- Email: [email protected]
In vitro evaluation of early embryo viability and development in summer heat-stressed, superovulated dairy cows
THERIOGENOLOGY
IN VITRO EVALUATION OF EARLY EMBRYO VIABILITY AND DEVELOPMENT IN SUMMER HEAT-STRESSED, SUPEROVULATED DAIRY COWS D.E. Monty, 1Department
of Veterinary
*Department
of Zoology, Received
Jr.1
and C. Racowsky2
Science, Arizona
University of Arizona, Tucson, 85721 State University, Tempe, AZ 85287
for publication: Accepted:
AZ
July 24, 1986 August
5, 1987
ABSTRACT Our study evaluated the viability and the development in vitro of embryos flushed from superovulated heat-stressed (hot season) and Holstein cows 6 to 8 d after artificial unstressed (cool season) insemination (AI). Plasma progesterone (P4) levels were measured in all The incidence of unfertilized ova was cows. significantly increased in hot-season cows (P < 0.05). There was no seasonal effect on the highly variable P4 levels 6 to 8 d after AI. Morulae and blastocysts flushed from hot-season cows were significantly less viable in culture than morulae or blastocysts flushed from cool-season cows (P < 0.001 and P < 0.0025, respectively). Furthermore, there was a significant seasonal effect both on blastulation (P < 0.0025) and hatching of blastocysts developed from morulae in culture (P < 0.005) and on expansion of blastocysts placed in culture at the blastocyst stage (P < 0.05). These data lead us to suggest that the reduced fertility of summer heat-stressed dairy cows may result from decreased viability and developmental capacity of Day-6 to Day-8 embryos. In addition, the results indicate that the efficiency of embryo transfer procedures may be significantly lowered by the reduced embryo viability associated with hot weather and that reduced embryo viability may be the cause of the well-documented seasonal reduction in the efficiency of AI. Key words:
heat
stress,
infertility,
embryo,
development,
cow
Acknowledgments: This research was funded by U.S. Dept. of Agriculture, Animal Health and Disease Research, PL95-113, Section 1433, Project No. ARZ ARZT360436-A-02-03. The number assigned by the publications editor is 586. We thank Mary Humphries, Mike Mecca, and Robin Hendricks for their able technical assistance; Amy Rosenhaus for typing the manuscript; Dr. Lee Kelley for assistance with the statistical analyses; Dr. Mark Wise for steroid RIA analyses; Dr. Tom Fuhrmann, and the and Jim Tappan, herdsmen at the Arizona Dairy Company for their excellent cooperation.
OCTOBER
1987 VOL. 28 NO. 4
451
THERIOGENOLOGY
INTRODUCTION A significant reduction in fertility occurs in dairy cows exposed to intense summer heat (l-7). Previous studies (l), and the experience of Arizona dairymen (personal communications), show that artificial insemination performed in Central Arizona during the intensely hot SummeK months results in less than 20% live births. Intense heat and solar radiation is particularly stressful to the lactating cow, which becomes hyperthermic during the hot afternoon and early evening hours (8,9). She responds by reducing sexual activity (reduced length and intensity of estrus), fertility, food consumption, and milk production, thereby reducing the metabolic heat she must dissipate to the environment to maintain thermoneutrality. The mechanisms underlying the well-documented reduction in fertility in heat-stressed cows remain unknown. The results of several studies in Arizona (1,3,8) have shown that under local conditions, a normal luteinizing hormone (LH) surge precedes a normal ovulation and that corpus luteum development and regression, and estrous cycle length, are not altered by summer heat stress. Nevertheless, progesterone levels are elevated and estradiol levels are depressed during the luteal phase of the estrous cycle in heat-stressed cows (8,lO). Numerous studies with several other species have shown an association between increased embryo mortality and heat stress (ll-14), and an increase in embryo mortality has been implicated in heat-stressed cows (15). In OUK study, we investigated this implication in lactating Holstein-Fresian cows at a large commercial dairy in Central Arizona. The influence of summer heat stress on early embryo viability and development was investigated in superovulated cows. The developmental capacities in culture of 6- to 8-d old embryos obtained from unstressed (cool season) and heat-stressed (hot season) superovulated cows were compared and contrasted.
MATERIALS AND METHODS Maintenance of Cows Adult, lactating Holstein-Fresian cows at the Arizona Dairy Company in Higley, Arizona, were used for this study. The cows were maintained in corrals (maximum capacity 126 to 160 cows) that have high shades, but not shade cooler systems, and were milked three times a day in semi-open milking parlors. The shades provided the only protection against the intense solar radiation. However, the cows were cooled by water sprays and fans as they passed through the holding pens of the milking parlors prior to milking. They were fed alfalfa silage, green chop and cubes, cottonseed oil meal, grain screening pellets, and vitamin-mineral mix in the corrals and milo and barley grain in the milking parlors. White salt was continuously available in the corrals. The water troughs in the corrals were shaded. Six of the experimental
OCTOBER
1987 VOL. 28 NO. 4
THERIOGENOLOGY
cows were fed 3 to 4 oz. Vita FermR Formulaa each day during one of the hot-season study periods as part of another investigation. The milk production of all experimental cows was approximated by recording the monthly Dairy Herd Improvement Association test measurement closest to the date of uterine flushing for embryos. Preparation of Cows and Collection of Material All cows selected for this study were chosen after palpation per rectum for reproductive tract abnormalities and following examination of their reproductive histories for previous infertility not related to heat stress. Only cows without abnormalities were used. The study was conducted during two consecutive years and used superovulated cows. Each cow was examined by palpation per rectum 4 to 7 d after a natural, normal estrus to verify the existence of a normal corpus luteum and the absence of reproductive tract abnormalities. The induction of superovulation was begun 9 to 13 d after estrus. Follicle stimulating hormone-pituitary (FSH-P, 64 mg)b was injected S.C. in divided, progressively decreasing dosages over 5 d; 750 mg of cloprostenol sodium (Estrumate)c was injected i.m. in two divided doses (early morning and late afternoon) on Day 4 of the procedure. The cows came into estrus approximately 48 h after the last injection of cloprostenol. They were artificially inseminated twice (24 h apart) with two vials of commercial frozen semen. Six to eight days after the first insemination, the cows were flushed for embryos. Nonsurgical uterine flushings were performed during early morning hours when air temperatures were relatively low. Each uterine horn was flushed separately with Dulbecco's phosphate buffered salined containing 1% filtered, heat-treated steer serum, penicillin G (100 III/ml), and streptomycin sulfate (50 pg/ml). The flushings were collected in washed, sterile silicone-coated flasks. The ovaries were palpated and the number of corpora lutea were counted. The size of the ovary was estimated. In some cases, the corpora lutea (CL) were so great in number that they could not be accurately counted, and these ovaries were designated "multiple CL ovaries" and assigned an estimated CL number of 20 for statistical comparison. Rectal temperatures, heart rates, and respiratory rates were measured concurrently. Blood was collected from the coccygeal vein into sterile tubes containing heparin. The tubes were chilled on ice until the plasma could be separated and frozen in the laboratory. The uterine flushings were transported to the laboratory, incubated at 37"C, allowed to settle for 1 h, and examined for embryos. Recovered embryos were washed several times in, and subsequently aVita FermR Formula, Bio-zyme Enterprises, Inc., 1231 Alabama, St. Joseph, MO 64504. bFSH-P, Burns-Biotec Laboratories, Inc., Omaha, NE 68103. CEstrumate, Miles Laboratories, Inc., Shawnee, KS 66201. dDulbecco's phosphate buffered saline, Gibco Laboratories, Grand Island Biological Co., Grand Island, NY 14072.
OCTOBER
1987 VOL. 28 NO. 4
453
THERIOGENOLOGY
cultured in, Eagle's Minimum Essential Medium (MEM)e containing 15% filtered, heat-treated steer serum, penicillin G (100 IU/ml), and streptomycin sulfate (50 tig/ml). They were cultured in wells of Lab-Tek chamber slides (No. 4838) in 0.2 ml medium at 37°C under a humidified atmosphere of 5% 02, 5% C02, and 90% N2. The embryos were examined daily and developmental stages were recorded until it was clear that they were degenerating. Some of the embryos that were cultured were obtained from superovulated cows that were sent to slaughter 6 to 8 d after they were artificially inseminated. The uterus was recovered at slaughter and taken to the laboratory where the uterine horns were flushed and the embryos cultured as described above. Uterine secretions were collected from a heat-stressed (hot season) cow and an unstressed (cool season) cow that had been sent to slaughter 6 to 8 d after a natural estrus. The cows were not bred. The uterus was removed and flushed immediately with physiological saline. The flushings were chilled on ice and transported to the laboratory where they were centrifuged at 3,100 rpm for 20 min, filtered through a 0.45urn filter, and frozen. They were maintained at -20°C until thawed, lyophilized, and analysed for protein concentration, as described by Lowry et al. (16). Subsequently, these uterine secretions were added to the culture medium in which 23 embryos from 8 hot-season cows and 15 embryos from 8 cool-season cows were maintained at a concentration of 10 a uterine secretion protein per milliliter of culture medium. Season Designation The hot season was designated as June through September and the cool season was designated as October through May. The climatologic measurements used to depict summer heat stressor were monthly average daily high and daily low temperatures and the monthly average relative humidity at 5:00 p.m. These data were obtained from the National Weather Service Forecast Office, Sky Harbor International Airport, Phoenix, Arizona. When the study included the same month in two hot or cool seasons, the meteorological data for the same month were averaged. From these data, the seasonal mean high and low temperatures and the relative humidities were calculated. Hormone Assays Plasma progesterone levels were measured by radioimmunoassay in the laboratory of Dr. Mark Wise (Department of Animal Science, University of Arizona, Tucson, AZ, 85721), as described by Niswender (17). Statistical Analyses The significance of seasonal differences and differences between treatment groups was determined by analysis of variance and G test (18) or Student's paired t-test, with differences greater than P < 0.05 eMinimum Essential Medium (Eagle), Gibco Laboratories, Grand Island Biological Co., NY 14072.
454
OCTOBER
1987 VOL. 28 NO. 4
THERIOGENOLOGY
relationships of The statistically significant. considered experimental measurements to meteorological data, and to each other, were evaluated by bivariate regression analysis.
RESULTS Meteorological Measurements The mean low temperature for the hot season was almost identical to Typical the mean high temperature of the cool season (Table 1). patterns of seasonal climatic changes in Central Arizona are described by Monty and Wolff (1,3).
Meteorological conditionsa
Table 1.
Seasonal mean measurements
Seasonb
Parameter measured
Cool
low temperature ("C) high temperature ("C) relative humidity at 5:00 p.m. (%)
12.1 f 0.8 25.3 f 1.3 27.7 f 4.5
Hot
low temperature ("C) high temperature ("C) relative humidity at 5:00 p.m. (%)
26.4 + 0.4 39.2 f 0.5 24.9 f 4.4
aEach value represents the monthly mean f SEM of daily measurements. bCool season = October through May; hot season = June through September.
Study Measurements The milk production of individual cows varied considerably in both seasons (Table 2), depending on individual production capacities and the duration of lactation. However, the average production level was similar in the two groups of cows. The number of CL on the ovaries, and therefore the response to superovulation, was similar in the cows
OCTOBER
1987 VOL. 28 NO. 4
455
THERIOGENOLOGY
Table 2.
Reproductive history of superovulated cows and development of embryos flushed from uterus during the cool and hot season
Parameter measured
Cool season (mean + SEM)
Hot season (mean + SEM)
No.
30
29
NSa
Age of cows (yr) Range
5.2 f 1.8 2.5 - 9.0
6.4 f 2.0 3.0 - 10.0
NS
No. of calves/cow Range
3.2 + 0.3 1.0 - 8.0
4.4 f 0.4 1.0 - 8.0
NS
Milk production (lb/d) Range
61.4 + 4.2 14.5 - 81.8
58.5 f 3.5 19.4 - 109.3
NS
Length of lactation (mo) Range
6.1 f 0.7 2.0 - 19.0
7.7 It 0.8 2.0 - 15.0
NS
No. of CL/cow
6.7 f 0.6
7.5 f 0.9
NS
Day after breeding that embryos were retrieved
7.3 f. 0.1
7.1 f 0.1
NS
No. of embryos recovered/cow Range
3.7 + 0.3 1.0 - 14.0
2.8 + 0.3 1.0 - 12.0
P < 0.05
No. cows with unfertilized ova
2 (6.7)b
8 (27.6)
P < 0.05
No. embryos at:
1 (1.0) 63 (57.3) 46 (41.8)
2 (2.4) 49 (59.8) 31 (37.8)
NS NS NS
of cows
premorula morula blastocyst
P value
aNot significant. bThe numbers in parentheses are the percentages of the total number of embryos examined in each season.
of both seasons (Table 2). There was no significant difference (P > 0.05) between the number of hot- and cool-season cows that had multiple CLs (7 and 9, respectively). The average number of embryos obtained from each cow was significantly reduced by heat stress, and more embryos were collected in the cool season (n = 110) than in the hot season (n - 82). There were large variations in the numbers of embryos flushed from individual cows in both seasons. Only those cows from which at least one embryo was flushed were included in the data; those that responded to superovulation induction but failed to yield embryos
456
OCTOBER
1987 VOL. 28 NO. 4
THERIOGENOLOGY
were eliminated. Compared with cool-season cows, significantly more There were no hot-season cows had one or more unfertilized ova. statistically significant differences between the two groups of cows with respect to the proportions of premorula, morula, and blastocyst embryos recovered from the uterus (Table 2). Nevertheless, a very marked seasonal effect was observed in the developmental capacity of Compared with morulae these embryos in culture (Tables 3 and 4). obtained from cool-season cows, significantly more morulae flushed from hot-season cows degenerated before any progression in development the proportions of morulae that blastulated and (Table 3). Moreover, underwent hatching were significantly fewer in the hot-season cows than Similar trends were observed for those in the cool-season cows. embryos placed in culture at the blastocyst stage of development (Table compared with blastocysts from cool-season cows, significantly 4): more blastocysts obtained from hot-season cows degenerated prior to expansion and significantly fewer blastocysts underwent expansion. However, there was no significant seasonal effect on the proportion of blastocysts that hatched in culture. The addition of uterine secretions from hot- and cool-season cows to the culture medium did not affect embryo development (data not shown), and so those embryos exposed to uterine flushings were included in the data presented in Tables 3 and 4.
Table 3.
Developmental capacity of morulae superovulated cows 6 to 8 d postbreedinga
flushed
from
Stage after culture
Cool seasonb (mean % + SEM)
Hot seasonb (mean % + SEM)
Degenerate
30.7 A 7.7
87.7 + 5.6
Blastocyst
28.3 + 8.0
1.5 + 1.2
P < 0.0025
Expanded blastocyst
4.9 + 2.3
2.3 AZ 2.3
NSC
Hatched blastocyst
36.1 + 8.7
8.2 f 5.1
P < 0.005
P value
P < 0.001
aEach value represents the mean 2 SEM of the percentage of morulae from each cow that degenerated, developed into a blastocyst, progressed to an expanded blastocyst, or hatched from the zona pellucida in culture. bThe total number of morulae cultured from cool-season cows was 63, whereas 47 were cultured from hot-season cows. 'Not significant.
OCTOBER
1987 VOL. 28 NO. 4
457
THERIOGENOLOGY
Table 4.
Developmental capacity of blastocysts superovulated cows 6 to 8 d post-breedinga
Stage after culture
Cool seasonb (mean % f SEM)
flushed
Hot seasonb (mean % f SEM)
from
P value
Degenerate
34.8 ?r 10.7
Expanded blastocyst
36.5 +
9.7
9.0 f 5.2
P < 0.05
Hatched blastocyst
28.7 k 10.2
7.7 f 5.5
NSC
83.3 f 6.8
P < 0.0025
aEach value represents the mean ?I SEM of the percentage of blastocysts from each cow that degenerated, expanded, or hatched from the zona pellucida in culture. bThe total number of blastocysts cultured from cool-season cows was 45, whereas 29 were cultured from hot-season cows. 'Not significant.
Table 5.
Physiologic responses and plasma progesterone concentrations in superovulated, artificially inseminated, lactating cows 6 to 8 d after breeding
Parameter measured
Cool season (mean + SRM)
Hot season (mean + SFM)
P value
Rectal temp ("F)
101.9 + 0.1 (28)a
102.0 + 0.1 (19)
NSb
Respiratory rate (breaths/min)
33.8 t 2.1 (27)
45.9 IL 3.1 (17)
P < 0.001
Heart rate (beats/min)
91.4 + 2.8 (25)
86.9 rf: 2.9 (19)
NSC
Progesterone (ng/ml) Range
15.6 5 2.6 (21) 2.3 - 47.9
17.2 + 4.0 (27) 2.6 - 66.2
NS=
aThe number of replicates is indicated in parentheses. bNot significant.
Table 5 shows the physiologic responses and hormone levels of hotand cool-season cows. The hot season induced a significant increase in respiratory rate, even though measurements were made during the There were significant (P < 0.01) moderate early morning hours.
458
OCTOBER
1987 VOL. 28 NO. 4
THERIOGENOLOGY
correlations observed between respiratory rate of all cows and morning and afternoon air temperature (r - 0.448 and 0.450, respectively), rectal temperature (r = 0.442), and plasma progesterone concentration (r = 0.544). Rectal temperatures were slightly higher and heart rates were slightly lower in hot-season cows than in cool-season cows, although the differences were not statistically significant (P > 0.05). Plasma progesterone concentrations varied widely in both hot- and coolseason superovulated cows, with no significant seasonal effects being observed. While plasma progesterone concentrations were not correlated with air temperatures or rectal temperatures, they were significantly correlated with the number of CL present (P < 0.05). Analysis of the hormone results and embryo viability and development data showed that there were no significant differences between data collected during the two hot seasons and the data collected during the two cool seasons. DISCUSSION The studies reported in this article, and others (l-3,10,15,19), demonstrate the ideal conditions in central Arizona for investigating the pathogenesis of heat-stress infertility in dairy cows. The ability of the dairy cow to adapt to intense summer heat is greatly reduced by the metabolic stresses of pregnancy and lactation (3). Adaptation to chronic heat stress has been shown to reduce fertility and milk production in order to lessen the body heat load which must be dissipated to maintain thermoneutrality (1-7). During the intense afternoon heat in central Arizona, lactating cows often become hyperthermic (3). Bligh (20) reported that the rectal temperature of the bovine begins to rise when the environmental temperature exceeds 30°C. Wolff and Monty (3) found this to be true in lactating Holstein cows but not in nongravid, nonlactating cows. Respiration increases greatly in heat-stressed cows and must play an important role in thermoregulation (3) since Bovidae have a limited capacity for heat dissipation by sweating and body surface evaporation. Our results are consistent with heat-induced respiratory compensation (Table 5). Under the conditions of our study, the use of increased respiratory evaporative cooling maintained basal (early morning) rectal temperatures at cool-season levels, whereas a previous study in Arizona (3) identified a seasonal rise. Plasma progesterone levels varied drastically during both seasons among individual superovulated cows and were positively correlated with the number of CL on the ovaries. It is likely, therefore, that the tremendous variation in ovarian progesterone output, induced by the gonadotropins used in the superovulation procedure, obscured any smaller variation in progesterone secretion that may have resulted from The effect of heat stress on peripheral climatic conditions. progesterone levels in naturally ovulating dairy cows is unsettled. Previous studies in Arizona (8) reported elevated progesterone levels during the luteal phase of the estrous cycle in hyperthermic, lactating that remained not in nongravid, nonlactating cows cows but
OCTOBER 1987 VOL. 28 NO. 4
459
THERIOGENOLOGY
thermoneutral. Further studies report increased progesterone levels associated with high temperatures (21), while others report decreased levels (19,22). Shaded cows have been shown to have lower progesterone levels than unshaded cows (23). Acute exposure to high temperatures for the first 72 h after breeding has been reported to raise plasma progesterone levels significantly in Hereford heifers (24). The ovulation rate of superovulated cows was unaffected by summer heat. There were, however, significantly fewer embryos flushed from the uteri of hot-season cows than from the uteri of cool-season cows. The reason for this is not known; however, the flushing procedure rarely retrieves all of the embryos in the uterus (as estimated by the Other studies have reported no effect of number of CL palpated). season on ovulation in naturally ovulating cows (1,3) and sheep (25), although a high incidence of anovulation and delayed ovulation has been reported in Israeli cows (26). The number of unfertilized ova recovered from superovulated cows was significantly greater during the hot season than during the cool season. The reason for this is unknown; however it may have been related to the high peripheral progesterone levels in those superovulated cows that yielded unfertilized ova. The mean plasma progesterone concentration in all superovulated cows was 16.5 ng/ml, whereas the average concentration in those superovulated cows that yielded unfertilized ova was 28.8 ng/ml. Progesterone and estradiol control genital tract secretion and motility, and sperm movement up the oviduct or ova movement down the oviduct may have been disrupted, preventing fertilization. The quality of the ova released by superovulated ovaries may be either inferior or more susceptible to the adverse effects of heat stress. The results of a recent study with hamster oocytes revealed that oocytes obtained from the normal preovulatory pool differ, electrophysiologically, from those that have been stimulated with pregnant mare's serum gonadotropin (27). These findings raise the interesting possibility that gonadotropinstimulated bovine oocytes may not be truly identical to those destined to be ovulated in animals that are cycling normally. Results of a previous study at the Arizona Dairy Co. on a limited number of normally ovulating cows in late lactation (10 cool-season and 9 hot-season cows, at an average ll~, of lactation) suggested that embryo development is retarded by summer heat stress (unpublished data). The development of embryos flushed from the oviducts of cows sent to slaughter 1.5 to 4.0 d after AI indicated retardation; however, the differences between seasons were not statistically significant. There was a high incidence of anovulation during both seasons. The indication of developmental retardation suggested an early sign of metabolic perturbation preceding death of the embryo. Analysis of the developmental stage of embryos flushed from the uteri of superovulated cows 6 to 8 d after breeding (Table 2) failed to reveal a significant effect of heat stress on embryonic development in vivo. However, the data presented in Tables 3 and 4 show a marked seasonal effect on the in vitro developmental capacities of morulae and blastocysts flushed from the uteri of superovulated cows. The development of embryos from heat-stressed cows in culture was significantly retarded. The results
460
OCTOBER 1987 VOL. 28 NO. 4
THERIOGENOLOGY
suggest that heat stress drastically reduces the capacity of bovine embryos to undergo blastulation and hatching from the zona pellucida. Although superovulation is reported to increase peripheral progesterone levels and reduce embryo quality (28), the predominant loss of viability and developmental capacity occurred in the summer season and may be attributed to seasonal heat stress. In Israel, it has been found that embryo mortality is greatest in repeat-breeder cows on Day 7 of the estrous cycle (29,30), at a time shortly after the embryo arrives in the horn of the uterus and when it is undergoing blastulation. The results of our study suggest that the problematic survival of embryos at this precarious stage of their development is Reduced embryo compounded by the adverse effects of heat stress. viability at this stage of development is consistent with previous findings that the estrous cycle is not prolonged in summer-season, repeat-breeding cows in Arizona (1). Since 7-d-old bovine embryos are not luteotropic (31) but may begin to be as early as Day 10 (32), heat stress-induced inhibition of blastulation and the demise of the embryo would preclude maintenance of the CL and prolongation of the estrous cycle. Heat stress in the ewe is also associated with early embryo mortality, which does not alter the length of the estrous cycle (11). Other studies with bred cows, however, report an association between heat stress and prolongation of the estrous cycle (2), suggesting that under certain conditions, death of the embryo may occur after maternal recognition of pregnancy, which occurs between Day 15 and 17 postbreeding (33); thus, the life of the degenerating embryo is prolonged, postponing luteolysis. The mechanisms underlying these observed adverse effects of heat stress on survivability of Day 6 to 8 cow embryos are unknown but may be due to a direct action of heat on the embryo or to an indirect effect that is mediated by the uterine environment. In support of a direct action, intrauterine temperatures are higher than rectal temperatures in hyperthermic lactating cows (3): high intrauterine temperatures may affect spermatozoa, ova, or embryos and have been related to infertility (34). Heat-damaged spermatozoa that fertilize ova may produce abnormal embryos which subsequently die (35). Ulberg and Sheehan (36) have reported that a slight increase in temperature can directly disorient embryo development, producing embryos which grow and may appear morphologically normal, but which subsequently die. The embryo is most susceptible to thermal stress during the first cleavage divisions. In support of an indirect action are the reports (described above) of abnormal sex steroid levels in heat-stressed animals. Progesterone and estrogen are essential to the control of uterine secretion and motility. It is reasonable to assume that abnormal steroid levels could produce a uterine environment that either is unable to support embryo development or is toxic to the embryo. Progesterone levels have been reported to influence the protein composition of uterine secretions of cattle (37), and heat stress has been reported to change the amino acid composition of oviductal secretions in rabbits (38).
OCTOBER
1987 VOL. 28 NO. 4
461
I-HERIOGENOLOGY
Either indirect or direct mechanisms may impair development and lead to the death of the embryo before maternal recognition of pregnancy, explaining one of the signs of summer heat stress infertility in Arizona dairy cows. Further studies are needed on larger numbers of non-superovulated cows in order to confirm the role of early embryo viability in summer heat stress infertility in dairy cows. In addition, the efficiency of embryo transfer procedures may be significantly lowered by the reduced embryo viability associated with hot weather.
REFERENCES 1.
Monty, D.E. and Wolff, L.K. Summer heat stress and reduced fertility in Holstein-Friesian cows in Arizona. Am. J. Vet. Res. 2:1495-1499 (1974).
2.
stott, G.H. and Williams, R.J. Causes of low breeding efficiency in dairy cattle associated with seasonal high temperatures. J. Dairy Sci. a:1369-1375 (1962).
3.
Wolff, L.K. and Monty, D.E. Physiologic response to intense summer heat and its effect on the estrous cycle of nonlactating and lactating Holstein-Friesian cows in Arizona. Am J. Vet. Res. =:187-192 (1974).
4.
Dunlap, S.E. and Vincent, C.K. Influence of postbreeding thermal stress on conception rate in beef cattle. J. Anim. Sci., z:1216-1218 (1971).
5.
Kelly, M.S. and Hurst, V. The effect of season on fertility of the dairy bull and diary cow. J. Am. Vet. Med. Assoc. 143:40-43 (1963).
6.
Hafez, E.S.E. Reproductive capacity of farm animals in relation to climate and nutrition. J. Am. Vet. Med. ASSOC. m:606-614 (1959).
7.
Heiman, M.M. Results and conclusions drawn from breeding. Dtsch. Tierarztl, Wschr. &:206-209 (1977).
8.
Wolff-Vaught, L., Monty, D.E. and Foote, W.C Effect of summer heat stress on serum luteinizing hormone and progesterone values in Holstein-Friesian cows in Arizona. Am. J. Vet. Res. %:10271030 (1977).
9.
Behavioral and physiologic Monty, D.E. and Garbareno, J.L. responses of Holstein-Friesian cows to high environmental temperatures and artificial cooling in Arizona. Am. J. Vet. Res. 2:877-882 (1978).
462
OCTOBER
A.I.
1987 VOL. 28 NO. 4
THERIOGENOLOGY
10.
Monty, D.E. Early embryo death in cattle during thermal La Reproduction De stress. Reunion Internationale sur: Ruminants En Zone Tropical. pp. 285-300. Jan 8 to 10, 1983. Pointe-a-Pitre (Guadaloupe) Antilles, Francaises.
11.
Dutt, R.H. Critical period for early embryo mortality in ewes exposed to high ambient temperature. J. Anim. Sci. 2:713-719 (1963).
12.
Edwards, R.L., Omtvedt, J.T., Turman, J., Stevens, D.F. and Mahoney, J.W. Reproductive performance in gilts following heat stress prior to breeding and in early gestation. J. Anim. Sci. =:1634-1637 (1968).
13.
Pennvciuk. P.R. The effects on rats of chronic exposure to 34°C: Iv. Reproduction. Aust. J. Biol. Sci. c:245-260 (1964).
14.
Sod-Moriah, U.A. Reproduction in the heat-acclimatized female rat as affected by high ambient temperature. J. Reprod. Fertil. 2&:209-218 (1971).
15.
Wiersma, F. and Stott, G.H. New concepts in the physiology of heat stress in cattle of interest to engineers. Tr. Am. Sot. Agric. Engineers Q:130-132 (1969).
16.
Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. Protein measurements with Folin phenol reagents. J. Biol. Chem. m:265-275 (1951).
17.
Niswender, G.D. Influence of the site of conjugation on the specificity of antibodies to progesterone. Steroids =:413-424 (1973).
18.
Sokal, R.R. and Rohlf, F.J. 1981, pp. 731-746.
19.
stott, G.H., Wiersma, F., Wood, J.M. Reproductive health program for cattle subjected to high environmental temperatures. J. Am. Vet. Med. Assoc. m:1339-1344 (1972).
20.
Bligh, J. The relationship between the temperature of the rectum and of the blood in the bicarotid trunk of the calf during exposure to heat stress. J. Physiol. m:393-403 (1957).
21.
Abilay, T.A., Johnson, H.D. and Madan, M. Influence of environmental heat on peripheral plasma progesterone and cortisol during the bovine estrous cycle. J. Dairy. Sci. 58: 1836-1840 (1975).
22.
Seasonal Rosenberg, M., Herz, Z., Davidson, M. and Zolman, Y. variations in post-partum plasma progesterone levels and
OCTOBER
1987 VOL. 28 NO. 4
Biometry.
Freeman, San Francisco,
I-HERIOGENOLOGY
conceptions
in
primiparous
J. Reprod. Fertil. =:363-367
and
multiparous
dairy
cows.
(1977).
23.
Hormonal Roman-Ponce, H., Thatcher, W.W. and Wilcox, E.J. interrelationships and physiological responses of lactating dairy cows to a shade management system in a subtropical environment. Theriogenology &:139-154 (1981).
24.
Mills, A.C., Thatcher, W.W., Dunlap, S.E. and Vincent, C.K. Influence of postbreeding thermal stress on peripheral plasma progestin concentrations in heifers. J. Dairy. Sci. a:1 abstr. (1979).
25.
Ulberg, L.C. and Burfening, P.J. Embryo death resulting from adverse environment on spermatozoa or ova. J. Anim. Sci. x:571-577 (1967). ’
26.
Ayalon, N., Feingold, D. and Almeida, A. Disturbed ovulation and summer infertility in Friesian cows in Israel. Refuah Vet. 39:1-2 (1982).
27.
Racowsky, C. and Satterlie, R.A. Metabolic, fluorescent dye and electrical coupling between hamster oocytes and cumulus cells during meiotic maturation b m and & m. Dev. Biol. m:191-202 (1985).
28.
Jensen, A.M., Greve, T., Madej, A. and Edqviat, L.E. Endocrine profiles and embryo quality in the PMSG-PGF2 treated cow. Theriogenology u:33-44 (1982).
29.
Fertility losses in normal cows and repeat Ayalon, N. breeders. Proc. 7th Int. Congr. Anim. Reprod. and A.I., Munich, Vol. 1, p. 741 abstr. (1972).
30.
Comparative Ayalon, N. Annual Report of Research, No. 2: studies of repeat breeders and normal cows. Kimron Veterinary Institute, Beit Dagan (Israel). pp. 1-48 (1973).
31.
Linares, T., Larsson, K. and Edquist, L. Plasma progesterone levels from oestrus through day 7 after A.I. in hiefers carrying embryos with normal or deviating morphology. Theriogenology =:125-132 (1982).
32.
Hansel, W. Plasma hormone concentrations associated with early embryo mortality in heifers. J. Reprod. Fertil. Suppl. x:231-239 (1981).
33.
Northey, D.L. and French, L.R. Effect of embryo removal and intrauterine infusion of embryonic homogenates on the lifespan of the bovine corpus luteum. J. Anim. Sci. s:298-302 (1980).
464
OCTOBER
1987 VOL. 28 NO. 4
THERIOGENOLOGY
34.
W.W. and Wilcox, C.J. Thatcher Gwazdaukas, F.C., factors at and hormonal environmental Physiological, insemination which affect conception. J. Dairy. Sci. 561873-877 (1973).
35.
Burfening, P.J. and Ulberg, L.C. Embryonic survival subsequent to culture of rabbit spermatozoa at 38" and 40°C. J. Reprod. Fertil. =:87-92 (1968).
36.
Ulberg, L.C. and Sheehan, L.A. Early development of mammalian embryos in elevated ambient temperatures. J. Reprod. Fertil. Suppl. =:155-161 (1973).
37.
Bartol, T.F., Thatcher, W.W., Lewis, G.S., Bliss, E.L., Drost, Effects of estradiol-17 beta on PGF and M. and Boyer, F.W. total protein content in bovine uterine flushings and peripheral plasma concentrations of 13,14-dihydro-15-keto PGF2e. Theriogenology u:345-358 (1981).
38.
Clark, D.A. The effects of heat stress and pregnancy on selected constituents of rabbit oviductal fluid. Thesis, Univ. of Missouri, Columbia. (1979).
OCTOBER 1987 VOL. 28 NO. 4
465
IN VITRO EVALUATION OF EARLY EMBRYO VIABILITY AND DEVELOPMENT IN SUMMER HEAT-STRESSED, SUPEROVULATED DAIRY COWS D.E. Monty, 1Department
of Veterinary
*Department
of Zoology, Received
Jr.1
and C. Racowsky2
Science, Arizona
University of Arizona, Tucson, 85721 State University, Tempe, AZ 85287
for publication: Accepted:
AZ
July 24, 1986 August
5, 1987
ABSTRACT Our study evaluated the viability and the development in vitro of embryos flushed from superovulated heat-stressed (hot season) and Holstein cows 6 to 8 d after artificial unstressed (cool season) insemination (AI). Plasma progesterone (P4) levels were measured in all The incidence of unfertilized ova was cows. significantly increased in hot-season cows (P < 0.05). There was no seasonal effect on the highly variable P4 levels 6 to 8 d after AI. Morulae and blastocysts flushed from hot-season cows were significantly less viable in culture than morulae or blastocysts flushed from cool-season cows (P < 0.001 and P < 0.0025, respectively). Furthermore, there was a significant seasonal effect both on blastulation (P < 0.0025) and hatching of blastocysts developed from morulae in culture (P < 0.005) and on expansion of blastocysts placed in culture at the blastocyst stage (P < 0.05). These data lead us to suggest that the reduced fertility of summer heat-stressed dairy cows may result from decreased viability and developmental capacity of Day-6 to Day-8 embryos. In addition, the results indicate that the efficiency of embryo transfer procedures may be significantly lowered by the reduced embryo viability associated with hot weather and that reduced embryo viability may be the cause of the well-documented seasonal reduction in the efficiency of AI. Key words:
heat
stress,
infertility,
embryo,
development,
cow
Acknowledgments: This research was funded by U.S. Dept. of Agriculture, Animal Health and Disease Research, PL95-113, Section 1433, Project No. ARZ ARZT360436-A-02-03. The number assigned by the publications editor is 586. We thank Mary Humphries, Mike Mecca, and Robin Hendricks for their able technical assistance; Amy Rosenhaus for typing the manuscript; Dr. Lee Kelley for assistance with the statistical analyses; Dr. Mark Wise for steroid RIA analyses; Dr. Tom Fuhrmann, and the and Jim Tappan, herdsmen at the Arizona Dairy Company for their excellent cooperation.
OCTOBER
1987 VOL. 28 NO. 4
451
THERIOGENOLOGY
INTRODUCTION A significant reduction in fertility occurs in dairy cows exposed to intense summer heat (l-7). Previous studies (l), and the experience of Arizona dairymen (personal communications), show that artificial insemination performed in Central Arizona during the intensely hot SummeK months results in less than 20% live births. Intense heat and solar radiation is particularly stressful to the lactating cow, which becomes hyperthermic during the hot afternoon and early evening hours (8,9). She responds by reducing sexual activity (reduced length and intensity of estrus), fertility, food consumption, and milk production, thereby reducing the metabolic heat she must dissipate to the environment to maintain thermoneutrality. The mechanisms underlying the well-documented reduction in fertility in heat-stressed cows remain unknown. The results of several studies in Arizona (1,3,8) have shown that under local conditions, a normal luteinizing hormone (LH) surge precedes a normal ovulation and that corpus luteum development and regression, and estrous cycle length, are not altered by summer heat stress. Nevertheless, progesterone levels are elevated and estradiol levels are depressed during the luteal phase of the estrous cycle in heat-stressed cows (8,lO). Numerous studies with several other species have shown an association between increased embryo mortality and heat stress (ll-14), and an increase in embryo mortality has been implicated in heat-stressed cows (15). In OUK study, we investigated this implication in lactating Holstein-Fresian cows at a large commercial dairy in Central Arizona. The influence of summer heat stress on early embryo viability and development was investigated in superovulated cows. The developmental capacities in culture of 6- to 8-d old embryos obtained from unstressed (cool season) and heat-stressed (hot season) superovulated cows were compared and contrasted.
MATERIALS AND METHODS Maintenance of Cows Adult, lactating Holstein-Fresian cows at the Arizona Dairy Company in Higley, Arizona, were used for this study. The cows were maintained in corrals (maximum capacity 126 to 160 cows) that have high shades, but not shade cooler systems, and were milked three times a day in semi-open milking parlors. The shades provided the only protection against the intense solar radiation. However, the cows were cooled by water sprays and fans as they passed through the holding pens of the milking parlors prior to milking. They were fed alfalfa silage, green chop and cubes, cottonseed oil meal, grain screening pellets, and vitamin-mineral mix in the corrals and milo and barley grain in the milking parlors. White salt was continuously available in the corrals. The water troughs in the corrals were shaded. Six of the experimental
OCTOBER
1987 VOL. 28 NO. 4
THERIOGENOLOGY
cows were fed 3 to 4 oz. Vita FermR Formulaa each day during one of the hot-season study periods as part of another investigation. The milk production of all experimental cows was approximated by recording the monthly Dairy Herd Improvement Association test measurement closest to the date of uterine flushing for embryos. Preparation of Cows and Collection of Material All cows selected for this study were chosen after palpation per rectum for reproductive tract abnormalities and following examination of their reproductive histories for previous infertility not related to heat stress. Only cows without abnormalities were used. The study was conducted during two consecutive years and used superovulated cows. Each cow was examined by palpation per rectum 4 to 7 d after a natural, normal estrus to verify the existence of a normal corpus luteum and the absence of reproductive tract abnormalities. The induction of superovulation was begun 9 to 13 d after estrus. Follicle stimulating hormone-pituitary (FSH-P, 64 mg)b was injected S.C. in divided, progressively decreasing dosages over 5 d; 750 mg of cloprostenol sodium (Estrumate)c was injected i.m. in two divided doses (early morning and late afternoon) on Day 4 of the procedure. The cows came into estrus approximately 48 h after the last injection of cloprostenol. They were artificially inseminated twice (24 h apart) with two vials of commercial frozen semen. Six to eight days after the first insemination, the cows were flushed for embryos. Nonsurgical uterine flushings were performed during early morning hours when air temperatures were relatively low. Each uterine horn was flushed separately with Dulbecco's phosphate buffered salined containing 1% filtered, heat-treated steer serum, penicillin G (100 III/ml), and streptomycin sulfate (50 pg/ml). The flushings were collected in washed, sterile silicone-coated flasks. The ovaries were palpated and the number of corpora lutea were counted. The size of the ovary was estimated. In some cases, the corpora lutea (CL) were so great in number that they could not be accurately counted, and these ovaries were designated "multiple CL ovaries" and assigned an estimated CL number of 20 for statistical comparison. Rectal temperatures, heart rates, and respiratory rates were measured concurrently. Blood was collected from the coccygeal vein into sterile tubes containing heparin. The tubes were chilled on ice until the plasma could be separated and frozen in the laboratory. The uterine flushings were transported to the laboratory, incubated at 37"C, allowed to settle for 1 h, and examined for embryos. Recovered embryos were washed several times in, and subsequently aVita FermR Formula, Bio-zyme Enterprises, Inc., 1231 Alabama, St. Joseph, MO 64504. bFSH-P, Burns-Biotec Laboratories, Inc., Omaha, NE 68103. CEstrumate, Miles Laboratories, Inc., Shawnee, KS 66201. dDulbecco's phosphate buffered saline, Gibco Laboratories, Grand Island Biological Co., Grand Island, NY 14072.
OCTOBER
1987 VOL. 28 NO. 4
453
THERIOGENOLOGY
cultured in, Eagle's Minimum Essential Medium (MEM)e containing 15% filtered, heat-treated steer serum, penicillin G (100 IU/ml), and streptomycin sulfate (50 tig/ml). They were cultured in wells of Lab-Tek chamber slides (No. 4838) in 0.2 ml medium at 37°C under a humidified atmosphere of 5% 02, 5% C02, and 90% N2. The embryos were examined daily and developmental stages were recorded until it was clear that they were degenerating. Some of the embryos that were cultured were obtained from superovulated cows that were sent to slaughter 6 to 8 d after they were artificially inseminated. The uterus was recovered at slaughter and taken to the laboratory where the uterine horns were flushed and the embryos cultured as described above. Uterine secretions were collected from a heat-stressed (hot season) cow and an unstressed (cool season) cow that had been sent to slaughter 6 to 8 d after a natural estrus. The cows were not bred. The uterus was removed and flushed immediately with physiological saline. The flushings were chilled on ice and transported to the laboratory where they were centrifuged at 3,100 rpm for 20 min, filtered through a 0.45urn filter, and frozen. They were maintained at -20°C until thawed, lyophilized, and analysed for protein concentration, as described by Lowry et al. (16). Subsequently, these uterine secretions were added to the culture medium in which 23 embryos from 8 hot-season cows and 15 embryos from 8 cool-season cows were maintained at a concentration of 10 a uterine secretion protein per milliliter of culture medium. Season Designation The hot season was designated as June through September and the cool season was designated as October through May. The climatologic measurements used to depict summer heat stressor were monthly average daily high and daily low temperatures and the monthly average relative humidity at 5:00 p.m. These data were obtained from the National Weather Service Forecast Office, Sky Harbor International Airport, Phoenix, Arizona. When the study included the same month in two hot or cool seasons, the meteorological data for the same month were averaged. From these data, the seasonal mean high and low temperatures and the relative humidities were calculated. Hormone Assays Plasma progesterone levels were measured by radioimmunoassay in the laboratory of Dr. Mark Wise (Department of Animal Science, University of Arizona, Tucson, AZ, 85721), as described by Niswender (17). Statistical Analyses The significance of seasonal differences and differences between treatment groups was determined by analysis of variance and G test (18) or Student's paired t-test, with differences greater than P < 0.05 eMinimum Essential Medium (Eagle), Gibco Laboratories, Grand Island Biological Co., NY 14072.
454
OCTOBER
1987 VOL. 28 NO. 4
THERIOGENOLOGY
relationships of The statistically significant. considered experimental measurements to meteorological data, and to each other, were evaluated by bivariate regression analysis.
RESULTS Meteorological Measurements The mean low temperature for the hot season was almost identical to Typical the mean high temperature of the cool season (Table 1). patterns of seasonal climatic changes in Central Arizona are described by Monty and Wolff (1,3).
Meteorological conditionsa
Table 1.
Seasonal mean measurements
Seasonb
Parameter measured
Cool
low temperature ("C) high temperature ("C) relative humidity at 5:00 p.m. (%)
12.1 f 0.8 25.3 f 1.3 27.7 f 4.5
Hot
low temperature ("C) high temperature ("C) relative humidity at 5:00 p.m. (%)
26.4 + 0.4 39.2 f 0.5 24.9 f 4.4
aEach value represents the monthly mean f SEM of daily measurements. bCool season = October through May; hot season = June through September.
Study Measurements The milk production of individual cows varied considerably in both seasons (Table 2), depending on individual production capacities and the duration of lactation. However, the average production level was similar in the two groups of cows. The number of CL on the ovaries, and therefore the response to superovulation, was similar in the cows
OCTOBER
1987 VOL. 28 NO. 4
455
THERIOGENOLOGY
Table 2.
Reproductive history of superovulated cows and development of embryos flushed from uterus during the cool and hot season
Parameter measured
Cool season (mean + SEM)
Hot season (mean + SEM)
No.
30
29
NSa
Age of cows (yr) Range
5.2 f 1.8 2.5 - 9.0
6.4 f 2.0 3.0 - 10.0
NS
No. of calves/cow Range
3.2 + 0.3 1.0 - 8.0
4.4 f 0.4 1.0 - 8.0
NS
Milk production (lb/d) Range
61.4 + 4.2 14.5 - 81.8
58.5 f 3.5 19.4 - 109.3
NS
Length of lactation (mo) Range
6.1 f 0.7 2.0 - 19.0
7.7 It 0.8 2.0 - 15.0
NS
No. of CL/cow
6.7 f 0.6
7.5 f 0.9
NS
Day after breeding that embryos were retrieved
7.3 f. 0.1
7.1 f 0.1
NS
No. of embryos recovered/cow Range
3.7 + 0.3 1.0 - 14.0
2.8 + 0.3 1.0 - 12.0
P < 0.05
No. cows with unfertilized ova
2 (6.7)b
8 (27.6)
P < 0.05
No. embryos at:
1 (1.0) 63 (57.3) 46 (41.8)
2 (2.4) 49 (59.8) 31 (37.8)
NS NS NS
of cows
premorula morula blastocyst
P value
aNot significant. bThe numbers in parentheses are the percentages of the total number of embryos examined in each season.
of both seasons (Table 2). There was no significant difference (P > 0.05) between the number of hot- and cool-season cows that had multiple CLs (7 and 9, respectively). The average number of embryos obtained from each cow was significantly reduced by heat stress, and more embryos were collected in the cool season (n = 110) than in the hot season (n - 82). There were large variations in the numbers of embryos flushed from individual cows in both seasons. Only those cows from which at least one embryo was flushed were included in the data; those that responded to superovulation induction but failed to yield embryos
456
OCTOBER
1987 VOL. 28 NO. 4
THERIOGENOLOGY
were eliminated. Compared with cool-season cows, significantly more There were no hot-season cows had one or more unfertilized ova. statistically significant differences between the two groups of cows with respect to the proportions of premorula, morula, and blastocyst embryos recovered from the uterus (Table 2). Nevertheless, a very marked seasonal effect was observed in the developmental capacity of Compared with morulae these embryos in culture (Tables 3 and 4). obtained from cool-season cows, significantly more morulae flushed from hot-season cows degenerated before any progression in development the proportions of morulae that blastulated and (Table 3). Moreover, underwent hatching were significantly fewer in the hot-season cows than Similar trends were observed for those in the cool-season cows. embryos placed in culture at the blastocyst stage of development (Table compared with blastocysts from cool-season cows, significantly 4): more blastocysts obtained from hot-season cows degenerated prior to expansion and significantly fewer blastocysts underwent expansion. However, there was no significant seasonal effect on the proportion of blastocysts that hatched in culture. The addition of uterine secretions from hot- and cool-season cows to the culture medium did not affect embryo development (data not shown), and so those embryos exposed to uterine flushings were included in the data presented in Tables 3 and 4.
Table 3.
Developmental capacity of morulae superovulated cows 6 to 8 d postbreedinga
flushed
from
Stage after culture
Cool seasonb (mean % + SEM)
Hot seasonb (mean % + SEM)
Degenerate
30.7 A 7.7
87.7 + 5.6
Blastocyst
28.3 + 8.0
1.5 + 1.2
P < 0.0025
Expanded blastocyst
4.9 + 2.3
2.3 AZ 2.3
NSC
Hatched blastocyst
36.1 + 8.7
8.2 f 5.1
P < 0.005
P value
P < 0.001
aEach value represents the mean 2 SEM of the percentage of morulae from each cow that degenerated, developed into a blastocyst, progressed to an expanded blastocyst, or hatched from the zona pellucida in culture. bThe total number of morulae cultured from cool-season cows was 63, whereas 47 were cultured from hot-season cows. 'Not significant.
OCTOBER
1987 VOL. 28 NO. 4
457
THERIOGENOLOGY
Table 4.
Developmental capacity of blastocysts superovulated cows 6 to 8 d post-breedinga
Stage after culture
Cool seasonb (mean % f SEM)
flushed
Hot seasonb (mean % f SEM)
from
P value
Degenerate
34.8 ?r 10.7
Expanded blastocyst
36.5 +
9.7
9.0 f 5.2
P < 0.05
Hatched blastocyst
28.7 k 10.2
7.7 f 5.5
NSC
83.3 f 6.8
P < 0.0025
aEach value represents the mean ?I SEM of the percentage of blastocysts from each cow that degenerated, expanded, or hatched from the zona pellucida in culture. bThe total number of blastocysts cultured from cool-season cows was 45, whereas 29 were cultured from hot-season cows. 'Not significant.
Table 5.
Physiologic responses and plasma progesterone concentrations in superovulated, artificially inseminated, lactating cows 6 to 8 d after breeding
Parameter measured
Cool season (mean + SRM)
Hot season (mean + SFM)
P value
Rectal temp ("F)
101.9 + 0.1 (28)a
102.0 + 0.1 (19)
NSb
Respiratory rate (breaths/min)
33.8 t 2.1 (27)
45.9 IL 3.1 (17)
P < 0.001
Heart rate (beats/min)
91.4 + 2.8 (25)
86.9 rf: 2.9 (19)
NSC
Progesterone (ng/ml) Range
15.6 5 2.6 (21) 2.3 - 47.9
17.2 + 4.0 (27) 2.6 - 66.2
NS=
aThe number of replicates is indicated in parentheses. bNot significant.
Table 5 shows the physiologic responses and hormone levels of hotand cool-season cows. The hot season induced a significant increase in respiratory rate, even though measurements were made during the There were significant (P < 0.01) moderate early morning hours.
458
OCTOBER
1987 VOL. 28 NO. 4
THERIOGENOLOGY
correlations observed between respiratory rate of all cows and morning and afternoon air temperature (r - 0.448 and 0.450, respectively), rectal temperature (r = 0.442), and plasma progesterone concentration (r = 0.544). Rectal temperatures were slightly higher and heart rates were slightly lower in hot-season cows than in cool-season cows, although the differences were not statistically significant (P > 0.05). Plasma progesterone concentrations varied widely in both hot- and coolseason superovulated cows, with no significant seasonal effects being observed. While plasma progesterone concentrations were not correlated with air temperatures or rectal temperatures, they were significantly correlated with the number of CL present (P < 0.05). Analysis of the hormone results and embryo viability and development data showed that there were no significant differences between data collected during the two hot seasons and the data collected during the two cool seasons. DISCUSSION The studies reported in this article, and others (l-3,10,15,19), demonstrate the ideal conditions in central Arizona for investigating the pathogenesis of heat-stress infertility in dairy cows. The ability of the dairy cow to adapt to intense summer heat is greatly reduced by the metabolic stresses of pregnancy and lactation (3). Adaptation to chronic heat stress has been shown to reduce fertility and milk production in order to lessen the body heat load which must be dissipated to maintain thermoneutrality (1-7). During the intense afternoon heat in central Arizona, lactating cows often become hyperthermic (3). Bligh (20) reported that the rectal temperature of the bovine begins to rise when the environmental temperature exceeds 30°C. Wolff and Monty (3) found this to be true in lactating Holstein cows but not in nongravid, nonlactating cows. Respiration increases greatly in heat-stressed cows and must play an important role in thermoregulation (3) since Bovidae have a limited capacity for heat dissipation by sweating and body surface evaporation. Our results are consistent with heat-induced respiratory compensation (Table 5). Under the conditions of our study, the use of increased respiratory evaporative cooling maintained basal (early morning) rectal temperatures at cool-season levels, whereas a previous study in Arizona (3) identified a seasonal rise. Plasma progesterone levels varied drastically during both seasons among individual superovulated cows and were positively correlated with the number of CL on the ovaries. It is likely, therefore, that the tremendous variation in ovarian progesterone output, induced by the gonadotropins used in the superovulation procedure, obscured any smaller variation in progesterone secretion that may have resulted from The effect of heat stress on peripheral climatic conditions. progesterone levels in naturally ovulating dairy cows is unsettled. Previous studies in Arizona (8) reported elevated progesterone levels during the luteal phase of the estrous cycle in hyperthermic, lactating that remained not in nongravid, nonlactating cows cows but
OCTOBER 1987 VOL. 28 NO. 4
459
THERIOGENOLOGY
thermoneutral. Further studies report increased progesterone levels associated with high temperatures (21), while others report decreased levels (19,22). Shaded cows have been shown to have lower progesterone levels than unshaded cows (23). Acute exposure to high temperatures for the first 72 h after breeding has been reported to raise plasma progesterone levels significantly in Hereford heifers (24). The ovulation rate of superovulated cows was unaffected by summer heat. There were, however, significantly fewer embryos flushed from the uteri of hot-season cows than from the uteri of cool-season cows. The reason for this is not known; however, the flushing procedure rarely retrieves all of the embryos in the uterus (as estimated by the Other studies have reported no effect of number of CL palpated). season on ovulation in naturally ovulating cows (1,3) and sheep (25), although a high incidence of anovulation and delayed ovulation has been reported in Israeli cows (26). The number of unfertilized ova recovered from superovulated cows was significantly greater during the hot season than during the cool season. The reason for this is unknown; however it may have been related to the high peripheral progesterone levels in those superovulated cows that yielded unfertilized ova. The mean plasma progesterone concentration in all superovulated cows was 16.5 ng/ml, whereas the average concentration in those superovulated cows that yielded unfertilized ova was 28.8 ng/ml. Progesterone and estradiol control genital tract secretion and motility, and sperm movement up the oviduct or ova movement down the oviduct may have been disrupted, preventing fertilization. The quality of the ova released by superovulated ovaries may be either inferior or more susceptible to the adverse effects of heat stress. The results of a recent study with hamster oocytes revealed that oocytes obtained from the normal preovulatory pool differ, electrophysiologically, from those that have been stimulated with pregnant mare's serum gonadotropin (27). These findings raise the interesting possibility that gonadotropinstimulated bovine oocytes may not be truly identical to those destined to be ovulated in animals that are cycling normally. Results of a previous study at the Arizona Dairy Co. on a limited number of normally ovulating cows in late lactation (10 cool-season and 9 hot-season cows, at an average ll~, of lactation) suggested that embryo development is retarded by summer heat stress (unpublished data). The development of embryos flushed from the oviducts of cows sent to slaughter 1.5 to 4.0 d after AI indicated retardation; however, the differences between seasons were not statistically significant. There was a high incidence of anovulation during both seasons. The indication of developmental retardation suggested an early sign of metabolic perturbation preceding death of the embryo. Analysis of the developmental stage of embryos flushed from the uteri of superovulated cows 6 to 8 d after breeding (Table 2) failed to reveal a significant effect of heat stress on embryonic development in vivo. However, the data presented in Tables 3 and 4 show a marked seasonal effect on the in vitro developmental capacities of morulae and blastocysts flushed from the uteri of superovulated cows. The development of embryos from heat-stressed cows in culture was significantly retarded. The results
460
OCTOBER 1987 VOL. 28 NO. 4
THERIOGENOLOGY
suggest that heat stress drastically reduces the capacity of bovine embryos to undergo blastulation and hatching from the zona pellucida. Although superovulation is reported to increase peripheral progesterone levels and reduce embryo quality (28), the predominant loss of viability and developmental capacity occurred in the summer season and may be attributed to seasonal heat stress. In Israel, it has been found that embryo mortality is greatest in repeat-breeder cows on Day 7 of the estrous cycle (29,30), at a time shortly after the embryo arrives in the horn of the uterus and when it is undergoing blastulation. The results of our study suggest that the problematic survival of embryos at this precarious stage of their development is Reduced embryo compounded by the adverse effects of heat stress. viability at this stage of development is consistent with previous findings that the estrous cycle is not prolonged in summer-season, repeat-breeding cows in Arizona (1). Since 7-d-old bovine embryos are not luteotropic (31) but may begin to be as early as Day 10 (32), heat stress-induced inhibition of blastulation and the demise of the embryo would preclude maintenance of the CL and prolongation of the estrous cycle. Heat stress in the ewe is also associated with early embryo mortality, which does not alter the length of the estrous cycle (11). Other studies with bred cows, however, report an association between heat stress and prolongation of the estrous cycle (2), suggesting that under certain conditions, death of the embryo may occur after maternal recognition of pregnancy, which occurs between Day 15 and 17 postbreeding (33); thus, the life of the degenerating embryo is prolonged, postponing luteolysis. The mechanisms underlying these observed adverse effects of heat stress on survivability of Day 6 to 8 cow embryos are unknown but may be due to a direct action of heat on the embryo or to an indirect effect that is mediated by the uterine environment. In support of a direct action, intrauterine temperatures are higher than rectal temperatures in hyperthermic lactating cows (3): high intrauterine temperatures may affect spermatozoa, ova, or embryos and have been related to infertility (34). Heat-damaged spermatozoa that fertilize ova may produce abnormal embryos which subsequently die (35). Ulberg and Sheehan (36) have reported that a slight increase in temperature can directly disorient embryo development, producing embryos which grow and may appear morphologically normal, but which subsequently die. The embryo is most susceptible to thermal stress during the first cleavage divisions. In support of an indirect action are the reports (described above) of abnormal sex steroid levels in heat-stressed animals. Progesterone and estrogen are essential to the control of uterine secretion and motility. It is reasonable to assume that abnormal steroid levels could produce a uterine environment that either is unable to support embryo development or is toxic to the embryo. Progesterone levels have been reported to influence the protein composition of uterine secretions of cattle (37), and heat stress has been reported to change the amino acid composition of oviductal secretions in rabbits (38).
OCTOBER
1987 VOL. 28 NO. 4
461
I-HERIOGENOLOGY
Either indirect or direct mechanisms may impair development and lead to the death of the embryo before maternal recognition of pregnancy, explaining one of the signs of summer heat stress infertility in Arizona dairy cows. Further studies are needed on larger numbers of non-superovulated cows in order to confirm the role of early embryo viability in summer heat stress infertility in dairy cows. In addition, the efficiency of embryo transfer procedures may be significantly lowered by the reduced embryo viability associated with hot weather.
REFERENCES 1.
Monty, D.E. and Wolff, L.K. Summer heat stress and reduced fertility in Holstein-Friesian cows in Arizona. Am. J. Vet. Res. 2:1495-1499 (1974).
2.
stott, G.H. and Williams, R.J. Causes of low breeding efficiency in dairy cattle associated with seasonal high temperatures. J. Dairy Sci. a:1369-1375 (1962).
3.
Wolff, L.K. and Monty, D.E. Physiologic response to intense summer heat and its effect on the estrous cycle of nonlactating and lactating Holstein-Friesian cows in Arizona. Am J. Vet. Res. =:187-192 (1974).
4.
Dunlap, S.E. and Vincent, C.K. Influence of postbreeding thermal stress on conception rate in beef cattle. J. Anim. Sci., z:1216-1218 (1971).
5.
Kelly, M.S. and Hurst, V. The effect of season on fertility of the dairy bull and diary cow. J. Am. Vet. Med. Assoc. 143:40-43 (1963).
6.
Hafez, E.S.E. Reproductive capacity of farm animals in relation to climate and nutrition. J. Am. Vet. Med. ASSOC. m:606-614 (1959).
7.
Heiman, M.M. Results and conclusions drawn from breeding. Dtsch. Tierarztl, Wschr. &:206-209 (1977).
8.
Wolff-Vaught, L., Monty, D.E. and Foote, W.C Effect of summer heat stress on serum luteinizing hormone and progesterone values in Holstein-Friesian cows in Arizona. Am. J. Vet. Res. %:10271030 (1977).
9.
Behavioral and physiologic Monty, D.E. and Garbareno, J.L. responses of Holstein-Friesian cows to high environmental temperatures and artificial cooling in Arizona. Am. J. Vet. Res. 2:877-882 (1978).
462
OCTOBER
A.I.
1987 VOL. 28 NO. 4
THERIOGENOLOGY
10.
Monty, D.E. Early embryo death in cattle during thermal La Reproduction De stress. Reunion Internationale sur: Ruminants En Zone Tropical. pp. 285-300. Jan 8 to 10, 1983. Pointe-a-Pitre (Guadaloupe) Antilles, Francaises.
11.
Dutt, R.H. Critical period for early embryo mortality in ewes exposed to high ambient temperature. J. Anim. Sci. 2:713-719 (1963).
12.
Edwards, R.L., Omtvedt, J.T., Turman, J., Stevens, D.F. and Mahoney, J.W. Reproductive performance in gilts following heat stress prior to breeding and in early gestation. J. Anim. Sci. =:1634-1637 (1968).
13.
Pennvciuk. P.R. The effects on rats of chronic exposure to 34°C: Iv. Reproduction. Aust. J. Biol. Sci. c:245-260 (1964).
14.
Sod-Moriah, U.A. Reproduction in the heat-acclimatized female rat as affected by high ambient temperature. J. Reprod. Fertil. 2&:209-218 (1971).
15.
Wiersma, F. and Stott, G.H. New concepts in the physiology of heat stress in cattle of interest to engineers. Tr. Am. Sot. Agric. Engineers Q:130-132 (1969).
16.
Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. Protein measurements with Folin phenol reagents. J. Biol. Chem. m:265-275 (1951).
17.
Niswender, G.D. Influence of the site of conjugation on the specificity of antibodies to progesterone. Steroids =:413-424 (1973).
18.
Sokal, R.R. and Rohlf, F.J. 1981, pp. 731-746.
19.
stott, G.H., Wiersma, F., Wood, J.M. Reproductive health program for cattle subjected to high environmental temperatures. J. Am. Vet. Med. Assoc. m:1339-1344 (1972).
20.
Bligh, J. The relationship between the temperature of the rectum and of the blood in the bicarotid trunk of the calf during exposure to heat stress. J. Physiol. m:393-403 (1957).
21.
Abilay, T.A., Johnson, H.D. and Madan, M. Influence of environmental heat on peripheral plasma progesterone and cortisol during the bovine estrous cycle. J. Dairy. Sci. 58: 1836-1840 (1975).
22.
Seasonal Rosenberg, M., Herz, Z., Davidson, M. and Zolman, Y. variations in post-partum plasma progesterone levels and
OCTOBER
1987 VOL. 28 NO. 4
Biometry.
Freeman, San Francisco,
I-HERIOGENOLOGY
conceptions
in
primiparous
J. Reprod. Fertil. =:363-367
and
multiparous
dairy
cows.
(1977).
23.
Hormonal Roman-Ponce, H., Thatcher, W.W. and Wilcox, E.J. interrelationships and physiological responses of lactating dairy cows to a shade management system in a subtropical environment. Theriogenology &:139-154 (1981).
24.
Mills, A.C., Thatcher, W.W., Dunlap, S.E. and Vincent, C.K. Influence of postbreeding thermal stress on peripheral plasma progestin concentrations in heifers. J. Dairy. Sci. a:1 abstr. (1979).
25.
Ulberg, L.C. and Burfening, P.J. Embryo death resulting from adverse environment on spermatozoa or ova. J. Anim. Sci. x:571-577 (1967). ’
26.
Ayalon, N., Feingold, D. and Almeida, A. Disturbed ovulation and summer infertility in Friesian cows in Israel. Refuah Vet. 39:1-2 (1982).
27.
Racowsky, C. and Satterlie, R.A. Metabolic, fluorescent dye and electrical coupling between hamster oocytes and cumulus cells during meiotic maturation b m and & m. Dev. Biol. m:191-202 (1985).
28.
Jensen, A.M., Greve, T., Madej, A. and Edqviat, L.E. Endocrine profiles and embryo quality in the PMSG-PGF2 treated cow. Theriogenology u:33-44 (1982).
29.
Fertility losses in normal cows and repeat Ayalon, N. breeders. Proc. 7th Int. Congr. Anim. Reprod. and A.I., Munich, Vol. 1, p. 741 abstr. (1972).
30.
Comparative Ayalon, N. Annual Report of Research, No. 2: studies of repeat breeders and normal cows. Kimron Veterinary Institute, Beit Dagan (Israel). pp. 1-48 (1973).
31.
Linares, T., Larsson, K. and Edquist, L. Plasma progesterone levels from oestrus through day 7 after A.I. in hiefers carrying embryos with normal or deviating morphology. Theriogenology =:125-132 (1982).
32.
Hansel, W. Plasma hormone concentrations associated with early embryo mortality in heifers. J. Reprod. Fertil. Suppl. x:231-239 (1981).
33.
Northey, D.L. and French, L.R. Effect of embryo removal and intrauterine infusion of embryonic homogenates on the lifespan of the bovine corpus luteum. J. Anim. Sci. s:298-302 (1980).
464
OCTOBER
1987 VOL. 28 NO. 4
THERIOGENOLOGY
34.
W.W. and Wilcox, C.J. Thatcher Gwazdaukas, F.C., factors at and hormonal environmental Physiological, insemination which affect conception. J. Dairy. Sci. 561873-877 (1973).
35.
Burfening, P.J. and Ulberg, L.C. Embryonic survival subsequent to culture of rabbit spermatozoa at 38" and 40°C. J. Reprod. Fertil. =:87-92 (1968).
36.
Ulberg, L.C. and Sheehan, L.A. Early development of mammalian embryos in elevated ambient temperatures. J. Reprod. Fertil. Suppl. =:155-161 (1973).
37.
Bartol, T.F., Thatcher, W.W., Lewis, G.S., Bliss, E.L., Drost, Effects of estradiol-17 beta on PGF and M. and Boyer, F.W. total protein content in bovine uterine flushings and peripheral plasma concentrations of 13,14-dihydro-15-keto PGF2e. Theriogenology u:345-358 (1981).
38.
Clark, D.A. The effects of heat stress and pregnancy on selected constituents of rabbit oviductal fluid. Thesis, Univ. of Missouri, Columbia. (1979).
OCTOBER 1987 VOL. 28 NO. 4
465