L.A. Johnson

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma.

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.

Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation

In this study, the effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was evaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC‐pisum sativum agglutenin and propidium iodide (PI). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR‐14 and PI.

Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency.

Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X-bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.

Birth of Piglets After Transfer of Embryos Cryopreserved by Cytoskeletal Stabilization and Vitrification

Abstract Pig embryos suffer severe sensitivity to hypothermic conditions, which limits their ability to withstand conventional cryopreservation. Research has focused on high lipid content of pig embryos and its role in hypothermic sensitivity, while little research has been conducted on structural damage. Documenting cytoskeletal disruption provides information on embryonic sensitivity and cellular response to cryopreservation. The objectives of this study were to document microfilament (MF) alterations during swine embryo vitrification, to utilize an MF inhibitor during cryopreservation to stabilize MF, and to determine the developmental competence of cytoskeletal-stabilized and vitrified pig embryos. Vitrified morulae/early blastocysts displayed MF disruptions and lacked developmental competence after cryopreservation; hatched blastocysts displayed variable MF disruption and developmental competence. Cytochalasin-b did not improve morula/early blastocyst viability after vitrification; however, it significantly (P < 0.05) improved survival and development of expanded and hatched blastocysts. After embryo transfer, we achieved pregnancy rates of almost 60%, and litter sizes improved from 5 to 7.25 piglets per litter. This study shows that the pig embryo cytoskeleton can be affected by vitrification and that MF depolymerization prior to vitrification improves blastocyst developmental competence after cryopreservation. After transfer, vitrified embryos can produce live, healthy piglets that grow normally and when mature are of excellent fecundity.

Glutaraldehyde fixation of boar spermatozoa for acrosome evaluation

Abstract The acrosome morphology, as assessed by phase-contrast microscopy, was not altered by fixing boar spermatozoa with 0.1 to 2% glutaraldehyde in comparison to spermatozoa inhibited with NaF. The acrosome morphology of glutaraldehyde-fixed samples remained unchanged for 14 days. Boar semen diluted and stored in Beltsville L1 (BL1), egg-yolk-glucose-bicarbonate (EGB), and Beltsville F5 (BF5) could be fixed with glutaraldehyde for acrosome evaluation if the supernatant solution containing egg yolk was removed immediately after glutaraldehyde addition. Spermatozoa extended in Beltsville F3 (BF3) extender could not be microscopically evaluated after glutaraldehyde fixation because of casein precipitation. Acrosome morphology of glutaraldehyde-fixed boar spermatozoa remained unchanged for at least 19 days holding at room temperature or shipping by parcel post.

PRODUCTION OF PIGLETS PRESELECTED FOR SEX FOLLOWING IN VITRO FERTILIZATION WITH X AND Y CHROMOSOME-BEARING SPERMATOZOA SORTED BY FLOW CYTOMETRY

In vivo-matured porcine oocytes were fertilized in vitro with X and Y chromosome-bearing spermatozoa, and sorted for sex on the basis of DNA content by flow cytometry. Developmental competence of the sexed embryos was determined through established pregnancies after embryo transfer. Spermatozoa were stained with Hoechst 33342 and sorted using a flow cytometry cell sorter. Purity of sorting was 83% for Y spermatozoa and 92% for X spermatozoa. A total of 387 mature cumulus-oocyte-complexes (COC) was collected from 18 superovulated prepuberal gilts shortly before ovulation. In vitro fertilization with sorted spermatozoa was performed in 4 replicates. After 18 h of sperm- oocyte co-culture at 39 degrees C, the zygotes were placed into culture medium (NCSU-23) for another 24 h. The average cleavage rate was 56.2%. Ninety-two embryos produced from X-sorted sperm cells were transferred surgically into the uterus of 2 recipients. Two gilts farrowed and delivered 6 and 4 healthy female piglets, respectively. Additionally, 2 gilts were inseminated intratubally via surgical laparotomy with either X or Y sorted spermatozoa (2 x 10(5)) per oviduct. The 2 sows farrowed producing 15 piglets. Thirteen of the 15 piglets were of the predicted gender (85%).

Uterine horn insemination of heifers with very low numbers of nonfrozen and sexed spermatozoa

Abstract Experiments were conducted to determine 1) pregnancy rates of heifers inseminated with very low numbers of spermatozoa under ideal field conditions, and 2) pregnancy rates with low doses of sexed spermatozoa. In Experiment 1, semen from 3 Holstein bulls was extended to 1 × 105 or 2.5 × 105 sperm/0.1 ml; 2.5 × 106 total sperm/0.21 ml were used for the control. Semen was cooled to 5 °C, packaged into modified 0.25-ml French straws, and used 26 to 57 h after collection. Spermatozoa were inseminated 24 h after detection of estrus into the uterine horn of Holstein heifers ipsilateral to the ovary with the largest follicle, as determined by ultrasound 12 h after estrus was detected; side of ovulation was verified by detection of a corpus luteum (CL) by ultrasound 7 to 9 d post estrus. Pregnancy was determined 40 to 45 d post estrus. The side of ovulation was determined correctly in 262 of 286 heifers (92%), and pregnancy rates were nearly identical for ipsilateral and contralateral inseminations. Pregnancy rates were 48/118 (41%), 56/111 (50%), and 35/57 (61%) for 1 × 105, 2.5 × 105 and 2.5 × 106 sperm per insemination (P .05) among the heifers for the 3 AI technicians or the 3 bulls. In Experiment 2, freshly collected semen was transported from Lancaster, Pennsylvania to Beltsville, Maryland, and sorted into X- and Y-sperm populations based on DNA difference using a flow cytometer/cell sorter over a 6-h period. Sorting rates were about 100 sperm/sec of each sex at ~90% purity. Sorted spermatozoa were shipped ~2600 km by air, and in most cases cooled to 5 °C during shipping over 6 h in an Equitainer. Heifers were inseminated with 1 to 2 × 105 sorted X- or Y-spermatozoa in 0.1 ml within 9 to 29 h of sorting. The inseminate was either deposited into the uterine horn ipsilateral to the ovary with the largest follicle as determined by ultrasound at the time of insemination, or half of the inseminate was deposited into each uterine horn. None of 10 heifers became pregnant when inseminated with sexed spermatozoa shipped at ambient temperature. Of the 155 heifers inseminated with sexed spermatozoa cooled to 5 °C, 15 of 67 females (22.4%) inseminated 9 to 13 h post sorting calved, but only 2 of 78 (2.6%) inseminated at 17 to 29 h calved. Fourteen of the 17 calves bom (82%) were of the selected sex.

Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry

The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.

A novel nozzle for more efficient sperm orientation to improve sorting efficiency of X and Y chromosome-bearing sperm†

Efficient high-resolution detection of DNA for flow cytometric sorting of X and Y chromosome-bearing sperm is dependent on effectively orientating the sperm head to the laser beam in orthogonally configured flow systems. Normally, a beveled needle is required to enlarge the fraction of properly orientated sperm (flat side facing the laser beam). In this report, a modification to a standard jet-in-air nozzle for improved sperm orientation is presented. Inside the modified nozzle (novel nozzle), orientation forces are applied lower in the nozzle than in the current beveled injection needle system. The nozzle was tested with sperm heads from several species. This study shows that use of the nozzle to orientate cattle, swine, rabbit, mouse, and human sperm effectively improves the percentage of sperm that are properly oriented. The percentage of sperm heads oriented by use of the former system (beveled needle) ranges around 30% for most species. With the newly designed nozzle, that percentage ranges around 60%. At least a twofold increase in analysis is achieved. It was found that, unlike results with the beveled needle, the percentage of properly oriented sperm was independent of the sample rate. The introduced nozzle is a significant improvement over the beveled needle system for the analysis and sorting of sperm on the basis of DNA content. In addition to the improvement in sorted sperm production brought about by the novel nozzle when fitted to standard-speed cell sorters, it clearly also has significant potential for improving the efficiency of the Beltsville Sperm Sexing Technology for separating X and Y chromosome-bearing sperm when adapted to high-speed cell-sorting systems.

In vitro production of pig embryos: Comparisons of culture media and boars

The utilization of in vitro produced pig embryos for commercial production or research is dependent upon the development of improved methodology. Our objective was to establish a consistent in vitro embryo production (IVP) system and subsequently utilize the procedures to evaluate culture system components and boar effects. To summarize the IVP system, 403 inseminated oocytes from a total of 2243 were analyzed across 17 replicates for maturation and fertilization efficiency, while 1838 zygotes were cultured in 26 replicates for developmental data. Penetration, cleavage and blastocyst development rates were determined at 18, 44 and either 144 or 168 h post insemination, respectively. Monospermic penetration averaged 31.8+/-7.3% while polyspermy was 30.8+/-17.2%. Cleavage rate was 44.9+/-16.1%, with 21.8+/-7.5% of fertilized oocytes and 51.9+/-15.9% of cleaved embryos developing to blastocysts. For culture medium comparison, fertilized oocytes were cultured in either BECM-6, BECM-7, NCSU-23 or NCSU-23aa and supplemented on Day 5 post insemination (pi) with 10% FCS. These treatments resulted in 4.0, 4.9, 19.8 and 13.6% (+/-3.2%) blastocysts by Day 7 pi, with an average cell number of 44.4+/-9.0, 65.1+/-8.2, 61.3+/-4.5 and 64.4+/-4.8, respectively. These IVP procedures consistently produced zygotes from semen of several different boars, capable of forming blastocysts in vitro. Comparison of developmental rates among the boars indicated that this system is variable among boars but not strictly boar-dependent. Culture media comparisons suggest that NCSU-23 yielded a higher percentage of blastocysts than the other media in this IVP system.