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Effects of Scrotal Insulation on Viability Characteristics of Cryopreserved Bovine Semen1
Effects of Scrotal Insulation on Viability Characteristics of Cryopreserved Bovine Semen'
c. J.
VOGLER, R. G. SAACKE,~J. H. BAME, J. M. DWARNETTE,~ and Y. L. MCGlLLlARD Department of Dairy Sdence Virginia Polytechnic Institute and State University Blacksburg 24061
both viability measurements, but only after incubation for 3 h at 37'C postthaw (P < .05). We conclude that epididymal sperm are adversely affected by elevated testicular temperatures, as noted by their decreased ability to maintain motility and acrosomal integrity following cryopreservation. (Key words: bovine, thermal stress, semen viability, frozen semen)
ABSTRACT
The effect of a 48-h scrotal insulation on spermatozoal viability (motility and acrosomal integrity), before and after semen cryopreservation, was studied in six young Holstein bulls whose semen was collected twice in succession at 3 d intervals. Motility and acrosomal integrity were measured before and after incubation of semen at 37'C for 3 h. For assessment of results, collection days were grouped: period 1 (control) = d -6, -3, and 0, where d 0 = initiation of scrotal insulation after semen collection; period 2 = d 3,6, and 9 (sperm presumed in the epididymis or rete testis during scrotal insulation); period 3 = d 12, 15,. . . 3 9 (sperm presumed in spermatogenesis during scrotal insulation). Semen was cryopreserved each collection day until morphologically abnormal cells exceeded 50% of the ejaculate (d 12 to 21). Semen viability before and after freezing was lower in period 3 than in period 1 (P < .05). These differences coincided with the appearance in period 3 of abnormal sperm morphology and depressed undiluted semen motility, which began on d 12 (P < .01). Semen collected during period 2 that was extended but unfrozen did not differ from that collected during period 1 in morphology or viability. However, for frozen semen, period 2 was significantly poorer than period 1 for
Abbreviation key: PIA = percentage of intact acrosomes, SI = scrotal insulation. INTRODUCTION
The classical studies of Williams and Savage (18) and Lagerlof (6) drew sharp attention
Received M a y 9, 1991. Accepted June 14, 1991. 'Research supported by Select Sires Inc.. Plain City, OH and the National Association of Animal Breeders, Columbia, MO. %o whom reprint requests should be addressed. 3 ~ c n address: t Select sires ~nc.,plain city, OH 43064. 1991 J Dairy Sci ?4:3827-3835
to the currently supported concept that fertile bulls have more viable sperm and a consistently lower incidence of morphologically abnormal sperm than sterile or subfertile bulls. The adverse effects of elevated temperatures on sptrmatogenesis have been well documented for the bull (3, 4, 7, 11, 14, 16) and consist of impaired efficiency of spermatogenesis, as reflected by increased sperm abnormalities and reduced sperm o u t p t and viability. Although previous reports indicated that epididymal sperm were not affected by elevated environmental or testicular temperatures (1, 11, 14), in these studies, only the undiluted semen from the stressed animals was evaluated. On the contrary, Wildeus and Entwistle (17) revealed a possible effect of heat on morphology of caput epididymal sperm. For semen that is to be used in AI, it is important to know whether heat stress renders sperm in the epididymis at the time of the stress more susceptible to injury from subsequent semen extension and cryopreservation. The overall objective of this study was to determine the effects of heat insult to the bull testis by scrotal insulation (SI) on ability of spermatozoa to be cryopreserved.
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the sack was installed (immediately after readjustment) and just before removal. Obtaining Anlmals and Semen Collection scrotal surface temwratures before emplacement of the sack was deemed uninfoAative Six Holstein bulls, individually penned in a because of interference from the variable amcold-housing, counter-slope barn, were se- bient temperatures throughout the experiment lected on their ability to produce semen having and resulting differences in the state of scrotal greater than 70% morphologically normal retraction and thickness. sperm with 60% or greater estimated progresFollowing final placement of the sack, bulls sive motility. Five bulls ranged in age from 14 were partially restrained by halter for 48 h SO to 17 mo, and one bull was 34 mo of age. that they had freedom to lie down, stand, eat, Collection of semen was by artificial vagina. and drink. This partial restraint was designed Two ejaculates were collected in succession, to prevent premature removal of the sacks and and each ejaculate was preceded by a period of ensure proper bedding, dry conditions, and sexual preparation consisting of two false general comfort during the thermal insult. mounts separated by 2 min of restraint. MATERIALS AND METHODS
Thermal Insult to the Testes
The purpose of the SI was to create a thermal insult to the testes, mimicking a naturally occurring environmental interference with testicular thermoregulation. To minimize potential stress from excessive intervention, no effort was made to establish or maintain a specific elevation in testicular temperature during the SI. Thermal insult of the testis was induced by enclosing the scrotum with a sack constructed of insulated material held in place by VELCRO@ Brand fasteners (VJ5LCRO@' Brand Fastening Systems, Manchester, NH) and medical tape. Scrotal sacks were fashioned from two layers of waterproofed nylon taffeta filled with a l c m insulating layer of polyester batting. The layers were machine quilted together and then sewn into a sack. After the first 30 min of SI, allowing for thermal response of the scrotum to the elevated temperature, the sack was readjusted to ensure complete coverage of the scrotum and scrotal neck up to the body wall. The sack was fitted to be loose enough not to interfere with circulation but sufficiently secure to avoid removal or slippage. The SI was maintained for 48 h. Bulls were placed on experiment at different times of the year, specifically, June, September, and October 1989 and February 1990. These months were chosen to avoid excessively high summer temperatures. Scrotal surface temperatures at the caudal midpoint of the testes were recorded using a type k flat thermistor probe, attached to a digital thermocouple thermometer (Cole-Pmer Instruments, Chicago, IL).Measurements were made when Journal of Dairy Science Vol. 74. No. 11, 1991
Experimental Design
Throughout the study, semen was collected at a frequency of two ejaculates in succession at 3-d intervals. Collection at this frequency began 2 wk prior to initiation of the experiment in order to stabilize epididymal sperm reserves and semen characteristics. Evaluation of undiluted semen characteristics (viability and morphology) and sperm output per collection day began 6 d prior to SI and continued until d 39 after initiation of SI. To evaluate the vulnerability to heat stress of spermatozoa in the epididymis and rete testis compared with those in stages of spermatogenic development (seminiferous tubules), three periods of semen collection were defined: period 1 = preinsult, i.e., collections on d -6, -3, and 0, where d 0 was the day of scrotal sack installation after semen collection; period 2 = postinsult, d +3, +6, and +9 after installation of the sack; period 3 = postinsult, d +12, +15, and +18 through +39. The basis for these three groups was to separate semen collections into a control period (period l), a period in which sperm present in the epididymis or rete testis at the time of heat insult would be ejaculated (period 2), and a period in which cells undergoing spermatogenesis at the time of the insult would be ejaculated (period 3). Semen was frozen from each collection day throughout periods 1 and 2 and up to the point when greater than 50% abnormal sperm were obtained in period 3. of Undiluted Semen
Most important was the assessment of response of bulls to the thermal stress imposed
HEAT STRESS AND FROZEN SEMEN
by SI. This was particularly important because neither accurate control nor measurement of intratesticular temperature was possible. In addition, response of bulls to a given thermal stress would also be expected to vary. On this basis, the undiluted semen characteristics and sperm output per collection day were the criteria for establishing the functional response of the bull to the thermal insult. Immediately after collection, the volume, concentration, and progressive motility of each ejaculate were measured. Concentration was determined using a spectrophotometer calibrated for bovine sperm using a hemacytometer. Motility was estimated to the nearest 10% after viewing several microscopic fields at 25Ox magnification using a phase contrast microscope equipped with a heated stage (37°C). Sperm abnormalities were quantified using differential interference contrast microscopy at 1250x magnification. One hundred microliters of semen from a pool of the two ejaculates of each collection day were fixed in 1 ml of Karnovsky’s fixative (5) and placed in a 1.5-ml Eppendorf tube. On the following day, differential counts of 100 cells from each of two wet smears of the fixed sample were averaged. Cells with multiple abnormalities were only counted once, but each abnormality of a particular cell was recorded. Thus, preemption of one cell abnormality by another was avoided. On this basis, actual percentages of abnormal heads, protoplasmic droplets, and abnormal tails were reported and could be collectively greater than the total percentage of abnormal cells in the sample. Specific cell abnormalities were identified by the classification of Mitchell et al. (8) and Barth and Oko (2). Semen Freezing
On each collection day, the two ejaculates of each bull were pooled and prepared in a conventional manner (10) for cryopreservation with egg yolk-citrate extender containing 7% glycerol (voVvol) at 50 x 106 celldml. Prior to semen dilution, extender was clarified by filtering through a glass prefilter connected to a .45-pm Acrodisc filter (Gelman Sciences, Ann Arbor, MI). Semen was packaged in .5-ml French straws (Instruments de MWecine
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V&6rinaire, l’Aigle, France), frozen 24 f 2 h after collection in static liquid nitrogen vapor for 10 min, and then plunged into liquid nitrogen. Freeze rates during the vapor freeze were monitored and were within the optimal range reported by Robbins et al. (10) for egg yolkcitrate-glycerol extended semen packaged in .5-ml straws. Straws were stored in liquid nitrogen until semen evaluation. Evaluatlon of Extended Semen Before and After Freezing
Prior to freezing (23 f 3 h after collection), the contents of three straws per bull from each collection day were pooled into a 1.5-mlEppendorf tube (Brinkman Instruments, Inc., Westbury, NY), warmed from 5 to 37°C using a dry bath, and evaluated for viability @ogressive motility and acrosomal integrity). Evaluation was conducted within 2 min of warming (0 h) and again after a 3-h incubation at 37°C. Percentage of progressive motility was estimated to the nearest 10% at 250x magnification using a phase contrast microscope equipped with a heated stage at 37°C. Acrosomal integrity was determined at the same incubation intervals and on the same smears using a differential interference contrast microscope at 1 2 5 0 ~magnification. Percentage of intact acrosomes (PIA) was based on the presence of the apical ridge of the acrosome (12) and was determined by averaging two direct counts of 100 cells from separate wet smears. After freezing and storage of semen at -196°C for at least 2 wk, three straws per bull from each collection day were thawed in water at 35°C for 45 s, and the contents were pooled and evaluated for viability using the same procedure as described for prefrozen extended semen. Frozen semen was coded so that the evaluator did not know the bull or collection day. For unfrozen semen, the bull was unknown to the evaluator. Statistical Analysis
All statistical analyses were conducted with programs available from SAS (13). Data were analyzed by the general linear models proceJournal of Dairy Science Vol. 74, No. 11, 1991
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VOGLW ET AL.
dure as well as nonorthogonal contrasts of period 1 with period 2 and period 1 with period 3 to detect changes in viability measurements, morphology, and sperm output. Because semen was frozen only until total sperm abnormalities reached 5096, for extended semen before and after cryopreservation, period 3 consisted of d y d 12 for two bulls, d 12 and 15 for two bulls, d 12, 15, and 18 for one bull, and d 12 through 21 for one bull. The model used for analysis of variance for undiluted semen characteristics, initial motility, and morphology, as well as sperm output per collection day, was as follows:
Pj = the fixed effect of the period j (j = 1, 2, or 3), Fk = the fixed effect of freeze k (k = 1, frozen or 2, unfrozen), HI = the fixed effect of hour 1 of incubation (l= 1 , 0 h or 2, 3 h), and q p ~= random error. Interactions with bull served as error terms for fixed effects. RESULTS
Mean testicular surface temperature, obtained at the caudal midpoint of the testis after adjustment of the sack and again before sack removal, was 34.8'C and ranged from 33.3 to 36.4'C. Ambient temperature and relative humidity during the experiment ranged from 4.9 to 33.7'C and from 29.7 to 93.9%,respectivewhere ly. Response of bulls to the thermal insult Yi,k = percentage of initial motility, per- based upon undiluted semen quality and sperm centage of abnormal sperm, or output is presented in Figure 1. There was a sperm output per collection day notable increase in total sperm abnormalities as well as a decrease in sperm motility in (x 104; period 3 compared with period 1 (P < .01). p = overall mean; Bi = the random effect of the bull i (i The motility was depressed 10 to nearly 20 percentage units, most apparent on d 15 and 18 = 1, 2, 3 , . .6); Pj = the fixed effect of the period j (j postinsult. The abnormal sperm content (abnormal heads) was elevated beginning d 12, = 1, 2, or 3); peaked at d 18 (nearly 70% above base), and Dg)k = the fixed effect of the day k persisted longer than did the depressed motiliwithin period j (k = 1,2,3, or 1, ty. Ejaculate content of abnormal sperm was 2, 3 , . . 10); and approaching preinsult levels at the termination eijk = random error. of the experiment, d 39 postinsult. Periods 1 and 3 did not differ in sperm output per collecThe model used for analysis of variance for tion day. Periods 1 and 2 did not differ in PIA and percentage of progressive motility of sperm morphology, motility, or sperm output extended semen evaluated before and after per collection day. cryopreservation was The types of sperm abnormalities associated with the significant increase in the abnormal population during period 3 were predominantly those involving the sperm head as contrasted with spermatozoa with abnormal tails or protoplasmic droplets (Figure 2). The head abnormalities most frequently encountered were decapitated sperm, diadem defects, cratered where sperm, and pyriform heads. Of importance was the relative stability of sperm morphology up Yiw = percentage of motile spermatozoa to d 9 postinsult, followed by an abrupt inor PIA, crease in abnormal heads on d 12. The apparp = overall mean, ent increase in seminal content of sperm with Bi = the random effect of the bull i (i abnormal tails between d 21 and 30 was not = 1, 2 , 3 ,... 6), significant.
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Journal of Dairy Science Vol. 74. No. 11, 1991
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HEAT STRESS AND FROZEN SEMEN ~~
I I
90 8C 70
I I
I
I I I
T T
A
60 50 40 30
20 IO 90
gl + i 8 7
I
I I
I
C
I
I I
I
6 5 4 3
2
I Day ,-6 -3 0 I 3 6 9 (12 15 18 21 2427 3 0 3 3 3 6 3g1 Period I Period 2 Period 3 pigru.e 1. Effects of 48-h scrotal insalation (d 0, p d o d 1) on undiluted semen sperm morphology (A),motility @). and sperm output p a collection day (C). Means f SE. n = 6.
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L Q)
80 70 60
n m
-
0 0 0 c c
n c
a a
50
2 40 oal 6 2 30
5 1 t
LL
Thermal insult
20 IO 0
Day
';6'-3' 0 ' 3' 6' 9'12'15'18~24~7'30'33'36'39' I I I Period I
Period 3
Period2
Abnormal heads
Protoplasmic droplets
I Abnormal fails
Figure 2. Effect of 48-h scrotal insulation (d 0. period 1) on incidence of abnormal sperm heads, protoplasmic droplets, and abnormal sperm tails. Only the abnomal sperm beads differed sigdicantly during peaiod 3 (P i.01).
The effect of the heat insult on the viability characteristics (sperm motility and acrosomal integrity) of unfrozen and frozen extended semen am presented in Tables 1 and 2, respectively. For unfrozen semen, a decrease in percentage of viable sperm was observed for period 3 relative to period 1 for both parameters at 0 h and again at 3 h of 37'C incubation (P < .05). On the contrary, unfrozen sperm ejaculated during period 2 were not different in viability from those ejaculated during period 1 for either measurement or incubation period. For frozen semen, period 3 was
again expectedly inferior to period 1 relative to percentage of motile cells and PIA at both incubation intervals. In addition, and differently from unfrozen semen, the frozen semen obtained during period 2 exhibited lower motility and acrosomal integrity after 3 h of incubation at 37'C than did semen collected during period 1 (P < .OS). Although the depression of viability during period 2 was Iimited to the frozen semen incubated for 3 h postthaw, the effect of the thermal insult appeared to become evident in an additive fashion as semen was stressed. To
TABLE 1. Effect of scrotal insulation (SI) for 48 h (d 0, period 1) on percentage of motility and percentage of intact acrosomes (F'IA) of unfrozen extendcd semen after storage for 1 d at 5'C (0h) and incubation at 37'C for 3 h (n = 6). Incubation time at 37'C Oh
3h
Period
Motility
PLA
Motility
PIA
1 2 3
66.4 62.5 41.1"
86.0 822 62R
56.1 47.1 25.la
79.6 74.6 49.9
~
~~~~
%Berent from pcriod 1 (P c .05); SEM motility = 2.9; SEM PIA = 1.6. 'Period 1 = d -6, -3.0 @reinsult) with 48 h of SI besinningon d 0. Period 2 = d 3,6,9(postinsult). Period 3 = d 12, 15, 18,and 21 or until ejaculate contained greater than 50% abnormal sperm. Day 12 only (2 bulls); d 12 and 15 (two bulls); d 12, 15, and 18 (one bull); d 12, 15, 18, and 21 (one boll). Journal of Dairy Science Vol. 74, No. 11. 1991
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HEAT STRESS AND FROZEN SEMEN
TABLE 2. Effect of scrotal insulation (SI) for 48 h (d 0, period 1) on percentage of motility and percentage of hlact acrosomes (PIA) of frozen semen after tbawing (0 h) and 3 h of incubation at 37'C (n = 6). Incubation time at 37'C ~~
~
Oh
3 h
Period1
Motility
PIA
Motility
PIA
1 2 3
55.3 48.6 37.8'
79.3 73.8 54.3'
46.4 30.8' 12.1"
73.O 62.8' 4Q.7a
%ifferent from period 1 (P < .05); S E M motility = 2.9; SEM PIA = 1.6. 'Period 1 = d -6, -3.0 @reinsalt) with 48 h of SI beginningon d 0. period 2 = d 3 . 6 . 9 (postinsult). Period 3 = d 12, 15, 18, and 21 or until ejaculate contained greater than 50% abnormal spenn. Day 12 only (mballs); d 12 and 15 (two bulls); d 12, 15, and 18 (one bull); d 12, 15, 18, and 21 (om ball).
aid in visualizing this additive effect, the data have been summarized in Table 3. As may be noted, progressing from the difference in viability of sperm ejaculated in period 2 versus period 1 for undiluted semen to that for extended unfrozen and frozen semen with and without incubation at 37'C for 3 h, the greater the stress factors applied to the semen (i.e., extension, freezing, and incubation), the greater the difference in viability of semen ejaculated during period 2 relative to period 1.
DISCUSSION
The response to SI achieved in this experiment based on undiluted semen is best described in Figures 1 and 2. The insult was characterized by no change in sperm output per collection day throughout the experiment (all periods), a relatively small decrease in motility (10 to nearly 20%) 12 to 18 d postinsult (at the beginning of period 3), and a distinct increase in abnormal heads beginning at the same time (d 12) and peaking at d 18 (nearly 70% above
TABLE 3. Comparative effect of 48-hscrotal insulation administered at the end of period 1 on percentage of viability of semen ejaculated during the following 9 d (period 2) based upon evaluation of undiluted semen and extended semen before and after freezing with (3 h) and without (0 h) incubation at 37'C.
Diffennce Semen
Incubation at 37'C
Viabdity measurement1
paid 12
Period 2
(period 1 period 2)
~ndilntd unfrozafl
0 h (only) Oh
Motility Motility
70.0 66.4
67.8 62.5 82.2 48.6 73.6 47.1 74.6 30.8 62.8
-2.2 -3.9 -3.8 -6.7 -5.5 -9.0 -5 .o -15.6 -10.2
1 7 ~ ~ ~ 5 Oh
PIA Motility
PIA UnfroZen
3 h
Prom
3 h
Motility PIA Motilitv
PIA
86.0 55.3 79.3 56.1 79.6 46.4 73.0
'Viability measurement, percentage of motility (estimated). and percentage of intact acrosomes (PIA), direct count. *Period 1 = d -6, -3. 0 @reinsult) with 48 h of scrotal iosnlation beginning on d 0. Period 2 = d +3, +6, +9 (postinsult). 3~0tilityevaluated immediately after collection. %Jnfrozen extended semen evaluated after 22 to 24 h of storage at 5'C (just prior to freezing). h o z e n extended semen evaluated after 2 wk of storage at -196'12
J o d of Dairy Science Vol. 74, No. 11, 1991
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VOGLER ET AL.
base values). Predominant types of abnormalities were decapitated heads, pyriform-shaped heads, and diadem and cratered defects. Abnormal tails and protoplasmic droplets were not more frequent than at base levels (established during period 1). Thus, the thermal insult achieved was considered relatively mild and, in general, thought to mimic that observed during the wium months at AI stations in North America for the Holstein breed (15). Considering that epididymal transport is approximately 8 to 11 d in the bull (9), the evaluation of undiluted semen in the present experiment indicates that the thermal stress interfered with the spermatogenic process r e p resented by sperm collected in period 3, but not with sperm maturation in the epididymis, represented by sperm collected in period 2. On the contrary, after freezing and incubation for 3 h at 37.C. spermatozoa collected in period 2 did differ from that of the preinsult spermatozoa (period 1) when judged by either viability measurement, motility, or acrosomal integrity Vable 2). This clearly indicated that spermatozoa in the epididymis were adversely afTected by the thermal stress to the testes. There also was a clear additive effect of the stresses imposed upon the ejaculated semen, which permitted resolution of the thermal insult to the epididymal sperm. In Table 3, the difference realized between semen collected in period 1 versus period 2 is organized from the least stressed, i.e., undiluted semen examined for motility immediately after collection, to that exposed to freezing and incubation. As may be noted from these data, extension of semen, incubation, and freezing all appeared to contribute individually to the differential that ultimately became sufficiently great to declare that spermatozoa collected during period 2 were inferior to those collected during period 1. Although past studies utilizing SI could not show an effect of the thermal insult on epididymal spermatozoa, only undiluted or unstressed semen was evaluated (1, 11). On the contrary, Wildeus and Entwistle (17) reported an effect of SI on the caput epididymis based on appearance of decapitated sperm 6 d later. They did not report responses in viability except for acrosomal alteration @resumably a measure of acrosomal integrity), which first occurred on d 10 postinsult. On this basis, OUT Journal of Dairy Science Vol. 74, No. 11, 1991
study would not refute published data utilizing the SI appmach. Rather, we would only conclude that spermatozoa stored in the epididym i s were adversely affected by thermal stress created by SI, but, to resolve the effect, the stress of Greezing semen along with subsequent incubation was required. The practical significance of these findings is that heat stress to bulls results in a decrease in the quality of cryopreserved semen prior to the appearance of abnormal spermatozoa or in a decline in undiluted semen viability, both of which have classically signified response of a male to elevated ambient temperature. The early effect of SI on ability of sperm to be cryopreserved suggests that epididymal sperm are vulnerable to the elevated testicular temperatures. ACKNOWLEDGMENTS
The authors wish to thank June Mullins for the graphic illustrations. REFERENCES
1 Awtia, J. W., E. W. Hum, and R L. M q k . 1961. E!ffect of scrotal insulation on semen of Hereford bulls. J. Anim. Sci. 2&3M. 2 Barth, A. D., and R.J. Ob.1989. Abnormal morphology of bovine spermatozoa. Iowa state univ. Ress, Ames. 3 Cassady,R B.,R M.Myers, and J. E. Legates. 1953. The effect of exposure to high ambient temperatme on spamatogenesis in the dairy ball. J. Dairy Sci. 36:14. 4 Jolmton, J. E., H.NaeLapaa, and J. B. me,Jr. 1%3. Physiologica! responses of Holstein, Brown Swiss and Red sindhi crossbred bulls exposed to high tempaahires and humidities. J. Anim. Sci 22:432. 5KarnovsLy. M. J. 1965. A formaldehyde glutadds hyde fmtive of high osmolality for use in elechun microscopy. J. Cell Biol. 273137A. 6Lagalof. N. 1934. Morphological studies on the changes in sperm structure and in the testes of bull with dccreasbd or abolished f&ty (translated title). Acta Wthol. Microbiol. S d . 19254. 7Meyerhoeffw, D. C.,R. P. Wetteanann, S. W. Cole man, and M.E. Wells. 1985. Reproductive criteria of beef bnlls during and after exposure to imxtased ambient tempenuure. J. Anim. Sci. m.352. SMitchell, J. R., R D. Hanson. and W. N. Fleming. 1978. Utiliziog difiermtial interferemx contrast microscopy for evaluating ebnormal spamatozoa Page 64 in Roc.7th Tech. Cod. AI Reprod., NaU. Assoc. Anim. Breeders, Columbia, MO. 90rgebin-Crisf M. C. 1962. Recbnchts exp&hmk tales SRI l a w de passage des spermatozoidcs dans I’tpididyme da tamu. AM. Biol. Anim. Biochim. Biophys. 2:51. lORobbins, R K., R. G. Saacke, and P. T. Chandler. 1976. Influeme of freeze rate, thaw rate and glycaol level on acrosomal retention and sunrivll of bovine spermatozoa frozen in French straws. J. Anim. Sci.
HEAT STRESS AND FROZEN SEMEN 42:145. 11 Ross, A. D., and K. W. Entwistle. 1979. The e f f d of scrotal insulation on spermatozoal morphology and the rates of spermatogenesis and epididymal passage of spermatozoa in the ball. Theniogenology 11:111. 12 Saacke, R. G., and C. E. Marshall. 1968. Observations 011 the Bctosomal cap of fmed and rmfixcd bovine spermatozoa. J. Reprod. Fertil. 16511. 13SASQDUser’s Guide: Statistics, Version 5 Edition. 1985. SAS h t . , Inc., Cary, NC. 14 Skinaer, J. D., and G. N. Loow. 1966. Heat stress and spermatogenesis in Bos indicuc and 90s lourus d e . J. Appl. Physiol. 21:1784. 15 Sullivan, I. J. 1970. Sperm numbers required for
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optimum breeding &ciemy in cattle. Page 36 in Roc. 3rd Tech. Cod. AI Reprod.,Natl. Assoc. Anim. Breeders, Columbia, MO. 16 Sulliven. J. J. 1978. Morphology and molility of spermatozoa. Page 286 in physiology of reproduction and artificial insemination of cattle.. 2nd ed. G.W. Salisbury,N.L. VanDemark, and J.R. Lodge, ed.Freeman and Co., San Francisco, CA. 17 Wildw, S., and K. W. Entwistle. 1983. Spemnigram and spenn reserves m hybrid Bos indim x Bos taurus bulls after scrotal insuhtion. J. Reprod. F d . 69:711. 18 Williams, W. W., and A. Savage. 1925. Observations on the semioal micropathology of bulls. Cornell Vet. 15:353.
Journal of Dairy Science Vol. 74, No. 11, 1991
c. J.
VOGLER, R. G. SAACKE,~J. H. BAME, J. M. DWARNETTE,~ and Y. L. MCGlLLlARD Department of Dairy Sdence Virginia Polytechnic Institute and State University Blacksburg 24061
both viability measurements, but only after incubation for 3 h at 37'C postthaw (P < .05). We conclude that epididymal sperm are adversely affected by elevated testicular temperatures, as noted by their decreased ability to maintain motility and acrosomal integrity following cryopreservation. (Key words: bovine, thermal stress, semen viability, frozen semen)
ABSTRACT
The effect of a 48-h scrotal insulation on spermatozoal viability (motility and acrosomal integrity), before and after semen cryopreservation, was studied in six young Holstein bulls whose semen was collected twice in succession at 3 d intervals. Motility and acrosomal integrity were measured before and after incubation of semen at 37'C for 3 h. For assessment of results, collection days were grouped: period 1 (control) = d -6, -3, and 0, where d 0 = initiation of scrotal insulation after semen collection; period 2 = d 3,6, and 9 (sperm presumed in the epididymis or rete testis during scrotal insulation); period 3 = d 12, 15,. . . 3 9 (sperm presumed in spermatogenesis during scrotal insulation). Semen was cryopreserved each collection day until morphologically abnormal cells exceeded 50% of the ejaculate (d 12 to 21). Semen viability before and after freezing was lower in period 3 than in period 1 (P < .05). These differences coincided with the appearance in period 3 of abnormal sperm morphology and depressed undiluted semen motility, which began on d 12 (P < .01). Semen collected during period 2 that was extended but unfrozen did not differ from that collected during period 1 in morphology or viability. However, for frozen semen, period 2 was significantly poorer than period 1 for
Abbreviation key: PIA = percentage of intact acrosomes, SI = scrotal insulation. INTRODUCTION
The classical studies of Williams and Savage (18) and Lagerlof (6) drew sharp attention
Received M a y 9, 1991. Accepted June 14, 1991. 'Research supported by Select Sires Inc.. Plain City, OH and the National Association of Animal Breeders, Columbia, MO. %o whom reprint requests should be addressed. 3 ~ c n address: t Select sires ~nc.,plain city, OH 43064. 1991 J Dairy Sci ?4:3827-3835
to the currently supported concept that fertile bulls have more viable sperm and a consistently lower incidence of morphologically abnormal sperm than sterile or subfertile bulls. The adverse effects of elevated temperatures on sptrmatogenesis have been well documented for the bull (3, 4, 7, 11, 14, 16) and consist of impaired efficiency of spermatogenesis, as reflected by increased sperm abnormalities and reduced sperm o u t p t and viability. Although previous reports indicated that epididymal sperm were not affected by elevated environmental or testicular temperatures (1, 11, 14), in these studies, only the undiluted semen from the stressed animals was evaluated. On the contrary, Wildeus and Entwistle (17) revealed a possible effect of heat on morphology of caput epididymal sperm. For semen that is to be used in AI, it is important to know whether heat stress renders sperm in the epididymis at the time of the stress more susceptible to injury from subsequent semen extension and cryopreservation. The overall objective of this study was to determine the effects of heat insult to the bull testis by scrotal insulation (SI) on ability of spermatozoa to be cryopreserved.
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the sack was installed (immediately after readjustment) and just before removal. Obtaining Anlmals and Semen Collection scrotal surface temwratures before emplacement of the sack was deemed uninfoAative Six Holstein bulls, individually penned in a because of interference from the variable amcold-housing, counter-slope barn, were se- bient temperatures throughout the experiment lected on their ability to produce semen having and resulting differences in the state of scrotal greater than 70% morphologically normal retraction and thickness. sperm with 60% or greater estimated progresFollowing final placement of the sack, bulls sive motility. Five bulls ranged in age from 14 were partially restrained by halter for 48 h SO to 17 mo, and one bull was 34 mo of age. that they had freedom to lie down, stand, eat, Collection of semen was by artificial vagina. and drink. This partial restraint was designed Two ejaculates were collected in succession, to prevent premature removal of the sacks and and each ejaculate was preceded by a period of ensure proper bedding, dry conditions, and sexual preparation consisting of two false general comfort during the thermal insult. mounts separated by 2 min of restraint. MATERIALS AND METHODS
Thermal Insult to the Testes
The purpose of the SI was to create a thermal insult to the testes, mimicking a naturally occurring environmental interference with testicular thermoregulation. To minimize potential stress from excessive intervention, no effort was made to establish or maintain a specific elevation in testicular temperature during the SI. Thermal insult of the testis was induced by enclosing the scrotum with a sack constructed of insulated material held in place by VELCRO@ Brand fasteners (VJ5LCRO@' Brand Fastening Systems, Manchester, NH) and medical tape. Scrotal sacks were fashioned from two layers of waterproofed nylon taffeta filled with a l c m insulating layer of polyester batting. The layers were machine quilted together and then sewn into a sack. After the first 30 min of SI, allowing for thermal response of the scrotum to the elevated temperature, the sack was readjusted to ensure complete coverage of the scrotum and scrotal neck up to the body wall. The sack was fitted to be loose enough not to interfere with circulation but sufficiently secure to avoid removal or slippage. The SI was maintained for 48 h. Bulls were placed on experiment at different times of the year, specifically, June, September, and October 1989 and February 1990. These months were chosen to avoid excessively high summer temperatures. Scrotal surface temperatures at the caudal midpoint of the testes were recorded using a type k flat thermistor probe, attached to a digital thermocouple thermometer (Cole-Pmer Instruments, Chicago, IL).Measurements were made when Journal of Dairy Science Vol. 74. No. 11, 1991
Experimental Design
Throughout the study, semen was collected at a frequency of two ejaculates in succession at 3-d intervals. Collection at this frequency began 2 wk prior to initiation of the experiment in order to stabilize epididymal sperm reserves and semen characteristics. Evaluation of undiluted semen characteristics (viability and morphology) and sperm output per collection day began 6 d prior to SI and continued until d 39 after initiation of SI. To evaluate the vulnerability to heat stress of spermatozoa in the epididymis and rete testis compared with those in stages of spermatogenic development (seminiferous tubules), three periods of semen collection were defined: period 1 = preinsult, i.e., collections on d -6, -3, and 0, where d 0 was the day of scrotal sack installation after semen collection; period 2 = postinsult, d +3, +6, and +9 after installation of the sack; period 3 = postinsult, d +12, +15, and +18 through +39. The basis for these three groups was to separate semen collections into a control period (period l), a period in which sperm present in the epididymis or rete testis at the time of heat insult would be ejaculated (period 2), and a period in which cells undergoing spermatogenesis at the time of the insult would be ejaculated (period 3). Semen was frozen from each collection day throughout periods 1 and 2 and up to the point when greater than 50% abnormal sperm were obtained in period 3. of Undiluted Semen
Most important was the assessment of response of bulls to the thermal stress imposed
HEAT STRESS AND FROZEN SEMEN
by SI. This was particularly important because neither accurate control nor measurement of intratesticular temperature was possible. In addition, response of bulls to a given thermal stress would also be expected to vary. On this basis, the undiluted semen characteristics and sperm output per collection day were the criteria for establishing the functional response of the bull to the thermal insult. Immediately after collection, the volume, concentration, and progressive motility of each ejaculate were measured. Concentration was determined using a spectrophotometer calibrated for bovine sperm using a hemacytometer. Motility was estimated to the nearest 10% after viewing several microscopic fields at 25Ox magnification using a phase contrast microscope equipped with a heated stage (37°C). Sperm abnormalities were quantified using differential interference contrast microscopy at 1250x magnification. One hundred microliters of semen from a pool of the two ejaculates of each collection day were fixed in 1 ml of Karnovsky’s fixative (5) and placed in a 1.5-ml Eppendorf tube. On the following day, differential counts of 100 cells from each of two wet smears of the fixed sample were averaged. Cells with multiple abnormalities were only counted once, but each abnormality of a particular cell was recorded. Thus, preemption of one cell abnormality by another was avoided. On this basis, actual percentages of abnormal heads, protoplasmic droplets, and abnormal tails were reported and could be collectively greater than the total percentage of abnormal cells in the sample. Specific cell abnormalities were identified by the classification of Mitchell et al. (8) and Barth and Oko (2). Semen Freezing
On each collection day, the two ejaculates of each bull were pooled and prepared in a conventional manner (10) for cryopreservation with egg yolk-citrate extender containing 7% glycerol (voVvol) at 50 x 106 celldml. Prior to semen dilution, extender was clarified by filtering through a glass prefilter connected to a .45-pm Acrodisc filter (Gelman Sciences, Ann Arbor, MI). Semen was packaged in .5-ml French straws (Instruments de MWecine
3829
V&6rinaire, l’Aigle, France), frozen 24 f 2 h after collection in static liquid nitrogen vapor for 10 min, and then plunged into liquid nitrogen. Freeze rates during the vapor freeze were monitored and were within the optimal range reported by Robbins et al. (10) for egg yolkcitrate-glycerol extended semen packaged in .5-ml straws. Straws were stored in liquid nitrogen until semen evaluation. Evaluatlon of Extended Semen Before and After Freezing
Prior to freezing (23 f 3 h after collection), the contents of three straws per bull from each collection day were pooled into a 1.5-mlEppendorf tube (Brinkman Instruments, Inc., Westbury, NY), warmed from 5 to 37°C using a dry bath, and evaluated for viability @ogressive motility and acrosomal integrity). Evaluation was conducted within 2 min of warming (0 h) and again after a 3-h incubation at 37°C. Percentage of progressive motility was estimated to the nearest 10% at 250x magnification using a phase contrast microscope equipped with a heated stage at 37°C. Acrosomal integrity was determined at the same incubation intervals and on the same smears using a differential interference contrast microscope at 1 2 5 0 ~magnification. Percentage of intact acrosomes (PIA) was based on the presence of the apical ridge of the acrosome (12) and was determined by averaging two direct counts of 100 cells from separate wet smears. After freezing and storage of semen at -196°C for at least 2 wk, three straws per bull from each collection day were thawed in water at 35°C for 45 s, and the contents were pooled and evaluated for viability using the same procedure as described for prefrozen extended semen. Frozen semen was coded so that the evaluator did not know the bull or collection day. For unfrozen semen, the bull was unknown to the evaluator. Statistical Analysis
All statistical analyses were conducted with programs available from SAS (13). Data were analyzed by the general linear models proceJournal of Dairy Science Vol. 74, No. 11, 1991
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VOGLW ET AL.
dure as well as nonorthogonal contrasts of period 1 with period 2 and period 1 with period 3 to detect changes in viability measurements, morphology, and sperm output. Because semen was frozen only until total sperm abnormalities reached 5096, for extended semen before and after cryopreservation, period 3 consisted of d y d 12 for two bulls, d 12 and 15 for two bulls, d 12, 15, and 18 for one bull, and d 12 through 21 for one bull. The model used for analysis of variance for undiluted semen characteristics, initial motility, and morphology, as well as sperm output per collection day, was as follows:
Pj = the fixed effect of the period j (j = 1, 2, or 3), Fk = the fixed effect of freeze k (k = 1, frozen or 2, unfrozen), HI = the fixed effect of hour 1 of incubation (l= 1 , 0 h or 2, 3 h), and q p ~= random error. Interactions with bull served as error terms for fixed effects. RESULTS
Mean testicular surface temperature, obtained at the caudal midpoint of the testis after adjustment of the sack and again before sack removal, was 34.8'C and ranged from 33.3 to 36.4'C. Ambient temperature and relative humidity during the experiment ranged from 4.9 to 33.7'C and from 29.7 to 93.9%,respectivewhere ly. Response of bulls to the thermal insult Yi,k = percentage of initial motility, per- based upon undiluted semen quality and sperm centage of abnormal sperm, or output is presented in Figure 1. There was a sperm output per collection day notable increase in total sperm abnormalities as well as a decrease in sperm motility in (x 104; period 3 compared with period 1 (P < .01). p = overall mean; Bi = the random effect of the bull i (i The motility was depressed 10 to nearly 20 percentage units, most apparent on d 15 and 18 = 1, 2, 3 , . .6); Pj = the fixed effect of the period j (j postinsult. The abnormal sperm content (abnormal heads) was elevated beginning d 12, = 1, 2, or 3); peaked at d 18 (nearly 70% above base), and Dg)k = the fixed effect of the day k persisted longer than did the depressed motiliwithin period j (k = 1,2,3, or 1, ty. Ejaculate content of abnormal sperm was 2, 3 , . . 10); and approaching preinsult levels at the termination eijk = random error. of the experiment, d 39 postinsult. Periods 1 and 3 did not differ in sperm output per collecThe model used for analysis of variance for tion day. Periods 1 and 2 did not differ in PIA and percentage of progressive motility of sperm morphology, motility, or sperm output extended semen evaluated before and after per collection day. cryopreservation was The types of sperm abnormalities associated with the significant increase in the abnormal population during period 3 were predominantly those involving the sperm head as contrasted with spermatozoa with abnormal tails or protoplasmic droplets (Figure 2). The head abnormalities most frequently encountered were decapitated sperm, diadem defects, cratered where sperm, and pyriform heads. Of importance was the relative stability of sperm morphology up Yiw = percentage of motile spermatozoa to d 9 postinsult, followed by an abrupt inor PIA, crease in abnormal heads on d 12. The apparp = overall mean, ent increase in seminal content of sperm with Bi = the random effect of the bull i (i abnormal tails between d 21 and 30 was not = 1, 2 , 3 ,... 6), significant.
.
.
Journal of Dairy Science Vol. 74. No. 11, 1991
3831
HEAT STRESS AND FROZEN SEMEN ~~
I I
90 8C 70
I I
I
I I I
T T
A
60 50 40 30
20 IO 90
gl + i 8 7
I
I I
I
C
I
I I
I
6 5 4 3
2
I Day ,-6 -3 0 I 3 6 9 (12 15 18 21 2427 3 0 3 3 3 6 3g1 Period I Period 2 Period 3 pigru.e 1. Effects of 48-h scrotal insalation (d 0, p d o d 1) on undiluted semen sperm morphology (A),motility @). and sperm output p a collection day (C). Means f SE. n = 6.
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VOGLER ET AL.
L Q)
80 70 60
n m
-
0 0 0 c c
n c
a a
50
2 40 oal 6 2 30
5 1 t
LL
Thermal insult
20 IO 0
Day
';6'-3' 0 ' 3' 6' 9'12'15'18~24~7'30'33'36'39' I I I Period I
Period 3
Period2
Abnormal heads
Protoplasmic droplets
I Abnormal fails
Figure 2. Effect of 48-h scrotal insulation (d 0. period 1) on incidence of abnormal sperm heads, protoplasmic droplets, and abnormal sperm tails. Only the abnomal sperm beads differed sigdicantly during peaiod 3 (P i.01).
The effect of the heat insult on the viability characteristics (sperm motility and acrosomal integrity) of unfrozen and frozen extended semen am presented in Tables 1 and 2, respectively. For unfrozen semen, a decrease in percentage of viable sperm was observed for period 3 relative to period 1 for both parameters at 0 h and again at 3 h of 37'C incubation (P < .05). On the contrary, unfrozen sperm ejaculated during period 2 were not different in viability from those ejaculated during period 1 for either measurement or incubation period. For frozen semen, period 3 was
again expectedly inferior to period 1 relative to percentage of motile cells and PIA at both incubation intervals. In addition, and differently from unfrozen semen, the frozen semen obtained during period 2 exhibited lower motility and acrosomal integrity after 3 h of incubation at 37'C than did semen collected during period 1 (P < .OS). Although the depression of viability during period 2 was Iimited to the frozen semen incubated for 3 h postthaw, the effect of the thermal insult appeared to become evident in an additive fashion as semen was stressed. To
TABLE 1. Effect of scrotal insulation (SI) for 48 h (d 0, period 1) on percentage of motility and percentage of intact acrosomes (F'IA) of unfrozen extendcd semen after storage for 1 d at 5'C (0h) and incubation at 37'C for 3 h (n = 6). Incubation time at 37'C Oh
3h
Period
Motility
PLA
Motility
PIA
1 2 3
66.4 62.5 41.1"
86.0 822 62R
56.1 47.1 25.la
79.6 74.6 49.9
~
~~~~
%Berent from pcriod 1 (P c .05); SEM motility = 2.9; SEM PIA = 1.6. 'Period 1 = d -6, -3.0 @reinsult) with 48 h of SI besinningon d 0. Period 2 = d 3,6,9(postinsult). Period 3 = d 12, 15, 18,and 21 or until ejaculate contained greater than 50% abnormal sperm. Day 12 only (2 bulls); d 12 and 15 (two bulls); d 12, 15, and 18 (one bull); d 12, 15, 18, and 21 (one boll). Journal of Dairy Science Vol. 74, No. 11. 1991
3833
HEAT STRESS AND FROZEN SEMEN
TABLE 2. Effect of scrotal insulation (SI) for 48 h (d 0, period 1) on percentage of motility and percentage of hlact acrosomes (PIA) of frozen semen after tbawing (0 h) and 3 h of incubation at 37'C (n = 6). Incubation time at 37'C ~~
~
Oh
3 h
Period1
Motility
PIA
Motility
PIA
1 2 3
55.3 48.6 37.8'
79.3 73.8 54.3'
46.4 30.8' 12.1"
73.O 62.8' 4Q.7a
%ifferent from period 1 (P < .05); S E M motility = 2.9; SEM PIA = 1.6. 'Period 1 = d -6, -3.0 @reinsalt) with 48 h of SI beginningon d 0. period 2 = d 3 . 6 . 9 (postinsult). Period 3 = d 12, 15, 18, and 21 or until ejaculate contained greater than 50% abnormal spenn. Day 12 only (mballs); d 12 and 15 (two bulls); d 12, 15, and 18 (one bull); d 12, 15, 18, and 21 (om ball).
aid in visualizing this additive effect, the data have been summarized in Table 3. As may be noted, progressing from the difference in viability of sperm ejaculated in period 2 versus period 1 for undiluted semen to that for extended unfrozen and frozen semen with and without incubation at 37'C for 3 h, the greater the stress factors applied to the semen (i.e., extension, freezing, and incubation), the greater the difference in viability of semen ejaculated during period 2 relative to period 1.
DISCUSSION
The response to SI achieved in this experiment based on undiluted semen is best described in Figures 1 and 2. The insult was characterized by no change in sperm output per collection day throughout the experiment (all periods), a relatively small decrease in motility (10 to nearly 20%) 12 to 18 d postinsult (at the beginning of period 3), and a distinct increase in abnormal heads beginning at the same time (d 12) and peaking at d 18 (nearly 70% above
TABLE 3. Comparative effect of 48-hscrotal insulation administered at the end of period 1 on percentage of viability of semen ejaculated during the following 9 d (period 2) based upon evaluation of undiluted semen and extended semen before and after freezing with (3 h) and without (0 h) incubation at 37'C.
Diffennce Semen
Incubation at 37'C
Viabdity measurement1
paid 12
Period 2
(period 1 period 2)
~ndilntd unfrozafl
0 h (only) Oh
Motility Motility
70.0 66.4
67.8 62.5 82.2 48.6 73.6 47.1 74.6 30.8 62.8
-2.2 -3.9 -3.8 -6.7 -5.5 -9.0 -5 .o -15.6 -10.2
1 7 ~ ~ ~ 5 Oh
PIA Motility
PIA UnfroZen
3 h
Prom
3 h
Motility PIA Motilitv
PIA
86.0 55.3 79.3 56.1 79.6 46.4 73.0
'Viability measurement, percentage of motility (estimated). and percentage of intact acrosomes (PIA), direct count. *Period 1 = d -6, -3. 0 @reinsult) with 48 h of scrotal iosnlation beginning on d 0. Period 2 = d +3, +6, +9 (postinsult). 3~0tilityevaluated immediately after collection. %Jnfrozen extended semen evaluated after 22 to 24 h of storage at 5'C (just prior to freezing). h o z e n extended semen evaluated after 2 wk of storage at -196'12
J o d of Dairy Science Vol. 74, No. 11, 1991
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VOGLER ET AL.
base values). Predominant types of abnormalities were decapitated heads, pyriform-shaped heads, and diadem and cratered defects. Abnormal tails and protoplasmic droplets were not more frequent than at base levels (established during period 1). Thus, the thermal insult achieved was considered relatively mild and, in general, thought to mimic that observed during the wium months at AI stations in North America for the Holstein breed (15). Considering that epididymal transport is approximately 8 to 11 d in the bull (9), the evaluation of undiluted semen in the present experiment indicates that the thermal stress interfered with the spermatogenic process r e p resented by sperm collected in period 3, but not with sperm maturation in the epididymis, represented by sperm collected in period 2. On the contrary, after freezing and incubation for 3 h at 37.C. spermatozoa collected in period 2 did differ from that of the preinsult spermatozoa (period 1) when judged by either viability measurement, motility, or acrosomal integrity Vable 2). This clearly indicated that spermatozoa in the epididymis were adversely afTected by the thermal stress to the testes. There also was a clear additive effect of the stresses imposed upon the ejaculated semen, which permitted resolution of the thermal insult to the epididymal sperm. In Table 3, the difference realized between semen collected in period 1 versus period 2 is organized from the least stressed, i.e., undiluted semen examined for motility immediately after collection, to that exposed to freezing and incubation. As may be noted from these data, extension of semen, incubation, and freezing all appeared to contribute individually to the differential that ultimately became sufficiently great to declare that spermatozoa collected during period 2 were inferior to those collected during period 1. Although past studies utilizing SI could not show an effect of the thermal insult on epididymal spermatozoa, only undiluted or unstressed semen was evaluated (1, 11). On the contrary, Wildeus and Entwistle (17) reported an effect of SI on the caput epididymis based on appearance of decapitated sperm 6 d later. They did not report responses in viability except for acrosomal alteration @resumably a measure of acrosomal integrity), which first occurred on d 10 postinsult. On this basis, OUT Journal of Dairy Science Vol. 74, No. 11, 1991
study would not refute published data utilizing the SI appmach. Rather, we would only conclude that spermatozoa stored in the epididym i s were adversely affected by thermal stress created by SI, but, to resolve the effect, the stress of Greezing semen along with subsequent incubation was required. The practical significance of these findings is that heat stress to bulls results in a decrease in the quality of cryopreserved semen prior to the appearance of abnormal spermatozoa or in a decline in undiluted semen viability, both of which have classically signified response of a male to elevated ambient temperature. The early effect of SI on ability of sperm to be cryopreserved suggests that epididymal sperm are vulnerable to the elevated testicular temperatures. ACKNOWLEDGMENTS
The authors wish to thank June Mullins for the graphic illustrations. REFERENCES
1 Awtia, J. W., E. W. Hum, and R L. M q k . 1961. E!ffect of scrotal insulation on semen of Hereford bulls. J. Anim. Sci. 2&3M. 2 Barth, A. D., and R.J. Ob.1989. Abnormal morphology of bovine spermatozoa. Iowa state univ. Ress, Ames. 3 Cassady,R B.,R M.Myers, and J. E. Legates. 1953. The effect of exposure to high ambient temperatme on spamatogenesis in the dairy ball. J. Dairy Sci. 36:14. 4 Jolmton, J. E., H.NaeLapaa, and J. B. me,Jr. 1%3. Physiologica! responses of Holstein, Brown Swiss and Red sindhi crossbred bulls exposed to high tempaahires and humidities. J. Anim. Sci 22:432. 5KarnovsLy. M. J. 1965. A formaldehyde glutadds hyde fmtive of high osmolality for use in elechun microscopy. J. Cell Biol. 273137A. 6Lagalof. N. 1934. Morphological studies on the changes in sperm structure and in the testes of bull with dccreasbd or abolished f&ty (translated title). Acta Wthol. Microbiol. S d . 19254. 7Meyerhoeffw, D. C.,R. P. Wetteanann, S. W. Cole man, and M.E. Wells. 1985. Reproductive criteria of beef bnlls during and after exposure to imxtased ambient tempenuure. J. Anim. Sci. m.352. SMitchell, J. R., R D. Hanson. and W. N. Fleming. 1978. Utiliziog difiermtial interferemx contrast microscopy for evaluating ebnormal spamatozoa Page 64 in Roc.7th Tech. Cod. AI Reprod., NaU. Assoc. Anim. Breeders, Columbia, MO. 90rgebin-Crisf M. C. 1962. Recbnchts exp&hmk tales SRI l a w de passage des spermatozoidcs dans I’tpididyme da tamu. AM. Biol. Anim. Biochim. Biophys. 2:51. lORobbins, R K., R. G. Saacke, and P. T. Chandler. 1976. Influeme of freeze rate, thaw rate and glycaol level on acrosomal retention and sunrivll of bovine spermatozoa frozen in French straws. J. Anim. Sci.
HEAT STRESS AND FROZEN SEMEN 42:145. 11 Ross, A. D., and K. W. Entwistle. 1979. The e f f d of scrotal insulation on spermatozoal morphology and the rates of spermatogenesis and epididymal passage of spermatozoa in the ball. Theniogenology 11:111. 12 Saacke, R. G., and C. E. Marshall. 1968. Observations 011 the Bctosomal cap of fmed and rmfixcd bovine spermatozoa. J. Reprod. Fertil. 16511. 13SASQDUser’s Guide: Statistics, Version 5 Edition. 1985. SAS h t . , Inc., Cary, NC. 14 Skinaer, J. D., and G. N. Loow. 1966. Heat stress and spermatogenesis in Bos indicuc and 90s lourus d e . J. Appl. Physiol. 21:1784. 15 Sullivan, I. J. 1970. Sperm numbers required for
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optimum breeding &ciemy in cattle. Page 36 in Roc. 3rd Tech. Cod. AI Reprod.,Natl. Assoc. Anim. Breeders, Columbia, MO. 16 Sulliven. J. J. 1978. Morphology and molility of spermatozoa. Page 286 in physiology of reproduction and artificial insemination of cattle.. 2nd ed. G.W. Salisbury,N.L. VanDemark, and J.R. Lodge, ed.Freeman and Co., San Francisco, CA. 17 Wildw, S., and K. W. Entwistle. 1983. Spemnigram and spenn reserves m hybrid Bos indim x Bos taurus bulls after scrotal insuhtion. J. Reprod. F d . 69:711. 18 Williams, W. W., and A. Savage. 1925. Observations on the semioal micropathology of bulls. Cornell Vet. 15:353.
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