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Effect of different processing techniques on motility and acrosomal integrity of cold-stored stallion spermatozoa
EFFECT OF DIFFERENT PROCESSING TECHNIQUES ON MOTILITY AND ACROSOMAL INTEGRITY OF COLD-STORED STALLION SPERMATOZOA George R. Dawson, MS1; Gary W. Webb, PhD3; Jane A. Pruitt, PhD4; Tom M. Loughin, PhD2; Mark J. Arns, PhD ~
SUMMARY Two experiments were conducted to test whether stallion and/or semen processing techniques influenced spermatozoal motility and acrosomal status following cold storage. Ejaculates from each of 18 stallions (N=54) were collected and split. In Experiment I, a skim milk-glucose extender (SKMG) was added to the semen following a 5, t5 or 30 minute delay post-collection. Following each delay, sperm were packaged at a final concentration of 25 million progressively motile sperm per ml (PMS/mt) in a commercially available skim milk-glucose extender (SKMG). In Experiment II, sperm were packaged at concentrations of 25, 50, and 75 million PMS/ml both in the presence and absence of seminal plasma (SP) utilizing SKMG and SKMG plus PBS, respectively. In both experiments, aliquots were cooled, stored, and the percentage of progressively motile and acrosome intact spermatozoa were determined at 24 and 48 hours post-collection. In Experiment I, delayed dilution resulted in a lower recovery of PMS. In Experiment II, removal of SP resulted in higher percentages of PMS following cold storage. Increasing the concentration of spermatozoa during packaging decreased the percentage of PMS; however, removal of SP reduced the harmful effects on spermatozoa motility. These data suggest that reducing the time that spermatozoa remain in an undiluted state and removal of SP maximize recovery of progressively motile, acrosome-intact spermatozoa. In addition, individualizing the processing techniques for each stallion may enhance spermatozoal survival following cold storage.
INTRODUCTION Despite the research in the area of transported-cooled Author's addresses: 1Department of Animal Sciences and Industry,
2Department of Statistics, Kansas State University, Weber Hall, Manhattan, KS 66506.3Colby Community College, 1255 South Range, Colby, KS 67701. 4Department of Agriculture, Southwest Missouri State University, 901 S. National, Springfield, MO 65804.
Volume 20, Number 3, 2000
stallion semen, confusion still exists as to how stallion spermatozoa should be handled and processed when cooled and stored for 24 to 48 hours. Currently, spermatozoa harvested for cooling are diluted in commercially available skim milk-based extenders at a final packaging concentration of 25 million progressively motile sperm (PMS) per ml. 1 Application of these standards allows many stallions to be utilized efficiently in a transported-cooled semen program; however, these parameters do not optimize spermatozoa survival for all stallions. The donor variability in spermatozoan response to short-term preservation has been addressed. 2,3,4 Moreover, numerous studies have alluded to the negative influences of seminal plasma and especially its detrimental impact on survivability of equine spermatozoa in vitro. 5,6,7 Subsequent studies have shown that SP removal and the incorporation of specific cooling extenders 3,8-1~ are beneficial for post-storage spermatozoa motility, However, these previous studies were done with a limited number of stallions. Therefore, our purpose was to use a large number of stallions to test if either stallion and/or semen processing procedures influence the percentage of progressively motile, acrosomal-intact spermatozoa following 24 and 48 hours of cold storage.
MATERIALS AND METHODS Three ejaculates were collected from each of 18 stallions, all of light horse breeds. Stallions were housed at six different sites. Those stallions that were in an active breeding program were considered to have extragonadal reserves stabilized. For those individuals not currently in an active breeding program, ejaculates were collected either twice in one day or on consecutive days, with one day of sexual rest prior to the experiment. Semen was extended using a commercially available skim milk-glucose extender containing amikacin sulfate." Sterile deionized water was warmed to 37~ and combined with the extender not more than 15 minutes prior to collection. For Experiment II, the SKMG was combined with a commercially available PBS containing glucose and pyruvate.10 Extender was maintained at 37~ until use, and if 191
collection was delayed more than 90 minutes, fresh extender was prepared. The gel-free portion of the ejaculate was harvested and sperm concentration determined manually with a hemacytometer. Noncentrifuged and post-centrifuged semen samples were diluted 1:20 and 1:40, respectively, with formalin. Progressive motility was estimated by phase contrast microscopy (200x). Following processing for both experiments, aliquots were placed into a static cooling device b for 24 hours, removed for evaluation, and then stored in a refrigerator (4-6~ for an additional 24 hours.
Experiment I Ejaculates were split, and aliquots of raw semen were held for 5, 15, and 30 minutes post-collection, with time zero indicating time of artificial vagina removal. After each delay, progressive motility was estimated, and spermatozoa were packaged immediately at a concentration of 25 million PMS/ml in a whirl pak bag. Processed aliquots were held at room temperature until all treatments were complete.
Experiment II Following the 5-minute delay (Experiment I), spermatozoa were packaged at concentrations of 50 and 75 x 106pMS/ml in the SKMG extender. Then, 20 ml of raw semen were divided among four centrifuge tubes (15 ml, conical) each containing an equal volume of SKMG extender. The aliquots were centrifuged for 15 minutes at 300-400 x g. The supernatant was discarded, and each pellet was resuspended and further diluted with SKMG-PBS to yield final concentrations of 25, 50, and 75 x 10 6 PMS/ml. At 24 and 48 hour intervals, aliquots of each respective treatment were diluted in fresh SKMG extender. Dilutions were 1:1, 1:2, or 1:3 (semen:extender), depending on concentration at packaging time. Samples were warmed (37~ for 2 minutes, and progressive motility was estimated using a double-blind procedure. Once motility had been estimated, an additional 250 lal of sample was added to each aliquot to increase the number of sperm available. The aliquots then were centrifuged for 1 minute at 1000 x g. Sperm pellets were resuspended, diluted in 95 % ethanol and incubated at 4-6~ for 30 min to permeabilize membranes. Sperm smears were made on microscope slides, in duplicate, and stored in a dark environment at 4-6~ until evaluation. Dried acrosomal smears were labeled with approximately 30 lat of fluorescein isothiocyanate-labeled Pisum sativum agglntinin (FITC-PSA). c Samples were incubated for 20-30 min at 4-6~ Upon removal, slides were rinsed with distilled water and approximately 25 lal of 1,4-diazabicyclo (2,2,2) octane (DABCO) c was applied. Slides then were refrigerated (4-6 ~C) until evaluation. A total of 100 spermatozoa per slide were evaluated in a double blind procedure. The percentage of spermatozoa with intact acrosomes (PIA) was determined by evaluating the number of sperm that exhibited even fluorescence over the acrosomal region.
192
Statistical Analysis Mixed models were fit to the variables using PROC MIXED in Statistical Analysis System 11 and tested using the appropriate random statement for each experiment. Ejaculates were used as blocks in a split-plot design with repeated measures. Tests for variability of stallion and of various treatment effects across stallion were carried out using Likelihood Ratio tests. F-tests were used for fixed effects. Pairwise comparisons were performed using t-tests. Contrasts comparing linear effects of treatment across SP were computed separately for each period and tested using F-tests.
RESULTS Experiment I Three stallions were removed from this study because of insufficient spermatozoal concentration in the ejaculate (n=l) or failure to recover any PMS following storage (n=2). Therefore, the mean (_+SE) percentages of PMS for 15 stallions following cold storage for 24 and 48 hours are summarized in Table 1. Spermatozoa packaged 5 minutes post-collection exhibited the highest percentage of PMS compared to spermatozoa packaged following a 15 (P<.02) or 30 (P<.005) minute delay. However, no difference was detected (P>.4) between a delay of 15 and 30 minutes. Likewise, immediate processing (5 minutes) tended (P<.06) to support a higher percentage (80_+4.2) of PIA after cold storage than did a delay of 15 minutes (76_+4.2) and did result in a higher (P<.02) percentage of PIA compared to a 30 minute delay (75_+4.2). The percentages of both PMS and PIA declined (P_<.0001) between 24 and 48 hours of cold storage. No differences were detected among farms for PMS (P>.05); however, variations were detected among ejaculates (P<.05) and stallions (P<.05). In addition, differences in PIA occurred among farms and ejaculates (P<.05), but not among stallions (P>.05). Experiment II Table 2 illustrates the effects of spermatozoal concentration and the presence (+SP) or absence (-SP) of seminal plasma on the mean PMS (-+SE) following 24 and 48 hours of cold storage. Overall, the removal of SP was beneficial
Table 1. The percentage (Ismean_+SE) of progressively motile stallion spermatozoa (PMS) following delayed dilution and cold storage for 24 and 48 hours Treatment (min) Storage Period 5 15 30 24 x 30+2.6 a 24+2.6 b 21 +2.8 b 48 y 11+1.7 a 8+1.7 b 9_+1.9 b abMeans within storage period with different superscripts differ (P<.05). XyMean PMS declined within all treatments (P_<.O001).
JOURNAL OF EQUINE VETERINARY SCIENCE
Table 2 , The effects of s p e r m a t o z o a concentration and seminal p l a s m a (SP) on p e r c e n t a g e (Ismean _+SE) of progressively motile stallion spermatozoa (PMS) after 24 and 48 hours of cold storage
Storage Period Seminal Plasma (+) 24x 48Y Seminal Plasma (-) 24 • 48Y
60-
40~ Q_
25
Concentration x 106 50
31 +3.6 a 12_+3.6"
19-+3.7b 2_+3.7b
8-+3.90 0+3.9b
39_+3.6~ 24-+3.6~
36+3.6 ~ 22-+3.6~
35+3.6a 15+3.6 b
75
I
s0J
30 20 10
1
abcMeans within SP (+ or ~) and storage interval with different superscripts differ (P<.05). ~Mean PMS declined (P_<. 0001) within all treatments.
2
3
4
5
6
7
8 9 10 11 12 13 14 15 16 17 18 Stallion
Figure 1, Influence of s t a l l i o n and s e m i n a l p l a s m a on progressively motile s p e r m a t o z o a (PMS) following 24 hours of storage. aSpermatozoa packaged at a concentration of 50 million PMS/ml
(P<.0001). More importantly, after 24 hours of cold storage in the absence of SP, PMS was not adversely (P>.10) affected by increasing spermatozoa concentration. However, concentrations lower than 75 x 106 PMS/ml were beneficial (P<.05) when the storage interval was prolonged. In contrast, increasing the concentration of spermatozoa during packaging depressed (P<.003) PMS in the +SP during the first storage interval. Although the recovery of PMS was low after 48 hours of cold storage in the +SP, more motile spermatozoa were observed at a concentration of 25 x 106 PMS/ml (P<.002) than at higher concentrations. A significant treatment by SP interaction was detected (P=.0016). Figure 1 shows the mean (n=3 ejaculates) percentages of PMS for all stallions following storage for 24 hours in the +SP and - S P when spermatozoa were packaged at a final concentration of 50 x 106 PMS/mi. Mean PIA declined (P-<.0001) with increased storage interval for all treatments (Table 3). In the presence of SP, increasing the concentration of spermatozoa from 25 to 75 x 106 PMS/ml lowered (P<.002) the PIA, but the PIA did not differ (P>.20) between concentrations of 50 and 75 x 106 PMS/ml following either 24 or 48 hours of storage. In the absence of SP, the PIA was similar (P>.3) across treatments after 24 hours of storage. However, after extended storage, more intact acrosomes (78+_4.1) were present when spermatozoa were packaged at a concentration of 25 x 106 Table 3 , The effects of s p e r m a t o z o a concentration and seminal plasma (SP) on percentage (Ismean _+SE) of intact acrosomes (PIA) after 24 and 48 hours of cold storage Storage Period 25 Seminal Plasma (+) 24X 84-+4.1a 48Y 75-+4.1a
Concentration x 106 50
75
77+4.2 b 66_+4.2b
63+4.3 b
82-+4.1a 75-+4.1ab
83-+4.1~ 73-+4.1b
Seminal Plasma (-) 24x 84+4.1 a 48Y 78-+4.1a
76-+4.3 b
abMeans within SP (+ or -) and storage interval with different superscripts differ (P<.05). XYMean PIA decffned (P<-.0001) within all treatments.
Volume 20, Number 3, 2000
PMS/mt (P<.02) compared to a concentration of 75 x 106 PMS/ml (73+_4.1). A significant treatment by SP interaction was detected (P<.004).
DISCUSSION
These results substantiate earlier studies showing that removal of SP is beneficial for the survivability of spermatozoa subjected to cold storage. ~,6,7Traditionally, seminal influences have been minimized through dilution of semen to a concentration of 25 million PMS/ml) Although data in this study, as well as data we have reported previously, 6,8-1~support this dilution rate, a simple dilution to a set concentration does not optimize sperm survival for all stallions, nor does it maximize the efficiency of the technology. ~2 Moreover, previous investigations have shown that removal, or at least reduction, of SP has a beneficial effect on recovery of spermatozoa following cold storage for 24 hours 5,6 or 48 hours. 6 Further, when SP was reduced through centrifugation and the supernatant replaced with a skim milk-salt diluent containing energy substrates, spermatozoal survival, as estimated by progressive motility, was enhanced. 3,8-1~ Moreover, these data suggest that when SP is removed, the negative effects of increasing spermatozoal concentration are reduced. Although the beneficial effect of the modified-skim milk extender has not been fully elucidated, our laboratory has shown that inclusion of energy substrates delivered with a salt mixture is necessary for enhanced recovery. 8,1~Recent work 13 supports our previous findings and further elucidates the beneficial components in skim milk. The data from this study indicate that not all stallions produce spermatozoa capable of withstanding cold storage. However, spermatozoa from some stallions benefited substantially from alternative processing techniques. Further research is needed to investigate the fertility of cold-stored equine spermatozoa when such alternate processing techniques are used.
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FOOTNOTES a EZ Mixin CST, Animal Reproductive Supplies, Chino, CA. b Lane STS, Denver, CO. ~ Chemicals, St. Louis, MO.
REFERENCES 1. Varner DD, Blanchard TL, Love CL, Garcia MC, Kenney RM: Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters. Theriogenology 1987;28(5):709-723. 2. Francl AT, Amann RP, Squires EL, Pickett BW: Motility and fertility of equine spermatozoa in a milk extender after 12 or 24 hours at 20~ Theriogenology 1987;27(3):517-525. 3. Padilla AW, Foote RH: Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa. J Anim Sci 1991 ;69:3308-3313. 4. Katila T: Procedures for handling fresh stallion semen. Theriogenology 1997;48:1217-1227. 5. Jasko DJ, Moran DM, Farlin ME, Squires EL: Effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled stallion semen. Theriogenology
1991 ;35(6):1059-1067. 6. Pruitt JA, Arns-MJ, Pool KC: Seminal plasma influences recovery of equine spermatozoa following in vitro culture (37 ~ C) and cold-storage (5~ C). Theriogenology1993;39:291 (Abstr.). 7. Braun J, Torres-Boggino F, Hochi S, Oguri N: Effect of seminal plasma on motion characteristics of epididymal and ejaculated stallion spermatozoa during storage at 5~ Dtsch tierarztl. Wschr 1994; 101:319-322. 8. Webb GW, Arns MJ: Influence of modified Tyrode's media on motility of cold-stored stallion spermatozoa. J Equine Vet Sci 1995;15:441-444. 9. Webb GW, Arns MJ: Influence of NaVK + ratios on cold-stored equine spermatozoa. Theriogenology 1996:45:312 (Abstr.). 10. Webb GW, Arns MJ: Effect of different salt solutions on the motility of cold-stored stallion spermatozoa. Proc Fifteenth Equine Nutr Physiol Symp, Ft. Worth, TX 1997:283-284. 11. SAS: SAS/STAT ~ Software: Changes and enhancements through release 6.12. 1997 SAS Inst. Inc., Cary, NC. 12. Webb GW, Arns MJ, Pool KC: Sperm concentration influences recovery of motile sperm and number of inseminations shipped in conventional containers. Proc Thirteenth Equine Nutr Physiot Syrup, Gainesville, FL 1993:389-390. 13. Batellier F, Magistrini M, Fauquant J, Palmer E: Effect of milk fractions on survival of equine spermatozoa. Theriogenology 1997;48:391-410.
ACUTE ALTITUDE EXPOSURE (3800 METERS) AND METABOLIC CAPACITY IN THE MIDDLE GLUTEAL MUSCLE OF EQUIDS Holly M. Greene, MS; Steven J. Wickler, DVM, PhD
SUMMARY
Even relatively short exposure to high altitude can produce changes in the metabolic capacity of muscle. This study examined the changes in citrate synthase (CS), 6-hydroxyacyl-CoA-dehydrogenase (HOAD), lactate dehydrogenase (LDH) and total protein (TP) activity of skeletal muscle in equids after acute high altitude exposure (3800 m). The middle gluteal muscles of trained equids (one Quarter Horse, one Shetland pony, and four Arabians) were Authors' addresses: The University of California White Mountain Research Station and California State Polytechnic University, Pomona. Equine Research Center, Pomona, California 91768. Acknowledgements: This project was supported by the Center for Equine Health (formerly the Equine Research Laboratory) with funds provided by the Oak Tree Racing Association, the State of California pari-mutuel wagering fund and contributions by private donors (SJW): Fellowship grant from the University of California White Mountain Research Station (HMG) and a Cal Poly Pomona, Research, Scholarship and Creative Activities Grant.
194
sampled before altitude exposure (225 m) and after nine days of sub-maximal exercise at altitude (3800 m). Muscle biopsies were take from a location 10 cm dorsocaudal to the tuber coxae at an angle of 45 ~ The insertion site was standardized to a depth of 8 cm. Tissue homogenates were assayed for TP and maximal activity of CS, HOAD, and LDH. All samples were run in duplicates and comparisons performed using a paired Student's t-test (significance set at P < 0.05). Altitude did not change CS, HOAD or TP. CS for pre-altitude and altitude averaged 31.2 _+ 2.9 and 32.6 + 4.4 ~moles/g/min, respectively (P = 0.51). HOAD values averaged 17.9 _+ 2.1 and 18.2 + 2.9 tamoles/g/min, respectively (P = 0.85). Altitude acclimatization decreased LDH activity. Pre-altitude and altitude LDH averaged 725.4 + 43.4 and 672.7 + 51.5 ~moles/g/min, respectively (P = 0.04). The decrease in LDH is consistent with decreases in skeletal muscle observed in other mammals at high altitude and suggests that muscles do not become more glycolyctic in altitude hypoxia. JOURNAL OF EQUINE VETERINARY SCIENCE
SUMMARY Two experiments were conducted to test whether stallion and/or semen processing techniques influenced spermatozoal motility and acrosomal status following cold storage. Ejaculates from each of 18 stallions (N=54) were collected and split. In Experiment I, a skim milk-glucose extender (SKMG) was added to the semen following a 5, t5 or 30 minute delay post-collection. Following each delay, sperm were packaged at a final concentration of 25 million progressively motile sperm per ml (PMS/mt) in a commercially available skim milk-glucose extender (SKMG). In Experiment II, sperm were packaged at concentrations of 25, 50, and 75 million PMS/ml both in the presence and absence of seminal plasma (SP) utilizing SKMG and SKMG plus PBS, respectively. In both experiments, aliquots were cooled, stored, and the percentage of progressively motile and acrosome intact spermatozoa were determined at 24 and 48 hours post-collection. In Experiment I, delayed dilution resulted in a lower recovery of PMS. In Experiment II, removal of SP resulted in higher percentages of PMS following cold storage. Increasing the concentration of spermatozoa during packaging decreased the percentage of PMS; however, removal of SP reduced the harmful effects on spermatozoa motility. These data suggest that reducing the time that spermatozoa remain in an undiluted state and removal of SP maximize recovery of progressively motile, acrosome-intact spermatozoa. In addition, individualizing the processing techniques for each stallion may enhance spermatozoal survival following cold storage.
INTRODUCTION Despite the research in the area of transported-cooled Author's addresses: 1Department of Animal Sciences and Industry,
2Department of Statistics, Kansas State University, Weber Hall, Manhattan, KS 66506.3Colby Community College, 1255 South Range, Colby, KS 67701. 4Department of Agriculture, Southwest Missouri State University, 901 S. National, Springfield, MO 65804.
Volume 20, Number 3, 2000
stallion semen, confusion still exists as to how stallion spermatozoa should be handled and processed when cooled and stored for 24 to 48 hours. Currently, spermatozoa harvested for cooling are diluted in commercially available skim milk-based extenders at a final packaging concentration of 25 million progressively motile sperm (PMS) per ml. 1 Application of these standards allows many stallions to be utilized efficiently in a transported-cooled semen program; however, these parameters do not optimize spermatozoa survival for all stallions. The donor variability in spermatozoan response to short-term preservation has been addressed. 2,3,4 Moreover, numerous studies have alluded to the negative influences of seminal plasma and especially its detrimental impact on survivability of equine spermatozoa in vitro. 5,6,7 Subsequent studies have shown that SP removal and the incorporation of specific cooling extenders 3,8-1~ are beneficial for post-storage spermatozoa motility, However, these previous studies were done with a limited number of stallions. Therefore, our purpose was to use a large number of stallions to test if either stallion and/or semen processing procedures influence the percentage of progressively motile, acrosomal-intact spermatozoa following 24 and 48 hours of cold storage.
MATERIALS AND METHODS Three ejaculates were collected from each of 18 stallions, all of light horse breeds. Stallions were housed at six different sites. Those stallions that were in an active breeding program were considered to have extragonadal reserves stabilized. For those individuals not currently in an active breeding program, ejaculates were collected either twice in one day or on consecutive days, with one day of sexual rest prior to the experiment. Semen was extended using a commercially available skim milk-glucose extender containing amikacin sulfate." Sterile deionized water was warmed to 37~ and combined with the extender not more than 15 minutes prior to collection. For Experiment II, the SKMG was combined with a commercially available PBS containing glucose and pyruvate.10 Extender was maintained at 37~ until use, and if 191
collection was delayed more than 90 minutes, fresh extender was prepared. The gel-free portion of the ejaculate was harvested and sperm concentration determined manually with a hemacytometer. Noncentrifuged and post-centrifuged semen samples were diluted 1:20 and 1:40, respectively, with formalin. Progressive motility was estimated by phase contrast microscopy (200x). Following processing for both experiments, aliquots were placed into a static cooling device b for 24 hours, removed for evaluation, and then stored in a refrigerator (4-6~ for an additional 24 hours.
Experiment I Ejaculates were split, and aliquots of raw semen were held for 5, 15, and 30 minutes post-collection, with time zero indicating time of artificial vagina removal. After each delay, progressive motility was estimated, and spermatozoa were packaged immediately at a concentration of 25 million PMS/ml in a whirl pak bag. Processed aliquots were held at room temperature until all treatments were complete.
Experiment II Following the 5-minute delay (Experiment I), spermatozoa were packaged at concentrations of 50 and 75 x 106pMS/ml in the SKMG extender. Then, 20 ml of raw semen were divided among four centrifuge tubes (15 ml, conical) each containing an equal volume of SKMG extender. The aliquots were centrifuged for 15 minutes at 300-400 x g. The supernatant was discarded, and each pellet was resuspended and further diluted with SKMG-PBS to yield final concentrations of 25, 50, and 75 x 10 6 PMS/ml. At 24 and 48 hour intervals, aliquots of each respective treatment were diluted in fresh SKMG extender. Dilutions were 1:1, 1:2, or 1:3 (semen:extender), depending on concentration at packaging time. Samples were warmed (37~ for 2 minutes, and progressive motility was estimated using a double-blind procedure. Once motility had been estimated, an additional 250 lal of sample was added to each aliquot to increase the number of sperm available. The aliquots then were centrifuged for 1 minute at 1000 x g. Sperm pellets were resuspended, diluted in 95 % ethanol and incubated at 4-6~ for 30 min to permeabilize membranes. Sperm smears were made on microscope slides, in duplicate, and stored in a dark environment at 4-6~ until evaluation. Dried acrosomal smears were labeled with approximately 30 lat of fluorescein isothiocyanate-labeled Pisum sativum agglntinin (FITC-PSA). c Samples were incubated for 20-30 min at 4-6~ Upon removal, slides were rinsed with distilled water and approximately 25 lal of 1,4-diazabicyclo (2,2,2) octane (DABCO) c was applied. Slides then were refrigerated (4-6 ~C) until evaluation. A total of 100 spermatozoa per slide were evaluated in a double blind procedure. The percentage of spermatozoa with intact acrosomes (PIA) was determined by evaluating the number of sperm that exhibited even fluorescence over the acrosomal region.
192
Statistical Analysis Mixed models were fit to the variables using PROC MIXED in Statistical Analysis System 11 and tested using the appropriate random statement for each experiment. Ejaculates were used as blocks in a split-plot design with repeated measures. Tests for variability of stallion and of various treatment effects across stallion were carried out using Likelihood Ratio tests. F-tests were used for fixed effects. Pairwise comparisons were performed using t-tests. Contrasts comparing linear effects of treatment across SP were computed separately for each period and tested using F-tests.
RESULTS Experiment I Three stallions were removed from this study because of insufficient spermatozoal concentration in the ejaculate (n=l) or failure to recover any PMS following storage (n=2). Therefore, the mean (_+SE) percentages of PMS for 15 stallions following cold storage for 24 and 48 hours are summarized in Table 1. Spermatozoa packaged 5 minutes post-collection exhibited the highest percentage of PMS compared to spermatozoa packaged following a 15 (P<.02) or 30 (P<.005) minute delay. However, no difference was detected (P>.4) between a delay of 15 and 30 minutes. Likewise, immediate processing (5 minutes) tended (P<.06) to support a higher percentage (80_+4.2) of PIA after cold storage than did a delay of 15 minutes (76_+4.2) and did result in a higher (P<.02) percentage of PIA compared to a 30 minute delay (75_+4.2). The percentages of both PMS and PIA declined (P_<.0001) between 24 and 48 hours of cold storage. No differences were detected among farms for PMS (P>.05); however, variations were detected among ejaculates (P<.05) and stallions (P<.05). In addition, differences in PIA occurred among farms and ejaculates (P<.05), but not among stallions (P>.05). Experiment II Table 2 illustrates the effects of spermatozoal concentration and the presence (+SP) or absence (-SP) of seminal plasma on the mean PMS (-+SE) following 24 and 48 hours of cold storage. Overall, the removal of SP was beneficial
Table 1. The percentage (Ismean_+SE) of progressively motile stallion spermatozoa (PMS) following delayed dilution and cold storage for 24 and 48 hours Treatment (min) Storage Period 5 15 30 24 x 30+2.6 a 24+2.6 b 21 +2.8 b 48 y 11+1.7 a 8+1.7 b 9_+1.9 b abMeans within storage period with different superscripts differ (P<.05). XyMean PMS declined within all treatments (P_<.O001).
JOURNAL OF EQUINE VETERINARY SCIENCE
Table 2 , The effects of s p e r m a t o z o a concentration and seminal p l a s m a (SP) on p e r c e n t a g e (Ismean _+SE) of progressively motile stallion spermatozoa (PMS) after 24 and 48 hours of cold storage
Storage Period Seminal Plasma (+) 24x 48Y Seminal Plasma (-) 24 • 48Y
60-
40~ Q_
25
Concentration x 106 50
31 +3.6 a 12_+3.6"
19-+3.7b 2_+3.7b
8-+3.90 0+3.9b
39_+3.6~ 24-+3.6~
36+3.6 ~ 22-+3.6~
35+3.6a 15+3.6 b
75
I
s0J
30 20 10
1
abcMeans within SP (+ or ~) and storage interval with different superscripts differ (P<.05). ~Mean PMS declined (P_<. 0001) within all treatments.
2
3
4
5
6
7
8 9 10 11 12 13 14 15 16 17 18 Stallion
Figure 1, Influence of s t a l l i o n and s e m i n a l p l a s m a on progressively motile s p e r m a t o z o a (PMS) following 24 hours of storage. aSpermatozoa packaged at a concentration of 50 million PMS/ml
(P<.0001). More importantly, after 24 hours of cold storage in the absence of SP, PMS was not adversely (P>.10) affected by increasing spermatozoa concentration. However, concentrations lower than 75 x 106 PMS/ml were beneficial (P<.05) when the storage interval was prolonged. In contrast, increasing the concentration of spermatozoa during packaging depressed (P<.003) PMS in the +SP during the first storage interval. Although the recovery of PMS was low after 48 hours of cold storage in the +SP, more motile spermatozoa were observed at a concentration of 25 x 106 PMS/ml (P<.002) than at higher concentrations. A significant treatment by SP interaction was detected (P=.0016). Figure 1 shows the mean (n=3 ejaculates) percentages of PMS for all stallions following storage for 24 hours in the +SP and - S P when spermatozoa were packaged at a final concentration of 50 x 106 PMS/mi. Mean PIA declined (P-<.0001) with increased storage interval for all treatments (Table 3). In the presence of SP, increasing the concentration of spermatozoa from 25 to 75 x 106 PMS/ml lowered (P<.002) the PIA, but the PIA did not differ (P>.20) between concentrations of 50 and 75 x 106 PMS/ml following either 24 or 48 hours of storage. In the absence of SP, the PIA was similar (P>.3) across treatments after 24 hours of storage. However, after extended storage, more intact acrosomes (78+_4.1) were present when spermatozoa were packaged at a concentration of 25 x 106 Table 3 , The effects of s p e r m a t o z o a concentration and seminal plasma (SP) on percentage (Ismean _+SE) of intact acrosomes (PIA) after 24 and 48 hours of cold storage Storage Period 25 Seminal Plasma (+) 24X 84-+4.1a 48Y 75-+4.1a
Concentration x 106 50
75
77+4.2 b 66_+4.2b
63+4.3 b
82-+4.1a 75-+4.1ab
83-+4.1~ 73-+4.1b
Seminal Plasma (-) 24x 84+4.1 a 48Y 78-+4.1a
76-+4.3 b
abMeans within SP (+ or -) and storage interval with different superscripts differ (P<.05). XYMean PIA decffned (P<-.0001) within all treatments.
Volume 20, Number 3, 2000
PMS/mt (P<.02) compared to a concentration of 75 x 106 PMS/ml (73+_4.1). A significant treatment by SP interaction was detected (P<.004).
DISCUSSION
These results substantiate earlier studies showing that removal of SP is beneficial for the survivability of spermatozoa subjected to cold storage. ~,6,7Traditionally, seminal influences have been minimized through dilution of semen to a concentration of 25 million PMS/ml) Although data in this study, as well as data we have reported previously, 6,8-1~support this dilution rate, a simple dilution to a set concentration does not optimize sperm survival for all stallions, nor does it maximize the efficiency of the technology. ~2 Moreover, previous investigations have shown that removal, or at least reduction, of SP has a beneficial effect on recovery of spermatozoa following cold storage for 24 hours 5,6 or 48 hours. 6 Further, when SP was reduced through centrifugation and the supernatant replaced with a skim milk-salt diluent containing energy substrates, spermatozoal survival, as estimated by progressive motility, was enhanced. 3,8-1~ Moreover, these data suggest that when SP is removed, the negative effects of increasing spermatozoal concentration are reduced. Although the beneficial effect of the modified-skim milk extender has not been fully elucidated, our laboratory has shown that inclusion of energy substrates delivered with a salt mixture is necessary for enhanced recovery. 8,1~Recent work 13 supports our previous findings and further elucidates the beneficial components in skim milk. The data from this study indicate that not all stallions produce spermatozoa capable of withstanding cold storage. However, spermatozoa from some stallions benefited substantially from alternative processing techniques. Further research is needed to investigate the fertility of cold-stored equine spermatozoa when such alternate processing techniques are used.
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FOOTNOTES a EZ Mixin CST, Animal Reproductive Supplies, Chino, CA. b Lane STS, Denver, CO. ~ Chemicals, St. Louis, MO.
REFERENCES 1. Varner DD, Blanchard TL, Love CL, Garcia MC, Kenney RM: Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters. Theriogenology 1987;28(5):709-723. 2. Francl AT, Amann RP, Squires EL, Pickett BW: Motility and fertility of equine spermatozoa in a milk extender after 12 or 24 hours at 20~ Theriogenology 1987;27(3):517-525. 3. Padilla AW, Foote RH: Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa. J Anim Sci 1991 ;69:3308-3313. 4. Katila T: Procedures for handling fresh stallion semen. Theriogenology 1997;48:1217-1227. 5. Jasko DJ, Moran DM, Farlin ME, Squires EL: Effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled stallion semen. Theriogenology
1991 ;35(6):1059-1067. 6. Pruitt JA, Arns-MJ, Pool KC: Seminal plasma influences recovery of equine spermatozoa following in vitro culture (37 ~ C) and cold-storage (5~ C). Theriogenology1993;39:291 (Abstr.). 7. Braun J, Torres-Boggino F, Hochi S, Oguri N: Effect of seminal plasma on motion characteristics of epididymal and ejaculated stallion spermatozoa during storage at 5~ Dtsch tierarztl. Wschr 1994; 101:319-322. 8. Webb GW, Arns MJ: Influence of modified Tyrode's media on motility of cold-stored stallion spermatozoa. J Equine Vet Sci 1995;15:441-444. 9. Webb GW, Arns MJ: Influence of NaVK + ratios on cold-stored equine spermatozoa. Theriogenology 1996:45:312 (Abstr.). 10. Webb GW, Arns MJ: Effect of different salt solutions on the motility of cold-stored stallion spermatozoa. Proc Fifteenth Equine Nutr Physiol Symp, Ft. Worth, TX 1997:283-284. 11. SAS: SAS/STAT ~ Software: Changes and enhancements through release 6.12. 1997 SAS Inst. Inc., Cary, NC. 12. Webb GW, Arns MJ, Pool KC: Sperm concentration influences recovery of motile sperm and number of inseminations shipped in conventional containers. Proc Thirteenth Equine Nutr Physiot Syrup, Gainesville, FL 1993:389-390. 13. Batellier F, Magistrini M, Fauquant J, Palmer E: Effect of milk fractions on survival of equine spermatozoa. Theriogenology 1997;48:391-410.
ACUTE ALTITUDE EXPOSURE (3800 METERS) AND METABOLIC CAPACITY IN THE MIDDLE GLUTEAL MUSCLE OF EQUIDS Holly M. Greene, MS; Steven J. Wickler, DVM, PhD
SUMMARY
Even relatively short exposure to high altitude can produce changes in the metabolic capacity of muscle. This study examined the changes in citrate synthase (CS), 6-hydroxyacyl-CoA-dehydrogenase (HOAD), lactate dehydrogenase (LDH) and total protein (TP) activity of skeletal muscle in equids after acute high altitude exposure (3800 m). The middle gluteal muscles of trained equids (one Quarter Horse, one Shetland pony, and four Arabians) were Authors' addresses: The University of California White Mountain Research Station and California State Polytechnic University, Pomona. Equine Research Center, Pomona, California 91768. Acknowledgements: This project was supported by the Center for Equine Health (formerly the Equine Research Laboratory) with funds provided by the Oak Tree Racing Association, the State of California pari-mutuel wagering fund and contributions by private donors (SJW): Fellowship grant from the University of California White Mountain Research Station (HMG) and a Cal Poly Pomona, Research, Scholarship and Creative Activities Grant.
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sampled before altitude exposure (225 m) and after nine days of sub-maximal exercise at altitude (3800 m). Muscle biopsies were take from a location 10 cm dorsocaudal to the tuber coxae at an angle of 45 ~ The insertion site was standardized to a depth of 8 cm. Tissue homogenates were assayed for TP and maximal activity of CS, HOAD, and LDH. All samples were run in duplicates and comparisons performed using a paired Student's t-test (significance set at P < 0.05). Altitude did not change CS, HOAD or TP. CS for pre-altitude and altitude averaged 31.2 _+ 2.9 and 32.6 + 4.4 ~moles/g/min, respectively (P = 0.51). HOAD values averaged 17.9 _+ 2.1 and 18.2 + 2.9 tamoles/g/min, respectively (P = 0.85). Altitude acclimatization decreased LDH activity. Pre-altitude and altitude LDH averaged 725.4 + 43.4 and 672.7 + 51.5 ~moles/g/min, respectively (P = 0.04). The decrease in LDH is consistent with decreases in skeletal muscle observed in other mammals at high altitude and suggests that muscles do not become more glycolyctic in altitude hypoxia. JOURNAL OF EQUINE VETERINARY SCIENCE