Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14

ELSEVIER

PORCINE SPERM VIABILITY, OOCYTE FERTILIZATION AND EMBRYO DEVELOPMENT AFTER STAINING SPERMATOZOA WITH SYBR-14 D.L. Gamer,” J.R. Dobrinsky,’ G.R. Welch’ and L.A. Johnson’ ‘School of Veterinary Medicine, University ofNd Rena, NV 89557 *Germplasm&GametePhysio1ogyLaboratory~AgriculturalResearchService US Department of Agriculture, Behsville, MD 20705 Received for publication: August 30, 1995 Accepted: October 18, 1995 ABSTRACT The objective of these experiments was to determine the efficacy of the new membrane permeant nucleic acid stain, SYBR-14, for assessing boar sperm viability and to determine it’s effect on fertilization and early embryonic development using the pig as a model. We examined the staining patterns of SYBR-14 and another vital stain, Hoechst 33342, both in combination with the dead cell stain, propidium iodide (PI), to quantify the proportion of living and dead spermatozoa in ejaculated and epididymal semen. Flow cytometry analyses of semen from 4 boars revealed significant differences among boars for the proportion of SYBR-1Cstained spermatozoa in both epididymal and ejaculated samples, but not for Hoechst 33342 or PI stained spermatozoa. Gilts were inseminated with unstained spermatozoa or spermatozoa stained with 2 levels of SYBR-14 or 2 levels of the reference stain, Hoechst 33342. Embryos recovered at 42 to 48 h postinsemination were morphologically evaluated, and only 4 to 8-cell embryos were continued in culture. Overall, fluorescent staining of boar spermatozoa with SYBR-14 or Hoechst 33342 neither affected their ability to fertilize oocytes, nor the developmental competence of the resultant embryos. Key words: spermatozoa, boar, fertility, embryonic development, fluorescent staining INTRODUCTION The fluorometric assessment of sperm viability targeted on sperm DNA has been enhanced by the availability of new stains and staining approaches to identify viable cells. The most effective method has been to combine these nucleic acid stains with dead cell stains for determining sperm viability ($6) and for increasing the efficiency of spermatozoa flow cytometric sorting protocols (9). The most effective approach in either case is to use 2 stains which react with the same cellular constituent, one stain to identifjl only living spermatozoa and a second that only stains dead spermatozoa. This concept of dual staining DNA to quantify viable versus dead Acknowledgments The authors are gratetil to Dr. R. P. Haugland of Molecular Probes, Eugene, Oregon for providing the SYBR-14. We are also indebted to Lori Schreier, Douglas Nate, Don Palencia and Scott Chambers for their technical assistance. This work was supported, in part, by USDA Specific Cooperative Agreement 58-1265-3-050 and by the Nevada Agricultural Experiment Station. aCorrespondence and reprints requests. Thetiogenology 45:i 103-l 113, 1996 0 1996 by Elseviet Science Inc. 655 Avenue of the Americas, New York, NY 10010

0093-691X/96/$15.00 PII SOO93-691X(96)00067-6

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spermatozoa for monitoring cryopreservation other mammals (6).

effects has been tested recently in the bull (5) and

Sperm viability assessments using dual staining with enzyme-based staining combinations (carboxyfluorescein diacetate and propidium iodide, PI) have also been used. However, they are too variable for routine use compared with the newer nucleic acid permeant stains (1, 7). Moreover, sperm DNA is a more appropriate cellular target due to its stainability and staining uniformity. The new membrane permeant nuclear stain, SYBR-14, which brightly fluoresces the nuclei of living cells, has been used in combination with PI to determine the proportion of living spermatozoa in semen Corn several different mammals (6) and has been proven effective for assessing sperm viability (5, 6). The SYBR-14 system excites with visible light (488 nm) and is, thus, more adaptable to clinical flow cytometry than the bisbenzimide stains, Hoechst 33342 and 33258, which excite in ultraviolet light (UV, 351, 364 nm) and require UV laser-equipped flow cytometry systems. Hoechst 33342 has been proven effective for staining living spermatozoa for purposes of flow cytometric separation of X- and Y-chromosome bearing spermatozoa and for production of normal sexed offspring in rabbits (1 l), pigs (10) and cattle (2, 3). We sought to compare the staining efficacy of SYBR-14 and Hoechst 33342 in combination with PI for living epididymal and ejaculated boar spermatozoa and to determine if SYBR-14 would allow for normal fertilization and early embryonic development using the pig as a model. The experiments were designed to compare the fertilizing capacity of ejaculated boar spermatozoa stained with 2 levels of SYBR-14, and 2 levels of Hoechst 33342 as well as control spermatozoa (unstained). The resultant embryos were cultured and monitored for early development. METHODS AND MATERIALS Ejaculated Semen, Epididymal Spermatozoa Recovery and Sample Preparation Four mature crossbred boars, aged 5 to 6 yr, that had been used for regular weekly semen collections for several years provided the ejaculated and epididymal semen for the study. Semen collections were made using the gloved-hand technique. Sperm concentration was determined using a hemocytometer. Spermatozoa were evaluated microscopically for seminal quality (sperm motility and morphology) after dilution to 10 x lo6 spermatozoa/ml in Beltsville TS (BTS, 8). To obtain epididymal spermatozoa, these same boars were slaughtered the day following ejaculation, their caudal epididymides recovered and flushed retrograde with BTS via the vas deferens, using a blunt 18-gauge needle and syringe. To aid in the flush, connective tissue was trimmed from the epididymides so that adequate pressure could be applied to flush the epididymal contents. The resulting epididymal samples were diluted with BTS to a concentration of approximately 10 x lo6 spermatozoa/ml. Viability assessments of both ejaculated and epididymal spermatozoa were made using 500 pl of diluted sperm samples contained in 1.5 ml Eppendorf tubes. The semen used for artificial insemination (AI) was collected from 5 other mature boars over a 90-d period. On each insemination day, the semen from 3 boars was pooled and diluted to a concentration of 50 x lo6 spermatozoa/ml. The diluted semen was then divided into aliquots for fluorescent staining of each insemination dose as described below.

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1105

Fluorescent Staining of Spermatozoa for Flow Cytometry The SYBR-14 (M W 565.1, FertrLight Kit)’ was prepared in anhydrous methyl sulfoxide @MSO)b at a concentration of 1 mg/rnl (9 x 1U3 CLM). Six 500~ul aliquots each of epididymal and ejaculated semen from each boar were allocated at random to each of 2 treatments (SYBR-14 /PI and Hoechst 33342/PI. One set of 3 aliquots was stained at 36°C with 0.27 ul of the stock solution of SYBR-14 and 2 ul of the PI stock solution (2.99 mM-2 mg/ml in Tyrodes solution’). These small volumes were carefully added using P-2 Pipetmand and FluoroPel pipette tips’. An identical triplicate set of aliquots was stained with 3.5 ul of a stock solution containing 500 ug Hoechst 33342’/ml of water (9 x lo5 @I) and 2 ul of PI stock. The samples were incubated for a minimum of 15 min at 32’C before examination, as has been previously shown to be an effective minimum staining for detection of DNA differences and for production gender preselected offspring (10). The living spermatozoa that were stained with the SYBR-14 combination fluoresced bright green, while the dead spermatozoa fluoresced red when excited at 488 nm. When the Hoechst 33342 and PI-stained samples were excited in the UV, the living spermatozoa fluoresced bright blue while the dead spermatozoa were a pinkish red. Fluorescent staining was monitored and photographed using a Zeiss Axiophot epifluorescent microscopeg equipped with FITC (Zeiss #487909) and W (Zeiss # 477702) filter sets. Flow Cytometry The SYBR-14 and PI-stained boar sperm populations were quantified using an Epics Profile II,” while the Hoechst 33342 and PI-stained samples were run on a FACS 440 flow cytometer’ modified for sperm sorting (14). The Epics Profile II, an air-cooled 15 mW Argon laser based (488 nm) flow cytometer, was equipped with the PowerPak option. Spermatozoa were analyzed for fluorescence intensity after gating on side and forward light scatter parameters to exclude debris, free cytoplasmic droplets, doublets and somatic cells. For the SYBR-14 stained samples, the log of the green fluorescence (LFLl) was collected through a 525-nm band pass filter, while the logs of the red fluorescence parameters, fluorescence 2 (LFL2) and fluorescence 3 (LFL3) were gathered through a 575~nm band and 635~nm band pass filters, respectively. The spillover of green fluorescence into the 635~nm red channel (LFW) was minimized by compensation (25%). For each sample, 10,000 spermatozoa were analyzed for the log of their fluorescence. For the data gathered with the Epics Profile, the Coulter Histogram Analysis program was used to analyze the a Commercial Kit, Molecular Probes, Eugene, OR. b Aldrich Chemcial Company, Milwaukee, WI. ’ Sigma Chemical Co., St. Louis, MO. d Rainin Instrument Co., Emeryville, CA. e No. T30510F, Baxter Diagnostics, McGaw Park, IL. f Hoechst 33342, Calbiochem, La Jolla, CA. g Carl Zeiss, Inc., Hanover, MD. h Coulter Corporation, Inc., Hialeah, FL. i Becton Dickinson Immunocytometry

Systems, San Jose, CA.

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cytograms for relative fluorescence of the LFLl and LFL3 channels. The dual stained (Hoechst 33342/PI) samples, which were analyzed using the modified FACS 440 for the logs of blue and red fluorescence, were quantified using the Consort 30 (Version G) program. Fluorescent Staining of Spermatozoa for Insemination Two stain concentrations were utilized in these experiments because we sought to stain the spermatozoa at the level that has been used for flow sorting of X- and Y-chromosome-bearing boar spermatozoa to produce offspring (10). Sperm sorting has been routinely carried out at a sperm concentration of 10 x lo6 spermatozoa/ml, whereas the insemination dose was 50 x 10” spermatozoa/ml. It was, therefore, also necessary to increase the stain concentration to reach a stoichiometric equilibrium with the higher number of spermatozoa in the insemination dose. The 80-ml insemination doses of semen were either unstained spermatozoa (Group A), stained with 2 levels of Hoechst 33342 (Group B at 5.4 pM and Group C at 10.8 pM), or stained with 2 levels of SYBR-14 (Group D at 9 x lo5 pM and Group E at 19 x 105 pM). For the Hoechst 33342stained samples, 560 pl of the 5OOyg/ml and 1000~@ml stock solutions were added for final concentrations of 5.4 and 10.8 pM, respectively. A stock solution of SYBR-14 (1 ms/ml) was diluted 1: 10 and I:5 with DMSO before use. Two 80-ml aliquots of the diluted semen were stained by adding 43.1 ~1 of the 1:10- and l:S-dilutions to attain concentrations of 9 x lo” pM and 19 x lo5 pM for SYBR-14, respectively. After staining the 80 ml-doses at 22 to 24°C (room temperature), they were incubated for 30 min at 32°C before being inseminated. Artificial Insemination Crossbred gilts were artificially inseminated twice during a 24-h period when they were in standing estrus, using the Melrose catheter (8) with ejaculated, pooled semen that was diluted with BTS and stained as described above for the 80-ml insemination doses. The number of gilts employed for each treatment was determined by the number of gilts that exhibited esttus on any given day. Embryo Recovery All embryos in these studies were produced from crossbred gilts that were 7 mo of age or older, weighed at least 120 kg at the time of breeding, and had exhibited at least 1 normal estrous cycle. In each replicate, 3 to 4 donors were used per day of recovery (Day 2, onset of es&us = Day 0). Embryos were recovered from the excised reproductive tracts (maintained at 37°C) within 10 min from the time that the tracts were recovered after slaughter. Embryos were recovered by flushing the excised uterine horns with sterile modified Dulbecco’s phosphate buffered saline (mDPB$ supplemented with 3 mg bovine serum albumin Fraction V (BSA-V, Sigma A2153)’ and 5.5 mM glucose (4).

j3 lo-404OAJ, Gibco BRL Life Technologies, Gaithersburg, MD

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Theriogenology Embryo Culture

Embryos were evaluated for quality and developmental status at recovery. Only morphologically normal 4 to S-cell embryos were placed in culture to determine their developmental competence. Embryos were cultured for 6 d in BECM-3 (4) in 5% CO2 and air with 100% humidity. The embryos were examined morphologically on Day 7 for development to blastocysts using a Zeiss SV-11 Sterozoom microscope.g Statistical Analyses Differences among the sperm populations, both within and among boar semen samples, were examined by analysis of variance (ANOVA) using the least squares procedure and the general linear models procedure of the Statistical Analysis System (SAS, 13). The data, which were acquired as percentages, were arcsine-transformed before analysis. The interrelationships among the sperm populations were evaluated using correlation coefficients generated by SAS. Chi-square was used to compare the data on embryonic development. RESULTS Epididymal and Ejaculated Boar Spermatozoa Two major populations and a minor one were identified when spermatozoa were stained with either SYBR-14 or Hoechst 33342 in combination with PI. Both SYBR-14 and Hoechst 33342 stained the nuclei of living spermatozoa very brightly as determined by simultaneous microscopic examination of fluorescence intensity and sperm motility. Although the fluorescent sperm populations were situated somewhat differently in the cytograms, the quantitative results from both epididymal and ejaculated semen indicated that the staining was essentially identical. The variation in placement of the sperm populations was due not only to the fact that they were stained using different viability stains, SYBR-14 or Hoechst 33342; but also that they had been run on different flow cytometers. A comparison of epididymal and ejaculated semen from Boar 3 when stained with either SYBR-14 and PI (A and B) or with Hoechst 33342 and PI (C and D) is illustrated in Figure 1. The proportion of living cells in the samples of epididymal spermatozoa tended to be slightly less for 2 of the boars than for the other boars for the Hoechst 33342 stained samples (Table 1). This boar variation was also evident for the samples stained with SYBR-14. Although variation was evident for both stains in combination with PI, the ranking of boars was not consistent between stains or sources of semen. The 2 boars with the lowest proportion of stained epididymal sperm cells were not necessarily those with the lowest proportion of stained spermatozoa in the ejaculated samples. The proportion of ejaculated spermatozoa stained with SYBR-14, which ranged from 83.4 + 0.2 % to 91.7 f 0.2% (Table l), differed among the 4 boars (PcO.01). The boars also differed (P
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Table 1.

Theriogenology The proportions of SYBR-14 and Hoechst 33342 stained sperm populations in epididymal and ejaculated semen of 4 boars when used in combination with propidium iodide as determined by flow cytometry analyses (n = 3).

Semen sample

Boar 1

Boar 2

Boar 3

Boar 4

Hoechst 33342 stain Epididymal

79.2 + 1.4”

80.0 f O.#

83.8 + 1.4a

70.3 f 1.8b

Ejaculated

87.4 + 2.3a

78.2 f 1.Sb

78.2 + 0.4b

84.6 f 0.4’

SYBR- 14 stain Epididymal 92.2 + 0.4’ 91.8 f 0.4a’b 88.7 f 0.6’ 90.4 f o.2b Ejaculated 90.7 f 0.3b 83.4 f 0.2’ 91.7 f 0.2b 88.4 f 0.1’ a. b*c*d Boars with the same letter did not differ for the proportion of Hoechst 33342 or SYBR-14 stained spermatozoa (PC 0.05). The proportion of epididymal spermatozoa that stained with Hoechst 33342 in combination with PI (Table 1) differed among the boars (P
Theriogenofogy

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Epididymal Gtained

Spermatozoa

Ejaculated Spermatozoa _Ai

spennatozoa

B

a j

Seminal debris

1; i 1

1

i SYBR-14-stained spermatozoa,; ._________.--____..L

3

Log of Green Fluorescence

s

C’

r= b)

z

2

s-

c

y PI-stained spermatozoa

Hoechst 33342-

Log of Blue Fluorescence Figure 1. A comparison of one of the 3 replicate cytograms from epididymal (A and C) and ejaculated (B and D) semen from Boar 3 when spermatozoa were stained with SYBR14 and propidium iodide (PI, A and B) or with Hoechst 33342 and PI (C and D). Two major spermatozoa populations (1 and 3) and a doubly stained population (2) were identified and quantified for each stain. Seminal debris, which is evident as the scattered dots in the lower left quadrant of A and B, was not quantified. For this particular sample, the proportions of spermatozoa stained with PI (Population l), doubly stained (Population 2), and SYBR-14-stained (Population 3) were, respectively, 4.9, 0.2 and 89.9% for epididymal semen (A) and 6.7, 1.3 and 88.6% for ejaculated semen (B). The proportions of spermatozoa stained with PI, doubly-stained and Hoechst 33342 stained (Population 3) were, respectively, 5.5, 0.4 and 85.8% for epididymal semen and 9.5, 0.3 and 83.1% for ejaculated semen.

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Developmental Competence of Embryos Developmental competence of embryos produced from SYBR-14- and Hoechst 33342stained spermatozoa was determined in vitro. Embryos produced from normal AI with untreated spermatozoa served as controls, These control embryos showed modest development (55%, Table 3) to blastocysts. The percentage of embryos that progressed to the blastocyst stage varied among the treatment groups and averaged 40% for the treatment groups. Embryos produced from Al with Hoechst 33342~stained spermatozoa showed no difference in development to blastocysts, irrespective of the concentration of stain (Croup B, 64%; Croup C, 51%), when compared with the controls (Table 3). Embryos produced from AI with SYBR-14 stained sperm cells, however, showed a differential effect on embryo development. Those embryos resulting from spermatozoa stained with the lower level of SYBR-14 (Croup D) developed at a lower rate (12%, P
The effect of staining of porcine spermatozoa on subsequent development of the resultant embryos. Gilts were inseminated with 80 ml of semen diluted to 50 x 10’ spermatozoa/ml in BTS which had been unstained or stained at 2 levels with SYBR-14 (I, 9 x 105 pM and II, 19 x lo5 ClM) or Hoechst 33342 (B, 5.4 @I and C, 10.8 pM). Blastocyst Blastocyst Treatment Gilts Embryos cultured development (%) (n) (n) development (n) group 55 22 A - Control 4 40 64 36 B - Hoechst 33342-B 5 56 51 32 C - Hoechst 33342-C 5 65 5 D - SYBR-14-I 4 41 12a 56 E - SYBR-14-B 5 52 29 “X2 of the 4 individual treatments and of the totals vs the control (P
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1111

Differences were noted among boars for the proportion of SYBR-1Cstained spermatozoa in both epididymal and ejaculated samples but not for the proportion of PI-stained sperm cells for either the Hoechst 33342/PI or SYBR/PI combinations. Only 1 of the animals (Boar 4) differed in the relatively lower proportion of epididymal sperm cells that stained with Hoechst 33342. Boar 4, however, was one of the boars with a high proportion of ejaculated sperm cells that stained with Hoechst 33342. Thus, it is not possible to draw any final conclusions concerning the predictive value of either data set from the epididymal or ejaculated samples for estimating the corresponding epididymal or ejaculated value for that same parameter. The proportion of spermatozoa that stained with SYBR-14 for the individual boars was higher for epididymal samples compared with that of the companion ejaculate samples for 2 of the boars with either of the stains. This variation in boars and in the source of semen may have resulted from the recovery procedure and from processing and staining methods rather than from actual biological differences. There was less variation among epididymal sperm samples with the SYBR-14 stain than with the Hoechst 33342 stain. The proportion of sperm cells that stained with SYBR-14 varied only 3.5% among the boars compared with 12.5% for the Hoechst 33342 stain. Variation in the proportions of live spermatozoa among the ejaculate samples from the 4 boars was also greater for the Hoechst 33342~stained spermatozoa than for the SYBR-14 stained sperm cells (9.2 and 8.3 %, respectively). The fluorescent staining patterns were similar for all boars for both epididymal and ejaculated sperm samples for the two stain combinations. To our knowledge this is the first use of a combination of Hoechst 33342 and PI for assessing the viability of spermatozoa in porcine semen, the procedure has been used previously to improve the efficiency of flow cytometric sorting of X and Y-chromosome-bearing spermatozoa in the bull (9) and boar (9). The fluorescent sperm populations, representing living sperm cells (i.e., stained with either SYBR-14 and Hoechst 33342), were positioned slightly differently in the cytograms because we were not able to adjust the photomultiplier gains to identical positions for each instrument. The variation in placement of the sperm populations was due to the fact that they were stained with different viability-indicating stains (SYRR-14 or Hoechst 33342) and that the samples had been run on different flow instruments. The quantitative data obtained from both epididymal and ejaculated semen samples of these populations, however, indicated that the 2 staining combinations yielded similar results. The advantage of using SYBR-14 is that it can be used to assess sperm viability in clinical flow cytometers equipped with non-UV light sources. In our study, spermatozoa that had been stained with either Hoechst 33342 or SYBR-14 fertilized oocytes in vivo at rates similar to those previously reported for Hoechst 33342-stained spermatozoa (10, 11, 12). The embryonic development of oocytes resulting from fertilization with SYBR-14 or Hoechst 33342-stained spermatozoa appeared normal, with the exception of sperm cells stained with the lower level of SYBR-14 (9 x 10” PM), when compared with embryos resulting from unstained spermatozoa. The controls exhibited only modest development, and embryos resulting from Hoechst 33342-stained spermatozoa did not differ in developmental competence from those of the other sources. The SYBR-14 data for the higher concentration of fluorochrome (19 x 1U3 pM) showed no detrimental effect on embryonic development in vitro, and was similar to the data from the controls and the embryos that resulted from Hoechst 33342treated spermatozoa. These results suggest the absence of serious harmful effects of SYBR-14 on embryonic development. However, the effect of staining spermatozoa with the lower level of SYBR-14 (9 x lU3 PM) can not be explained in the design of this experiment. The 12%

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development competence of the embryos resulting from the lower SYBR-I4 (Group D - SYBR14 at 9 x IO” pM) stained spermatozoa does not support the conclusion that this low level supports normal embryonic development. Clarification of this point will need to be determined in future studies. REFERENCES 1.

2.

3. 4. 5

6. 7. 8.

9.

10.

11. 12. 13. 14.

Almlid T, Johnson LA. Effects of glycerol concentration, equilibrium time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws. 3 Anim Sci 1988; 66:2899-2905. Cran DG, Johnson LA, Miller NGA, Cochrane D., Polge C. Production of bovine calves following separation of X- and Y-chromosome bearing sperm and in vitro fertilization. Vet Ret 1993; 132:40-41. Cran DG, Johnson LA, Polge C. Sex preselection in cattle: a field trial. Vet Ret 1995; 136:495-496. Dobrinsky JR, Johnson LA. Cryopreservation of porcine embryos by vitrification: A study on in vitro development. Theriogenology 1994; 42125-35. Garner DL, Johnson LA, Yue ST, Roth BL, Haugland RP. Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. J Androl 1994; 16:620-629. Garner DL, Johnson LA. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 1995;53:276-284. Garner DL, Pinkel D, Johnson LA, Pace MM. Assessment of spermatozoal finction using dual fluorescent staining and flow cytometric analyses. Biol Reprod 1986;34: 127-138. Johnson LA, Aalbers JG, Grooton HJG. Artificial insemination of swine: fecundity of boar semen stored in Beltsville TS (BTS), modified Modena (MM) or M&A and inseminated on one, three and four days after collection. Zuchthygiene 1988;23:49-55. Johnson LA, Welch GR, Garner DL. Improved flow sorting resolution of X- & Ychromosome bearing viable sperm separation using dual staining and dead cell sorting. Cytometry 1994; 7 (Suppl):83, abstract 476D. Johnson LA. Gender preselection in swine: Altered sex ratios in offspring following surgical insemination of flow sorted X- and Y-bearing sperm. Reprod Dom Anim 1992;26:309-314. Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 1989; 4 1: 199-203. McNutt TL, Johnson LA. Flow cytometric sorting of sperm: Influence on fertilization and embryo/fetal development in the rabbit. Mol Reprod Dev 1995; 41 :(in press). SAS User’s Guide, Statistics. SAS. Cary, NC: Statistical Analysis System Institute, Inc, 1985. Welch, GR, Houck DW, Johnson LA. Fluidic and optical modifications to a FACS IV for flow sorting of X- and Y-chromosome bearing sperm based on DNA. Cytometry 1994;7 (Suppl):74, abstract 398B.

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Theriogenology

development competence of the embryos resulting from the lower SYBR-14 (Group D - SYBR14 at 9 x 10” pM) stained spermatozoa does not support the conclusion that this low level supports normal embryonic development. Clarification of this point will need to be determined in future studies. REFERENCES 1.

2.

3. 4. 5

6. 7. 8.

9.

10.

11. 12. 13. 14.

Almlid T, Johnson LA. Effects of glycerol concentration, equilibrium time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws. J Anim Sci 1988; 66:2899-2905. Cran DG, Johnson LA, Miller NGA, Cochrane D., Polge C. Production of bovine calves following separation of X- and Y-chromosome bearing sperm and in vitro fertilization. Vet Ret 1993; 132:40-41. Cran DG, Johnson LA, Polge C. Sex preselection in cattle: a field trial. Vet Ret 1995; 136:495-496. Dobrinsky JR, Johnson LA. Cryopreservation of porcine embryos by vitrification: A study on in vitro development. Theriogenology 1994; 42:25-3 5. Garner DL, Johnson LA, Yue ST, Roth BL, Haugland RP. Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. J Androl 1994; 16:620-629. Gamer DL, Johnson LA. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 1995;53:276-284. Gamer DL, Pinkel D, Johnson LA, Pace MM. Assessment of spermatozoal hmction using dual fluorescent staining and flow cytometric analyses, Biol Reprod 1986;34:127-138. Johnson LA, Aalbers JG, Grooton HJG. Artificial insemination of swine: fecundity of boar semen stored in Beltsville TS (BTS), modified Modena (MM) or MR-A and inseminated on one, three and four days after collection. Zuchthygiene 1988;23:49-55. Johnson LA, Welch GR, Gamer DL. Improved flow sorting resolution of X- & Ychromosome bearing viable sperm separation using dual staining and dead cell sorting. Cytometry 1994; 7 (Suppl):83, abstract 476D. Johnson LA. Gender preselection in swine: Altered sex ratios in offspring following surgical insemination of flow sorted X- and Y-bearing sperm, Reprod Dom Anim 1992;26:309-314. Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 1989; 41: 199-203 McNutt TL, Johnson LA. Flow cytometric sorting of sperm: Influence on fertilization and embryo/fetal development in the rabbit. Mol Reprod Dev 1995; 41 :(in press). SAS User’s Guide, Statistics. SAS. Gary, NC: Statistical Analysis System Institute, Inc, 1985. Welch, GR, Houck DW, Johnson LA. Fluidic and optical modifications to a FACS IV for flow sorting of X- and Y-chromosome bearing sperm based on DNA. Cytometry 1994;7 (Suppl):74, abstract 398B.