Cryopreservation of Rooster Sperm Using Methyl Cellulose1

Cryopreservation of Rooster Sperm Using Methyl Cellulose1 J. J. PHILLIPS, R. K. BRAMWELL, and J. K. GRAHAM2 Department of Physiology, Colorado State University, Fort Collins, Colorado 80523

(Key words: cryopreservation, rooster, spermatozoa, methyl cellulose) 1996 Poultry Science 75:915-923

INTRODUCTION

effective rooster sperm cryoprotectant, is contraceptive when comprising >1% (vol/vol) of the sample, even though motility is nearly equivalent to prefreeze values (Tajima et al, 1989). Consequently, the removal of glycerol is necessary prior to insemination. Glycerol reduction can be accomplished by diluting semen with media containing no glycerol and reconcentrating the sperm using centrifugation (Lake et al, 1981), or by dialysis (Polge, 1951; Buss, 1993). Both methods are deleterious to the sperm and result in fertilization rates and durations of egg fertility that are unacceptable for commercial purposes (Mitchell and Buckland, 1976; Wishart, 1985). In addition, these procedures are cumbersome and time consuming, making them impractical in the field (Lake and Ravie, 1984). Glycerol and other cell-penetrating cryoprotectants protect cells from cryodamage, in part, by entering the cell and displacing intracellular water. This displacement causes cellular dehydration or binds intracellular water, preventing intracellular ice formation, which can damage the cell (Mazur, 1984; Amann and Pickett, 1987). High molecular weight compounds have been used in combination with glycerol as ram sperm cryoprotectants (Schmehl et al, 1986). These compounds cannot enter

Tremendous genetic progress has been made in the dairy industry since the advent of artificial insemination with frozen-thawed semen, permitting the use of the most genetically superior bulls in breeding programs (Foote, 1982). To date, the poultry industry has been unable to take advantage of using frozen semen because highly fertile cryopreserved poultry semen is not available. Cryopreservation of rooster sperm has been accomplished but success has been highly variable (Graham et al., 1984; Lake, 1986; Hammerstedt and Graham, 1992; Buss, 1993). Preservation of rooster sperm requires 6 to 9% cryoprotectant in the freezing medium for recovery of motile sperm (Smith and Polge, 1950; Clark and Shaffner, 1960; Shaffner, 1964; Lake and Ravie, 1984; Lake, 1986; Tajima et al, 1989). Glycerol, the most

Received for publication January 23, 1996. Accepted for publication April 5, 1996. financial support provided by Colorado State University Experiment Station. 2 To whom correspondence should be addressed.

915

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subsequent centrifugation exhibited 59, 30, 35, 60, and 69% viable cells, respectively; and 65, 38, 46, 69, and 65% motile sperm, respectively. Sperm cryopreserved with MC and either 4 or 9% glycerol exhibited similar numbers of sperm binding to chicken perivitelline layers in vitro as did fresh sperm, whereas sperm frozen with MC and 3% glycerol bound oocytes with only 31% efficiency (P < 0.05). The extent to which cryopreserved sperm penetrated the perivitelline layer in vitro was independent of glycerol concentration, but was four times more efficient than that of fresh sperm (P < 0.05). The fertility rates of fresh semen, semen frozen in 9% glycerol with the cryoprotectant removed after thawing, and semen frozen in MC with either 3 or 4% glycerol were 87.4, 27.6, 0.8, and 0.5%, respectively (P < 0.05). The MC reduces the contraceptive effects of glycerol when inseminated with fresh sperm, but does not maintain fertilizing capacity in frozen-thawed sperm when used in combination with 3 or 4% glycerol.

ABSTRACT Experiments were designed to determine when, during the cryopreservation process, sperm lose fertilizing capacity and whether the cryoprotectant, methyl cellulose (MC), could be used in combination with glycerol to cryopreserve sperm and remain in the inseminate without reducing fertility. Semen diluted in Minnesota Avian extender (MNA) and inseminated immediately had greater fertility (75%) than semen processed for cryopreservation (12 to 60%). The largest decreases in fertility were due to addition of glycerol to sperm and to cryopreservation. In another experiment, fertility of inseminates containing 0, 1, and 2% glycerol were 82, 29, and 21%, respectively, for eggs collected 2 to 5 d after insemination. When 0.5% MC was added to the same three treatments, fertility rates were 88, 63, and 69%, respectively. Semen cryopreserved in MNA containing 9% glycerol; MC + 3% glycerol; MC + 4% glycerol; MC + 9% glycerol; or 9% glycerol with the cryoprotectant removed post-thaw by dilution and

916

PHILLIPS ET AL.

MATERIALS AND METHODS Birds of a Leghorn strain were used. All birds were individually caged, exposed to 15 h light and 9 h dark, and provided ad libitum access to a commercial layer (hens) or commercial breeder (roosters) feed.

Experiment 1. Steps During Cryopreservation That Affect

Fertility

This experiment was conducted to determine which steps in the cryopreservation process reduce the fertilizing potential of rooster sperm. Semen was collected by abdominal massage from 6 to 10 roosters and pooled. The pooled semen was then split four ways and diluted 1:2 (vol:vol) with: 1) Minnesota Avian extender (MNA; Tajima et ri., 1989); 2) MNA, further diluted with 6.5 vol of MNA and centrifuged at 300 x g for 25 min at 5 C to reconcentrate the sperm; 3) MNA + 13.5% glycerol (final glycerol level of 9%), further diluted with 6.5 vol of MNA and centrifuged; or 4) MNA + 13.5% glycerol, frozen, thawed, diluted with 6.5 vol of MNA, and centrifuged prior to insemination. Semen was frozen as described by Tajima et ol. (1989). Briefly, semen was diluted 1:2 (vol semen:vol extender) with extender at 5 C, and packaged in 0.25-mL straws 3 at 5 C. One hour after dilution, semen was frozen in static liquid nitrogen vapors by placing the straws 6.0 cm above liquid nitrogen for 15 min. Straws were then plunged into liquid nitrogen for storage until thawing in a 5 C water bath for 5 min.

3IMV, Minneapolis, MN 55430.

To remove glycerol prior to insemination, 2 mL of fresh semen diluted in MNA containing glycerol or 2 mL of frozen-thawed sperm were placed into 15-mL centrifuge tubes and MNA added to the sperm in increments (10 x 0.1 mL, 10 x 0.2 mL, 10 x 0.5 mL, and 5 x 1.0 mL) each added 1 min apart, allowing time for sperm to equilibrate with the decreasing glycerol levels between each MNA addition (Tajima et ah, 1989) and resulting in a final dilution of 1:6.5 (volume of sperm per volume of MNA). The sperm were then centrifuged at 300 x g for 25 min at 5 C to reconcentrate the cells. The supernatant was removed and the sperm pellet resuspended in an equal volume of MNA and the sperm concentration determined. This procedure results in a final glycerol concentration of approximately 0.6%. The concentration of sperm in each sample was determined spectrophotometrically (Hammerstedt, 1975) and a total of 100 x 10 6 fresh sperm or 200 x 10 6 frozenthawed sperm (inseminate volumes were between 30 and 100 iiL) was inseminated into each of 10 hens per treatment once a week for 4 consecutive wk. Eggs were collected beginning 2 d following the initial insemination (1st d of fertilized eggs) and set daily. The percentage of fertilized eggs was determined by candling eggs 4 to 7 d following the start of incubation. Data were analyzed by chi-square analysis (SAS Institute, 1985).

Experiments 2 and 3. Addition of MC to Fresh Semen In the second experiment, semen was collected as described in Experiment 1. The pooled semen was split and diluted 1:2 (semen:extender) in Beltsville Poultry Semen Extender (BPSE; Sexton, 1977) or BPSE plus 0.5% MC containing 0, 0.75, 1.5, 2.25, or 3% glycerol (resulting in final glycerol levels of 0, 0.5, 1, 1.5, and 2%, respectively). A total of 100 x 106 sperm were inseminated intravaginally into six hens per treatment once a week for 3 consecutive wk. To evaluate potential differences in the interactions between MC, glycerol, and the constituents within the semen extender, the third experiment investigated the effects of MC when used with MNA on egg fertility when the glycerol concentration ranged up to 4%. Semen was diluted in MNA with 0.5% MC and 0, 1, 2, 3, or 4% glycerol. A volume of 100 x 106 sperm was inseminated intravaginally into eight hens per treatment once a week for 4 consecutive wk. Eggs were collected and set daily and the percentage of fertilized eggs was determined by candling eggs 4 to 7 d following the start of incubation. Data for these two experiments were analyzed using chisquare analysis (SAS Institute, 1985).

Experiment 4. Effects of MC on Sperm Viability and Motion Semen was collected as described previously and diluted in: 1) MNA + 9% glycerol; 2) MNA + 0.5% MC + 9% glycerol; 3) MNA + 0.5% MC + 4% glycerol; or 4) MNA

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cells, but may induce formation of ice crystal lattices or clathrates as external shields around the cell membrane (Karow and Webb, 1965). In addition, these agents may enable cellular membranes to reversibly leak, without sustaining damage, with the osmotic stresses imposed during freezing (Meryman, 1971). Roy and Djerassi (1965) reported that a combination of penetrating (dimethylsulfoxide) and nonpenetrating (sucrose) cryoprotectants had a synergistic effect for freezing of blood platelets. Combinations of penetrating agents with sugars have been used to cryopreserve sperm from several species (Graham et ah, 1984). A combination of a high molecular weight compound and glycerol, using glycerol at a level sufficiently low to be noncontraceptive by minimal dilution, may provide a technique that would allow the use of frozen semen in the poultry industry. These studies were designed to determine 1) when during the cryopreservation process sperm loses its fertilizing potential, 2) how methyl cellulose (MC), a high molecular weight compound, affects sperm function, and 3) whether MC, in combination with glycerol at levels of 3 to 4%, can adequately cryopreserve rooster sperm.

ROOSTER SPERM CRYOPRESERVATION USING METHYL CELLULOSE

Experiment 5. Evaluation of Sperm Binding to and Penetration of the Oocyte Perivitelline Layer In Vitro To evaluate sperm binding in vitro, chicken oocyte perivitelline layers were prepared as described by Cramer et al. (1994). Briefly, yolks from unfertilized chicken eggs were separated from the egg whites and the yolk perivitelline layer punctured to allow the yolk contents to drain from the membrane. The perivitelline layer was placed into a 10-mL glass vial filled with MNA and shaken several times to remove adhering yolk. The MNA was decanted from the membrane and new MNA added. This

4

Sigma Chemical Co., St. Louis, MO 63178-9916. Coulter Electronics, Miami, FL 33116. Stromberg-Mika, Bad Feilnbach, Germany D-8201. TDCA-YVorks, Inc., Cincinnati, OH 45240. 5

procedure was repeated until decanted MNA was clear. The membrane was then spread evenly upon a glass slide and covered with a latex template (1 mm thick) having holes cut into it (4 mm diameter). This was clamped to the slide using an additional template made of stainless steel (with identical holes cut into it). A 15-/iL vol of MNA was added to each well and the slide placed into a humidified incubator at 41 C in an atmosphere of 5% CO2 in air. Sperm were collected as described, and diluted 1:2 (sememextender) in MNA plus 0.5% MC and glycerol at levels resulting in final glycerol levels of 3, 4, or 9%. A subsample of treated sperm was incubated with chicken oocyte membranes immediately after dilution whereas the remainder of the sperm were cryopreserved, as described above, and incubated with oocyte membranes after thawing. A 10-^L vol of chicken semen (200,000 cells), was added to individual wells and incubated at 41 C for 3 h. A fresh semen sample diluted in MNA without MC or glycerol served as a control in each replicate. After incubation, slide chambers were rinsed with MNA to remove unbound sperm from the membrane and 10 /iL of fixative (3:1 ethanol:glacial acetic acid) was added to each well. A 10-/iL vol of 4,6-diamidino-2-phenylindole (DAPI; 1 m g / mL in water) was added to each well, incubated for 5 min at 25 C, and the number of spermatozoa remaining attached to the membrane determined by visual enumeration using fluorescence microscopy at 400x magnification, with an excitation wavelength of 350 nm and an emissions cut-off of 450 to 500 nm. A total of six fields, three fields in each of two replicate wells, were enumerated for each sperm treatment. This experiment was replicated four times. In order to compensate for possible differences in oocyte membranes used in the different replicates, the relative number of spermatozoa binding the membrane for each treatment is reported. This was determined as the ratio of the number of spermatozoa binding the oocyte membrane for each treatment and the number of control spermatozoa (not exposed to MC or glycerol) binding the membrane. The ratios of the number of spermatozoa binding to the oocyte membrane for each treatment were analyzed by ANOVA, and treatment means were separated using Student-Newman-Keuls multiple range test (SAS Institute, 1985). The in vitro sperm penetration assay used was a modification of that employed by Bramwell and Howarth (1992). The perivitelline layer was obtained from an oviposited egg, cut into -1.0 cm 2 sections, with each section placed in 5.0 mL 5% NaCl solution and placed on an agitator. 7 After 1 h of agitation, the perivitelline layer sections were placed in fresh NaCl solution and agitated for 1 additional h. Each section was then thoroughly rinsed with MNA to remove the NaCl, and placed in a 1.5-mL Eppendorf tube containing 300 /xL MNA. Sperm was collected and diluted in MNA plus MC as previously described (Experiment 4). A total of 2 x 10 6 cells were added to the Eppendorf tubes containing the

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+ 0.5% MC + 3% glycerol. A 50-^L aliquot of each fresh sample was reserved for analysis but the majority of each treatment was frozen as described above. Treatment 1 was frozen in duplicate so that one sample could be assayed immediately after thawing and the other could be assayed after the cryoprotectant was removed by dilution and subsequent centrifugation as described above. This experiment was repeated seven times. Analysis of Viability Using Flow Cytometry. Cells from each treatment were diluted to 3 x 106 cells per milliliter in respective extender containing 0.1% BSA. A 10-/iL vol of propidium iodide 4 (PI) at 1 m g / m L in water was added to a 400-/iL sperm suspension and the sample was incubated for 10 min. Sperm samples were filtered through 40 /un nylon mesh immediately prior to analysis to remove any large debris. The percentage of dead cells (Pi-positive) was determined by flow cytometry using an Epics 753 flow cytometer. 5 The PI was excited at 488 nm by an argon laser at 100 mW of power. Fluorescence emission was measured with a 515-nm long pass filter and with a 610-nm long pass filter for PI detection. Percentages of dead cells in treatments were transformed by arc sine, analyzed using ANOVA, and treatment means separated using Student-Newman-Keuls multiple range test (SAS Institute, 1985). Evaluation of Motion Parameters. Cells from each treatment were diluted to 100 x 106 cells per milliliter in respective extender containing 0.1% BSA to facilitate analysis of the percentage of motile sperm using a Stromberg-Mika cell motion analyzer. 6 The percentage of motile cells was determined, and for those spermatozoa that were motile, the straight line velocities and the average path velocities were determined. The values for the percentages of motile sperm were transformed by arc sine prior to statistical analysis. Differences between treatments for each motion parameter were determined using ANOVA and means separated using StudentNewman-Keuls multiple range test (SAS Institute, 1985).

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PHILLIPS ET AL. TABLE 1. The percentage of fertilized eggs after insemination with sperm at the various steps during cryopreservation. Treatments include the initial dilution of the semen in Minnesota avian extender (MNA), additional dilution in medium and subsequent centrifugation (Cent), addition of 9% glycerol, and cryopreservation in the presence of 9% glycerol (Frozen-thawed) Period eggs were collected 2 to 8 d

2 to 5 d Treatment

Percentage fertile

Number of eggs

Percentage fertile

Number of eggs

MNA MNA + Cent MNA + 9% glycerol + Cent Frozen-thawed + Cent

75' 60" 35= 12d

127 132 130 137

64a 49b 27c

220 236 228 235

9

d

a-^Values within columns with no common superscript differ significantly (P < 0.05).

Experiment 6. Fertility of Semen Cryopreserved with MC Semen was collected and split into four treatments: 1) MNA (fresh); 2) MNA + 9% glycerol, frozen-thawed, diluted, and centrifuged as described previously; 3) MNA + 0.5% MC + 4% glycerol; or 4) MNA + 0.5% MC + 3% glycerol (both MC treatments were frozen-thawed and had glycerol present in the inseminate). An aliquot of 100 x 106 sperm (fresh) or 200 x 106 sperm (frozen-thawed) was inseminated into 10 hens per treatment once a week for 3 consecutive wk. Eggs were collected the 2nd d following insemination and set daily. The percentages of fertilized eggs, laid on Days 2 to 5 after insemination and Days 2 to 8 (weekly fertility) were determined by candling eggs 4 to 7 d following the start of incubation. Data were statistically analyzed by chi-square analysis (SAS Institute, 1985).

RESULTS Experiment

1

The percentage of fertilized eggs laid was reduced by each step of the cryopreservation process (Table 1). Dilution and centrifugation of fresh sperm reduced the

percentage of fertilized eggs from 75 to 60%. Addition of 9% glycerol to fresh sperm prior to dilution and centrifugation decreased the percentage of fertilized eggs to 35%. Cryopreservation, including all the steps of the previous treatments resulted in a fertility rate of only 12%.

Experiment

2

The addition of MC to BPSE resulted in improved fertility rates at each glycerol concentration when used with fresh sperm (Table 2). In the absence of MC, the fertility of eggs collected over a 7-d period following insemination declined from 82% when no glycerol was added to 13% when 2% glycerol was added to the semen. In contrast, when MC was added to the media, egg fertility was not different when 2% glycerol was added to the sperm.

Experiment

3

The second fertility trial examined the fertility of inseminates containing glycerol levels up to 4%. The fertility rates of eggs from hens inseminated with sperm diluted in MNA plus 0.5% MC and containing either 0, 1, 2, 3, or 4% glycerol were 91, 91, 48, 42, and 24%, respectively. Fertility rates for sperm inseminated with 2 and 3% glycerol were lower than those for sperm inseminated with 0 or 1% glycerol, but higher than those for sperm inseminated with 4% glycerol (P < 0.05).

Experiment

4

Sperm viability, as determined by flow cytometry, was not different between treatments for cooled cells treated in MNA + 9% glycerol, MC + 9% glycerol, MC + 4% glycerol, or MC + 3% glycerol but decreased after freezing and thawing (Table 3). Freezing cells in MNA plus MC + 3% glycerol or MC + 4% glycerol resulted in fewer live cells following cryopreservation. Similarly, the percentages of motile cells were not different between treatments after cells were cooled to 5 C, but differences between treatments existed after

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perivitelline layer sections and incubated at 41 C for 30 min. Sperm treatments included fresh sperm diluted in MNA, which acted as a control, and sperm treated with MC and glycerol both prior to and after cryopreservation, as described above. Each pooled ejaculate (fresh and frozen samples) was tested on perivitelline layer sections collected from eggs laid by two different hens. Following a 30-min incubation period with sperm, the perivitelline layer sections were removed, fixed, and stained as described by Bramwell and Howarth (1992). Sperm penetration holes in six different 0.1 mm 2 areas of each perivitelline layer section were counted using bright field microscopy at 400x magnification. Values from the six fields of both perivitelline layers were averaged into single mean value for each treatment from each ejaculate. Differences between treatments were determined using ANOVA as described for sperm binding above.

ROOSTER SPERM CRYOPRESERVATION USING METHYL CELLULOSE

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TABLE 2. The percentage of fertilized eggs collected 2 to 5 d and 2 to 8 d after insemination of rooster sperm diluted in Beltsville Poultry Semen Extender (BPSE) with and without 0.5% Methyl cellulose (MC) and 0, 0.5, 1, 1.5, or 2% glycerol (GLYC) in the inseminate. Number of eggs per treatment in parentheses Eggs collected 2 to 5 d Treatr neiat BPSE BPSE BPSE BPSE BPSE

+ + + + +

0% GLYC 0.5% GLYC 1.0% GLYC 1.5% GLYC 2.0% GLYC

Eggs collected 2 to 13 d

0% MC

0.5% MC

0% MC

0.5% MC

82* 49b 29c 22' 21=

88" 87** 63b* 62b* 69b*

82* 39b 23' 16= 13=

83* 78"* 56b* 59b* 72'*

(66) (60) (62) (55) (63)

(60) (61) (52) (61) (64)

(114) (102) (106) (96) (104)

(106) (110) (99) (112) (112)

a_

=Values within columns with no common superscript differ significantly (P < 0.05). 'Indicates treatment with MC is different from treatment having no MC (P < 0.05).

(Table 5). No treatment differences were detected in the ability of cryopreserved sperm to penetrate the perivitelline layer. However, cryopreserved sperm penetrated the perivitelline layer four times more efficiently than fresh sperm (P < 0.05).

Experiment 6 The percentage of fertilized eggs was reduced from 87.4% for fresh semen to 27.6% when sperm were frozen in MNA + 9% glycerol, then thawed, diluted, and centrifuged. The MC with 4% glycerol and MC with 3% glycerol resulted in extremely low fertility of 0.8 and 0.5%, respectively (Table 6).

Experiment 5

DISCUSSION

The number of spermatozoa capable of binding to the oocyte vitelline membrane in vitro did not differ between fresh sperm treated with MC and glycerol up to 9% and control sperm diluted in MNA (Table 4). Similarly, rooster sperm frozen in the presence of MC and either 9 or 4% glycerol did not differ from control samples. However, when the glycerol level was reduced to 3%, fewer frozenthawed spermatozoa bound to the oocyte membrane in vitro (Table 4). Similarly, the number of spermatozoa penetrating the perivitelline layer in vitro were not different for fresh sperm samples, regardless of MC or glycerol treatment

Each step of the cryopreservation process reduced the fertilizing potential of rooster sperm. We were not able to separate the effects of diluting sperm, centrifuging sperm, or removing the seminal plasma from the sperm in this experiment. However, the combination of diluting sperm and subsequently centrifuging it decreased fertility rates 15% compared to fresh sperm. It is likely that mechanical damage during centrifugation was a major factor in this step. Centrifugation has been found to be detrimental to ram (Graham, 1994), bull (Hammer-

TABLE 3. The percentage of viable and motile rooster sperm prior to and after cryopreservation in Minnesota Avian medium (MNA) containing 0 or 0.5% methyl cellulose (MC) and 3, 4, or 9% Glycerol (GLYC). For motile cells the line velocity (VSL) and average path velocity (VAP) are also reported, (n = 10) Cooled Treatment

Viable

MNA + 9% GLYC MNA + 9% GLYC MNA + MC + 9% MNA + MC + 4% MNA + MC + 3% Pooled SEM

97 . . . 97 99 99 1

Frozen-thawed

MOT

VSL

VAP

Viable

MOT

VSL

88

69"

72*

71 *b

65> 65* 69" 46b 39 b

76» 106 b

77° 73» 73"

59 b 69» 60b 35= 30=

90 93 93 1

74b

1

1

3

1

(%)

a_

(c)i GLYC GLYC GLYC

fcan/s)

7 1 ab

(%)

G*m/s)

=Values within columns with no common superscript differ significantly (P < 0.05). (c) denotes treatment prepared by dilution and centrifugation post-thaw to reduce glycerol concentration.

l

VAP

77a

72" 76» 1

78» 108 b 80* 74a 79a 1

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cryopreservation (Table 3). Sperm frozen in MNA plus MC + 3% glycerol or MC + 4% glycerol exhibited fewer motile cells than other treatments. Cooled cells treated with MC + 9% glycerol exhibited higher straight line (VSL) and average path (VAP) velocities than cells treated with MNA + 9% glycerol. However, after cryopreservation, sperm cell velocities were similar between these treatments. Samples treated with MNA + 9% glycerol that had been diluted and centrifuged did not have more motile cells than samples that were not centrifuged, but cells exhibited higher velocities than cells in other cryopreserved treatments (Table 3).

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PHILLIPS ET AL. TABLE 4. The binding of rooster sperm to the chicken oocyte vitelline when sperm were diluted in Minnesota Avian extender (MNA) containing and different levels of glycerol, prior to (fresh) and after cryopreservation sperm bound (± standard error) and the binding efficiency compared (sperm diluted in MNA) are presented, (n = 4)

membrane in vitro methyl cellulose (MC) (F-T). The number of to control sperm

Treatment

Number of sperm bound

Percentage of control bound

MNA (fresh; control) MNA MC + 9% glycerol (fresh) MNA MC + 4% glycerol (fresh) MNA MC + 3% glycerol (fresh) MNA MC + 9% glycerol (F-T) MNA MC + 4% glycerol (F-T) MNA MC + 3% glycerol (F-T)

605 682 651 683 512 441 202

100= 110= 101= 108=

± 158 ± 215 ± 197 ± 198 ± 144 ± 119 ± 65

Pooled SEM

85= 73= 31>>

12

=-bMeans within a column with no common superscript differ significantly (P < 0.05).

alone, and many cells were ruptured. Likewise, Westfall and Howarth (1978) reported increased quantities of glutamic oxaloacetic transaminase (an indicator of cell membrane damage) released from rooster sperm following centrifugation and resuspension of frozen-thawed cells. High molecular weight compounds have been used in semen extenders to improve cryopreservation (Graham et al, 1978; 1984) and it has been suggested that these compounds, when used in conjunction with other cryoprotective agents, provide additional protection against freeze-thaw damage to sperm (Salamon et al, 1973; Schmehl et al, 1986). The addition of 0.5% methyl cellulose to BPSE in the first fertility trial (Table 2) enhanced the fertility of eggs following insemination with fresh semen and 0.5, 1, 1.5, and 2% glycerol. The MC apparently dampens the contraceptive effects of glycerol. Previous findings by Smith and Polge (1950) and Neville et al (1971) reported that >1% glycerol is detrimental to fertility of eggs. The addition of MC to extender containing 2% glycerol resulted in 72% fertilized eggs compared to 13% fertilized eggs when MC was absent from BPSE, and indicates that 2% glycerol

TABLE 5. Sperm penetration of the chicken perivitelline layer in vitro when sperm were diluted in Minnesota Avian Extender (MNA) containing 0.5% methyl cellulose (MC) and different levels of glycerol, prior to (fresh) and after cryopreservation (F-T). The number of sperm penetration holes (± standard error) and holes expressed as a percentage of control sperm (sperm diluted in MNA). (n = 6) Treatment

Number of sperm penetration holes

Percentage of control

MNA (fresh; control) MNA MC + 9% glycerol (fresh) MNA MC + 4% glycerol (fresh) MNA MC + 3% glycerol (fresh) MNA MC + 9% glycerol (F-T) MNA MC + 4% glycerol (F-T) MNA MC + 3% glycerol (F-T)

19.3 20.3 17.9 16.6 89.0 87.9 77.9

100= 107=

Pooled SEM a b

± ± ± ± ± ± ±

2.3 5.4 4.4 4.6 20.0 13.4 12.5

94= 87= 463b 460b 408b

26

- Means within a column with no common superscript differ significantly (P < 0.05).

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stedt, 1975; Garcia and Graham, 1987), and stallion sperm (Pickett et al, 1975; Padilla and Foote, 1991). A d d i t i o n of glycerol to the s p e r m p r e p a r a t i o n resulted in only 58% (35- vs 60%) of the fertilizing potential of semen not treated with glycerol, but diluted and centrifuged. Glycerol, therefore, had a detrimental effect on sperm even though it was removed prior to insemination. The exact mechanisms by which glycerol affects the sperm were not determined in these studies, but are likely due to damage to the sperm membrane, glycocalyx, or intracellular compartments that could not be repaired after the glycerol was removed (Hammerstedt and Graham, 1992). The reduction in fertility caused by freezing the sperm was most likely due to ice crystal damage to the subcellular components of the sperm. Electron microscopic investigations of turkey sperm have shown that ultrastructural changes in the cells occur during the cryopreservation process and following centrifugation to remove glycerol (Marquez and Ogasawara, 1977). These authors reported that addition of glycerol induced a loosening of the plasma membrane. Subsequent centrifugation of frozen-thawed turkey sperm resulted in greater structural damage than did the freezing process

ROOSTER SPERM CRYOPRESERVATION USING METHYL CELLULOSE

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TABLE 6. The percentage of fertilized eggs after insemination with sperm diluted in Minnesota Avian extender (MNA) or cryopreserved in MNA containing 9% glycerol, the glycerol being removed by dilution and centrifugation prior to insemination (Cent) or cryopreserved in MNA with 0.5% methyl cellulose (MC) with 3 or 4% glycerol and the sperm inseminated without glycerol removal Period eggs were collected 2 to 5 d

2 to 8 d

Treatment

Percentage fertile

Number of eggs

Percentage fertile

Number of eggs

MNA MNS MNA MNA

88.4a 36.0b 0.9c 0.4c

225 211 213 230

87.4= 27.6b 0.8c 0.5^

389 370 370 399

(Fresh) + 9% glycerol + Cent + MC + 4% glycerol + MC + 3% glycerol

'^Values within columns with no common superscript differ significantly (P < 0.05).

The number of sperm binding to chicken oocyte membranes in vitro suggested that glycerol does not affect the ability of sperm to bind to the oocyte. We have seen similar results for semen diluted in medium not containing MC, when u p to 9% glycerol was added to the sperm (unpublished data). Following cryopreservation, MNA extender containing MC was able to maintain the sperm's ability to bind to the oocyte in the presence of only 4% glycerol. When sperm were cryopreserved with 3% glycerol, however, the number of sperm capable of binding to the oocyte was reduced. The number of sperm penetrating the perivitelline layer in vitro was not affected by either MC or glycerol. However, cryopreservation of the sperm resulted in more sperm penetrating the perivitelline layer. Unlike data presented by Bramwell et al. (1995), who demonstrated that increased numbers of holes in the perivitelline layer were associated with increased fertility of an inseminate, the current data more likely indicate that cryopreserved sperm sustained membrane damage during cryopreservation sufficient for the acrosome reaction to occur more readily in these sperm than in noncryopreserved sperm. This damage may affect the membrane (Hammerstedt and Graham, 1992) and glycocalyx (Froman and Engel, 1989), which might reduce sperm storage in the utero-vaginal glands. In either case, the net effect might reduce the fertilizing potential of cryopreserved sperm. The fertility trial further confirmed the viability and motion data, which indicate that 3 and 4% glycerol did not adequately protect sperm from freeze-thaw damage nor provide an acceptable level of fertility. Previous studies performed in our laboratory and several other laboratories indicated that 1% glycerol is the maximum level of glycerol that can be inseminated with semen and still maintain fertility (Smith and Polge, 1950; Neville et «/., 1971). However, preliminary studies in this laboratory using MC suggested that semen that was extended and frozen in MNA with MC + 3% glycerol survived the cryopreservation process and might be inseminated without removing the glycerol. The results from the current fertility trial were disap-

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can be inseminated with fresh semen without dramatically compromising the percentage of fertilized eggs obtained. Experiment 3 revealed that the fertility of eggs, for hens inseminated with fresh rooster sperm diluted in MNA extender containing MC, was not different between treatments containing 2 vs 3% glycerol. However, treatments with 2% glycerol resulted in lower egg fertility than treatments with 0 or 1% glycerol. It is evident that MNA and BPSE interact differently with glycerol in alleviating the anti-fertility effects of glycerol, as the percentages of fertilized eggs differ between the two extenders for identical glycerol levels (0, 1, and 2% glycerol). This result may be due to differences in extender ingredients, resulting in different interactions between extender components and MC. Results from the viability experiment suggest that the addition of MC to extender does not affect the percentage of cells with intact plasma membranes in sperm that has been cooled. The percentage of live cells after cryopreservation using MC with 3 or 4% glycerol, however, is reduced to only half the number of spermatozoa surviving freezing at higher glycerol concentrations. This indicates that 3 to 4% glycerol does not provide sufficient protection to cells during freezing, even in combination with MC. Semen frozen with MNA and 9% glycerol, which were diluted and centrifuged, had higher percentages of live cells than each of the other frozen-thawed treatments; this is probably due to removal of dead cells during centrifugation. The percentages of motile sperm, regardless of MC treatment or glycerol concentration, were not different prior to freezing. Following cryopreservation, the percentages of motile sperm frozen in low levels of glycerol (3 to 4%) were inferior to that of sperm frozen with 9% glycerol, regardless of the addition of MC. This result further suggests that neither 3 nor 4% glycerol protects the cells sufficiently from cryodamage. Sperm velocity of the motile cells was not affected by glycerol level. Centrifugation and resuspension of sperm frozen in MNA plus 9% glycerol resulted in higher sperm velocities than sperm in media containing MC; this is probably due to viscosity differences between the media, as glycerol is removed from the centrifuged treatments.

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pointing but not unexpected, as the viability of cells frozen in MC plus 3% and MC plus 4% glycerol were low. In addition to the reduced viability of cells, it is also possible that the physiology of frozen-thawed sperm cells has been altered, and that even with the presence of MC, glycerol may have to be reduced to lower levels prior to insemination. Although our initial hypothesis, that sperm could effectively be frozen using only 3% glycerol, has not yet been achieved, results from these studies are very encouraging. Methyl cellulose is able to lessen the contraceptive effects of glycerol and this information may prove very useful in the development of cryopreserved rooster sperm that can be used in the field without additional processing after thawing. It may be possible to cryopreserve sperm in medium containing MC with levels of glycerol that can be adequately reduced by simple dilution of the sperm after thawing.

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Graham, E. F., M. L. Schmehl, and R.C.M. Deyo, 1984. Cryopreservation and fertility of fish, poultry and mammalian spermatozoa. Pages 4-29 in: Proceedings of the 10th Technical Conference on Artificial Insemination and Reproduction. National Association Animal Breeders. Milwaukee, WI. Graham, J. K., 1994. Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process. Theriogenology 41:1151-1162. Hammerstedt, R. H., 1975. Use of high speed dialysis to prepare bovine sperm for metabolic studies. Biol. Reprod. 13:389-396. Hammerstedt, R. H., and J. K. Graham, 1992. Cryopreservation of poultry sperm: The enigma of glycerol. Cryobiology 29: 26-38. Karow, A. M., and W. R. Webb, 1965. Tissue freezing: a theory for injury and survival. Cryobiology 2:99-108. Lake, P. E., 1986. The history and future of the cryopreservation of avian germ plasm. Poultry Sci. 65:1-15. Lake, P. E., and O. Ravie, 1984. An exploration of cryoprotective compounds for fowl spermatozoa. Br. Poult. Sci. 25: ACKNOWLEDGMENT 145-150. Lake, P. E., O. Ravie, and J. McAdam, 1981. Preservation of The authors would like to thank Carol Streiner for her fowl semen in liquid nitrogen: Application to breeding technical assistance with these studies. programmes. Br. Poult. Sci. 22:71-77. Marquez, B. J., and F. X. Ogasawara, 1977. Ultrastructural REFERENCES changes in turkey spermatozoa after immersion in glycerolyzed media and during various steps used for Amann, R. P., and B. W. Pickett, 1987. Principles of cryopreservation. Poultry Sci. 56:1806-1813. cryopreservation and a review of cryopreservation of Mazur, P., 1984. Freezing of living cells: mechanisms and stallion spermatozoa. Equine Vet. Sri. 7:145-173. implications. Am. J. Physiol. 247:C125-C142. Bramwell, R. K., and B. Howarth, Jr., 1992. Preferential Meryman, H. T., 1971. Cryoprotective agents. Cryobiology 8: attachment of cock spermatozoa to the perivitelline layer 173-183. directly over the germinal disc of the hen's ovum. Biol. Mitchell, R. L., and R. B. Buckland, 1976. Fertility of frozen Reprod. 47:1113-1117. semen after intravaginal and intrauterine inseminations Bramwell, R. K., H. L. Marks, and B. Howarth, Jr., 1995. using various concentrations and equilibration times of Quantitative determination of spermatozoa penetration of dimethylsulfoxide and a range of freezing and thawing the perivitelline layer of the hen's ovum as assessed on rates. Poultry Sci. 55:2195-2200. oviposited eggs. Poultry Sci. 74:1875-1883. Neville, W. J., J. W. Macpherson, and B. Reinhart, 1971. The Buss, E. G., 1993. Cryopreservation of rooster sperm. Poultry contraceptive action of glycerol in chickens. Poultry Sci. Sci. 72:944-954. 50:1411-1415. Clark, C. E., and C. S. Shaffner, 1960. The fertilizing capacity of Polge, C, 1951. Functional survival of fowl spermatozoa after frozen chicken sperm and the influence of related in vitro freezing to -79°C. Nature 167:949-950. processes. Poultry Sci. 39:1213-1220. Padilla, A. W., and R. H. Foote, 1991. Extender and Cramer, P. G., G. F. Barbato, and R. H. Hammerstedt, 1994. centrifugation effects on the motility patterns of slowSperm-egg binding assay assessing potential fertility of cooled stallion spermatozoa. J. Anim. Sci. 69:3308-3313. rooster sperm. Biol. Reprod. 50(Suppl. 1):128. (Abstr.) Pickett, B. W., J. J. Sullivan, M. S. Byers, M. M. Pace, and E. E. Foote, R. H., 1982. Cryopreservation of spermatozoa and Remmenga, 1975. Effect of centrifugation and seminal artificial insemination: past, present, and future. J. Androl. plasma on the motility and fertility of stallion and bull 3:85-100. spermatozoa. Fertil. Steril. 26:167-174. Froman, D. P., and H. N. Engel, Jr. 1989. Alteration of the Roy, A. J., and I. Djerassi, 1965. Prevention of freezing injury spermatozoal glycocalyx and its effect on duration of by simultaneous use of agents with different modes of fertility in the fowl (Gallus domesticus). Biol. Reprod. 40: action. Cryobiology 1 (Suppl. 1):20. (Abstr.) 615-621. Salamon, S., I. Wilmut, and C. Polge, 1973. Deep freezing of boar semen. I. Effects of diluent composition, protective Garcia, M. A., and E. F. Graham, 1987. Dialysis of bovine agents, and method of thawing on survival of spersemen and its effect on fresh and freeze-thawed spermatozoa. Aust. J. Biol. Sci. 26:219-230. matozoa. Cryobiology 24:446-454. SAS Institute, 1985. SAS® User's Guide: Statistics. Version 5 Graham, E. F., B. G. Crabo, and M. M. Pace, 1978. Current Edition. SAS Institute Inc., Cary, NC. status of semen preservation in the ram, boar and stallion. Schmehl, M. K., I. A. Vazquez, and E. F. Graham, 1986. The J. Arum. Sci. 47(Suppl. 2):80-119. effects of nonpenetrating cryoprotectants added to TEST-

ROOSTER SPERM CRYOPRESERVATION USING METHYL CELLULOSE yolk-glycerol extender on the post-thaw motility of ram spermatozoa. Cryobiology 23:512-517. Sexton, T. J., 1977. A new poultry semen extender. I. Effect of extension on the fertility of chicken semen. Poultry Sci. 56: 1143-1146. Shaffner, C. S., 1964. Observations on freezing chicken semen. Pages 426-429 in: Proceedings of the 5th International Congress on Animal Reproduction and Artificial Insemination. Volume 4. Trento, Italy. Smith, A. U., and C. Polge, 1950. Survival of spermatozoa at low temperatures. Nature 166:668-669.

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Tajima, A., E. F. Graham, and D. M. Hawkins, 1989. Estimation of the relative fertilizing ability of frozen chicken spermatozoa using a heterospermic competition method. J. Reprod. Fertil. 85:1-5. Westfall, F. D., and B. Howarth, Jr., 1978. The effect of glycerol and dilution on the release of glutamic oxaloacetic transaminase from rooster spermatozoa. Poultry Sci. 57: 1037-1041. Wishart G. J., 1985. Quantitation of the fertilising ability of fresh compared with frozen and thawed fowl spermatozoa. Br. Poult. Sci. 26:375-380.

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