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Effect of seminal extenders containing egg yolk and glycerol on motion characteristics and fertility of stallion spermatozoa
EFFECT OF SEMINAL EXTENDERS CONTAINING EGG YOLK AND GLYCEROL ON MOTION CHARACTERISTICS AND FERTILITY OF STALLION SPERMATOZOA S.J. Bedford, D.J. Jasko, J.K. Graham, R.P. Amann, E.L. Squires and B.W. Pickett Animal Reproduction and Biotechnology Laboratory Colorado State University Fort Collins, CO 80523 USA Received
for publication: Accepted:
June?14, 1994 Novemi~er 17, 1994
ABSTRACT 'Three experiments were conducted to evaluate the effects of egg yolk and(or) glycerol added to a nonfat dried skim milk-glucose and fertility of (NDSMG) extender on motion characteristics In Experiment 1, ejaculates from each of 8 stallion spermatozoa. stallions were exposed to each of 4 extender treatments: 1) NDSMG, 2) NDSMG + 4% egg yolk (EY), 3) NDSMG + 4% glycerol (GL), and 4) NDSMG + 4% egg yolk + 4% glycerol (EY+GL). Samples were cooled at -0.7OC/min from 37 to 20°C; subsamples were then cooled at -0.05 or -O.S°C/min from 20 to 5OC. Percentages of motile spermatozoa (MOT) and progressively motile spermatozoa (PMOT) were determined at 6, There was no overall 24 and 48 h after initiation of cooling. PMOT was highest (PcO.05) for effect (Pr0.05) of cooling rate. spermatozoa extended in NDSMG+GL at 48 h. At 24 and 48 h, MOT and PMOT were lowest (PcO.05) for spermatozoa extended in NDSMG+EY. In Experiment 2, ejaculates from 8 stallions were exposed to each of 4 treatments: 1) NDSMG, 2) NDSMG+EY, 3) semen centrifuged in NDSMG and resuspended in NDSMG, and 4) semen centrifuged in NDSMG and resuspended in NDSMG+EY. Samples were cooled from 20 to 5OC at each of 2 rates (-0.05, -O.S°C/min). A detrimental interaction between seminal plasma and egg yolk was noted for PMOT at 6 h and for both MOT and PMOT at ~24 h postcooling. Experiment 3 determined if egg yolk or glycerol affected fertility. The seminal treatments were 1) NDSMG, 2) NDSMG+EY with previous removal of seminal plasma, and 3) NDSMG+GL. All samples were cooled to 5OC and stored 24 h before insemination. Embryo recovery rates 7 d after ovulation were lower for mares inseminated with spermatozoa cooled in NDSMG+EY (17%, 4/24) or NDSMG+GL (13%, 3/24) extenders, than semen cooled in NDSMG (50%, 12/24). We concluded that egg yolk (with seminal plasma removal) or glycerol added to NDSMG extender did not depress MOT or PMOT of cooled stallion spermatozoa but adversely affected fertility. Key words: stallion, egg yolk, glycerol, cooled semen, fertility Acknowledgements This study was funded in part by the Arabian Horse Registry, Westminster, CO, and by private donations from Arabian breeders. S.J. Bedford was supportedby *'LaCaixa", a Spanish Financial Institution. Theriogenology 43:95!%967, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691X/95/$10.00 SSDI 0093-691X(95)00045-A
956
Theriogenology INTRODUCTION
Cooling and shipping semen for artificial insemination is common practice in the horse industry. Stallion semen usually is diluted in a non-fat dried skimmilk-glucose (NDSMG) extender prior to cooling (10-12.14.17.27.28). However, if stallion snermatozoa are cooled -rapidly,- they .undergo irreversible membrane changes Moran et al. (20) demonstrated that (301, termed cold shock. stallion spermatozoa were most sensitive to cold shock between 19 and 8OC. Kayser et al. (16) reported that a linear cooling rate of -0.05°C/min between 20 and 5OC maintained maximal percentage of Stallion spermatozoa cooled slowly (initial motile spermatozoa. rate between -0.2 and -0.4°C/min) over these temperatures provided acceptable fertility (10,111. Inclusion of glycerol in a milk-based extender prolonged the duration of satisfactory fertility of bull spermatozoa stored at 5OC from 2 to 14 d (2). Glycerol is also used in extenders for freezing bull and stallion spermatozoa (4,6-8,261. Egg yolk also exerts a protective effect against cold shock, primarily due to its phospholipid components (29). Jasko et al. (12) reported that 4% egg yolk in a NDSMG extender was beneficial for maintenance of spermatozoa1 motion characteristics after cooling. However, addition of glycerol to an egg yolk-Tris extender resulted in lowered fertility, although motility of spermatozoa was maintained during 2 h incubation at 38OC (22). It is not known whether egg yolk or glycerol would improve the fertility of stallion spermatozoa cooled in a milk-based extender. The objectives of these studies were to determine the effects of in a NDSMG and glycerol extender on motion egg yolk characteristics and fertility of stallion spermatozoa cooled to 5OC and stored for 48 h. MATERIALS AND METHODS Experiment
1
This experiment was designed to study the effects of 4 extender treatments and 2 cooling rates on spermatozoa1 motion characteristics. Extender was prepared previous to collection of semen; the exact composition is shown in Table 1. Semen was collected from 8 stallions having acceptable seminal characteristics (23). Only 1 ejaculate was used from each stallion. Aliguots for each ejaculate containing 500x106 spermatozoa (range 0.5-3 ml) were placed into 8 prewarmed 19-ml, screw-cap, disposable plastic cooling bottles. Two bottles were filled, without leaving an air space, with each of the following extender treatments: 1) NDSMG; 2) NDSMG + 4% glycerol (vol/vol; GL); 3) NDSMG + 4% egg yolk (vol/vol; EY); and 4) NDSMG + 4% glycerol + 4% egg yolk (vol/vol; GL+EY). Bottles were capped and gently inverted to mix the semen with the extender and were then
Theriogenology Table 1.
957
Composition of the nonfat dry skim milk-glucose used in Experiments 1 and 2
extender
Dry skim milka
2.4 g
D- (+)-Glucoseb
4.9 g
Ticarcillin
1 mg/ml
Deionized
disodiumC
sterile waterd to make 100 ml of extender
%analac, Beatrice/Hunt-Wesson, Inc., P.O. Box 4800, Fullerton, CA 92634. bSigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178. 'TicillinR, SmithKline Beacham, West Chester, PA 19380. dNANOpure-A Systems, The Barnstead Company, Boston, MA 02132. placed into 2 thermoelectric cooling unitsa (151, at an initial temperature of 37OC. Two linear cooling rates were evaluated for each extender treatment: 1) -0.7°C/min from 37 to 20°C, then O.OS°C/min from 20 to 5OC (Rate I); and, 2) -0.7°C/min from 37 to 20 OC, then -O.S°C/min from 20 to 5OC (Rate II). Motion characteristics of stallion spermatozoa were determined initially for only spermatozoa diluted in the NDSMG extender treatment, and for all treatments at 6, 24 and 48 h after The computerized systemb (CASA) used had initiation of cooling. been previously validated for stallion spermatozoa1 analysis (13). A minimum of 400 spermatozoa and 2 slides were evaluated for each sample in order to reduce bias. For each analysis, a 0.5-ml aliquot was removed from the center of each bottle after carefully mixing the sample. The aliquot was then introduced in a sample cup' placed on an aluminum block maintained at 37OC on a slide warmer, and the bottle immediately replaced in the cooler. Subsamples to be analyzed were warmed for 10 min prior to evaluation of motion characteristics. Samples were evaluated for the percentage of motile spermatozoa (MOT), the percentage of progressively motile spermatozoa (PMOT), and for curvilinear velocity (VCL,um/sec;3). Data for each time period were analyzed as a 4 (extender) by 2 (rate) by 8 (stallion) factorial experiment with stallions as a random effect using a General Linear Models program There was no (P>O.O5) (25). interaction between cooling rate and seminal treatment; therefore, aARS Model 584A Mod2 Thermoelectric Cooler, Animal Reproduction Systems, Inc., Chino, CA 91710. bHTM-S Motility Analyzer, Hamilton-Thorn Research, Inc., Danvers, MA 01923. 'Auto-analysis sample cups, VWRR, Ref# 2120-800 (0.5 ml), VWR ScientiEic Inc., P.O. Box 13645, Philadelphia, PA 19101-3645.
958
Theriogenology
means averaged across cooling rates were compared. Mean values for spermatozoa1 motion characteristics were separated using the Student-Newman-Keuls multiple range test. Experiment
2
The purpose of this experiment was to determine if there was a detrimental interaction between egg yolk and seminal plasma on spermatozoa1 motion characteristics. Ejaculates were collected from 8 stallions as described, split and exposed to each of 5 noncentrifuged; 2) NDSMG+EY, nontreatments: 1) NDSMG, centrifuged; 3) NDSMG, centrifuged, and the spermatozoa1 Pellet resuspended in NDSMG (NDSMGC); 4) NDSMG, centrifuged and the spermatozoa1 pellet resuspended in NBSMG+EY (EYc); and 5) NDSMG, centrifuged, but the supernatant not removed and the sample further diluted with 20 ml of NLXMG (NDSMGCS). The last treatment was used only to determine whether centrifugation itself adversely affected spermatozoa. To remove seminal plasma, aliquots of each sample (range 1 to 6 ml) were extended in NDSMG at a Concentration Of 50x106 spermatozoa/ml and centrifuged for 15 min at 400 g (12) in 50 ml non-disposable conical plastic tubes. The supernatant was then removed, and the pellet was resuspended in the appropriate extender. Non-centrifuged samples were maintained at room temperature until other samples had been centrifuged. For cooling, all samples were diluted in the appropriate extender to a concentration of 25~10~ spermatozoa/ml. Samples in all treatments were placed into 19-ml, screw-cap plastic cooling bottles. Bottles for each treatment were capped and placed into each of 2 thermoelectric cooling units and cooled as in Experiment 1, except that cooling began at room temperature (23OC). Motion characteristics of stallion spermatozoa were determined prior to cooling for spermatozoa in NDSMG and NDSMGcs, and for all treatments at 6, 24 and 48 h after initiation of cooling, as described in Experiment 1. To assess the centrifugation effect, data for NDSMG and NDSMGc were compared using a Studentized t-test. Means for motion characteristics for spermatozoa in NDSMG, NDSMG+EY, NDSMGc and EYc were analyzed as a 2 (centrifugation) by 2 (extender) by 8 (stallion) factorial experiment with the stallions as a random effect. The interaction means averaged over 2 cooling rates were compared by the Student-Newman-Keuls multiple range test. To assess whether egg yolk offered any beneficial effect when spermatozoa were cooled at the fast cooling rate, data for the treatments NDSMG and NDSMG+EYc at both cooling rates were analyzed by performing an ANOVA for the 4 extender/rate combinations. When significant differences were found, means were separated by a Student-Newman-Keuls' multiple range test.
959
Experiment
3
Semen from 2 stallions diluted in 3 extenders was used to The fertility trial was breed mares during 72 estrous cycles. performed during the breeding season (May and June), using 49 mares of predominantly Quarter Horse, Thoroughbred, Appaloosa or Arabian breeding, each weighing between 350 and 500 kg. Mares were teased daily and examined by ultrasonography per rectum daily, beginning When a follicle 235 mm in on the second day of estrus (18). diameter was observed, mares were initially inseminated, and subsequently reinseminated with the same treatment every other day until ovulation occurred. Inseminations consisted of semen from either stallion extended to 25~10~ spermatozoa/ml in a commercialized nonfat, dried skim milk-glucose-based extendera (control). The only difference from the extender used in this and in previous experiments was that this extender contained 0.01625 g of Polymyxin B Sulfate and 0.0625 g of Amikacin for every 125 ml of extender, instead of the antibiotic For this experiment, either egg yolk or ticarcillin disodium. glycerol was added to the NDSMG extender to achieve a final For semen extended in NDSMG+EY, concentration of 4% (vol/vol). seminal plasma was removed prior to cooling by centrifugation as in A series of 19-ml, screw cap, plastic cooling Experiment 2. bottles were filled with each extender treatment and placed in thermoelectric cooling units. Cooling began at 23'C and proceeded at -0.7OC/min to 20°C; cooling resumed at -0.05°C/min, from 20 to 5oc. Semen was stored for 24 h at 5OC prior to breeding the mares. The 6 treatments used (2 stallions and 3 extenders) were randomized before starting the experiment, and the same order maintained during inseminations throughout the experiment. As mares exhibited estrus, they were assigned to the corresponding treatment following the randomization order. Mares bred during the second and third cycles followed the randomized treatment order regardless of previous seminal treatments. In the first replicate of 6 treatments, mares were given an implant of GnRH agonist (2.2 mg, s.c., Desloreline) on the day of initial insemination. The GnRH implants were used on every other replicate of 6 semen treatments throughout the study. Fertility was based on embryo recovery by uterine lavage 7 d after ovulation (19). Three liters of a prewarmed (32 to 35OC) modified phosphate-buffered saline solutionf supplemented with 0.1% (w/v011 of bovine serum albuming were used to flush each mare. Immediately after flushing, mares were given a luteolytic d E-Z Mixin-K, Animal Reproduction Systems, Chino, CA 91710. F Deslorelin, Peptide Technology Ltd., Dee Why, Australia. Dulbecco's Phosphate Buffered Saline, Sigma Chemical Company, St. Louis, MO 63178. g Albumin Bovine, Fraction V, United States Biochemical Corp., Cleveland, OH 44122.
960
Theriogenology
dose of prostaglandin FZcrhto shorten estrous cycles. Motion characteristics for spermatozoa used in the breeding trial were analyzed immediately after collection for spermatozoa suspended in NDSMG. At 24 h after initiation of cooling and again after 3 additional hours of incubation at 37OC, motion analyses were conducted for all treatments. Data for motion characteristics of spermatozoa used in the breeding trial were analyzed as a 3 Whenever (extender) by 2 (stallion) factorial experiment. significant differences were found, the Student-Newman-Keuls test recovery data were compared by was used to compare means. Embryo a Chi-square analysis. RESULTS Experiment 1 Rate of cooling had no effect (P>O.O5) on motion characteristics at any time period, nor was there an interaction between cooling rate and seminal treatment. Thus, treatment means for motion characteristics were averaged across cooling rates. There was no effect of seminal treatment (P>O.O5) on motion characteristics at 6 h (Table 2). However, at 24 h, MOT, PMOT and VCL were lower (PaO.05) for spermatozoa in NDSMG+EY than for spermatozoa in other treatments. By 48 h, MOT, PMOT and VCL were less (~<0.05) for spermatozoa in both extenders containing egg yolk than for spermatozoa in extenders not containing egg yolk. The inclusion of glycerol in the NDSMG extender resulted in higher (~<0.05) PMOT than any other treatment for spermatozoa stored for 48 h. Experiment 2 Motion characteristics were similar (P>O.O5) for noncentrifuged spermatozoa extended in NDSMG and for spermatozoa in NDSMGcs (centrifugedwithout removal of seminal plasma). The 2 by 2 factorial analysis (NDSMG, NDSMG+EY vs NDSMGc, EYc), revealed an interaction between extenders and centrifugation, for data averaged across the 2 cooling rates. The interaction was significant (PcO.05) at 6 h for PMOT and VCL, and at 24 and 48 h after initiation of cooling for all parameters analyzed. Means for the 4 treatments are presented in Table 3. MOT and PMOT were lower at 24 and 48 h (PcO.05) for spermatozoa in NDSMG+EY, without seminal plasma removal, than for all other treatments. PMOT at 48 h was also lower (PcO.05) for spermatozoa in NDSMG in which seminal plasma had been removed by centrifugation, than for spermatozoa in NDSMG without centrifugation and spermatozoa in NDSMG+EY in which seminal plasma had been removed. At 24 h, VCL was higher for spermatozoa devoid of seminal plasma and cooled in NDSMG+EY, than for spermatozoa in h LutalYse, Upjohn, The Upjohn Company, Kalamazoo, MI 49001.
961
Theriogenology
most other treatments. In general, at both 24 and 48 h, VCL was less (P
Percentage of total motile spermatozoa (MGT, %I, progressively motile spermatozoa (PMOT, %) and curvilinear velocity (VCL, um/sec) of spermatozoa cooled to 5OC, extended in different diluents Motion
Storage interval (hours)
MOT
Extender
characteristic PMOT
VCL
0
NDSMGa
69
51
138
6
NDSMG NDSMG + EYb NDSMG + GLC NDSMG + EY + GL
56 54 56 55
39
113 111 106 111
24
NDSMG NDSMG + EY NDSMG + GL NDSMG + EY + GL
46d 35e 52d 45d
48
NDSMG NDSMG + EY NDSMG + GL NDSMG + EY + GL
Pooled SEM
2.2
35 37 36
113d 102e 114d 1lOd 2oe 5f 27d 12f
lOad 86e 112d 95e
2.3
3.5
Data were analyzed as a 4 (extender) by 2 (rate) by 8 (stallion) factorial experiment. There was no extender (P>O.O5) by rate interaction; therefore, means were averaged over 2 cooling rates. aNonfat dried skim milk-glucose. bNDSMG + 4% egg yolk (vol/vol). 'NDSMG + 4% glycerol (vol/vol). d-fMeans within time and column bearing different superscripts differ (PxO.05). Motion characteristics of spermatozoa extended in NDSMG but not centrifuged, and NDSMG+EY centrifuged and cooled at both slow and fast rates, are presented in Table 4. MOT was similar (P>O.O5) for spermatozoa among extenders at all time periods. At 48 h, PMOT was lower (P
3
Motion characteristics
of spermatozoa from ejaculates used to
Theriogenology
962
inseminate mares are presented in Table 5. The effect of stallion, but not stallion by extender, was significant (PO.O5) after incubation in the various seminal extenders at 37OC for 3 h. VCL was faster (P
Effect of nonfat dried skimmilk-glucose (NDSMG) extender with and without 4% egg yolk (EY) and removal of Seminal plasma on percentage of motile spermatozoa (MOT, %I, progressively motile spermatozoa (PMOT, %) and curvilinear velocity (VCL, pm/set) of cooled stallion spermatozoa
Storage interval (h)
Extender
Seminal plasma removal
Motion characteristic MOT
PMOT
VCL
0
NDSMG NDSMGcsa
No No
71 70
53 55
137 146
6
NDSMG
No Yes No Yes
57 49 51 52
40 33 31 37
129b= 119c 118c 133b
No Yes No Yes
49b 45b 28' 50b
33b 27b 15c 33b
122bC 114c 102d 131b
No Yes No Yes
4ob= 34c ga 46b
24b 16' 2d 27b
12Sb 110= 87a 127b
NDSMG + EY 24
NDSMG NDSMG + EY
48
NDSMG NDSMG + EY
Pooled SEM
2.5
2.3
3.2
Data were analyzed as a 2 (centrifugation) by 2 (extender) by 8 (stallion) factorial experiment (averaging over the 2 cooling rates). There were significant (P
Theriogenology
963
Administration of GnRH agonist had no effect on recovery rate of embryos (P>O.O5). Recovery of embryos was lower (PKO.05) in mares inseminated with spermatozoa in NDSMG+EY or extended Fertility rate per cycles inseminated, based on embryo NDSMG+GL. recovery 7 d after ovulation, were 50% (12/24), 17% (4/24) and 13% (3/24) for mares inseminated with spermatozoa in NDSMG, NDSMG+EY and NDSMG+GL, respectively. Embryo recovery rates for Stallions 1 and 2, respectively, were 50% (6/12) and 50% (6/12) when using NDSMG extender, 8% (l/12) and 25% (3/12) when using NDSMG+EY extender, and 0% (O/12) and 25% (3/12) when using NDSMG+GL extender. Table 4.
Effects of a non-fat dried skim milk-glucose (NDSMG) extender with and without 4% egg yolk (EY), and cooling rates on the percentages of total motile spermatozoa (MOT, %), progressively motile spermatozoa (PMOT, %) and on the curvilinear velocity (VCL, um/sec) of stallion spermatozoa
Storage interval Extender (h)
Motion characteristic
Seminal plasma removal
Rate
MOT
PMOT
VCL
__
71
53
137
0
NDSMG
NO
6
NDSMG
No No Yes Yes
1a IIb I II
58 56 56 49
41 40 41 33
128 129 136 130
No No Yes Yes
I II I II
52 47 45 54
35 31 31 35
119 126 130 132
No NO Yes Yes
I II I II
44 36 46 46
28c 19d 27c 2ac
127 124 122 131
2.5
2.3
3.5
NDSMG+EY 24
NDSMG NDSMG+EY
48
NDSMG NDSMG+EY
Pooled SEM
Data were analyzed as an ANOVA for the 4 combinations of extender and rate. Cooling began at 23OC and proceeded to 20°C at -0.7°C/min. aSemen was cooled at -O.OS°C/min from 20 to 5OC (Rate I). bSemen was cooled at -O.S°C/min from 20 to 5OC (Rate II). CfdMeans within time and column bearing different superscripts were different (P
964 DISCUSSION Previous studies have shown a significant reduction in motion characteristics of stallion spermatozoa when cooled rapidly over the temperatures of cold shock sensitivity (16,20,27). whenmotion characteristics were compared for spermatozoa in the four extenders (Experiment 11, there were no detrimental effects of rapid cooling. Table 5.
Percentage of total motile spermatozoa (MOT, %I, progressively motile spermatozoa (PMOT, %) and curvilinear velocity (VCL, pm/set) of cooled StalliOn spermatozoa in ejaculates used for insemination of mares (averaged over 2 stallions)
Storage interval (hours) 0 24
Motion Extender
MOT
characteristic PMOT
VCL
NDSMG
75
57
108
NDSMG NDSMG + EY NDSMG + GL
45h 59a 4lh
29b 46a 28b
113b 127a 121ah
NDSMG NDSMG + EY NDSMG + GL
41 41 37
14 15 9
61ab 66a 55h
3.0
1.6
Incubation period at 37OC (h) 3
Pooled SEM
2.3
Semen was extended to 25~10~ spermatozoa/ml in non-fat dried skim milk-glucose (NDSMG), NDSMG + 4% egg yolk (EY) or NDSMG + 4% glycerol (GL), and cooled at -O.OS°C/min (n=20 ejaculates per stallion). Semen extended in EY had been previously centrifuged in NDSMG at 50~10~ spermatozoa/ml for removal of seminal plasma. Data were analyzed as a 3 (extender) by 2 (stallion) factorial experiment. abMeans within time and column bearing different superscripts differ (P
Theriogenology
965
The addition of GL to the NDSMG did not protect spermatozoa increased spermatozoa1 motion cooling, except for during The addition of characteristics detected after 48 h of storage. glycerol to a milk-based extender has been shown beneficial for Province et al. (241, storage of bull spermatozoa at 5'C (1). however, reported no advantage for the addition of glycerol to an NUSMG extender when cooling stallion spermatozoa to 5OC. However, spermatozoa were only stored for 24 h in that study. Unlike Jasko et al. (14), we found that removal of seminal plasma and cooling in NDSMG alone resulted in a depression in PMOT of spermatozoa after storage for 48 h. In agreement with Padilla and Foote (211, we concluded that seminal plasma should be Present to provide protection for cooled storage of stallion spermatozoa in Perhaps if Jasko et al. (14) had evaluated a NDSMG extender. longer storage intervals or PMOT, results might have been similar to those of the present study. A level of 20% egg yolk has been previously added to extenders Jasko et al. (121, used to freeze stallion spermatozoa (7). demonstrated maintenance in motion characteristics of stallion spermatozoa when 4% egg yolk was added to a NDSMG extender and when seminal plasma had been removed. In our study, when spermatozoa were centrifuged to remove seminal plasma and NDSMG+EY was added, motion characteristics were higher than for spermatozoa devoid of seminal plasma and diluted in NJXMG alone. Thus, either seminal plasma or EY had to be present in NDSMG extender in order to maintain spermatozoa1 motion characteristics. In contrast, if both seminal plasma and EY were present in the NDSMG extender, motion characteristics of stallion spermatozoa were severely depressed. The mechanism whereby EY and seminal plasma interact to depress motion characteristics was not determined. Perhaps seminal plasma or components of the NDSMG extender resulted in lipid peroxidation and release of free radicals that damaged spermatozoa. Evidence of EY protection was only seen for PMOT after 48 h of storage. PMOT was higher for this treatment than for NDSMG alone when semen had been cooled at the fast rate. Embryo recovery rates of mares inseminated with spermatozoa extended in NDSMG+GL and NDSMG+EY were low. In contrast, fertility rates for mares inseminated with spermatozoa extended in NDSMG and cooled for 24 h were similar to those in previous studies (10,ll). The adverse effects of GL on fertility of mares has been reported Using 7% glycerol in cream-gel extender (9) and Tris extender containing 22.8% EY and 5.25% glycerol In the latter (22). experiment, the investigators did not assess the independent effects of glycerol and egg yolk. Clay et al. (5) reported lower fertility when using an extender containing 20% egg yolk for cooled stallion spermatozoa. Unfortunately, these studies were confounded by multiple factors that did not allow direct assessment of the effects of glycerol and egg yolk. However, based on the results of the Present study, GL or EY, when combined with JVDSMG, resulted in lower fertility. The mechanism for depression of fertility was not determined but may possibly be different for GL and EY. Based on
Theriogenology
966
a preliminary study in our laboratory, fertility rates (embryo recovery) were 69% (27/39) for mares inseminated with semen cooled in a modification of the freezing extender used in France (4). However, only 2% (vol/vol) EY was added to this extender. Whether the composition of the extender or lowered percentage of egg yolk accounted for the higher fertility remains undetermined. Motion characteristics of spermatozoa in ejaculates used to inseminate mares were similar or higher for spermatozoa in NDSMG+GL This or NDSMG+EY, respectively, than for spermatozoa in NDSMG. lack of correlation between spermatozoa1 motility at the time of insemination and fertility was also reported for mares inseminated with TRIS extender (22). In conclusion, although GL or EY (with removal of seminal plasma) added to NDSMG extender did not depress motion characteristics of stallion spermatozoa after cooling and storage for 24 to 48 h, fertility was extremely depressed. REFERENCES Albright JL, Ehlers MH, Erb RE. Motility of bovine spermatozoastored at 5OC when extended in mixtures of yolk-citrate,yolk-glycine,whole milk, skimmilk and alvcerol. J Dairy Sci 1957;41:524-529. 2. Almquist JO, Wickersham EW. Diluents for bovine semen. XII. Fertility and motility of spermatozoain skim-milk with various levels of glycerol and methods of glycerolization. J Dairy Sci 1962;45:782-787. 3. Amann RP. Can the fertility potential of a seminal sample be predicted accurately? J Androl 1989;10:89-98. 4. Amann RP, Pickett BW. Principles of cryopreservationand a review of cryopreservationof stallion spermatozoa. Equine Vet J 1987;7:145-173. 5. Clay CM, Slade NP, Amann RP, Squires EL. Effects of extenders, storage temperature and centrifugationon stallion spermatozoa1motility and fertility. Int Cong Anim Reprod and AI 19&34;10:186-188. 6. Cochran JD, Amann RP, Froman DP. Pickett BW. Effects of centrifuqation, glycerol level, cooling to 5OC, freezing rate and thawing rate on the post-thaw motility of equine sperm. Theriogenology1984:22:25- 38. 7. Cristanelli MJ, Amann RP, Squires EL, Pickett BW. Effects of egg yolk and glycerol levels in lactose-EDTA-eggyolk extender and of freezing rate on the motility of frozen-thawedstallion spermatozoa. Theriogenology 1985;24:681-686. 0. Cristanelli MJ, Squires EL, Amann RP, Pickett BW. Fertility of stallion semen processed, frozen and thawed by a new procedure. Theriogenology 1984;22:39-45. 9. Demick DS, Voss JL, Pickett BW. Effect of cooling, Storage, glycerolizationand spermatozoa1numbers on equine fertility. J Anim Sci 1976;43:633-637. 10. Douglas-HamiltonDH. Burns PJ. Driscoll DD. Viale EM. Fertility and characteristicsof slow-cooled stallion semen. J Reprod Fert Suppl 1987;35:649-650. 11. Douglas-HamiltonDH, 0~01 R, 0901 G, Driscoll D, Noble H. A field study of the fertility of-transportedequine semen. Theriogenology 1984;22:291-304. 1.
Theriogenology
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12. Jasko DJ, Hathaway JA, Schaltenbrand VL. Simper WD, Squires EL. Effect Of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa. Theriogenology 1992;37:1241-1252. 13. Jasko DJ, Lein, D.H. and Foote, R.H. A comparison of two computerautomated semen analysis instruments for the evaluation of sperm motion characteristics in the stallion. J. Androl. 11(5):453-459(1990). 14. Jasko DJ, Moran DM, Farlin ME, Squires EL. Effect of seminal plasma dilution or removal on spermatozoa1 motion characteristics of cooled stallion semen. Theriogenology 1991;35:1059-1067. 15. Kayser JPR. Effects of linear cooling rates on motion characteristics of stallion spermatozoa. M.S. Thesis. Colorado State Univ, Fort Collins, CO 80523, 1991. 16. Kayser JP, Amann RP, Shideler RK, Squires EL, Jasko DJ, Pickett BW. Effects of linear cooling rate on motion characteristics of stallion spermatozoa. Theriogenology 1984:22:291-304. 17. KeMev RM. Beraman RV. CooDer WL. Morse GW. Minimal contamination techn&ues for-breeding mares; techniques and preliminary findings. Proc AM Conv Am Assoc Eguine Pratt 1975;21:327-336. 18. McKinnon AO, Squires EL, Voss JL. Ultrasonic evaluation of the mare's reproductive tract-Part I. Compend Contin Educ PractVet1987;9:336-345. 19. McKinnon AO, Squires EL, Voss JL, Cook VM. Equine embryo transfer. Compend Contin Educ Pratt Vet 1988;10:343-353. 20. Moran DM, Jasko DJ, Squires EL, Amann RP. Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa. Theriogenology 1992;38:601-614. 21. Padilla AW. Foote RI-I. Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa. J Anim Sci 1991;69:3308-3313. 22. Pickett BW. Bunvash LD, Voss JL, Back DG. Effect of seminal extenders on equine fertility. J Anim Sci 1975;40:1136-1143. 23. Pickett BW. Squires EL, McKinnon AO. Procedures for Collection, Evaluation and Utilization of Stallion Semen for Artificial Insemination. Colorado State Univ Anim Reprod Lab Bull No 03, Fort Collins, 1987. 24. Province CA, Amann RP, Pickett BW, Squires EL. Extenders for preservation of canine and equine spermatozoa at 5%. Theriogenology 1984;22:409-415. 25. Statistical Analysis System - SAS User's Guide: Statistics. SAS Institute, Cary, NC, 1985;113-137. 26. Unal MB, Bemdtson WE, Pickett BW. Influence of sugars with glycerol on post-thaw motility of bovine spermatozoa in straws. J Dairy Sci 1978;61:83-89. 27. Vamer DD, Blanchard TL, Love CL, Garcia MC, Kenney RM. Effects of cooling rate and storage temperature on equine spermatozoa1 motility parameters. Theriogenology 1988;29:1044-1054. 20. Vamer DD, Blanchard TL, Meyers PJ, Meyers SA. Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20°C. Theriogenology 1989;32:515-525. 29. WatSOn PF. The roles of lipid and protein in the protection of ram spermatozoa at 5% by egg-yolk lipoprotein. J Reprod Fert 1981;62:483492. 30. Watson PF, Plummer JM, Allen WE. Quantitative assessment of membrane damage in cold-shocked spermatozoa of stallions. J Reprod Fert Suppl 1987;35:651-653.
for publication: Accepted:
June?14, 1994 Novemi~er 17, 1994
ABSTRACT 'Three experiments were conducted to evaluate the effects of egg yolk and(or) glycerol added to a nonfat dried skim milk-glucose and fertility of (NDSMG) extender on motion characteristics In Experiment 1, ejaculates from each of 8 stallion spermatozoa. stallions were exposed to each of 4 extender treatments: 1) NDSMG, 2) NDSMG + 4% egg yolk (EY), 3) NDSMG + 4% glycerol (GL), and 4) NDSMG + 4% egg yolk + 4% glycerol (EY+GL). Samples were cooled at -0.7OC/min from 37 to 20°C; subsamples were then cooled at -0.05 or -O.S°C/min from 20 to 5OC. Percentages of motile spermatozoa (MOT) and progressively motile spermatozoa (PMOT) were determined at 6, There was no overall 24 and 48 h after initiation of cooling. PMOT was highest (PcO.05) for effect (Pr0.05) of cooling rate. spermatozoa extended in NDSMG+GL at 48 h. At 24 and 48 h, MOT and PMOT were lowest (PcO.05) for spermatozoa extended in NDSMG+EY. In Experiment 2, ejaculates from 8 stallions were exposed to each of 4 treatments: 1) NDSMG, 2) NDSMG+EY, 3) semen centrifuged in NDSMG and resuspended in NDSMG, and 4) semen centrifuged in NDSMG and resuspended in NDSMG+EY. Samples were cooled from 20 to 5OC at each of 2 rates (-0.05, -O.S°C/min). A detrimental interaction between seminal plasma and egg yolk was noted for PMOT at 6 h and for both MOT and PMOT at ~24 h postcooling. Experiment 3 determined if egg yolk or glycerol affected fertility. The seminal treatments were 1) NDSMG, 2) NDSMG+EY with previous removal of seminal plasma, and 3) NDSMG+GL. All samples were cooled to 5OC and stored 24 h before insemination. Embryo recovery rates 7 d after ovulation were lower for mares inseminated with spermatozoa cooled in NDSMG+EY (17%, 4/24) or NDSMG+GL (13%, 3/24) extenders, than semen cooled in NDSMG (50%, 12/24). We concluded that egg yolk (with seminal plasma removal) or glycerol added to NDSMG extender did not depress MOT or PMOT of cooled stallion spermatozoa but adversely affected fertility. Key words: stallion, egg yolk, glycerol, cooled semen, fertility Acknowledgements This study was funded in part by the Arabian Horse Registry, Westminster, CO, and by private donations from Arabian breeders. S.J. Bedford was supportedby *'LaCaixa", a Spanish Financial Institution. Theriogenology 43:95!%967, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691X/95/$10.00 SSDI 0093-691X(95)00045-A
956
Theriogenology INTRODUCTION
Cooling and shipping semen for artificial insemination is common practice in the horse industry. Stallion semen usually is diluted in a non-fat dried skimmilk-glucose (NDSMG) extender prior to cooling (10-12.14.17.27.28). However, if stallion snermatozoa are cooled -rapidly,- they .undergo irreversible membrane changes Moran et al. (20) demonstrated that (301, termed cold shock. stallion spermatozoa were most sensitive to cold shock between 19 and 8OC. Kayser et al. (16) reported that a linear cooling rate of -0.05°C/min between 20 and 5OC maintained maximal percentage of Stallion spermatozoa cooled slowly (initial motile spermatozoa. rate between -0.2 and -0.4°C/min) over these temperatures provided acceptable fertility (10,111. Inclusion of glycerol in a milk-based extender prolonged the duration of satisfactory fertility of bull spermatozoa stored at 5OC from 2 to 14 d (2). Glycerol is also used in extenders for freezing bull and stallion spermatozoa (4,6-8,261. Egg yolk also exerts a protective effect against cold shock, primarily due to its phospholipid components (29). Jasko et al. (12) reported that 4% egg yolk in a NDSMG extender was beneficial for maintenance of spermatozoa1 motion characteristics after cooling. However, addition of glycerol to an egg yolk-Tris extender resulted in lowered fertility, although motility of spermatozoa was maintained during 2 h incubation at 38OC (22). It is not known whether egg yolk or glycerol would improve the fertility of stallion spermatozoa cooled in a milk-based extender. The objectives of these studies were to determine the effects of in a NDSMG and glycerol extender on motion egg yolk characteristics and fertility of stallion spermatozoa cooled to 5OC and stored for 48 h. MATERIALS AND METHODS Experiment
1
This experiment was designed to study the effects of 4 extender treatments and 2 cooling rates on spermatozoa1 motion characteristics. Extender was prepared previous to collection of semen; the exact composition is shown in Table 1. Semen was collected from 8 stallions having acceptable seminal characteristics (23). Only 1 ejaculate was used from each stallion. Aliguots for each ejaculate containing 500x106 spermatozoa (range 0.5-3 ml) were placed into 8 prewarmed 19-ml, screw-cap, disposable plastic cooling bottles. Two bottles were filled, without leaving an air space, with each of the following extender treatments: 1) NDSMG; 2) NDSMG + 4% glycerol (vol/vol; GL); 3) NDSMG + 4% egg yolk (vol/vol; EY); and 4) NDSMG + 4% glycerol + 4% egg yolk (vol/vol; GL+EY). Bottles were capped and gently inverted to mix the semen with the extender and were then
Theriogenology Table 1.
957
Composition of the nonfat dry skim milk-glucose used in Experiments 1 and 2
extender
Dry skim milka
2.4 g
D- (+)-Glucoseb
4.9 g
Ticarcillin
1 mg/ml
Deionized
disodiumC
sterile waterd to make 100 ml of extender
%analac, Beatrice/Hunt-Wesson, Inc., P.O. Box 4800, Fullerton, CA 92634. bSigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178. 'TicillinR, SmithKline Beacham, West Chester, PA 19380. dNANOpure-A Systems, The Barnstead Company, Boston, MA 02132. placed into 2 thermoelectric cooling unitsa (151, at an initial temperature of 37OC. Two linear cooling rates were evaluated for each extender treatment: 1) -0.7°C/min from 37 to 20°C, then O.OS°C/min from 20 to 5OC (Rate I); and, 2) -0.7°C/min from 37 to 20 OC, then -O.S°C/min from 20 to 5OC (Rate II). Motion characteristics of stallion spermatozoa were determined initially for only spermatozoa diluted in the NDSMG extender treatment, and for all treatments at 6, 24 and 48 h after The computerized systemb (CASA) used had initiation of cooling. been previously validated for stallion spermatozoa1 analysis (13). A minimum of 400 spermatozoa and 2 slides were evaluated for each sample in order to reduce bias. For each analysis, a 0.5-ml aliquot was removed from the center of each bottle after carefully mixing the sample. The aliquot was then introduced in a sample cup' placed on an aluminum block maintained at 37OC on a slide warmer, and the bottle immediately replaced in the cooler. Subsamples to be analyzed were warmed for 10 min prior to evaluation of motion characteristics. Samples were evaluated for the percentage of motile spermatozoa (MOT), the percentage of progressively motile spermatozoa (PMOT), and for curvilinear velocity (VCL,um/sec;3). Data for each time period were analyzed as a 4 (extender) by 2 (rate) by 8 (stallion) factorial experiment with stallions as a random effect using a General Linear Models program There was no (P>O.O5) (25). interaction between cooling rate and seminal treatment; therefore, aARS Model 584A Mod2 Thermoelectric Cooler, Animal Reproduction Systems, Inc., Chino, CA 91710. bHTM-S Motility Analyzer, Hamilton-Thorn Research, Inc., Danvers, MA 01923. 'Auto-analysis sample cups, VWRR, Ref# 2120-800 (0.5 ml), VWR ScientiEic Inc., P.O. Box 13645, Philadelphia, PA 19101-3645.
958
Theriogenology
means averaged across cooling rates were compared. Mean values for spermatozoa1 motion characteristics were separated using the Student-Newman-Keuls multiple range test. Experiment
2
The purpose of this experiment was to determine if there was a detrimental interaction between egg yolk and seminal plasma on spermatozoa1 motion characteristics. Ejaculates were collected from 8 stallions as described, split and exposed to each of 5 noncentrifuged; 2) NDSMG+EY, nontreatments: 1) NDSMG, centrifuged; 3) NDSMG, centrifuged, and the spermatozoa1 Pellet resuspended in NDSMG (NDSMGC); 4) NDSMG, centrifuged and the spermatozoa1 pellet resuspended in NBSMG+EY (EYc); and 5) NDSMG, centrifuged, but the supernatant not removed and the sample further diluted with 20 ml of NLXMG (NDSMGCS). The last treatment was used only to determine whether centrifugation itself adversely affected spermatozoa. To remove seminal plasma, aliquots of each sample (range 1 to 6 ml) were extended in NDSMG at a Concentration Of 50x106 spermatozoa/ml and centrifuged for 15 min at 400 g (12) in 50 ml non-disposable conical plastic tubes. The supernatant was then removed, and the pellet was resuspended in the appropriate extender. Non-centrifuged samples were maintained at room temperature until other samples had been centrifuged. For cooling, all samples were diluted in the appropriate extender to a concentration of 25~10~ spermatozoa/ml. Samples in all treatments were placed into 19-ml, screw-cap plastic cooling bottles. Bottles for each treatment were capped and placed into each of 2 thermoelectric cooling units and cooled as in Experiment 1, except that cooling began at room temperature (23OC). Motion characteristics of stallion spermatozoa were determined prior to cooling for spermatozoa in NDSMG and NDSMGcs, and for all treatments at 6, 24 and 48 h after initiation of cooling, as described in Experiment 1. To assess the centrifugation effect, data for NDSMG and NDSMGc were compared using a Studentized t-test. Means for motion characteristics for spermatozoa in NDSMG, NDSMG+EY, NDSMGc and EYc were analyzed as a 2 (centrifugation) by 2 (extender) by 8 (stallion) factorial experiment with the stallions as a random effect. The interaction means averaged over 2 cooling rates were compared by the Student-Newman-Keuls multiple range test. To assess whether egg yolk offered any beneficial effect when spermatozoa were cooled at the fast cooling rate, data for the treatments NDSMG and NDSMG+EYc at both cooling rates were analyzed by performing an ANOVA for the 4 extender/rate combinations. When significant differences were found, means were separated by a Student-Newman-Keuls' multiple range test.
959
Experiment
3
Semen from 2 stallions diluted in 3 extenders was used to The fertility trial was breed mares during 72 estrous cycles. performed during the breeding season (May and June), using 49 mares of predominantly Quarter Horse, Thoroughbred, Appaloosa or Arabian breeding, each weighing between 350 and 500 kg. Mares were teased daily and examined by ultrasonography per rectum daily, beginning When a follicle 235 mm in on the second day of estrus (18). diameter was observed, mares were initially inseminated, and subsequently reinseminated with the same treatment every other day until ovulation occurred. Inseminations consisted of semen from either stallion extended to 25~10~ spermatozoa/ml in a commercialized nonfat, dried skim milk-glucose-based extendera (control). The only difference from the extender used in this and in previous experiments was that this extender contained 0.01625 g of Polymyxin B Sulfate and 0.0625 g of Amikacin for every 125 ml of extender, instead of the antibiotic For this experiment, either egg yolk or ticarcillin disodium. glycerol was added to the NDSMG extender to achieve a final For semen extended in NDSMG+EY, concentration of 4% (vol/vol). seminal plasma was removed prior to cooling by centrifugation as in A series of 19-ml, screw cap, plastic cooling Experiment 2. bottles were filled with each extender treatment and placed in thermoelectric cooling units. Cooling began at 23'C and proceeded at -0.7OC/min to 20°C; cooling resumed at -0.05°C/min, from 20 to 5oc. Semen was stored for 24 h at 5OC prior to breeding the mares. The 6 treatments used (2 stallions and 3 extenders) were randomized before starting the experiment, and the same order maintained during inseminations throughout the experiment. As mares exhibited estrus, they were assigned to the corresponding treatment following the randomization order. Mares bred during the second and third cycles followed the randomized treatment order regardless of previous seminal treatments. In the first replicate of 6 treatments, mares were given an implant of GnRH agonist (2.2 mg, s.c., Desloreline) on the day of initial insemination. The GnRH implants were used on every other replicate of 6 semen treatments throughout the study. Fertility was based on embryo recovery by uterine lavage 7 d after ovulation (19). Three liters of a prewarmed (32 to 35OC) modified phosphate-buffered saline solutionf supplemented with 0.1% (w/v011 of bovine serum albuming were used to flush each mare. Immediately after flushing, mares were given a luteolytic d E-Z Mixin-K, Animal Reproduction Systems, Chino, CA 91710. F Deslorelin, Peptide Technology Ltd., Dee Why, Australia. Dulbecco's Phosphate Buffered Saline, Sigma Chemical Company, St. Louis, MO 63178. g Albumin Bovine, Fraction V, United States Biochemical Corp., Cleveland, OH 44122.
960
Theriogenology
dose of prostaglandin FZcrhto shorten estrous cycles. Motion characteristics for spermatozoa used in the breeding trial were analyzed immediately after collection for spermatozoa suspended in NDSMG. At 24 h after initiation of cooling and again after 3 additional hours of incubation at 37OC, motion analyses were conducted for all treatments. Data for motion characteristics of spermatozoa used in the breeding trial were analyzed as a 3 Whenever (extender) by 2 (stallion) factorial experiment. significant differences were found, the Student-Newman-Keuls test recovery data were compared by was used to compare means. Embryo a Chi-square analysis. RESULTS Experiment 1 Rate of cooling had no effect (P>O.O5) on motion characteristics at any time period, nor was there an interaction between cooling rate and seminal treatment. Thus, treatment means for motion characteristics were averaged across cooling rates. There was no effect of seminal treatment (P>O.O5) on motion characteristics at 6 h (Table 2). However, at 24 h, MOT, PMOT and VCL were lower (PaO.05) for spermatozoa in NDSMG+EY than for spermatozoa in other treatments. By 48 h, MOT, PMOT and VCL were less (~<0.05) for spermatozoa in both extenders containing egg yolk than for spermatozoa in extenders not containing egg yolk. The inclusion of glycerol in the NDSMG extender resulted in higher (~<0.05) PMOT than any other treatment for spermatozoa stored for 48 h. Experiment 2 Motion characteristics were similar (P>O.O5) for noncentrifuged spermatozoa extended in NDSMG and for spermatozoa in NDSMGcs (centrifugedwithout removal of seminal plasma). The 2 by 2 factorial analysis (NDSMG, NDSMG+EY vs NDSMGc, EYc), revealed an interaction between extenders and centrifugation, for data averaged across the 2 cooling rates. The interaction was significant (PcO.05) at 6 h for PMOT and VCL, and at 24 and 48 h after initiation of cooling for all parameters analyzed. Means for the 4 treatments are presented in Table 3. MOT and PMOT were lower at 24 and 48 h (PcO.05) for spermatozoa in NDSMG+EY, without seminal plasma removal, than for all other treatments. PMOT at 48 h was also lower (PcO.05) for spermatozoa in NDSMG in which seminal plasma had been removed by centrifugation, than for spermatozoa in NDSMG without centrifugation and spermatozoa in NDSMG+EY in which seminal plasma had been removed. At 24 h, VCL was higher for spermatozoa devoid of seminal plasma and cooled in NDSMG+EY, than for spermatozoa in h LutalYse, Upjohn, The Upjohn Company, Kalamazoo, MI 49001.
961
Theriogenology
most other treatments. In general, at both 24 and 48 h, VCL was less (P
Percentage of total motile spermatozoa (MGT, %I, progressively motile spermatozoa (PMOT, %) and curvilinear velocity (VCL, um/sec) of spermatozoa cooled to 5OC, extended in different diluents Motion
Storage interval (hours)
MOT
Extender
characteristic PMOT
VCL
0
NDSMGa
69
51
138
6
NDSMG NDSMG + EYb NDSMG + GLC NDSMG + EY + GL
56 54 56 55
39
113 111 106 111
24
NDSMG NDSMG + EY NDSMG + GL NDSMG + EY + GL
46d 35e 52d 45d
48
NDSMG NDSMG + EY NDSMG + GL NDSMG + EY + GL
Pooled SEM
2.2
35 37 36
113d 102e 114d 1lOd 2oe 5f 27d 12f
lOad 86e 112d 95e
2.3
3.5
Data were analyzed as a 4 (extender) by 2 (rate) by 8 (stallion) factorial experiment. There was no extender (P>O.O5) by rate interaction; therefore, means were averaged over 2 cooling rates. aNonfat dried skim milk-glucose. bNDSMG + 4% egg yolk (vol/vol). 'NDSMG + 4% glycerol (vol/vol). d-fMeans within time and column bearing different superscripts differ (PxO.05). Motion characteristics of spermatozoa extended in NDSMG but not centrifuged, and NDSMG+EY centrifuged and cooled at both slow and fast rates, are presented in Table 4. MOT was similar (P>O.O5) for spermatozoa among extenders at all time periods. At 48 h, PMOT was lower (P
3
Motion characteristics
of spermatozoa from ejaculates used to
Theriogenology
962
inseminate mares are presented in Table 5. The effect of stallion, but not stallion by extender, was significant (P
Effect of nonfat dried skimmilk-glucose (NDSMG) extender with and without 4% egg yolk (EY) and removal of Seminal plasma on percentage of motile spermatozoa (MOT, %I, progressively motile spermatozoa (PMOT, %) and curvilinear velocity (VCL, pm/set) of cooled stallion spermatozoa
Storage interval (h)
Extender
Seminal plasma removal
Motion characteristic MOT
PMOT
VCL
0
NDSMG NDSMGcsa
No No
71 70
53 55
137 146
6
NDSMG
No Yes No Yes
57 49 51 52
40 33 31 37
129b= 119c 118c 133b
No Yes No Yes
49b 45b 28' 50b
33b 27b 15c 33b
122bC 114c 102d 131b
No Yes No Yes
4ob= 34c ga 46b
24b 16' 2d 27b
12Sb 110= 87a 127b
NDSMG + EY 24
NDSMG NDSMG + EY
48
NDSMG NDSMG + EY
Pooled SEM
2.5
2.3
3.2
Data were analyzed as a 2 (centrifugation) by 2 (extender) by 8 (stallion) factorial experiment (averaging over the 2 cooling rates). There were significant (P
Theriogenology
963
Administration of GnRH agonist had no effect on recovery rate of embryos (P>O.O5). Recovery of embryos was lower (PKO.05) in mares inseminated with spermatozoa in NDSMG+EY or extended Fertility rate per cycles inseminated, based on embryo NDSMG+GL. recovery 7 d after ovulation, were 50% (12/24), 17% (4/24) and 13% (3/24) for mares inseminated with spermatozoa in NDSMG, NDSMG+EY and NDSMG+GL, respectively. Embryo recovery rates for Stallions 1 and 2, respectively, were 50% (6/12) and 50% (6/12) when using NDSMG extender, 8% (l/12) and 25% (3/12) when using NDSMG+EY extender, and 0% (O/12) and 25% (3/12) when using NDSMG+GL extender. Table 4.
Effects of a non-fat dried skim milk-glucose (NDSMG) extender with and without 4% egg yolk (EY), and cooling rates on the percentages of total motile spermatozoa (MOT, %), progressively motile spermatozoa (PMOT, %) and on the curvilinear velocity (VCL, um/sec) of stallion spermatozoa
Storage interval Extender (h)
Motion characteristic
Seminal plasma removal
Rate
MOT
PMOT
VCL
__
71
53
137
0
NDSMG
NO
6
NDSMG
No No Yes Yes
1a IIb I II
58 56 56 49
41 40 41 33
128 129 136 130
No No Yes Yes
I II I II
52 47 45 54
35 31 31 35
119 126 130 132
No NO Yes Yes
I II I II
44 36 46 46
28c 19d 27c 2ac
127 124 122 131
2.5
2.3
3.5
NDSMG+EY 24
NDSMG NDSMG+EY
48
NDSMG NDSMG+EY
Pooled SEM
Data were analyzed as an ANOVA for the 4 combinations of extender and rate. Cooling began at 23OC and proceeded to 20°C at -0.7°C/min. aSemen was cooled at -O.OS°C/min from 20 to 5OC (Rate I). bSemen was cooled at -O.S°C/min from 20 to 5OC (Rate II). CfdMeans within time and column bearing different superscripts were different (P
964 DISCUSSION Previous studies have shown a significant reduction in motion characteristics of stallion spermatozoa when cooled rapidly over the temperatures of cold shock sensitivity (16,20,27). whenmotion characteristics were compared for spermatozoa in the four extenders (Experiment 11, there were no detrimental effects of rapid cooling. Table 5.
Percentage of total motile spermatozoa (MOT, %I, progressively motile spermatozoa (PMOT, %) and curvilinear velocity (VCL, pm/set) of cooled StalliOn spermatozoa in ejaculates used for insemination of mares (averaged over 2 stallions)
Storage interval (hours) 0 24
Motion Extender
MOT
characteristic PMOT
VCL
NDSMG
75
57
108
NDSMG NDSMG + EY NDSMG + GL
45h 59a 4lh
29b 46a 28b
113b 127a 121ah
NDSMG NDSMG + EY NDSMG + GL
41 41 37
14 15 9
61ab 66a 55h
3.0
1.6
Incubation period at 37OC (h) 3
Pooled SEM
2.3
Semen was extended to 25~10~ spermatozoa/ml in non-fat dried skim milk-glucose (NDSMG), NDSMG + 4% egg yolk (EY) or NDSMG + 4% glycerol (GL), and cooled at -O.OS°C/min (n=20 ejaculates per stallion). Semen extended in EY had been previously centrifuged in NDSMG at 50~10~ spermatozoa/ml for removal of seminal plasma. Data were analyzed as a 3 (extender) by 2 (stallion) factorial experiment. abMeans within time and column bearing different superscripts differ (P
Theriogenology
965
The addition of GL to the NDSMG did not protect spermatozoa increased spermatozoa1 motion cooling, except for during The addition of characteristics detected after 48 h of storage. glycerol to a milk-based extender has been shown beneficial for Province et al. (241, storage of bull spermatozoa at 5'C (1). however, reported no advantage for the addition of glycerol to an NUSMG extender when cooling stallion spermatozoa to 5OC. However, spermatozoa were only stored for 24 h in that study. Unlike Jasko et al. (14), we found that removal of seminal plasma and cooling in NDSMG alone resulted in a depression in PMOT of spermatozoa after storage for 48 h. In agreement with Padilla and Foote (211, we concluded that seminal plasma should be Present to provide protection for cooled storage of stallion spermatozoa in Perhaps if Jasko et al. (14) had evaluated a NDSMG extender. longer storage intervals or PMOT, results might have been similar to those of the present study. A level of 20% egg yolk has been previously added to extenders Jasko et al. (121, used to freeze stallion spermatozoa (7). demonstrated maintenance in motion characteristics of stallion spermatozoa when 4% egg yolk was added to a NDSMG extender and when seminal plasma had been removed. In our study, when spermatozoa were centrifuged to remove seminal plasma and NDSMG+EY was added, motion characteristics were higher than for spermatozoa devoid of seminal plasma and diluted in NJXMG alone. Thus, either seminal plasma or EY had to be present in NDSMG extender in order to maintain spermatozoa1 motion characteristics. In contrast, if both seminal plasma and EY were present in the NDSMG extender, motion characteristics of stallion spermatozoa were severely depressed. The mechanism whereby EY and seminal plasma interact to depress motion characteristics was not determined. Perhaps seminal plasma or components of the NDSMG extender resulted in lipid peroxidation and release of free radicals that damaged spermatozoa. Evidence of EY protection was only seen for PMOT after 48 h of storage. PMOT was higher for this treatment than for NDSMG alone when semen had been cooled at the fast rate. Embryo recovery rates of mares inseminated with spermatozoa extended in NDSMG+GL and NDSMG+EY were low. In contrast, fertility rates for mares inseminated with spermatozoa extended in NDSMG and cooled for 24 h were similar to those in previous studies (10,ll). The adverse effects of GL on fertility of mares has been reported Using 7% glycerol in cream-gel extender (9) and Tris extender containing 22.8% EY and 5.25% glycerol In the latter (22). experiment, the investigators did not assess the independent effects of glycerol and egg yolk. Clay et al. (5) reported lower fertility when using an extender containing 20% egg yolk for cooled stallion spermatozoa. Unfortunately, these studies were confounded by multiple factors that did not allow direct assessment of the effects of glycerol and egg yolk. However, based on the results of the Present study, GL or EY, when combined with JVDSMG, resulted in lower fertility. The mechanism for depression of fertility was not determined but may possibly be different for GL and EY. Based on
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966
a preliminary study in our laboratory, fertility rates (embryo recovery) were 69% (27/39) for mares inseminated with semen cooled in a modification of the freezing extender used in France (4). However, only 2% (vol/vol) EY was added to this extender. Whether the composition of the extender or lowered percentage of egg yolk accounted for the higher fertility remains undetermined. Motion characteristics of spermatozoa in ejaculates used to inseminate mares were similar or higher for spermatozoa in NDSMG+GL This or NDSMG+EY, respectively, than for spermatozoa in NDSMG. lack of correlation between spermatozoa1 motility at the time of insemination and fertility was also reported for mares inseminated with TRIS extender (22). In conclusion, although GL or EY (with removal of seminal plasma) added to NDSMG extender did not depress motion characteristics of stallion spermatozoa after cooling and storage for 24 to 48 h, fertility was extremely depressed. REFERENCES Albright JL, Ehlers MH, Erb RE. Motility of bovine spermatozoastored at 5OC when extended in mixtures of yolk-citrate,yolk-glycine,whole milk, skimmilk and alvcerol. J Dairy Sci 1957;41:524-529. 2. Almquist JO, Wickersham EW. Diluents for bovine semen. XII. Fertility and motility of spermatozoain skim-milk with various levels of glycerol and methods of glycerolization. J Dairy Sci 1962;45:782-787. 3. Amann RP. Can the fertility potential of a seminal sample be predicted accurately? J Androl 1989;10:89-98. 4. Amann RP, Pickett BW. Principles of cryopreservationand a review of cryopreservationof stallion spermatozoa. Equine Vet J 1987;7:145-173. 5. Clay CM, Slade NP, Amann RP, Squires EL. Effects of extenders, storage temperature and centrifugationon stallion spermatozoa1motility and fertility. Int Cong Anim Reprod and AI 19&34;10:186-188. 6. Cochran JD, Amann RP, Froman DP. Pickett BW. Effects of centrifuqation, glycerol level, cooling to 5OC, freezing rate and thawing rate on the post-thaw motility of equine sperm. Theriogenology1984:22:25- 38. 7. Cristanelli MJ, Amann RP, Squires EL, Pickett BW. Effects of egg yolk and glycerol levels in lactose-EDTA-eggyolk extender and of freezing rate on the motility of frozen-thawedstallion spermatozoa. Theriogenology 1985;24:681-686. 0. Cristanelli MJ, Squires EL, Amann RP, Pickett BW. Fertility of stallion semen processed, frozen and thawed by a new procedure. Theriogenology 1984;22:39-45. 9. Demick DS, Voss JL, Pickett BW. Effect of cooling, Storage, glycerolizationand spermatozoa1numbers on equine fertility. J Anim Sci 1976;43:633-637. 10. Douglas-HamiltonDH. Burns PJ. Driscoll DD. Viale EM. Fertility and characteristicsof slow-cooled stallion semen. J Reprod Fert Suppl 1987;35:649-650. 11. Douglas-HamiltonDH, 0~01 R, 0901 G, Driscoll D, Noble H. A field study of the fertility of-transportedequine semen. Theriogenology 1984;22:291-304. 1.
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12. Jasko DJ, Hathaway JA, Schaltenbrand VL. Simper WD, Squires EL. Effect Of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa. Theriogenology 1992;37:1241-1252. 13. Jasko DJ, Lein, D.H. and Foote, R.H. A comparison of two computerautomated semen analysis instruments for the evaluation of sperm motion characteristics in the stallion. J. Androl. 11(5):453-459(1990). 14. Jasko DJ, Moran DM, Farlin ME, Squires EL. Effect of seminal plasma dilution or removal on spermatozoa1 motion characteristics of cooled stallion semen. Theriogenology 1991;35:1059-1067. 15. Kayser JPR. Effects of linear cooling rates on motion characteristics of stallion spermatozoa. M.S. Thesis. Colorado State Univ, Fort Collins, CO 80523, 1991. 16. Kayser JP, Amann RP, Shideler RK, Squires EL, Jasko DJ, Pickett BW. Effects of linear cooling rate on motion characteristics of stallion spermatozoa. Theriogenology 1984:22:291-304. 17. KeMev RM. Beraman RV. CooDer WL. Morse GW. Minimal contamination techn&ues for-breeding mares; techniques and preliminary findings. Proc AM Conv Am Assoc Eguine Pratt 1975;21:327-336. 18. McKinnon AO, Squires EL, Voss JL. Ultrasonic evaluation of the mare's reproductive tract-Part I. Compend Contin Educ PractVet1987;9:336-345. 19. McKinnon AO, Squires EL, Voss JL, Cook VM. Equine embryo transfer. Compend Contin Educ Pratt Vet 1988;10:343-353. 20. Moran DM, Jasko DJ, Squires EL, Amann RP. Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa. Theriogenology 1992;38:601-614. 21. Padilla AW. Foote RI-I. Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa. J Anim Sci 1991;69:3308-3313. 22. Pickett BW. Bunvash LD, Voss JL, Back DG. Effect of seminal extenders on equine fertility. J Anim Sci 1975;40:1136-1143. 23. Pickett BW. Squires EL, McKinnon AO. Procedures for Collection, Evaluation and Utilization of Stallion Semen for Artificial Insemination. Colorado State Univ Anim Reprod Lab Bull No 03, Fort Collins, 1987. 24. Province CA, Amann RP, Pickett BW, Squires EL. Extenders for preservation of canine and equine spermatozoa at 5%. Theriogenology 1984;22:409-415. 25. Statistical Analysis System - SAS User's Guide: Statistics. SAS Institute, Cary, NC, 1985;113-137. 26. Unal MB, Bemdtson WE, Pickett BW. Influence of sugars with glycerol on post-thaw motility of bovine spermatozoa in straws. J Dairy Sci 1978;61:83-89. 27. Vamer DD, Blanchard TL, Love CL, Garcia MC, Kenney RM. Effects of cooling rate and storage temperature on equine spermatozoa1 motility parameters. Theriogenology 1988;29:1044-1054. 20. Vamer DD, Blanchard TL, Meyers PJ, Meyers SA. Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20°C. Theriogenology 1989;32:515-525. 29. WatSOn PF. The roles of lipid and protein in the protection of ram spermatozoa at 5% by egg-yolk lipoprotein. J Reprod Fert 1981;62:483492. 30. Watson PF, Plummer JM, Allen WE. Quantitative assessment of membrane damage in cold-shocked spermatozoa of stallions. J Reprod Fert Suppl 1987;35:651-653.