Effect of various extenders and taurine on survival of stallion sperm cooled to 5 °C

EFFECT OF VARIOUS

EXTENDERS AND TAURINE ON SURVIVAL SPERM COOLED TO 5'C

OF STALLION

A. IjaiY and R. Ducharme Centre d' amelioration des Races Equines du Quebec, Division de la Cooperative des Inseminateurs du Quebec, St-Hyacinthe, Canada Received

forpublication: Febmary Accepted:JuZy 26, ABSTRACT

18, 1994 1995

Stallion semen was diluted in five different extenders tryode modified (dimitro-poulus onze (Dimitro's), Kenney's (Kenney's), modified INRA82 (INRA82), egg yolk-citrate-taurine (Citrate) and EZ-Mixin) and evaluated for motility after cooling and storage at 5OC for 0, 24, 48, 72 and 96 h. EZ-Mixin extender was used as control while 70 and 100 mM of taurine were added to Dimitro's, Kenney's and INRA82 to study its effect under conditions of storage at 5°C and varying processing modifications. Motility in INRA82 was 57.0, 58.4, 61.1, and 56.1% after 24, 48, 72 and 96 h, respectively and was higher (PqO.05) than in other extenders after 48, 72, and 96 h. Motility decreased over time in Dimitro's (P
extenders,

motility,

stallion,

sperm, taurine.

Acknowledgement: This project was funded by Les Inseminateurs en Recherche et Developpement (IRD) Inc. St-Hyacinthe, Quebec, Canada. 'Current address: Department of Animal Reproduction, College of Veterinary Sciences, Lahore - 54000, Pakistan. Theriogenology 44: 1039-l 050, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0093-691W95/$10.00 SSDI 0093-691X(95)00290-1

1040

Theriogenology

INTRODUCTION As in other livestock species, the success of artificial insemination in the stallion depends on maintenance of viability and fertility of spermatozoa during storage. To achieve this objective, a number of extenders and their modifications have been developed. Among milk-based extenders are those containing powdered skim milk, glucose, antibiotics, Kenney's extender, and ultra heat treated skim milk extender (16,26). Motility and fertilizing capacity of stallion spermatozoa, cooled to 5OC in Kenney's and INRA82 extenders and then stored for 24 h in these extenders, were maintained at pre-storage levels (5,7,8,16,36). Considerable variation in the capacity to survive the cooling process existed among spermatozoa from individual stallions. Fertility levels decreased with increasing storage time beyond 24 h (31). However, when Kenney's extender was supplemented with a high-potassium Tyrode's medium, the sperm motility was remarkably preserved up to 72 h (25). Similarly, by using modified INRA82, sperm motility of) 60% was achieved after 96 h of storage (20,27). Egg yolk based extendersare not commonlyused for dilution of fresh stallion semen. However, one extender, Dimitro-poulos Onze (Dimitro's) has been extensively used in Europe (17). Fertilizing capacity of stallion sperm cooled in this extender was generally maintained close to that seen in natural breeding when used within 24 h of collection (4,17); however, viability and motility of sperm decreased significantly after 18 h (28). High concentration of seminal plasma was shown to be deleterious during cooling and storage of extended stallion semen at 5OC (30). Extension of stallion semen in suitable extenders and then centrifugation to remove seminal plasma has been recommended to conserve sperm motility during storage at 5OC (15,25). Motility of hamster spennatozoawasmaintainedduringinvitro storage by inclusion of the free beta amino acid "taurine" (21,24). In this species taurine not only enhanced sperm progressive motility but also supported fertilization in vitro (18,19).

The objectiveof this study was to compareextendersroutinely used in the field and to determine whether inclusion of taurine in these extenders has any effect on stallion sperm viability and We found that stallion sperm motility during storage at 5'C. viability and motility was preserved effectively in INRA82 up to 96 h and viability and progressive motility were further improved by inclusion of taurine in extenders which were isotonic with stallion semen. MATERIALS AND METHODS The following extenders were prepared before each semen

1041

Theriogenology

collection using cell culture-tested chemicalsb. Dimitro-poulos onze Extender (Dimitro's) Consisted of two parts, part one containing glucose, 2 g; fructose, 2 g and deionized water to 100 ml and part 2 comprised of trisodium citrate dihydrate, 2 g; glycerol (85%), 940 mg; sulphanilamide, 350 mg and deionized water to 100 ml. Finally, 30 ml of part one and 50 ml of part 2 were combined with chicken egg yolk (20% v/v). The extender was centrifuged (3,000 x g; 20 min), and the supernatant fluid with the osmotic pressure of 311 mOsmol/kg and pH 7.1 was used for semen extension (17). Kenney's Modified Tyrode Extender (Kenney's) Kenney's extender was prepared by dissolving glucose 4.9 g; non-fat dry skim milk, 2.4 g; sodium penicillin, 1.5 x 10' units; dihydrostreptomycin, 150 mg to 100 ml water. This extender was modified to a high-potassium Tyrode's medium by combining 65% Kenney's extender with 35% (v/v) of Tyrode's medium (NaCl, 420 mg; KCl, 187 mg; Na2HC03,210 mg; Na lactate syrup (98%) 190 ul; CaCl,, 29 mg; MgCl,, 8 mg; Hepes, 238 mg; Na pyruvate, 11 mg; bovine serum albumin fatty acid free (BSABFF), 600 mg; and water to 100 ml). Final osmotic pressure was 315 mosmol/kg and the pH of this medium was adjusted to 7.2 (25). Modified INNA

Extender (INEA82)

Consistedof glucose, 5 g; lactose 300mg; raffinose, 300 mg; trisodium citrate dihydrate, 60 mg; potassium citrate, 82 mg; Hepes, 952 mg; penicillin, 10 I.U./ml; gentamycin, 10 ug/ml; water to 100 ml and UHT skimmed milk, 100 ml. Final osmotic pressure was 326 mOsmol/Kg and the pH of the prepared medium was adjusted to pH 7.1 (20,27). Egg Yolk-Citrate-Taurine Extender (Citrate) The 3.185% aquous solutionof trisodiumcitrate dihydratewas mixed 1:l (v/v) with a 4.1% aquous solution of taurine. Chicken egg yolk (20% v/v) was added to this mixture. Final osmotic pressure was 302 mOsmol/kg and pH was adjusted to 7.2 (11). EZ-Mixin Extender' Cjmotic pressure andpHof freshly prepared EZ-Mixinwas 345 mOsmol/kg and 7.2, respectively. EZ-Mixin served as a control in all the experiments having glucose and non-fat dry milk solids as its major components.

' Sigma

Chemicals,

' EZ-Mixin

with

St. Louis,

amikacine,

MO.

Animal

Reproduction

Systems,

Chino,

CA.

1042

Theriogenology

Semen Collection, Sperm Preparation and Motility Evaluation Extragonadalreserves of two stallions (Appaloosaaged 5 yr; Quarter Horse aged 4 yr) were stabilized by five daily collections before the onset of the experiments. Semen was obtained at intervals of 24 h from each stallion using an artificial vaginad, fitted with a milk filtere to remove gelatinous material and extraneous debris. Gel-free ejaculates kept at 37°C were evaluated immediately for volume, percentage motility and concentration of spermatozoa. Ejaculates showing > 55% progressively motile spermatozoa and 1 65 x lo6 spermatozoa/ml were processed further. All experiments were of the split ejaculate design allowing the study of the effect of each extender on spermatozoa from each ejaculate. Briefly, each ejaculate was split into 5 aliquots and one of the 5 extenders, warmed to 37"C, was added to each aliquot to make a dilution of 1:l (v/v). The samples were then centrifuged at 350 x g for 5 min. Most of the supernatant was decanted and the sperm concentration in the centrifugate was measured using a hemocytometer. The same extender was then added to each aliquot to obtain approximately 50 x lo6 sperm/ml, resulting in a total of 10 ml of extended semen for each extender; the semen was apportioned in 3.5 ml plastic tubes', capped and cooled slowly to 5°C. Cooling rates were approximately -0.12°C/min from 37 to 20°C and O.O3OC/min from 20 to 5OC. Progressivemotilityof spermwas evaluatedin diluted semen samples immediately after cooling to 5°C (0 h), and after StOrage at this temperature for 24, 48, 72 and 96 h. The dilution and storage of semen was achieved through the following experiments and effect of taurine on sperm motility was determined by taurine addition in the extenders used in experiments 2 and 3. Experiment 1 First ejaculates (n-5) of each stallionwere extended in nontaurine extenders, Dimitro's, INHAB2, Kenney's and EZ-Mixin, cooled, and were stored for 96 h at 5'C. After 24 h, an aliquot (3.5 ml) of each treatment was washed twice (350 x g, 5 min) at room temperature in 6.5 ml of SP-TALP medium supplemented with 0.6% BSABAFF (29). The pellet was resuspended at a concentration of 25 x lo6 sperm/ml in the SP-TALP, and was then incubated for 24 h (39OC, 5% CO,) in a humidifier incubator. This processing of semen was performed to determine the effect of washing on sperm motility. Motility was evaluated at specified time intervals as mentioned in d CSU Model, e Premium f Sarstedt,

Animal

Milk

Reproduction

Filter,

Nambrecht,

Agway

Systems,

Inc.,

Germany.

Chino,

Syracuse,

NY.

CA.

Theriogenology

1043

the results section. Experiment 2 Six ejaculates of each stallion were extended in taurinecontaining extenders, Citrate, Kenney's and INRA82 along with EZMixin extender, cooled and stored for 96 h at 5°C. In both INRA82 and Kenney's extenders, amount of glucose was reduced to one half and was substituted with 70 mM taurine. After 24 h an aliquot (3.5 ml) of each treatment was processed as described above in experiment 1. Experiment 3 Since the osmotic pressure of freshlycollectedejaculates (n=70) was (345f2.1), isotonic extenders were used in this experiment. Six ejaculates of each stallion were extended in isotonic (350 mOsmol/kg) extenders (INRA82 and Kenney's) with or without taurine. These were cooled and stored for 144 h at 5'C. Osmotic pressure of non-taurine extenders INRA82 and Kenney's was adjusted by manipulating the concentration of sodium citrate and potassium chloride, respectively. Osmotic pressure of INRA82 and Kenney's with taurine was adjusted by increasing taurine concentration from 70 mM to 100 mM. After 24 h storage at S'C, an aliquot (3.5 ml) of each treatment was processed as in experiment 1.

Statistical Analysis Data obtained were subjected to analysis of variance with General Linear Models options in the Statistical Analysis System (35). In the experiments, extenders were fixed effects, stallions and ejaculates were considered as replicates and were tested as random variable and each time period was analyzed separately. Means from analysis that were different at PcO.05 were tested using Fisher PLSD (35). RESULTS Motility of freshlycollectedsemen (2-3min after collection) ranged from 55 to 70% in all the experiments discussed below.

Experiment 1 In this experiment semen extended in four extenders without taurine (Dimitro's, INRA82, Kenney's and EZ-Mixin) was evaluated (Table 1). Motility in all 4 extenders was similar at 5OC (0 h). At 24 h, motility decreased (PsO.05) in all extenders but was the lowest (PcO.05) in Kenney's. After 48, 72 and 96 h, the highest (PcO.05) motility was maintained in INRA82 followed by EZ-Mixin. At prolonged intervals of storage (72, 96 h) sperm extended in Kenney's and Dimitro's showed the poorest motility (PcO.05).

1044

Theriogenology

Table 1. Effect of extenders on motility of stallion sperm stored at 5°C up to 96 h' % Snerm motilitv after storaae for: Extender

0 h

24 h

48 h

72 h

96 h

Dimitro's

74.7f9.1

56.0*7.7b

40.7*a.3b

28.6f9.gb

13.1f16.0b

INRA82

74.Sf3.0

57.0k4.2'

5S.4f9.BC

61.lf6.2'

56.lf4.6=

Kenney's

68.7f11.3

27.0f11.8C

16.5f12.2d

12.6i9.Sd

05.8*8.6b

ES-Mixin

72.7k0.3

47.0f7.1b

49.1f6.1bc

47.9f6.0'

40.5fa.sd

values shown are means f SD (n = 10). "C'd'%enote differences (PCO.05) within columns.

Experiment 2 Effects of taurine (70 mM) in three extenders (Citrate, Kenney's and INRA82) on motility are presented in Table 2. Extender EZ-Mixin acted as control. Motility in Citrate was lower Table 2.

Effect of taurine-containing extenders on motility stallion sperm stored at 5'C up to 96 h'

of

% Sperm motility after storage for: Extender

Oh

24 h

48 h

72 h

96 h

Citrate

60.2f11.3b

28.4f13.4b

1i3.4f11.Sb

013.5f9.0b

02.7*4.9b

INRA82 with taurine

74.6f7.SC

68.3f5.3=

54.2*12.8=

54.9*12.3=

54.2f9.6C

Kenney's with taurine

69.5*4.9&

57.5f8.0C

17.1f12.1b

07.5f9.3b

Ol.Sf3.3b

EZ-Mixin

72.0+5.8=

42.1i7.aW

43.5i7.44

40.0*10.44

3l.Of8.54

'Values shown are means + SD (n = 12). "C'%enote differences (PcO.05) within columns.

(PxO.05) at 0 h than other extenders. After 24 h, motility in INRA82 and Kenney's extenders with taurine was greater (PcO.05) than in Citrate and EZ-Mixin. Motility in EZ-Mixin decreased (P
1045

Theriogenology

Table 3.

Effect on motility of stallion sperm stored in various extenders at 5'C for 24 h, then washed and stored in SpTALP at 39OC' % Soerm motilitv

Extender

Before washing at 24 h

After 2x washing at 24 h

6 h

12 h

Dimitro's

56.0

50.0=

35.6&

09.09

Citrateb

28.4

30.6'

25-o=

20.9'

INRA82

61.0

60.0'

45.4d

14.0e

68.3

59.gc

55.5=

54.1=

21.0

24.8'

10.16

oo.od

33.7

30.9=

32.1'

28.7'

45.0

47.9=

35.1=

03.Td

INRA82

with

taurineb

Kenney's Kenney'a taurineb

with

EZ-Mixin

values shown are means (n = 10). Paurine IO mM. "d'%enote differences (PqO.05) within

rows

after

washing.

Washing had no effect on motility, and the pre- and post-washing results were essentially similar in all the extenders. Motility of spermatozoa was well maintained in all taurine containing extenders up to 12 h. However, motility significantly decreased (PcO.05) after 6 and 12 h in extenders without taurine. Experiment 3 Table 4 shows the effect on sperm motility of isotonic extenders (100 mMtaurine in extenders with taurine) during storage at 5OC. Except after 0 h, motility of spermatozoa was greater (PXO.05) in INNA with taurine than in INHA82, Kenney's, Kenney's with taurine and EZ-Mixin throughout storage. No decrease in motility was observed in INNA with taurine for 96 h and motility was well maintained in this extender without any appreciable loss even for 5-6 days in some instances. In Kenney's motility decreased (PcO.01) over time throughout the storage, where as motility in Kenney's with taurine decreased (P~0.05) after 24 h, remained stable for 48 h, and then decreased (PxO.05) throughout storage. No decrease of motility was observed after 6 h in all treatments when spermatozoa were first stored in isotonic extenders for 24 h and then washed, and stored in SP-TALP for further 24 h (Table 5). After 12 h, motility in extenders INRA82 and Kenney's with taurine was comparable to 6 h but motility in extenders INHA and Kenney's was lower (P
1046

Theriogenology

Table 4.

Effect of isotonic (345 mOsmol/Kg) extenders on stallion sperm motilfty stored at S°C" % Sperm motility after storage for:

Time

INRA82

INRA82 with Taurineb

Kenney's

Kenney's Taurineb

0

78.lf6.4

75.524.5

72.5f6.9

73.3f5.2

70.8f5.6

24

60.7il3.6'

72.2f7.3d

28.6*8.7e

58.9*4.1=

54.2*9.9=

48

60.6*10.5=

70.6f7.3d

18.3f10.3'

53.0*15.2Cf

48.4f15.0f

72

56.6*14.1=

70.6f9.24

08.7f13.5'

22.8f10.1e

41.5i13.bf

96

48.4i15.4=

65.0ilO.O*

02.5f4.2e

18.3i9.bf

34.2f15.2g

120

36.3f16.2C

55.2*11.7*

0

0

24.3f16.!Se

144

20.5f18.5=

40.4*7.1*

0

0

14.6f15.6=

x

51.6f13.5

64.1i8.1

18.6t8.7

32.1f7.9

41.2i13.0

(h)

'Values shown are means f SD (n = 12). %!aurine 100 mM. cSd'e~f'gDenote differences (PCO.05) within

Table 5.

rows excluding

with

overall

EZ-Mixin

means.

Effect on motility of stallion sperm stored in isotonic extenders for 24 h at S°C, then washed and stored in SpTALP at 39'C' % Snerm motilitv

Extender

Before washing at 24 h

After 2x washing at 24 h

6 h

12 h

INRA82

60.7

INRA82 with Taurineb

72.2

Kenney's Kenney's with taurineb EZ-Mixin

58.3'

54.4=

34.9*

05.1°

66.7'

60.0'

52.8'

27.9*

28.6

27.0'

30.5=

09.6*

OO.oe

57.6

56.7'

52.6'

50.0=

05.6*

54.2

48.1'

41.1=

11.96

03.78

'Values shown are means (n = 8). hTaurine 100 mM. "*'%enote differences (P~0.05) within

rows

after

24 h

_.

washing.

DISCUSSION Among the milk-based extenders tested, motility was well maintained in INKA and was above 56% after 96 h of storage (Table The observation that INRA82 was effective in maintaining 1). viability and motility for longer duration was consistent with previous reports (20,26,27). However, Kenney's extender which had been reported to preserve remarkable motility up to 72 h (25) gave

Theriogenology

1047

poor results even after 24 h of storage. Provinceet al. (33) comparedvarious egg yolk semen extenders with heated skim milk extenders for their ability to maintain stallion sperm motility in cooled semen and found that skim milk extenders were superior to the others. The fertilizing capacity of stallion spermatozoa stored at 5°C for 24 h in egg yolk containing extenders was found to be close to or even higher than that seen in natural breeding (4,17), while the sperm viability and motility in them decreased significantly after 18 h of storage (28). Present observation that motility decreased significantly after 24 h of storage in Dimitro's (Table 1) was in agreement with other studies (14,28,33). Similarly, Citrate which is capable of maintaining high motility of bovine sperm up to 48 h of storage (11) gave poor motility results even at 0 h (Table 2) in case of the stallion. The low motility in Citrate in this study may partly be explained because of species differences. The egg yolk had to be clarified before use as the large particulate material in egg yolk interfered with motility analysis because these particles are of the size of spermatozoa and could be wrongly interpreted as non motile sperm cells (14). Inclusionof 70 mMtaurineinextenders INKA andKenney's improved stallion sperm viability and motility after 24 h of storage (Table Z), while increasing this level to 100 mM in these extenders, to make these extenders isotonic with stallion semen, further improved these characteristics (Table 4). Earlier it had been reported that in the hamster, presence of taurine in the extenders enhanced progressive motility of spermatozoa in vitro (21,24). It had also been reported that motility of bovine sperm cooled to 5OC was maintained close to pre- storage levels in taurine-containing Citrate extender up to 48 h. This was not the case with other extenders (11). A close linear correlationbetweendecline of spermmotility and extent of lipid peroxidation is known to exist (1). Taurine reduces the rate of superoxide production of rabbit spermatozoa and concomitantly reduces inactivation of the superoxide dismutase in these cells. Since superoxide seems to be the major inducer of lipid peroxidation in rabbit spermatozoa, the protective effect of taurine, which readily permeates plasma membranes, may be ascribed to scavenging of intracellular superoxide. Thus taurine protects motility by inhibiting the rate of lipid peroxidation in spermatozoa (2). Taurine also decreases sperm-membraneenzyme Na+- K'- ATPase activity in a dose-dependent manner (23). Na'- K+- ATPase is a membrane-bound enzyme controlling the intracellular Na' and K' levels by exchanging extracellular K+ ions for intracellular Na' ions (34). LOSS of forward motility of rabbit epididymal spermatozoa in high K’ phosphate buffer is inhibited by taurine in vitro (2). Taurine is presentubiquitouslyinmanymammaliantissues (9).

1048

Theriogenology

is present at high concentrations in female reproductive tract Eluids of several mammalian species, especially in oviductal fluid (3,6,22). In the bovine, it is known that sperm are capable of naintaininq viability and fertilizing capacity for a longer duration while in the oviduct (32). Oviductal fluid or supernatant from in vitro cultured oviductal cells is also capable of naintaining sperm motility in vitro for extended time periods (12,13). The presence of taurine, at high concentrations, in the nammalian reproductive tract, with its inhibitory effect on the Va'- K'- ATPase (6,23), shows the potential beneficial effect of this amino acid on maintaining sperm motility in vivo. Since nontaurine isotonic extenders had no effect in enhancing sperm viability and motility and taurine containing extenders, which were iso-osmotic with stallion seminal plasma, were significantly (PcO.05) capable of enhancing and maintaining sperm viability and aotility (Table 4), we hypothesize that in our experiments taurine containing extenders showed this beneficial effect in vitro on the same principle as it does in vivo. Et

The exact mechanism by which the stallion sperm stored in taurine containing extenders for 24 h, was capable of maintaining viability and motility after washing and incubating in Sp-TALP for further 24 h (Table 3,5) is not known. Since taurine is involved in osmoregulation in tissues, stabilization of membranes, protection of cells from self-destruction during processes that generate oxidants, stimulation of certain metabolic actions such as glycolysis and ion regulation (9,10,37), it is hypothesized that the survival of taurine treated sperm was mainly due to 1) membrane stability. 2) inhibition of lipid peroxidation. 3) modulation of Na'- K'- ATPase activity causing subsequent higher intracellular K' concentrations and 4) combination of the aforementioned taurine effects. REFERENCES 1.

Alvarez.JG and Storey BT. Spontaneous lipid peroxidation in rabbit epididymal spermatozoa: its effects on sperm motility. Biol Reprod 1982;27:1102-1108. 2. Alvarez JG, Storey BT. The role of superoxide dismutase in protecting rabbit spermatozoa from 0, toxicity due to lipid peroxidation. Biol Reprod 1983;28:1129-1136. 3. Casslen BG. Free amino acids in human uterine fluids. J Reprod Med 1987;32:181-184. 4. De Vries PJ. Evaluation of the use of fresh extended, transported stall,ionsemen in the Netherland. J Reprod Fertil 1987;35(Suppl 1):641 abstr. 5. Douglas-Hamilton DH, Osol R, Osol G, Driscoll D, Noble H. A field study of the fertility of transported equine semen. Theriogenology 1984;22:291-304. 6. Dumoulin JCM, Evers JLH, Bras M, Pieters MHEC, Geraedts JPM. Positive effect of taurine on preimplantation development of mouse embryos in vitro. J Reprod Fertil 1992;94:373-380. 7. Franc1 AT, Aman RP, Squires EL, Pickett BW. Motility and fertility of equine spermatozoa in a milk extender after 12 or 24 hours at 20°c. Theriogenology 1987;27:517-525.

Ther-iogenology 8.

Heiskanen M, Prihonen A, Koskinen E, Maenpaa PH. Motility and ATP content of extended equine spermatozoa in different J Reprod Fertil 1987;35(Suppl l):lO3 storage conditions. abstr. 9. Huxtable RJ. Phsiological actions of taurinf?. Physiol Rev 1992;72:101-163. the and the heart: LA. Taurine 10. Huxtable RJ, Sebring In Iwata H, Lombardini JB, Segawa T. phospholipid connection. Boston: Kluwer Academic and the heart. feds), Taurine Publishers 1989;31-42. Identification of the EF. 11. Ijaz A, Hunter AG, Graham capacitating agent for bovine sperm in egg yolk-TEST semen extender. J Dairy Sci 1989;72:2700-2706. In vitro cultured granulosa 12. Ijaz A, Lambert RD, Sirard, MA. cell and oviductal cell secrete sperm motility maintaining factors. Mole Reprod Dev 1994;37:54-60. 13. Ijaz A, Sirard MA. Bovine granulosa and oviductal cell Theriogenology 1993;38:234 secretions support spermmotility. abstr. 14. Jasko DJ, Hathaway JA, Schaltenbrand VL, Simper WD, Squires EL. Effect of seminal plasma and egg yolk on motion spermatozoa. stallion characteristics cooled of Theriogenology 1992;37:1241-1252. 15. Jasko DJ, -Moran DM, Farlin ME, Squires EL. Effect of seminal motion spermatozoa1 removal on plasma dilution or characteristics of cooled stallion semen. Theriogenology 1991;35:1059-1067. 16. Kenney RM, Bergman RV, Cooper WL, Morse GW. Minimal mares: techniques contamination techniques for breedin onv Am Assoc Equine Proc Ann ?! and preliminary findings. Pratt 1975;21:327-335. 17. Kenney RM, Varner DD. Artificial Insemination in equines. Modern methods of Colloque reproduction in equine. International, Paris, 12 March 1986;2-37. 18. Leibfried ML, Bavister BD. The effects of taurine and hypotaurine on in vitro fertilization in the golden hamster. Gamete Res 1981;4:57-63. 19. Leibfried ML, Bavister BD. Effects of epinephrine and hypotaurine on in vitro fertilization in the golden hamster. J Reprod Fertil 1982;66:87-93. 20. Magistrini M, Couty I, Palmer E. Interaction between sperm packaging, gas environment, temperature and diluent on fresh stallion sperm survival. Acta Vet Stand 1992;88:97-110. 21. Meizel S, Lui CW, Working PK, Mrsny RJ. Taurine and hypotaurine; their effects on motility, capacitation, and the acrosome reaction of hamster sperm in vitro and their presence in sperm and reproductive tract fluids of several mammals. Dev Growth and Differ 1980;22:483-494. 22. Miller JGO, Schultz GA. Amino acid contents of preimplantation rabbit embryos and fluid of the reproductive tract. Biol Reprod 1987;36:125-129. 23. Mrsny RJ, and Meizel S. Inhibition of hamster sperm Na',K'ATPase activitybytaurine andhypotaurine. Life Sci 1985;36: 211-215.

1050

24.

25.

26.

27.

28.

29.

30. 31.

32.

33.

34. 35. 36.

37.

Theriogenology

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