The effects of cooling to 5\s=deg\Cand freezing and thawing on the ultrastructure of bull spermatozoa R. C. Jones and D. L. Stewart Wellcome Institute of Comparative Physiology, The Zoological Society of London, London NW1 4RY, and |Cattle Breeding Centre, Ministry ofAgriculture, Fisheries and Food, Shinfield, Reading, RG2 9B2, Berkshire, UK.
Summary. Semen from 6 bulls was examined under the transmission electron microscope immediately after collection, after dilution and cooling to 5\s=deg\Cand after freezing and thawing. Conception rates were determined following artificial insemination of the frozen and thawed semen. Dilution and cooling to 5\s=deg\Ccaused acrosomal swelling in about 50% of the spermatozoa. Subsequent freezing and thawing caused considerable ultrastructural changes to the acrosomes (disruption of the plasma and outer acrosomal membranes and dispersion of the acrosomal contents) and middle pieces (breakage of the plasma membrane and a reduction in the electron density of the mitochondrial matrix) of a high proportion of spermatozoa. The average non-return rate following insemination of semen from 5 of the bulls was 61\m=.\6%and higher (P < 0\m=.\001) than for the sixth bull (15%). Although this difference in semen viability was also demonstrated in the structural studies (acrosome, P < 0\m=.\05;middle piece, P < 0\m=.\001),more work is required to assess the relationship between structure and function of spermatozoa. Introduction
species progress has been slow in developing methods for the deep-frozen storage of (Jones, 1971). A major problem has been that spermatozoa lose viability during processing so that following artificial insemination they can no longer ascend and survive in the female reproductive tract in order to fertilize an ovum (Mattner, Entwistle & Martin, 1969; Polge, Salamon & Wilmut, 1970). As this loss in viability has been correlated with structural changes (Healey, 1969; Quinn, White & Cleland, 1969; Pedersen & Lebech, 1971; Nath, 1972) which cannot all be detected with the light microscope (Tasseron, Amir & Schindler, 1977), it is For many semen
necessary to examine further the ultrastructure of spermatozoa to assess the value of such an approach and to provide a basis for comparative studies. Earlier work on this project was carried out to quantitate and improve methods of preparing spermatozoa for transmission electron microscopy and to assess the effects of the preparative procedures on sperm structure (Jones, 1973a, b, c, 1975). These methods were used to determine the effects on sperm structure of diluting and cooling semen to 5°C and of freezing and thawing. Since, at this stage of the project, it is important to relate the structural changes to sperm viability the experiment was carried out using sub-samples of semen which were tested for fertilizing capacity (Linford, Glover, Bishop &
Stewart, 1976).
Materials and Methods The 3 Friesian and 3 Hereford bulls used in this study had, between them, previously produced a wide range of conception rates following artificial insemination. Semen was collected into an artificial vagina and processed by the procedures used routinely at the Cattle Breeding Centre, * Present address: Department of Australia.
Biological Sciences, University
of Newcastle, New South Wales
2308,
Reading. One ejaculate was collected from each bull and diluted at 30°C with an unglycerolated diluent before cooling to 5°C over 30 min. An equal volume of diluent containing 14% (v/v) glycerol was added in two stages 15 min apart to give a final diluent composition of 83% (v/v) ultra heat-treated skim milk, 10% (v/v) egg yolk, 7% v/v glycerol, 12-5 mg fructose/ml, 500 i.u. penicillin/ml and 500 pg streptomycin sulphate/ml. Before use the diluent was cleared of particulate matter by centrifugation at 5000 g for 30 min (5°C) and only the supernatant was collected for use. The semen was equilibrated at 5°C for 6 h and frozen in 0-5 ml Polyvinyl¬ IO6 chloride straws suspended 4 cm above liquid nitrogen. Each straw contained 20 spermatozoa and was stored at —196°C for at least 28 days before thawing in air for use. Semen was used only for first inseminations and conception rates were determined as non-returns to oestrus within 16 weeks of insemination. Sub-samples of semen were taken for microscopic examination before the initial dilution at 30°C, just before freezing, and after freezing and thawing. For electron microscopy samples were concentrated into about 0-3 ml by centrifugation at 700 g for 10 min, removal of the supernatant and mixing with a Pasteur pipette. Spermatozoa were fixed in PFG-cacodylate (Jones, 1973a) and prepared for examination according to the methods described by Jones (1973a). These included coding and randomization of samples and taking low power (initial magnification, 5000) electron micrographs of different areas of the Araldite sections prepared from each treatment. Prints (photographic enlargement, 2) were examined and all the sections of spermatozoa through the appropriate plane were scored according to the type and degree of structural change occurring in the head and middle piece. Usually 100 sections of heads and 100 of middle pieces were scored per treatment and the frequencies of each type are expressed as mean percentages in Tables 1—4. Heads of spermatozoa were scored as corresponding to one of the types shown in PI. 1, Figs 1 to 9 of Jones & Martin (1973) and described in the headings of Tables 1 and 3. Sections of middle pieces were classified into the types shown in PL 1, Figs 10 to 14 of Jones & Martin (1973) and described in the headings of Tables 2 and 4. The 2 analyses described by Claringbold (1961) were carried out on the frequency data and the statistical significance of treatment effects was assessed with an F-test using the error mean square calculated in the analysis. Table 1. Assessment of the structure of the heads of freshly collected bull spermatozoa (means of 6 ejaculates from different bulls) and the effects on this structure of cooling to 5°C and freezing and thawing Scored as type shown in Plate 1 of Jones & Martin (1973)
Fig. Plasma membrane: intact broken or lost
+
1
Fig. 2
Fig. 3
Fig. 4
+
+
+
—
—
Fig.
5
—
+
—
—
Fig. 6
Fig. 7
Fig. 8
+
—
—
—
+
+
Fig. 9
—
+
Acrosome:
swollen vesiculated vacuolated loss of outer membrane loss of contents Semen treatment
(1) Undiluted (2) Cooled to 5°C (3) Frozen and thawed 1 versus 2 2 versus 3
+
3
69 14 10
4 3 10
7 51 18 < 0-001 < 0001
13 18 45
Results
Tables 1 and 2 show the effect of processing on the structure of heads and middle pieces of spermatozoa. Cooling semen to 5°C caused acrosomal swelling in a high proportion of spermatozoa and freezing and thawing caused rupture of the outer acrosomal membrane and the plasma membrane covering the acrosome, and loss of the acrosomal contents (Table 1). However, there was a large error term in the 2 analysis ( 250 for the interaction of treatments, replicates and sperm type 261 -97, < 0-001). This was partly because the initial semen quality =
Table 2. Assessment of the structure of the middle pieces of freshly collected bull spermatozoa (means of 6 ejaculates from different bulls) and the effects on this structure of cooling to 5°C and freezing and
thawing
Scored
as
type shown in Plate 1 of Jones & Martin (1973)
Plasma membrane intact
Plasma membrane broken
Mitochondria Mitochondria Mitochondria Mitochondria Mitochondria Mitochondria normal normal condensed condensed* pale pale
(Fig. 10)
Semen treatment (1) Undiluted (2) Cooled to 5°C (3) Frozen and thawed 1 versus 2 2 versus 3
+
•Scored membrane.
(Fig. 12)
51 56 25
as
(Fig. 13)
(Fig. 14)
6 13 37
3
34 24 36 < 0-001 < 0-001
similar to type shown in PI. 1, Fig. 11 (Jones & Martin, 1973), but with
Table 3. Characteristics of unstained spermatozoa in
Scored
Fig. Plasma membrane: intact broken or lost Acrosome: swollen vesiculated vacuolated loss of outer membrane loss of contents Bull No. 1 2 3 4 5 6 Mean 1-5
(Fig. 11)
6
broken
plasma
semen: structure of heads, percentage of 16-week non-return rates after artificial
insemination
as
type shown in Plate 1 of Jones & Martin (1973)
Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 +
+
+
Conception rate No. of % % unstained cows non-return
+ +
+
+
+
+
+
+
1
12 6 12 4
+
—
—
9 6 9 15 20 0 versus
1
deep-frozen and thawed bull eosin-nigrosin smears and
a
31 19 15 14 30 0
13 9 4 4 0
Summary. Semen from 6 bulls was examined under the transmission electron microscope immediately after collection, after dilution and cooling to 5\s=deg\Cand after freezing and thawing. Conception rates were determined following artificial insemination of the frozen and thawed semen. Dilution and cooling to 5\s=deg\Ccaused acrosomal swelling in about 50% of the spermatozoa. Subsequent freezing and thawing caused considerable ultrastructural changes to the acrosomes (disruption of the plasma and outer acrosomal membranes and dispersion of the acrosomal contents) and middle pieces (breakage of the plasma membrane and a reduction in the electron density of the mitochondrial matrix) of a high proportion of spermatozoa. The average non-return rate following insemination of semen from 5 of the bulls was 61\m=.\6%and higher (P < 0\m=.\001) than for the sixth bull (15%). Although this difference in semen viability was also demonstrated in the structural studies (acrosome, P < 0\m=.\05;middle piece, P < 0\m=.\001),more work is required to assess the relationship between structure and function of spermatozoa. Introduction
species progress has been slow in developing methods for the deep-frozen storage of (Jones, 1971). A major problem has been that spermatozoa lose viability during processing so that following artificial insemination they can no longer ascend and survive in the female reproductive tract in order to fertilize an ovum (Mattner, Entwistle & Martin, 1969; Polge, Salamon & Wilmut, 1970). As this loss in viability has been correlated with structural changes (Healey, 1969; Quinn, White & Cleland, 1969; Pedersen & Lebech, 1971; Nath, 1972) which cannot all be detected with the light microscope (Tasseron, Amir & Schindler, 1977), it is For many semen
necessary to examine further the ultrastructure of spermatozoa to assess the value of such an approach and to provide a basis for comparative studies. Earlier work on this project was carried out to quantitate and improve methods of preparing spermatozoa for transmission electron microscopy and to assess the effects of the preparative procedures on sperm structure (Jones, 1973a, b, c, 1975). These methods were used to determine the effects on sperm structure of diluting and cooling semen to 5°C and of freezing and thawing. Since, at this stage of the project, it is important to relate the structural changes to sperm viability the experiment was carried out using sub-samples of semen which were tested for fertilizing capacity (Linford, Glover, Bishop &
Stewart, 1976).
Materials and Methods The 3 Friesian and 3 Hereford bulls used in this study had, between them, previously produced a wide range of conception rates following artificial insemination. Semen was collected into an artificial vagina and processed by the procedures used routinely at the Cattle Breeding Centre, * Present address: Department of Australia.
Biological Sciences, University
of Newcastle, New South Wales
2308,
Reading. One ejaculate was collected from each bull and diluted at 30°C with an unglycerolated diluent before cooling to 5°C over 30 min. An equal volume of diluent containing 14% (v/v) glycerol was added in two stages 15 min apart to give a final diluent composition of 83% (v/v) ultra heat-treated skim milk, 10% (v/v) egg yolk, 7% v/v glycerol, 12-5 mg fructose/ml, 500 i.u. penicillin/ml and 500 pg streptomycin sulphate/ml. Before use the diluent was cleared of particulate matter by centrifugation at 5000 g for 30 min (5°C) and only the supernatant was collected for use. The semen was equilibrated at 5°C for 6 h and frozen in 0-5 ml Polyvinyl¬ IO6 chloride straws suspended 4 cm above liquid nitrogen. Each straw contained 20 spermatozoa and was stored at —196°C for at least 28 days before thawing in air for use. Semen was used only for first inseminations and conception rates were determined as non-returns to oestrus within 16 weeks of insemination. Sub-samples of semen were taken for microscopic examination before the initial dilution at 30°C, just before freezing, and after freezing and thawing. For electron microscopy samples were concentrated into about 0-3 ml by centrifugation at 700 g for 10 min, removal of the supernatant and mixing with a Pasteur pipette. Spermatozoa were fixed in PFG-cacodylate (Jones, 1973a) and prepared for examination according to the methods described by Jones (1973a). These included coding and randomization of samples and taking low power (initial magnification, 5000) electron micrographs of different areas of the Araldite sections prepared from each treatment. Prints (photographic enlargement, 2) were examined and all the sections of spermatozoa through the appropriate plane were scored according to the type and degree of structural change occurring in the head and middle piece. Usually 100 sections of heads and 100 of middle pieces were scored per treatment and the frequencies of each type are expressed as mean percentages in Tables 1—4. Heads of spermatozoa were scored as corresponding to one of the types shown in PI. 1, Figs 1 to 9 of Jones & Martin (1973) and described in the headings of Tables 1 and 3. Sections of middle pieces were classified into the types shown in PL 1, Figs 10 to 14 of Jones & Martin (1973) and described in the headings of Tables 2 and 4. The 2 analyses described by Claringbold (1961) were carried out on the frequency data and the statistical significance of treatment effects was assessed with an F-test using the error mean square calculated in the analysis. Table 1. Assessment of the structure of the heads of freshly collected bull spermatozoa (means of 6 ejaculates from different bulls) and the effects on this structure of cooling to 5°C and freezing and thawing Scored as type shown in Plate 1 of Jones & Martin (1973)
Fig. Plasma membrane: intact broken or lost
+
1
Fig. 2
Fig. 3
Fig. 4
+
+
+
—
—
Fig.
5
—
+
—
—
Fig. 6
Fig. 7
Fig. 8
+
—
—
—
+
+
Fig. 9
—
+
Acrosome:
swollen vesiculated vacuolated loss of outer membrane loss of contents Semen treatment
(1) Undiluted (2) Cooled to 5°C (3) Frozen and thawed 1 versus 2 2 versus 3
+
3
69 14 10
4 3 10
7 51 18 < 0-001 < 0001
13 18 45
Results
Tables 1 and 2 show the effect of processing on the structure of heads and middle pieces of spermatozoa. Cooling semen to 5°C caused acrosomal swelling in a high proportion of spermatozoa and freezing and thawing caused rupture of the outer acrosomal membrane and the plasma membrane covering the acrosome, and loss of the acrosomal contents (Table 1). However, there was a large error term in the 2 analysis ( 250 for the interaction of treatments, replicates and sperm type 261 -97, < 0-001). This was partly because the initial semen quality =
Table 2. Assessment of the structure of the middle pieces of freshly collected bull spermatozoa (means of 6 ejaculates from different bulls) and the effects on this structure of cooling to 5°C and freezing and
thawing
Scored
as
type shown in Plate 1 of Jones & Martin (1973)
Plasma membrane intact
Plasma membrane broken
Mitochondria Mitochondria Mitochondria Mitochondria Mitochondria Mitochondria normal normal condensed condensed* pale pale
(Fig. 10)
Semen treatment (1) Undiluted (2) Cooled to 5°C (3) Frozen and thawed 1 versus 2 2 versus 3
+
•Scored membrane.
(Fig. 12)
51 56 25
as
(Fig. 13)
(Fig. 14)
6 13 37
3
34 24 36 < 0-001 < 0-001
similar to type shown in PI. 1, Fig. 11 (Jones & Martin, 1973), but with
Table 3. Characteristics of unstained spermatozoa in
Scored
Fig. Plasma membrane: intact broken or lost Acrosome: swollen vesiculated vacuolated loss of outer membrane loss of contents Bull No. 1 2 3 4 5 6 Mean 1-5
(Fig. 11)
6
broken
plasma
semen: structure of heads, percentage of 16-week non-return rates after artificial
insemination
as
type shown in Plate 1 of Jones & Martin (1973)
Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 +
+
+
Conception rate No. of % % unstained cows non-return
+ +
+
+
+
+
+
+
1
12 6 12 4
+
—
—
9 6 9 15 20 0 versus
1
deep-frozen and thawed bull eosin-nigrosin smears and
a
31 19 15 14 30 0
13 9 4 4 0
