (43.1+/-1.1%;), whereas in FERT-A unsorted sperm gave a cleavage rate of 17.9+/-4.4% and sorted sperm gave 30.4+/-1.4%. Additionally, cleavage rates were higher (P<.05) after fertilization with sorted sperm vs unsorted sperm, independent of fertilization medium. Cytogenetic analysis of ova revealed that more oocytes with unsorted than with sorted sperm remained in Metaphase 2 arrest (P<.05). This was also independent of the fertilization medium. Monospermic fertilization rates were the same for IVF with unsorted or sorted sperm, independent of the fertilization system, except FERT-A with unsorted sperm (P<.05). Polyspermic fertilization rates were highest in FERT-B (37.6+/-6.6). A total of 57 pigs were born from nine litters. Six litters from sexed sperm (X-sorted) produced 33 females (97%) and one male. Three litters from control transfers produced 23 pigs, 11 of which were female (48%). The sex ratio of the offspring was predicted based on the sort reanalysis of the sorted sperm for DNA content.     

分离和冷冻处理对牛性控精液线粒体膜电位的影响

采用JC-1染色结合流式细胞仪和激光共聚焦显微镜,分别对4组牛精液样品(鲜精、分离精液、普通冻精和分离冻精)的线粒体膜电位进行检测,研究分离和冷冻处理对牛精子线粒体膜电位的影响。结果表明:分离处理对A和B种公牛的精子线粒体膜电位影响不显著(P0.05),但对C种公牛影响显著(P0.05);而冷冻处理后,A、B和C种公牛的线粒体膜电位均显著变化(P0.05);且鲜精时线粒体膜电位无显著差异的A和B种公牛,分离后线粒体膜电位出现显著差异(P0.05)。当精液从采集到处理的间隔时间在24h以内时,分离处理后精子线粒体膜电位无显著性变化(P0.05),但同样时间间隔内冷冻处理后,精子线粒体膜电位显著变化(P0.05)。实验表明和分离处理相比,冷冻处理对精子线粒体膜电位的影响更为显著,且不同种公牛对分离和冷冻处理的耐受性不同。     

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.     

Effects of glutathione treatments during sperm washing and in vitro fertilization on the in vitro early development of embryos of Japanese Black cattle

This study was conducted to examine the effects of adding glutathione (1 mM) to media used for sperm washing and in vitro fertilization (IVF) on the improvement of early development of embryos produced using cryopreserved spermatozoa of the less IVF-competent bull (the one considered unqualified as spermatozoa supplier for the production of bovine blastocysts using IVF). The cryopreserved spermatozoa of this bull were characterized by normal motility and lower ATP content and blastocyst productivity than those of IVF-competent bulls. The addition of glutathione to the sperm washing medium was more effective in improving the productivity of blastocysts and ATP content than the addition of glutathione to the IVF medium or no glutathione addition at all (control). These results suggest that this simple method may be used to improve the potential of cryopreserved spermatozoa of less IVF-competent bulls to fertilize oocytes in vitro and to induce normal embryonic development after fertilization.     

Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure

This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF. PCV2 spiked semen did not reveal any reduction in sperm viability or motility but its ability to produce infected blastocysts was irrelevant as only one out of 15 blastocysts obtained by IVF were PCV2b; however two blastocysts were PCV2a positive. Furthermore, the presence of PCV2 was demonstrated also in embryos obtained by parthenogenesis (one out of 17 was PCV2b and one PCV2a positive). Even if PCV2 firmly attaches to the surface of spermatozoa, experimentally spiked sperm were not effective in infecting oocytes during IVF and in producing PCR positive embryos. The infected blastocysts we obtained derived most probably from infected oocytes recovered at the abattoir.     

Reduction of Oxidative Stress in Bovine Spermatozoa During Flow Cytometric Sorting

The goal of the study was to investigate the effect of antioxidant supplementation on the quality of frozen-thawed flow cytometrically sorted bull spermatozoa. Twelve ejaculates from two Holstein Friesian bulls were sorted according to the Beltsville Sperm Sexing Technology. Each ejaculate was divided into three parts and processed as (i) unsorted controls, (ii) according to a standard sorting protocol and (iii) in the presence of different antioxidants (S-AO). Cooling and freezing of the samples were performed in the same way for all three groups, except that antioxidants were added to the TRIS-egg-yolk freezing extender for those semen samples that were already sorted in the presence of antioxidants. The semen quality in frozen-thawed samples was determined by morphology analysis immediately after thawing, motility estimation in a thermo-resistance test after 0, 6, 12 and 24 h incubation at 37 degrees C and Fluorescein isothiocyanate conjugated PNA/propidium iodide (FITC-PNA/PI) staining after 0, 12 and 24 h of incubation at 37 degrees C. There was a significantly higher (p < 0.05) percentage of motile spermatozoa in S-AO samples in comparison to unsorted frozen-thawed control at 0, 6 and 24 h after thawing and compared with normally sorted samples at all times after thawing. The percentage of damaged acrosomes was significantly lower (p < 0.05) in S-AO samples than in the unsorted controls (20.8 +/- 6.9% vs 30.3 +/- 12.0%). The percentage of morphologically abnormal spermatozoa in this group was significantly lower (p < 0.05) than in the unsorted controls and normally sorted samples (25.8 +/- 5.2%, 36.0 +/- 12.5% and 35.1 +/- 7.4%, respectively). Analysis of frozen-thawed spermatozoa with FITC/PI revealed no significant difference in membrane integrity at 0 and 12 h after sorting, but after 24 h of incubation the S-AO samples had a significantly higher (p < 0.001) percentage of spermatozoa with intact membranes in comparison to unsorted controls and normally sorted semen (40.7 +/- 6.3%, 7.8 +/- 4.7% and 7.4 +/- 4.6%, respectively). The percentage of acrosome-reacted spermatozoa was significantly lower (p < 0.05) in the S-AO samples than in the unsorted controls (14.1 +/- 7.5%, 23.4 +/- 5.4% and 28.8 +/- 6.3% vs 25.9 +/- 14.4%, 38.5 +/- 16.7% and 79.8 +/- 4.1%, for 0, 12 and 24 h after thawing, respectively) and in comparison to normally sorted semen 24 h after thawing (67.3 +/- 10.0%). This study demonstrates the highly protective effects of antioxidants on the quality of flow cytometrically sorted frozen-thawed bull spermatozoa.     

Male and female effects on the in vitro production of bovine embryos

A 3-year study was carried out to evaluate male and female effects on the efficiency of an in vitro fertilization (IVF) programme. The semen of different bulls used for artificial insemination was tested for the in vitro production of transferable blastocysts. The fertilization capacity was recorded for each bull. Bovine oocytes were matured in vitro, fertilized with frozen/thawed semen of 63 individual bulls and cultured during 8 days. The semen of one bull was used as control. The percentage of cleavage (36.3-93.4%) and blastocysts on day 7 (6.9-51.2%) varied from bull to bull. Despite high variability, blastocysts were produced with the semen of all bulls in the first trial. Moreover, oocytes fertilized with 85% of tested bulls reached a blastocyst rate not different to the control bull. The correlation coefficients of six bulls showed no significant male effect but an influence of oocytes on the cleavage rate (F-value 0.38, P > 0.05, and 12.4, P < 0.001, respectively). The development to blastocysts on day 7 was significantly influenced by sperms and also oocytes and session (P < 0.01), but no combined interaction was observed between female and male. It is concluded that transferable embryos can be produced in vitro in the first trial with frozen/thawed semen of 63 tested bulls. The results show different capacities of bulls to produce embryos and high male and female effects on the efficiency of an IVF programme.     

Production of Lambs After the Transfer of Fresh and Cryopreserved in vitro Produced Embryos from Prepubertal Lamb Oocytes and Unsorted and Sex-sorted Frozen-thawed Spermatozoa

Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p <0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p <0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p >0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p >0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p >0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.     

Production of sexed bovine embryos in vitro can be improved by selection of sperm treatment and co-culture system

The study investigated the effects of sperm sorting, capacitation treatment and co-cultivation on sexed bovine in vitro embryo production. The effect of treatment and co-culture on production of embryos of the preferred sex from unsorted sperm was also studied. Sperm from five breeding bulls was used for fertilization of mature oocytes as follows : Experiment 1, sorted and unsorted sperm (bulls A-E) treated only with heparin in standard co-cultures; Experiment 2, sorted sperm (bulls A-E) treated with heparin-PHE (penicillamine, hypotaurine, and epinephrine) or heparin-caffeine in drop co-cultures; and Experiment 3, unsorted sperm (bull E) treated with either heparin-PHE or heparin-caffeine in both standard and drop co-cultures. In all bulls, treatment with heparin resulted in significantly (p < .05) reduced cleavage and blastocyst rates from sorted sperm, as compared with those from unsorted sperm. In bulls A, B, D and E, treatment of sorted sperm with heparin-PHE in drops significantly increased the blastocyst rate (p < .05). In unsorted sperm of bull E, heparin-PHE treatment in drops resulted in the XX/XY sex ratio inverse to that obtained by heparin-caffeine treatment in standard co-cultures (32.3%/67.7% and 66.7%/33.3%, respectively). In conclusion, the treatment of sorted sperm with heparin-PHE in modified drop co-cultures can be recommended for production of in vitro sexed embryos. The use of unsorted sperm for production of embryos of the preferred sex by selected capacitation treatment and co-culture can be the method of choice in bulls with low IVF yields from sorted sperm.     

Insemination with Sex Sorted Fresh Bovine Spermatozoa Processed in the Presence of Antioxidative Substances

Flow cytometrically sex sorted spermatozoa are reduced in their fertilizing capacity, particularly when stored either in cooling extender or after freezing in liquid nitrogen. So far, preservation methods for sorted spermatozoa have differed only marginally from procedures used for unsorted semen. In the present study, a TRIS extender was modified to balance major cell damage caused by the sorting process and by liquid storage of the sorted spermatozoa. The new extender, containing a combination of antioxidants (AO) and bovine serum albumin (BSA), significantly increased the lifespan and fertilizing capacity of sex sorted spermatozoa. No significant differences were observed between unsorted controls and sorted samples for motility and status of sperm membranes as tested by fluorescein-isothiocyanat-peanut agglutinin/propidium iodide (FITC-PNA/PI). Acrosome integrity of spermatozoa was significantly better when semen was stored at 15 degrees C for 24 and 48 h in an extender containing AO with or without BSA as compared with controls (p < 0.05). There were no significant differences, in pregnancy rates of heifers inseminated at a natural oestrus, between unsorted controls (16/24, 66.7%) and both sorted groups (AO + BSA: 18/31, 58.1% and AO-BSA: 12/22, 54.5%). Additionally, it was shown for the first time that artificial insemination (AI) with liquid sexed bull spermatozoa stored for 72 h after sorting can result in pregnancy rates similar to AI with non-sorted semen.     

Fertility in Holstein × Gyr cows in a subtropical environment after insemination with Gyr sex-sorted semen

Breeding records, including 649 inseminations during fall and winter at a dairy farm in a subtropical area of Western Mexico (24o N; 24°C, mean annual temperature 24°C) were analyzed to document effects of sex-sorted semen from commercial Gyr bullls, estrus synchronization protocol, inseminator, sire and environmental conditions on fertility of crossbred cows (Holstein × Gyr). Percentage of services resulting in pregnancies decreased sharply when sex-sorted semen was used (22.7 vs. 37.7%; P < 0.01). Although statistically not significant (P = 0.31), cows whose first insemination was in November experienced a numerically greater reduction (21 percentage points) in pregnancy rate compared to cows whose first insemination occurred in December. Substantial increases in services per pregnancy (4.71 ± 1.35 vs. 2.13–2.43; P < 0.01) were associated with the warmer month of the study period, November, compared to other winter months. Pregnancy rates of cows regardless of semen category (33%) were not affected by sire, temperature–humidity index and estrus synchronization protocol. Cows inseminated by one inseminator had higher pregnancy rates (P < 0.01) than cows inseminated by other two technicians. The sorted sperm produced 91% (142/156) female offspring. It was concluded that, under the field conditions of the present study, pregnancy rate with sexed semen was 15 percentage points lower than pregnancy rates using conventional semen, with 91% of female calves derived from sexed sperm.     

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.     

Post-thaw viability of european bison (Bison bonasus) semen frozen with extenders containing egg yolk or lipids of plant origin and examined with a heterologous in vitro fertilization assay.

Basic characteristics of European bison (Bison bonasus) semen were described and the efficacies of two extenders-Triladyl, containing egg yolk, and a synthetic extender, containing soybean lipids-were tested for semen cryopreservation. Seven ejaculates were collected by electroejaculation from a 10-yr-old, European bison bull. Each ejaculate was diluted at 37 degrees C to a final concentration of 200 x 10(6) sperm/ml with Triladyl or the synthetic extender. Extended semen samples were frozen according to a standard bull semen freezing protocol. After 2 wk of storage, one straw from each extender and ejaculate was thawed, and postthaw quality was evaluated by individual sperm motility and movement rate, numbers of sperm morphologic abnormalities and intact acrosomes, functional integrity of the sperm membranes determined by hypoosmotic swelling test (HOST), viability (live-dead, eosin-nigrosin stain), and a heterologous in vitro sperm penetration assay (SPA). A total of 600 in vitro-matured bovine oocytes were inseminated with 1 X 10(6) spermatozoa of Holstein semen frozen-thawed in Triladyl (control) or of European bison semen frozen in Triladyl or the synthetic extender. Nuclear status of the oocytes was determined after 18 h of sperm-oocyte coincubation. Extender had no effect on any evaluated parameters of semen after dilution and cooling (4 hr at 5 degrees C) or in postthaw individual motility, quality of movement, and sperm morphology. However, significantly (P < 0.05) higher numbers of spermatozoa with intact acrosomes, intact membranes (HOST), and viable sperm (P < 0.01) were in semen frozen in Triladyl than in the synthetic extender. Mean values for heterologous SPA for bull (control) and for bison semen frozen in the synthetic extender were very much alike-63.3+/-10.6% and 63.1 +/- 15.9%, respectively; bison semen frozen in Triladyl was lower, 43.0+/-24.2% but not significantly different. Cumulative results from a variety of viability assays of diluted/cooled and frozen-thawed semen, including the heterologous SPA, suggest that European bison semen can be successfully frozen in both extenders tested in this study.     

Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI

Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6%) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60% of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors.     

Effects of month of breeding on reproductive efficiency of Holstein cows and heifers inseminated with sex-sorted or conventional semen in a hot environment

The main objective of this study was to assess the effect of month of breeding on reproduction performance of Holstein heifers and cows inseminated with sex-sorted or conventional semen in a hot environment. Pregnancy per artificial insemination (P/AI; 64,666 services over an 8-year period) both in heifers (n?=?22,313) and cows (n?=?42,353) from a large dairy herd in northern Mexico (26°N) were evaluated with the GENMOD procedure of SAS, with respect to month of AI. Overall, P/AI with sex-sorted semen was greater (P?<?0.01) in heifers (41.6 %) than cows (17.3 %). P/AI for cows serviced with conventional semen was 10 % points higher (P?<?0.01) in January and December (31 vs. 21 %) than cows serviced with sex-sorted semen. While there was no difference in P/AI between the sex-sorted sperm and conventional semen in cows inseminated in July (16 and 18 %, respectively), P/AI plummeted for both groups of cows during the summer and fall (more severe heat stress). P/AI was not different between heifers serviced with sex-sorted or conventional semen during the hottest months of the year (July to October). However, during the coldest month of the year (January and February), P/AI was 10 percentage points greater (P?<?0.01) in heifers serviced with conventional than sex-sorted semen. It was concluded that in this hot climate cow and heifer fertility declined in the summer and fall when inseminated with conventional semen. However, the use of sex-sorted semen during summer and fall did not compromise the breeding success in heifers. Thus, this data suggest that sex-sorted semen promotes some embryonic thermoprotective mechanism, which leads to a marginal summer and fall fertility depression with this type of semen in this particular hot environment.     

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.     

Assessment of viral presence in semen and reproductive function of frozen-thawed spermatozoa from Pallas' cats (Otocolobus manul) infected with feline herpesvirus.

Although herpesviruses are known to contaminate the semen of several mammalian species, the occurrence of feline herpesvirus type 1 (FHV-1) in semen of infected cats has not been reported. Our objectives in this study were to investigate the presence of FHV-1 DNA in seminal fluid and frozen-thawed spermatozoa from FHV-1 infected Pallas' cats (Otocolobus manul) and assess the functionality of their frozen-thawed spermatozoa in vitro. Over a 3-yr period, semen (n = 33 ejaculates) was collected periodically via electroejaculation from four Pallas' cats chronically infected with FHV-1. Spermic ejaculates were frozen by pelleting on dry ice and stored in liquid nitrogen. After thawing, sperm motility and acrosome status were assessed over time during in vitro culture. For vitro fertilization (IVF), viable domestic cat (Felis silvestris catus) oocytes were inseminated with frozen-thawed Pallas' cat spermatozoa and evaluated for embryo cleavage. For FHV-1 polymerase chain reaction (PCR) analysis, DNA was extracted from seminal fluid, frozen-thawed spermatozoa, inseminated oocytes, heterologous IVF embryos, and conjunctival biopsies and analyzed for presence of a 322-base pair region of the FHV-1 thymidine kinase gene. Immediately post-thaw, sperm motility and percentage of intact acrosomes were decreased (P < 0.05) compared to fresh samples, and declined further (P < 0.05) during culture. However, all frozen-thawed IVF samples were capable of fertilizing domestic cat oocytes (overall, 46.1 +/- 6.0% cleavage). PCR analysis did not identify FHV-1 DNA in any reproductive sample despite the repeated detection of FHV-1 DNA in conjunctival biopsies. These results suggest that semen collected from Pallas' cats infected with FHV-1 does not contain cell-associated or non-cell-associated virus and that frozen-thawed spermatozoa exhibit adequate function for potential genetic rescue with minimal risk of FHV-1 transmission.     

Single layer centrifugation-selected boar spermatozoa are capable of fertilization in vitro

Background

Good quality spermatozoa are important to achieve fertilization, viable embryos and offspring. Single Layer Centrifugation (SLC) through a colloid (Androcoll-P) selects good quality spermatozoa. However, it has not been established previously whether porcine spermatozoa selected by this method maintain their fertility.

Methods

The semen was prepared either by SLC or by standard centrifugation (control) and used for in vitro fertilization (IVF) at oocyte:spermatozoa ratios of 1:50; 1:100 and 1:300 (or 4 x 10³, 8 x 10³ and 24 x 10³ spermatozoa/ml) to evaluate their subsequent ability to generate blastocysts. In addition, sperm motility was assessed by computer assisted sperm motility analysis.

Results

Total and progressive motility were significantly higher in sperm samples prepared by SLC compared to uncentrifuged samples. Sperm binding ability, polyspermy, cleavage and blastocyst rates were affected by the oocyte:sperm ratio, but not by sperm treatment.

Conclusion

The use of SLC does not adversely affect the in vitro fertilizing and embryo-generating ability of the selected spermatozoa compared to their unselected counterparts, but further modifications in the IVF conditions would be needed to improve the monospermy in IVF systems. Since SLC did not appear to have a negative effect on sperm fertilizing ability, and may in fact select for spermatozoa with a greater potential for fertilization, an in vivo trial to determine the usefulness of this sperm preparation technique prior to artificial insemination is warranted.     

白绒山羊性控胚胎生产及移植应用研究初探

应用X性控冷冻精液对超排供体白绒山羊进行人工授精,生产性控胚胎并进行鲜胚移植。结果发现,①用性控冻精和鲜精输精的供体母羊,平均卵子回收数分别为13.3和12.5枚,其中性控冻精组受精卵比例为29.6%（55/186）,极显著低于鲜精组94.0%（281/299）的比例（P<0.01）;②性控胚胎和普通胚胎移植受体母羊产羔率分别为42.2% 和58.1% ,二者差异极显著（P<0.01）;③性控胚胎移植受体母羊所产羔羊雌性所占百分数为100%,极显著高于普通胚胎移植（P<0.01）。表明通过白绒山羊X、Y精子分离、人工授精生产性控胚胎,同时结合胚胎移植技术,可以达到按照生产实际中的意愿得到性别控制子代白绒山羊的目的。     

水牛精液中X和Y精子比率的鉴定

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。