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Post-thawing survival of motile ram sperm after isolation by layering on protein columns
THERIOGENOLOGY
POST-THAWING
W.M.C.
SURVIVAL OF MOTILE RAM SPERM AFTER BY LAYERING ON PROTEIN COLUMNS
ISOLATION
2 Maxwell, l-3 G. Mendoza, and I.G. White'
1 2 Departments of Animal Husbandry and Veterinary Physiology, University of Sydney, Sydney, Australia 3
Present
address: Animal Breeding and Research Katanning, Western Australia 6317 Received
for publication: Accepted:
2006
Institute,
August 16, 1983 February 10, 1984
ABSTRACT -Post-thawing survival of ram sperm was examined after semen which had been layered on top of isolation columns containing solutions of bovine serum albumin (BSA) in Tris diluent was processed for freezing by the pellet method. Sperm isolated from the bottom of the BSA column had better post-thawing survival than sperm from the top of the column. The efficiency of sperm isolation was affected by the concentration of BSA in the column, the holding time of semen on the column and the concentration of sperm in the layered semen. The best post-thawing6surviva1 of sperm occurred when semen diluted to a concentration of 200 x 10 sperm/ml was layered on a column of 6% BSA in Tris diluent and the bottom layer of the column was isolated for freezing after two hours' holding time. INTRODUCTION It has been claimed that the fractionation of human sperm by layering on a column containing a solution of protein (particularly BSA) enables the isolation of an enriched Y-sperm fraction with enhanced motility and improved survival after storage (1, 2, 3, 4). The same principle has been applied to semen of other species (e.g. horse and pig) with similar enhancement of the motility of sperm recovered in selected fractions (5, 6). This investigation wss concerned with an extension of the technique to ram semen in order to examine the viability of sperm after removal from the column and freeze-thawing. MATERIALS
AND METHODS
Semen was collected by electroejaculation from three Merino rams. The individual ejaculates were extended to the required concentration of sperm with Diluent A (300 mM tris, 27.75 mM glucose, 95 mM citric acid). Two-milliliter aliquots of the diluted semen were carefully layered on top of solutions of BSA in Diluent A in glass isolation columns (10 x 1.4 cm) The columns contained either two layers (4 ml of 6% or 10% BSA above 2 ml of 12% or 20% BSA in Diluent A (Experiment 1))or a single layer of 6 ml of 6% BSA in Diluent A &Experiment 2). Semen was held on the columns at room temperature (21-23 C). After portions:
APRIL
one or two hours of holding, the columns were separated into top (2 ml), middle (4 ml) and bottom (2 ml) in Experiment 1,
1984 VOL. 21 NO. 4
601
and top (2 ml) and bottom (6 ml) In Experiment 2. The number of sperm in each portion was counted using a haemocytometer and the percentage penetration of sperm LPPS = 100 (concentration of sperm in diluted semen/ concentration of sperm in column)] was calculated. The portions of the isolation columns were then centrifuged (1000 g, 15 minutes, room temperature), washed twice with Diluent A (room temperature) and finally diluted 1:2 (semen:diluent,v/v) at 3OoC with Diluent B L360 mFl Tris, 33.3 mM glucose, 113.7 mM citric acid, 18% (v/v) egg yolk and 6% (v/v) glycerol (7)7. The diluted semen was cooled to 5'C in 1.5 hr and frozen on dry ice in pellet form (0.1 ml/pellet). The pellets were transferred to liquid nitrogen and stored for 24 to 72 hr before thawing for examination. The semen was thawed in dry test tubes which were shaken in a water bath at 37OC (2 to 3 pellets/tube), and resuspended (1:4 thawed semen: diluent, v/v) with Diluent A. The percentage of motile sperm was assessed under a coverslip on a warm stage (37'C) immediately after resuspension, and at intervals of 1 hr for 3 hr (Experiment 1) or intervals of 2 hr for 6 hr (Experiment 2) during incubation at 37OC. Data on percentage of motile sperm, following arcsin transformation, were examined by analysis of variance for a split-split-plot factorial experiment, with portion of column being the sub-plot and post-thawing incubation the sub-sub-plot. Data for PPS, after arcsin transformation, were examined by analysis of variance for a factorial experiment. Where significant first-order interaction was revealed between rams and other factors, the interaction mean square was used to test the relevant main effect. RESULTS Experiment 1 This experiment was of 2 x 2 x 3 x 3 factorial design and examined the following factors: 1.
Composition of isolation column: 6%/12% BSA in Tris vs 10%/20% BSA in Tris.
2.
Holding times of semen on column:
3.
Portion of column from which sperm were isolated: top vs middle vs bottom.
4.
Rams:
1 vs 2 hr.
ejaculates from three Merino rams.
A higher percentage of sperm penetrated the 6%f12% BSA columns than the 10%/20% BSA columns (p<0.05), and holding the diluted semen on the isolation column for two hours allowed more sperm to penetrate to the bottom layer than after one hour of holding on the column (p
602
APRIL 1984 VOL. 21 NO. 4
THERIOGENOLOGY
TABLE 1:
PERCENTAGE PENETRATION TO BOTTOM OF ISOLATION COLUMNS BY SPERM FROM THREE RAMS AFTER ONE- OR TWO-HOUR HOLDING ON COLLJMNSWITH DIFFERENT COMPOSITION.
Composition of column
Rams
Holding tine on column (hr) 1 2 -
6%,'127 0 BSA"
10%/20% BSA*
*
a
15.7
17.9
b
10.9
19.8
c
12.7
17.8
Means
13.1
18.5
a
5.1
14.6
b
11.2
13.1
c
8.0
13.2
Means
8.1
13.6
bovine serum albumin
Post-thawing motility of sperm was affected by holding tine of semen on the column (P(o.005) and by portion of the column from which sperm were isolated (P
APRIL
1984 VOL. 21 NO. 4
603
THERIOGENOLOGY
TABLE 2:
EFFECT OF COMPOSITION OF ISOLATION COLUMN, HOLDING TIME ON COLUMN, AND PORTION OF COLUMN FROM WHICH SPERM WAS ISOLATED ON PERCENTAGE OF MOTILE SPERM AFTER THAWING.
Composition of column
Holding time on column (hr)
6%/12% BSA*
1
35.5
49.4
45.0
2
44.3
50.2
57.1
1
43.9
44.5
45.1
2
49.5
53.4
51.5
Means
43.3
49.4
49.7
10%/20% BSA*
*
Portion of column from which sperm was isolated Middle Bottom Top
bovine serum albumin
Experiment 2 The factors included in this experiment (2 x 2 x 2 x 3 factorial) were the following: 1.
Concentratign of sperm in semen layered on columns: vs 200 x 10 /ml.
2.
Holding time of semen on column:
3.
Portion of column from which sperm were isolated: bottom.
4.
Rams:
100
1 vs 2 hr. top vs
ejaculates from three Merino rams.
Sperm isolated from the bottom of the column maintained higher post-thawing motility than sperm from the top (Table 3). There was a steeper decline in post-thawing motility of sperm isolated in the bottom portion of the columns after holding semen for 1 hr than after 2 hr, and a lower percentage of sperm were motile at a sperm concentration of 100 than at 200 x 10 /ml. These findings were reflected in significant second-order interactions between concentration of sperm in semen layered on columns, portion of column and post-thawing incubation time (P
APRIL
1984 VOL. 21 NO. 4
THERIOGENOLOGY TABLE 3:
RELATIONSHIP BETWEEN CONCENTRATION OF SPERMATOZOA, HOLDING TIME, AND PORTION OF COLUMN ON PERCENTAGE OF MOTILE SPERMATOZOA DURING POST-THAWING INCUBATION.
Toe
Concentration of sperm layered pn column (xl0 /ml)
Holding time on column
100
200
1
2
0
44.0
53.4
46.5
50.9
48.7
2
47.0
51.9
52.4
46.4
49.4
4
39.0
46.0
46.0
39.0
42.5
6
38.2
39.7
39.0
38.2
38.8
42.1
47.8
46.0
43.6
44.9
52.8
62.3
59.6
55.5
57.6
46.4
51.3
50.8
46.9
48.9
47.4
48.4
47.4
48.4
47.9
43.0
50.9
45.4
48.5
47.0
47.4
53.2
50.8
49.8
50.4
44.8
50.9
48.5
46.8
47.7
MEANS Bottom
0
& OVERALL
MEANS
-
Means
(hr)
-
DISCUSSION Isolation of motile sperm (in terms of post-thawing survival) was The efficiency of this effectively achieved using a BSA column. isolation was affected by the concentration of BSA in the column (Tables 1 and 2), the holding time of semen on the column (Tables 1, 2 and 3), and the concentration of sperm in the layered semen (Table 3). Isolation columns containing lower concentrations of BSA (viz 6%/12% or 6% BSA) allowed penetration of more sperm to the bottom of Longer the column and improved the post-thawing survival of sperm. holding time of semen on the column also resulted in a greater percentage of sperm penetrating into the column, and these sperm possessed higher post-thawing motility. A higher concentration of sperm in semen layered on top of the column provided better survival of sperm during post-thawing incubation, presumably because this procedure allowed the penetration of a greater proportion of viable sperm.
APRIL
1984 VOL. 21 NO. 4
605
THERIOGENOLOGY In general, the most satisfactory procedure for selecting highly motile ram sperm was to dilute the semen to a concentration of 200 x lo6 sperm/ml, layer the diluted semen on a column of 6% BSA in Tris diluent, and isolate the bottom layer of the column after two hours' holding time. When applied to human, bull or rabbit sperm (8), such a procedure apparently not only enhances motility and post-freezing survival but also eliminates abnormal forms and partially separates X- and Y-chromosomebearing sperm. The results presented in this paper point to the possibility of improving the fertility obtained after artificial insemination of sheep with frozen-thawed semen by using highly motile sperm isolated on a protein column. REFERENCES 1.
Ericsson, R.J., Langevin, C.N., Nishio, M. Isolation of fractions rich in human Y sperm. Nature, Lond. -246: 421-4 (1973).
2.
Ericsson, R.J. Isolation and storage of progressively motile human sperm. Andrologia, 2: 111-114 (1977).
3.
Glass, R.H. and Ericsson, R.J. Intra-uterine insemination of isolated motile spermatozoa. Feril. Steril., 9: 535-538
(1978)
4.
Dmowski, W.P., Gaynor, L., Lawrence, M., Rao, R. and Scommegna, A. Artificial insemination homologous with oligospermic semen separated on albumin columns. Fertil. Steril., -31: 58-62 (1979).
5.
Goodeaux, S.D. and Kreider, J.L. Motility and fertility of stallion spermatozoa isolated in bovine serum albumin. Theriogenology, -10: 405-414 (1978).
6.
Dixon, K.E., Songy, E.A.J., Thrasher, D.M. and Kreider, K.L. Effect of bovine serum albumin on the isolation of boar spermatozoa and their fertility. Theriogenology, $_: 437-444 (1980).
7.
Visser, D. and Salamon, S. Effect of composition of Tris-based diluent and of thawing solution on survival of ram spermatozoa frozen by the pellet method. Aust. J. Biol. Sci., -25: 605-18 (1972).
8.
Ericsson, R.J. and Glass, R.H. Functional differences between sperm bearing the X- and Y-chromosomes. In: Prospects for Sexing Mammalian Sperm, R.P. Amann and G.E Seidel, Eds. Colorado Association University Press, Boulder. 1982, pp. 201-211.
606
APRIL
1984 VOL. 21 NO. 4
POST-THAWING
W.M.C.
SURVIVAL OF MOTILE RAM SPERM AFTER BY LAYERING ON PROTEIN COLUMNS
ISOLATION
2 Maxwell, l-3 G. Mendoza, and I.G. White'
1 2 Departments of Animal Husbandry and Veterinary Physiology, University of Sydney, Sydney, Australia 3
Present
address: Animal Breeding and Research Katanning, Western Australia 6317 Received
for publication: Accepted:
2006
Institute,
August 16, 1983 February 10, 1984
ABSTRACT -Post-thawing survival of ram sperm was examined after semen which had been layered on top of isolation columns containing solutions of bovine serum albumin (BSA) in Tris diluent was processed for freezing by the pellet method. Sperm isolated from the bottom of the BSA column had better post-thawing survival than sperm from the top of the column. The efficiency of sperm isolation was affected by the concentration of BSA in the column, the holding time of semen on the column and the concentration of sperm in the layered semen. The best post-thawing6surviva1 of sperm occurred when semen diluted to a concentration of 200 x 10 sperm/ml was layered on a column of 6% BSA in Tris diluent and the bottom layer of the column was isolated for freezing after two hours' holding time. INTRODUCTION It has been claimed that the fractionation of human sperm by layering on a column containing a solution of protein (particularly BSA) enables the isolation of an enriched Y-sperm fraction with enhanced motility and improved survival after storage (1, 2, 3, 4). The same principle has been applied to semen of other species (e.g. horse and pig) with similar enhancement of the motility of sperm recovered in selected fractions (5, 6). This investigation wss concerned with an extension of the technique to ram semen in order to examine the viability of sperm after removal from the column and freeze-thawing. MATERIALS
AND METHODS
Semen was collected by electroejaculation from three Merino rams. The individual ejaculates were extended to the required concentration of sperm with Diluent A (300 mM tris, 27.75 mM glucose, 95 mM citric acid). Two-milliliter aliquots of the diluted semen were carefully layered on top of solutions of BSA in Diluent A in glass isolation columns (10 x 1.4 cm) The columns contained either two layers (4 ml of 6% or 10% BSA above 2 ml of 12% or 20% BSA in Diluent A (Experiment 1))or a single layer of 6 ml of 6% BSA in Diluent A &Experiment 2). Semen was held on the columns at room temperature (21-23 C). After portions:
APRIL
one or two hours of holding, the columns were separated into top (2 ml), middle (4 ml) and bottom (2 ml) in Experiment 1,
1984 VOL. 21 NO. 4
601
and top (2 ml) and bottom (6 ml) In Experiment 2. The number of sperm in each portion was counted using a haemocytometer and the percentage penetration of sperm LPPS = 100 (concentration of sperm in diluted semen/ concentration of sperm in column)] was calculated. The portions of the isolation columns were then centrifuged (1000 g, 15 minutes, room temperature), washed twice with Diluent A (room temperature) and finally diluted 1:2 (semen:diluent,v/v) at 3OoC with Diluent B L360 mFl Tris, 33.3 mM glucose, 113.7 mM citric acid, 18% (v/v) egg yolk and 6% (v/v) glycerol (7)7. The diluted semen was cooled to 5'C in 1.5 hr and frozen on dry ice in pellet form (0.1 ml/pellet). The pellets were transferred to liquid nitrogen and stored for 24 to 72 hr before thawing for examination. The semen was thawed in dry test tubes which were shaken in a water bath at 37OC (2 to 3 pellets/tube), and resuspended (1:4 thawed semen: diluent, v/v) with Diluent A. The percentage of motile sperm was assessed under a coverslip on a warm stage (37'C) immediately after resuspension, and at intervals of 1 hr for 3 hr (Experiment 1) or intervals of 2 hr for 6 hr (Experiment 2) during incubation at 37OC. Data on percentage of motile sperm, following arcsin transformation, were examined by analysis of variance for a split-split-plot factorial experiment, with portion of column being the sub-plot and post-thawing incubation the sub-sub-plot. Data for PPS, after arcsin transformation, were examined by analysis of variance for a factorial experiment. Where significant first-order interaction was revealed between rams and other factors, the interaction mean square was used to test the relevant main effect. RESULTS Experiment 1 This experiment was of 2 x 2 x 3 x 3 factorial design and examined the following factors: 1.
Composition of isolation column: 6%/12% BSA in Tris vs 10%/20% BSA in Tris.
2.
Holding times of semen on column:
3.
Portion of column from which sperm were isolated: top vs middle vs bottom.
4.
Rams:
1 vs 2 hr.
ejaculates from three Merino rams.
A higher percentage of sperm penetrated the 6%f12% BSA columns than the 10%/20% BSA columns (p<0.05), and holding the diluted semen on the isolation column for two hours allowed more sperm to penetrate to the bottom layer than after one hour of holding on the column (p
602
APRIL 1984 VOL. 21 NO. 4
THERIOGENOLOGY
TABLE 1:
PERCENTAGE PENETRATION TO BOTTOM OF ISOLATION COLUMNS BY SPERM FROM THREE RAMS AFTER ONE- OR TWO-HOUR HOLDING ON COLLJMNSWITH DIFFERENT COMPOSITION.
Composition of column
Rams
Holding tine on column (hr) 1 2 -
6%,'127 0 BSA"
10%/20% BSA*
*
a
15.7
17.9
b
10.9
19.8
c
12.7
17.8
Means
13.1
18.5
a
5.1
14.6
b
11.2
13.1
c
8.0
13.2
Means
8.1
13.6
bovine serum albumin
Post-thawing motility of sperm was affected by holding tine of semen on the column (P(o.005) and by portion of the column from which sperm were isolated (P
APRIL
1984 VOL. 21 NO. 4
603
THERIOGENOLOGY
TABLE 2:
EFFECT OF COMPOSITION OF ISOLATION COLUMN, HOLDING TIME ON COLUMN, AND PORTION OF COLUMN FROM WHICH SPERM WAS ISOLATED ON PERCENTAGE OF MOTILE SPERM AFTER THAWING.
Composition of column
Holding time on column (hr)
6%/12% BSA*
1
35.5
49.4
45.0
2
44.3
50.2
57.1
1
43.9
44.5
45.1
2
49.5
53.4
51.5
Means
43.3
49.4
49.7
10%/20% BSA*
*
Portion of column from which sperm was isolated Middle Bottom Top
bovine serum albumin
Experiment 2 The factors included in this experiment (2 x 2 x 2 x 3 factorial) were the following: 1.
Concentratign of sperm in semen layered on columns: vs 200 x 10 /ml.
2.
Holding time of semen on column:
3.
Portion of column from which sperm were isolated: bottom.
4.
Rams:
100
1 vs 2 hr. top vs
ejaculates from three Merino rams.
Sperm isolated from the bottom of the column maintained higher post-thawing motility than sperm from the top (Table 3). There was a steeper decline in post-thawing motility of sperm isolated in the bottom portion of the columns after holding semen for 1 hr than after 2 hr, and a lower percentage of sperm were motile at a sperm concentration of 100 than at 200 x 10 /ml. These findings were reflected in significant second-order interactions between concentration of sperm in semen layered on columns, portion of column and post-thawing incubation time (P
APRIL
1984 VOL. 21 NO. 4
THERIOGENOLOGY TABLE 3:
RELATIONSHIP BETWEEN CONCENTRATION OF SPERMATOZOA, HOLDING TIME, AND PORTION OF COLUMN ON PERCENTAGE OF MOTILE SPERMATOZOA DURING POST-THAWING INCUBATION.
Toe
Concentration of sperm layered pn column (xl0 /ml)
Holding time on column
100
200
1
2
0
44.0
53.4
46.5
50.9
48.7
2
47.0
51.9
52.4
46.4
49.4
4
39.0
46.0
46.0
39.0
42.5
6
38.2
39.7
39.0
38.2
38.8
42.1
47.8
46.0
43.6
44.9
52.8
62.3
59.6
55.5
57.6
46.4
51.3
50.8
46.9
48.9
47.4
48.4
47.4
48.4
47.9
43.0
50.9
45.4
48.5
47.0
47.4
53.2
50.8
49.8
50.4
44.8
50.9
48.5
46.8
47.7
MEANS Bottom
0
& OVERALL
MEANS
-
Means
(hr)
-
DISCUSSION Isolation of motile sperm (in terms of post-thawing survival) was The efficiency of this effectively achieved using a BSA column. isolation was affected by the concentration of BSA in the column (Tables 1 and 2), the holding time of semen on the column (Tables 1, 2 and 3), and the concentration of sperm in the layered semen (Table 3). Isolation columns containing lower concentrations of BSA (viz 6%/12% or 6% BSA) allowed penetration of more sperm to the bottom of Longer the column and improved the post-thawing survival of sperm. holding time of semen on the column also resulted in a greater percentage of sperm penetrating into the column, and these sperm possessed higher post-thawing motility. A higher concentration of sperm in semen layered on top of the column provided better survival of sperm during post-thawing incubation, presumably because this procedure allowed the penetration of a greater proportion of viable sperm.
APRIL
1984 VOL. 21 NO. 4
605
THERIOGENOLOGY In general, the most satisfactory procedure for selecting highly motile ram sperm was to dilute the semen to a concentration of 200 x lo6 sperm/ml, layer the diluted semen on a column of 6% BSA in Tris diluent, and isolate the bottom layer of the column after two hours' holding time. When applied to human, bull or rabbit sperm (8), such a procedure apparently not only enhances motility and post-freezing survival but also eliminates abnormal forms and partially separates X- and Y-chromosomebearing sperm. The results presented in this paper point to the possibility of improving the fertility obtained after artificial insemination of sheep with frozen-thawed semen by using highly motile sperm isolated on a protein column. REFERENCES 1.
Ericsson, R.J., Langevin, C.N., Nishio, M. Isolation of fractions rich in human Y sperm. Nature, Lond. -246: 421-4 (1973).
2.
Ericsson, R.J. Isolation and storage of progressively motile human sperm. Andrologia, 2: 111-114 (1977).
3.
Glass, R.H. and Ericsson, R.J. Intra-uterine insemination of isolated motile spermatozoa. Feril. Steril., 9: 535-538
(1978)
4.
Dmowski, W.P., Gaynor, L., Lawrence, M., Rao, R. and Scommegna, A. Artificial insemination homologous with oligospermic semen separated on albumin columns. Fertil. Steril., -31: 58-62 (1979).
5.
Goodeaux, S.D. and Kreider, J.L. Motility and fertility of stallion spermatozoa isolated in bovine serum albumin. Theriogenology, -10: 405-414 (1978).
6.
Dixon, K.E., Songy, E.A.J., Thrasher, D.M. and Kreider, K.L. Effect of bovine serum albumin on the isolation of boar spermatozoa and their fertility. Theriogenology, $_: 437-444 (1980).
7.
Visser, D. and Salamon, S. Effect of composition of Tris-based diluent and of thawing solution on survival of ram spermatozoa frozen by the pellet method. Aust. J. Biol. Sci., -25: 605-18 (1972).
8.
Ericsson, R.J. and Glass, R.H. Functional differences between sperm bearing the X- and Y-chromosomes. In: Prospects for Sexing Mammalian Sperm, R.P. Amann and G.E Seidel, Eds. Colorado Association University Press, Boulder. 1982, pp. 201-211.
606
APRIL
1984 VOL. 21 NO. 4