CRYOUlOLOCY

12, 93-97 (1975)

Rooster Spermatozoan Motility, Forward Progression, Fertility after Storage at 25, 5, or -196°C in Various Extenders R. L. AX Department

and

J. R. LODGE

AND

of Dairy Science, University of iIlinok at Urbana-Champaign, Urbana,

IEEinois

61801

Consistent satisfactory fertility after in datory for samples equihbrated in 8% vitro storage of rooster semen has been a glycero1 (3)) and Harris (6) reported that either dimethyl sulfoxide or polyvinylproblem. Polge (13) reported 54% fertility after storing semen in Ringer’s solution pyrrolidone could be used satisfactorily incontaining 30% glycerol for one hour at stead of glycerol and that egg yolk was toxic, Equilibration for 30 min and storage -79°C. No significant difference was noted in fertility between fresh semen or fresh for 12 days at -196°C resulted in approximately 30% fertility. Semen samples stored semen diluted 1:3 or 1: 10 (I7), and 2-770 at -79°C by Watanabe (16) for 30, 90, fertility from undiluted sperm compared to 50-80s from sperm diIuted I: 10 in a and 100 days produced 29.X, 25.0, and 14.3% fertility, respectively, in eggs colphosphate buffer stored 20-24 hr at 10°C lected for one week after insemination. has been observed (19). Semen diluted Cryopreservatives have been shown to be I : 3 in a glutamate-saline solution by Lake (10) yielded 64 and 47% fertility after stor- important for successful long-term rooster semen storage (7, 15). Pisteuma eCal. (12) age at 0-2°C for 24 and 48 hr, respectively. diluted sperm 1:2 in a modified Krebs or No loss in fertility after 8 hr storage of 1:2 1:30 in a gIycine-citrate buffer and equilior I : 10 diluted semen in phosphate buffer brated 120 min to 3°C. Storage for 1.5 hr was found by Wilcox (18); storage at 10°C resulted in higher fertility than 25”C, and and insemination of 200 x 10” sperm/hen resulted in SOY, fertility which was not sigegg yolk was deIeterious. Clark and Shaffner (5) added 7.5-8.5% glycero1 to semen di- nificantly different from controls. They also confirmed the detrimental effect of egg Iuted 1: 3 in a phosphate buffer. ,Controlled cooling and equilibration were found un- yolk. Investigators of most rooster sperm storage studies fail to report sperm numnecessary, so samples were rapidly frozen, bers inseminated or the percent of motile and then thawed in a 40°C water bath. sperm and rate of progressive motility of Glycerol was removed by centrifugation the sperm prior to and after storage. It has and the spermatozoa resuspended in buffer been shown that the percent of motile before insemination, Up to 40% fertility sperm in fresh and 5°C stored bovine was obtained in eggs coIIected 6-7 days semen collections is significantly, positiveIy postinsemination. Centrifugation for glyccorrelated with fertility ( 2). erol remova has been suggested not manThis paper reports a study conducted to Received June 12, 1974. determine the percent of motile sperm and 93

AX AND

94

rate of progressive motility of rooster sperm in fresh semen collections and the changes in these parameters after s,torage in various extenders at 25, 5, or -196°C. Fertility results were also obtained after artificially inseminating pullets with fresh sperm, sperm stored 24 hr at either 5 o,r -196°C in a mouse ova culture medium, or fresh sperm diluted with TC199. MATERIALS

AND

METHODS

Semen was collected by massage (9) from two fertile New Hampshire roosters, pooled, sperm numbers determined by hemacytometer, and the percent motility as well as forward progressive motility microscopically assessed ( 100 x ) . The pooled semen samples were left undiluted or diluted to 1 x lo9 motile sperm/ml at 25°C. Portions of the semen samples were kept at 25°C or slowly cooled in a water bath to 5°C and stored. Other portions were immediately frozen; or equilibrated with gIycero1 for 15, 30, or 60 min in a 5°C room prior to freezing in liquid nitrogen vapor in LO-ml ampules with subsequent immersion into liquid nitrogen a few minutes later. Glycerol (8%) was added at 25°C to all samples to be frozen to prevent freezing loss. Egg yolk was not used in the extenders since it has been shown to be harmful to rooster sperm (6, 12, X8). The extenders used were TCI99 or a pyruvatelactate mouse ova culture medium ( 1). At various times during storage sam.ples were microscopically examined to deterTABLE THE EFFECT percent of motile werm

95 75 50 25 0 ‘7t

OF 25°C STORAGE

LODGE

mine the percent motiIe sperm and progressive motility to evaluate the success of the extenders at the different temperatures. Samples for insemination were stored 24 hr at 5°C or -196°C in pyruvat+lactate. The frozen ampuIes were thawed in cold tap water and then placed into warm water. Damage to sperm stored at 5 or -196°C was determined microscopically (100x ) by percent of motile sperm recovered and forward progression. Pullets were inseminated with 1 X lo8 motile sperm in the afternoon to obtain highest fertility (8, 11). After a single insemination, eggs were collected Days 27. Inseminations were then repeated. Day 1 of fertile eggs was counted as the second day after insemination, because eggs laid within 24 hr of insemination would have been ovulated the morning of the insemination date (4). The eggs were incubated at 37.8”C for one week and then candIed to assess fertility. The contents of apparent infertile eggs were examined for signs of fertility and early embryonic death. The remaining eggs which were fertile were allowed to incubate through hatching, and the unhatched eggs examined for time of death. All fertility and embryonic mortaIity results were compared by Chi-square analysis using a 2 x 2 contingency table (14). RESULTS

Sperm in freshly colIected semen samples were approximateIy 95% motile with a 1

ON MOTILITY

Whole Vernon Time (W

0

1 2 3.5 5

pmss r

3.5 2.5 2.0 1.0 0.0

= 10 collections (45 aemplea/cdlection).

OF ROOSTER

SPERMATOZOAN

TC 199 Time (hr)

0 2 4 8 12

‘i”“Tm

3.5 3.0 2.0 2.0 0.0

0 6 15 20 30

3.5 3.0 2.5 2.5 0.0

ROOSTER

SPERM

FERTILITY

TABLE

2

THE EFFECT OF 5*C STORAGE ON MOTILITY Percent of motile sperm

Whole Yemen Time (hr)

-~

-

95

0

75 50 25 0 -

1 3 5.5 8

a n = 10 coilectionv

(-6

TC 199

r pi”,“z

Progress craw

0 3 6 11 16

3.5 3.0 2.5 2.5 0.0

3.5 2.5 2.0 1.0 0.0

TABLE

storage

whole

a 12 = 10 collections

(-6

3..? 3.0 3.0 2.5 0.0

and

Rooster spermatozoan motility, forward progression, and fertility after storage at 25, 5, or -196 degrees C in various extenders.

CRYOUlOLOCY 12, 93-97 (1975) Rooster Spermatozoan Motility, Forward Progression, Fertility after Storage at 25, 5, or -196°C in Various Extenders R...
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