ELSEV] ER

ADVERSE IMPACT OF HEAT STRESS ON EMBRYO PRODUCTION: CAUSES AND STRATEGIES FOR MITIGATION

P.J. Hansen t , M. Drost a, R.M. Rivera ~, F.F. Paula-Lopes t. Y.M. AI-Katananit,C.E. Krininger III ~ and C.C. Chase, Jr 3

~Department of Animal Sciences and 2 Department of Large Animal Clinical Sciences, University of Florida, Gainesville, Florida 32611-0920.3Subtropical Agricultural Research Station, USDA,

ARS, Brooksville, Florida 34601-4672

ABSTRACT

The production of embryos by superovulation is often reduced in periods of heat stress. The associated reduction in the number of transferable embryos is due to reduced superovulatory response, lower fertilization rate, and reduced embryo quality. There are also reports that success of in vitro fertilization procedures is reduced during warm periods of the year. Heat stress can compromise the reproductive events required for embryo production by decreasing expression of estrus behavior, altering follicular development, compromising oocyte competence, and inhibiting embryonic development. While preventing effects of heat stress can be difficult, several strategies exist to improve embryo production during heat stress. Among these strategies are changing animal housing to reduce the magnitude of heat stress, utilization of cows with increased resistance to heat stress (i.e., cows with lower milk yield or from thermally-adapted breeds), and manipulation of physiological and cellular function to overcome deleterious consequences of heat stress. Effects of heat stress on estrus behavior can be mitigated by use of estrus detection aids or utilization of ovulation synchronization treatments to allow timed embryo transfer. There is some evidence that embryonic survival can be improved by antioxidant administration and that pharmacological treatments can be developed that reduce the degree of hyperthermia experienced by cows exposed to heat stress. © 2000 by Elsevier Science Inc.

Key words: heat stress, superovulation, in vitro fertilization, oocyte, embryo, cattle

THE IMPACT OF HEAT STRESS ON EMBRYO PRODUCTION

Reproductive processes in the male and female mammal are very sensitive to disruption by hyperthermia with the most pronounced consequences being reduced quantity and quality of sperm production in males and decreased fertility in females (32). Problems of heat stress can be experienced over a wide geographical area: hyperthermia in lactating dairy cows can occur at air temperatures as low as 27 °C (12) and a summer depression in fertility (62) and oocyte quality (55) have been observed in the Upper Midwest region of the United States. Infertility in the male caused by heat stress can be eliminated through the utilization of artificial insemination (AI) with semen collected and frozen from males in cool environments. In females, the situation is more complicated.

Acknowledgements This is Journal Series No. R-07765 of the Florida Agricultural Experiment Station. The authors' research was supported in part by grants from the USDA NRICGP and TSTAR programs and by the Florida Milk Checkoff Program. Correspondence: Email: hansen@animal.ufl.edu

Theriogenology 55:91-103, 2001 0093-691YJO1/S-see front matter © 2000 Elsevier Science Inc. PII: S0093-691X(00)00448-9

92 Theriogenology

Embryo transfer represents a method analogous to AI in that embryos can be collected from non- stressed cows and transferred to heat-stressed recipients (56). Nonetheless, the donor cow is itself susceptible to heat stress. The production of embryos by superovulation is often reduced in periods of hot weather. The hot-weather associated reduction in the number of transferable embryos via superovulation is due to reduced superovulatory response [seen in some (2, 29) but not all experiments (42)], lower fertilization rate (29, 42), and reduced embryo quality (2, 29, 42). Experimental application of heat stress on days 1-7 after insemination can also lead to reduced embryonic development (48). There are also reports that success of in vitro fertilization (IVF) procedures is reduced during warm periods of the year. This has been shown for ooeytes recovered from slaughterhouse ovaries in Wisconsin (55) and collected from Angus crossbred and Holstein cows in Louisiana via ultrasound-guided follicle aspiration (54). However, there was no seasonal variation in IVF success using oocytes collected from a slaughterhouse in Florida where most slaughtered cows were beef cows and many had Bos indicus breeding (52). Similarly, oocytes from Brahman cows in Texas did not display seasonal variation in subsequent IVF performance (54). As will be detailed later, genotype can have major effects on the magnitude of heat stress effects on the whole animal and on tissue responses to heat shock.

The purpose of this review is to elucidate what is known about the mechanisms by which heat stress compromises embryo production via superovulation and in vitro fertilization and to discuss approaches for improvement of embryo production during hot weather that are based on the physiological, genetic, and environmental factors that determine the magnitude of the influence of heat stress on bovine reproduction.

ACTIONS OF HEAT STRESS ON REPRODUCTIVE PROCESSES IMPORTANT FOR EMBRYO PRODUCTION

Detection of Estrus

It is more difficult to detect estrus in donors and recipient cattle when cattle are subjected to heat stress. Estrus in Guernsey heifers maintained at 18.2 o C averaged 17.0 h in length vs. 12.5 h for heifers at 33.5 °C (1). Similarly, the number of mounting episodes per estrus for Holstein cows in Virginia was 4.5 mounts in summer vs 8.6 in winter (45). The mechanism by which heat stress reduces expression of estrus may be hormonal in part since there are reports that heat stress reduces circulating estradiol- 17~ concentrations and can sometimes increase adrenocorticotropin secretion, which itself can block estradiol-induced estrus behavior (see 33 for review). It is also likely that estrus expression is reduced by the physical lethargy experienced by heat-stressed cows.

Follicular Development

Seasonal effects on superovulatory responses probably reflect the disruption of follicular development that can occur concomitant with heat stress. Heat stress has been reported to decrease growth (10) and follicular fluid concentrations of estradiol-1713 (68) of the first-wave dominant follicle. In addition, heat stress seems to suppress the ability of the first-wave dominant follicle to exert dominance since heat stress beginning at day 1 of the estrous cycle caused an increase

Theriogenology 93

in number of follicles > 10 mm in diameter and earlier emergence of the dominant follicle of the second follicular wave (67). Initiation of heat stress at day 11 of the estrous cycle decreased the size of the second-wave dominant follicle in heifers (65) and circulating concentrations of estradiol-1713 in heifers and cows (65, 66). Heat stress beginning at day 11 either decreased (65), increased (61) or had no effect (66) on number of small follicles.

Table 1.

Breed

Breed differences in effect of season on production of embryos via in vitro maturation, fertilization and development in Louisiana."

Variable Cool season Hot season

Holstein

Brahman

No. of oocytes

Oocytes classified as normal (%)

Fertilization rate (%)

Oocytes developing to blastocyst (%)

No. of oocytes

Oocytes classified as normal (%)

Fertilization rate (%)

Oocytes developing to blastocyst (%)

67 28

80.0+ 19.1 24.6+6.3**

59.8 + 11.7 52.3 + 10.6

29.0 + 14.8 0"*

83 89

83.3 + 17.4 77.0 + 6.3

83.1 + 10.7 79.3 + 10.6

52.3 + 13.5 41.3 + 7.2

a from reference 54. ** P<0.01

Oocyte Competence

There is evidence that fertiliTation rate following AI is compromised by heat stress (58). Effects of heat stress on oocyte competence can also affect the success of embryo production technologies. Thus, fertilization rate in superovulated cows is sometimes lower in summer than in winter (29, 42, 51). In a study with a limited number of oocytes collected from Holstein cows in Louisiana via ttltrasound guided aspiration, the proportion of oocytes classified as morphologically normal and the rate of blnstocyst development following in vitro fertilization was lower in summer than winter (54; see Table 1). There was a also a decrease in the proportion of oocytes that developed to the blastocyst stage during July and August as compared to other months in a study (55) using oocytes recovexed from a Wisconsin abattoir that presumably represented a preponderance of Holsteins. In both the Louisiana and Wisconsin studies, fertilization rate was not affected by season but the lower development following fertilization during summer is indicative of oocyte damage. Furthermore, there were lower rates of both fertiliTation and embryonic development during summer than winter in another study in Louisiana using oocytes collected via ultrasound-g~_~ided aspiration from Holstein and crossbred Angus cows.

Further evidence for effects of heat stress on oocyte competence comes from a retrospective analysis of a large data set from Florida and south Georgia. Among cows exposed to cool temperatures from day 9 before estrus until breeding, there was a negative association between beat stress 10 days before breeding and subsequent 90-day non-return rate (3). Near estrus, the oocyte also appears sensitive to damage since exposure of superovulated cows to heat stress for 10 h beginning at the onset of estrus had no effect on fertilization rate but reduced the proportion of normal embryos recovered on day 7 after estrus (50).

94 Theriogenology

There are at least two possible mechanisms by which heat stress could compromise oocyte competence. First, disruption in patterns of follieu!ogenesis could lead to ovulation of an aged oocyte with lowered potential for fertiliT~tion (40). Consistent with this idea is the finding that heat stress beginning at day 1 oftbe estrous cycle caused earlier emergence oftbe dominant follicle of the second follicular wave (67). However, this explanation does not explain why season (and presumably heat stress) adversely affects mukiple oocytes in the cohort of follicles ovulated following superovulation (29), collected by transvaginal ultrasound-guided recovery (54) or aspirated from excised ovaries (55). Perhaps oocytes are analogous to male germs cells in that they have heightened sensitivity to elevated temperature. Unlike most cells, the oocyte in developing follicles is transcriptionally inactive after it reaches a diameter ~ 110 ~tm (i.e., at about the 3-mm follicle stage; 37). This means that the range of cellular adjustments to elevated temperature that are possible in the oocyte are limited to those not involving transcription. For example, bovine oocytes cannot undergo increased synthesis of heat shock protein 70 in response to elevated temperature (22, 23).

Damage to Sperm Placed in the Reproductive Tract

It is possible that exposure of spermatozoa to elevated temperatures while in the uterus or oviduct of a hyperthermic female could compromise sperm survival in utero or fertilizing capability. While survival in utero has not been examined, frozen-thawed spermatozoa are not greatly affected by heat shock as determined by simple measures of functionality such as motility and viability (15, 41). Such simple tests may not be adequate to detect sperm damage. Rabbit embryos formed from fertilization of oocytes with heat-shocked spermatozoa had reduced survival (14, 35).

Embryonic Development

Early embryonic development can be compromised by heat stress. Experimental application of heat stress from day 1 to 7 after estrus reduced developmental stage and morphological characteristics of embryos flushed from the reproductive tract of superovulated cows at day 7 after estrns (48). Similarly, embryos recovered from superovulated cows in Arizona from June through September were less able to develop in culture than similar embryos collected from superovulated cows in October through May (42). The mechanism by which heat stress decreases embryonic development is likely multifactorial. However, given that exposure of cultured embryos to temperatures characteristic of rectal temperatures of lactating cows in Florida during the summer can decrease embryonic development (53), it is likely that direct effects of elevated temperature on embryonic function contribute to the decrease in development. In sheep, a reciprocal embryo transfer scheme was utilized to show that both the embryo and reproductive tract are compromised by heat stress, with greater effects being exerted on the embryo itself (4).

One of the features of embryonic development is that the embryo becomes more resistant to maternal heat stress as pregnancy advances. Exposure to a no-shade environment in the summer reduced development and viability of embryos on day 8 after estrns ff superovulated cows were exposed to heat stress at day 1 after estrns but not if heat stress was imposed on days 3, 5 or 7 (21). In culture, morulae are less susceptible to disruption by elevated temperature than 2-cell embryos (23). If embryonic resistance to elevated temperature increases as development proceeds, one would

Theriogenology 95

expect that heat stress would have less effect on embryonic survival for recipient cows receiving an embryo at day 7 after estrus than for inseminated cows. This has been shown in several studies (5, 18, 49). Also, there was also no relationship between environmental temperature and pregnancy rate in a large data set from recipient cows in the southern United States (51). The application of embryo transfer as a scheme for improving pregnancy rates of heat-stressed cows is discussed elsewhere in this proceedings (56).

Though embryonic resistance to heat stress increases with advancing gestation, severe heat stress can inhibit embryonic development later in pregnancy. In a large data set of lactating Holsteins in Florida and Georgia, air temperature on day 10 after estrus was associated with reduced fertility among a subset of cows exposed to cooler temperatures from days 0-9 relative to estrus (Figure 1 and ref 3). In addition, heat stress from days 8-16 of pregnancy reduced embryonic size at day 17 (13).

~-- lOO

"-" 9 0

N 80

70 e- . L . .

~ 60

~ 50 i

t - 4 0 o ' - 30

!

o 20

lO

o Jan Feb Mar A p r M a y J u n Jul AugSep Oct Nov Dec

M o n t h o f f i r s t s e r v i c e

Figure 1. Seasonal variation in 90-day non-return rate in Holsteins in south Georgia and Florida as affected by milk yield. Data represent least-squares means + SEM for cows with 305-day milk yields less than 4536 kg (O), 4536-9072 kg (o) and > 9072 kg (&). Data are reproduced from ref. 3 with permission of Journal of Dairy Science.

APPROACHES FOR MITIGATING EFFECTSOF HEAT STRESS ON EMBRYO PRODUCTION

Heat stress reduces embryo production because physiological and cellular aspects of reproductive function are disrupted by either the increase in body temperature caused by heat stress or by the physiological adaptations engaged by the cow to reduce hyperthermia. It follows then that

96 Theriogenology

one can improve embryo production during heat stress by 1) reducing the magnitude of heat stress through environmental manipulation, 2) altering the cow genetically or physiologically to improve regulation of body temperature in the presence of heat stress or 3) manipulating the cow to prevent or overcome the disruption in cellular and physiological processes that compromise reproduction.

Reducing Degree of Heat Stress

One obvious method for reducing the effects of heat stress is to change the environment to reduce factors driving cows into hyperthermia. In dairy cattle, extensive facilities with shade, fans, and various evaporative cooling systems are often deployed. While cooling cows can increase pregnancy rate (see ref. 32 for review), large seasonal variation in reproductive function can persist even on farms that have made extensive use of such cooling systems (33).

Given that embryos are most sensitive to heat stress early in pregnancy (21, 23), providing cooling for a limited number of days during maximum sensitivity of the embryo to heat stress can moderatley improve pregnancy rates (see ref. 32). For example, pregnancy rate to AI for cows cooled for 8 days beginning after prostaglandin F,_~ (PGF2~) was 16% vs 6% for controls (19). Failure of limited cooling to increase pregnancy rate more dramatically is probably a reflection of heat stress on follicular or oocyte function before cooling was initiated or later in pregnancy after cooling had been terminated.

Altering Ability of Cows to Regulate Body Temperature

Genetic effects. There are clear genetic differences in resistance to heat stress, with tropically-adapted breeds

experiencing lower body temperatures during heat stress than non-adapted breeds (28, 30, 31). The summer-time reduction in oocyte quality observed in Holsteins in Louisiana was not observed for Brahman cattle in Texas (54; Table 1). Even in non-adapted breeds, it is probably possible to perform genetic selection for resistance to heat stress since the heritability estimate for rectal temperature in cattle is high (0.25-0.65, reviewed in ref. 28). There are also specific genes that could be selected which confer increased thermoregulatory ability, including those for coat color (39, 53) and the slick gene identified in Senepol cattle that causes short hair length (46).

It may be possible to identify genes that control cellular resistance to elevated temperature. The superior fertility of tropically-adapted breeds during heat stress is a function in large part of the enhanced ability of animals from these breeds to regulate body temperature in response to heat stress. However, certain tropically-adapted breeds are also more resistant to elevated temperature at the cellular level. For example, heat shock killed a smaller proportion of lymphocytes from Brahman and Senepol cows than from Holstein and Angus cows (38). Recently, it was demonstrated that the reduction in development caused by culturing embryos at 41 ° C for 6 h was less for embryos from Brahman cows than for embryos from Holstein and Angus cows (47). Identification of the genes responsible for enhanced cellular resistance to heat shock may allow these genes to be transferred into thermally-sensitive breeds through conventional or transgenic breeding techniques to produce an animal whose oocytes and embryos have increased resistance to elevated temperature.

Theriogenology 97

Milk yield. One of the most important determinants of thermoregulatory ability is milk yield because of the

metabolic heat production required for milk synthesis. The increase in body temperature upon exposure to heat stress is greater for luctating cows than non-lactating cows (16) and, among lactating cows, body temperature during heat stress hv~reases with increasing milk production (12). The seasonal variation in pregnancy rate per AI in lactating dairy cows in Florida was absent in non-

lactating heifers (11). Moreover, the summer depression in 90-day non-return rate in Florida and south Georgia was more pronounced in cows with high milk yield than in cows with lower yields.

Milk yield of an embryo or oocyte donor is likely to have a similar effect on the degree to which heat stress compromises embryo production. Data on the relationship between superovulation results for cows in the southwest United States and average maximum air temperature from day 0-7 after estrus are summarized in Table 2. Although numbers of observations are few, note the large decline in superovulation performance with increasing air temperature in dairy cattle. In a much larger data set of beef cattle, there was no effect of envirormaental temperature. This diffeamace probably reflects differences in milk yield as well as breed differences in thermoregulatory ability. However, even in a data set restricted to European cattle (>95% beef), there was no relationship between maximum air temperature and superovulation performance (51).

Table 2. Differences between dairy and beef cattle in the relationship between air temperature on days 0-7 after estrus and response to superovulation in the southwest United States."

Maximum air temperature (°C)

Variable <27 27-32 32-40 >40

Dairy cattle

Beef cattle

Number of cows 8 10 4 0

Ova collected/cow 9.0~3.4 9.5+3.3 1.3!-0.2"*

Ova fertilized (number/cow 7.8+2.6 7.0-!-_2.4 0.3_+0.2 and % of ova) (87.5%) (73.7%) (20.0%)**

Transferable embryos 6.5+2.2 4.3+1.4 0.3_+0.2 (number/cow and % of ova) (72.2%) (45.3%) (20.0%)**

Number of cows 893 904 1,260 301

Ova collected/cow 12.7+ .3 11.8_!-0.3 b 12.2_+0.3 11.8+0.6

Ova fertilized (number/cow 9.5_+0.3 8 .8_+0.3 9 .3_+0 .2 9.1_+0.5 and % of ova) (74.6%) (74.6%) (76.6%) (77.2%)

Transferable embryos 7.2_+0.2 7 .5_+0.2 7 .1_+0 .2 6.8_+0.4 (number/cow and % of ova) (56.1%) (55.2%) (58.2%) (57.4%)

"from ref. 51 b extrapolated from data ** P<0.01 for 32-40 vs other groups

98 Theriogenology

Bovine somatotroDin. Treatment of dairy cows with bovine somatotropin (BST) increases body temperature during

beat stress (16, 27, 64). Nonetheless, it is not clear wbether treatment of cows with BST will reduce or enhance fertility during heat stress. Recent data from Thatcher and colleagues (reviewed in this volume; ref. 60) indicate that treatment of cows with BST can increase pregnancy rates of cows bred via timed AI, increase the survivability of embryos recovered from donors, and increase the pregnancy rate of recipients treated with BST (43).

Pharmacolo2ical control of body temperat~¢, There has been little interest in cattle in identifying methods to pharmacologically regulate body

temperature during heat stress. There are reports that feeding culture extracts of the fungus Asner~illus o ryzae (36) and supplemental niacin (59) can decrease body temperatures of heat-stressed cows. In sedentary sheep, treatment with indomethacin lowered the rise in body temperature accompanying heat stress (57), presumably because indomethacin blocks prostaglandin-mediated actions of gastrointestinal endotoxins released during heat stress on hypothalamic thermoregulatory centers. Interestingly, physically fit sheep experienced lower degrees of hyperthermia following heat stress (ascribed to increased blood perfusion of the gastrointestinal tract due to increased cardiovascular output) than sedentary sheep and indomethacin did not affect body temperature in these sheep (57).

Overcoming Changes in Cellular and Physiological Processes that Cause Decreased Embryo Production

Estrus detection. Two approaches are available for overcoming the reduced expression of estrus in beat-stressed

cows. The first is to take advantage of one of the several estrus-deteetion aids available such as HeatWatch® transponders, pedometers, Kamar* detectors and tail paint as well as use of androgenized females or surgically-modifr, d bulls as beat-detector animals. Little research has been conducted to determine the efficacy of these approaches under heat stress conditions. In a study conducted during the summer in Florida, use of tail chalk increased the proportion of cows detected in estrus within 96 h following injection of PGF2~ from 24% to 43% (19).

The second approach is to take advantage of recent developments in ovulation synchronization to perform timed AI or embryo transfer in the summer. Utilization of a timed insemination program based on breeding cows at a fixed time using the OvSynch protocol [gonadotropin releasing hormone (GnRH) at day 0, prostaglandin F2a at day 7, GnRH at day 9 and insemination 16-24 h later] increased the pregnancy rate at fixed times postpartum (8, 17).

This same ovulation synchronization protocol has also been used to transfer embryos into receipients at a fixed time (5). Lactating cows during heat stress were subjected to the OvSynch protocol and received either a fresh or frozen-thawed, in vitro-produced embryo at 8 days after the last GnRH injection. Based on plasma progesterone concentrations, only 76% of cows responded successfully to the synchronization protocol. Among these cows, pregnancy rate was 6.7% for cows bred via AI, 6.1% for cows receiving a frozen embryo and 17.5% for cows receiving a fresh embryo. Improvements in the synchronization procedure itself, including inclusion of presynchronization

Theriogenology 99

treatments (63) and selection of cows based on body condition score (44), and improvements in culture systems and freezing systems to improve the freezability of in vitro produced embryos should make timed embryo transfer of in vitro produced embryos a useful tool for enhancing fertility in the summer (see ref 56 in these proceedings).

_Antioxidants. There is some evidence that the depression in embryo survival following exposure to elevated

temperature involves increased flee radical production. Addition of various antioxidants to culture medium can reduce the effects of heat shock on bovine (20) and mouse embryos (6, 7). Whether heat stress of cows is associated with increased free radical production is less clear. Exposure of cows to heat stress did not reduce circulating concentrations of the antioxidants ~-carotene or vitamin E or increase skeletal muscle content of malondialdehyde, a product of free radical oxidation of lipid membrane (61). However, heat stress was associated with reduced total antioxidant activity in blood plasma (34). Treatment of cows with antioxidants to improve fertility in summer has also given equivocal results. There was no beneficial effect of short-term treatment of heat-stressed cows with vitamin E (19) or I~-carotene (9) around the time of breeding. In contrast, long-term (~ 90 d) feeding of supplemental ~-carotene increased herd pregnancy rate for cows calving in Florida from May 2- August 5 (8). The proportion of cows pregnant by day 120 postpartum was 21% in controls and 35% for cows fed supplemental 13-carotene. Additional research regarding effects of long-term administration of antioxidants on fertility of lactating dairy cows is ongoing.

CONCLUSIONS

What Can Be Done to Maximize Embryo Production During Periods of Heat Stress? It is likely that reproductive function will be compromised in a cow whose body temperature increases approximately 1 °C or more. Environmental conditions to cause such a rise in body temperature occur frequently in many temperate areas of the world. One can argue that heat stress will have a greater effect on reproduction in the future than is the case currently because of continued increases in milk yield, which increases the susceptibility of cows to heat stress, as well as became of global climate changes. It has proven difticult to reduce effects of warm weather on fertility in dairy cattle and, when

possible, embryo production programs should be carried out in periods of the year when heat stress is not a concern. Nonetheless, there are specific steps that can be taken to mitigate effects of heat stress. Problems of diminished estrus behavior can be reduced by use of estrus detection aids or can be bypassed by utiliTation of ovulation synchronization treatments to allow for timed embryo transfer. Both donor and recipient animals should be housed in facilities that maximize cow comfort. In

addition, utiliT.ation of cows with increased resistance to heat stress is likely to improve success of embryo production programs during periods of hot weather. For example, embryos or oocytes can be recovered from donors when cows are on the descending portion of the lactation curve or are non- lactating. While not specifically tested experimentally, it is also likely that pregnancy rates following embryo transfer will be improved by using recipients with low milk yield or from thermally-adapted breeds. In the long-term, pharmacological approaches may be developed that involve use of embryotrophic drugs or antioxidants to overcome deleterious consequences of hyperthermia on embryonic survival or that use thermoregulatory molecules to reduce the magnitude of hyperthermia caused by heat stress.

1 O0 Theriogenology

REFERENCES

1. Abiiay TA, Johnson HD, Madan M. Influence of environmental heat on peripheral plasma progesterone and cortisol during the bovine estrous cycle. J Anim Sci 1975; 58:1836-1840.

2. Alfujairi MM, Alibrahim RM, Elnouty FD. Seasonal variations in superovulatory responses of Holstein cows treated with pregnant mare serum gonadotropin in Saudi Arabia. J Reprod Fertfl 1993; Abs Set 11:75 (abstr).

3. AI-Katauani YM, Webb DW, Hansen PL Factors affecting seasonal variation in 90 day non- return rate to first service in lactating Holstein cows in a hot climate. J Dan'y Sci 1999; 82:2611-2615.

4. Alliston CW, Ulherg LC. Early pregnancy loss in sheep at ambient temperatures of 70 ° and 90 ° F. as determined by embryo transfer. J Anim Sci 1961; 20:608-613.

5. Ambrose JD, Drost M, Monson RL, Rutledge JJ, Leibfried-Rutledge NIL, Thatcher M-J, Kassa T, Binelll M, Hansen PJ, Chenoweth PJ, Thatcher WW. Efficacy of timed embryo transfer with fresh and frozen in vitro produced embryos to increase pregnancy rates in heat- stressed dah'y cattle. J Dairy Sci1999; 82:2369-2376.

6. Ar~chi_ga CF, Ealy AD, Hansen PJ. Efficacy of vitamin E and glutathione for thermopmtection of murine morulae. Theriogenology 1994; 41:1545-1553.

7. Ar6chiga CF, Ealy AD, Hansen PJ. Evidence that glutathione is involved in thermotolerance of preimplantation mouse embryos. Biol Reprod 1995; 52:1296-1301.

8. Ar6~higa CF, Staples CR, McDowell LR, Hansen PJ. Effects of timed insemination and supplemental [3-carotene on reproduction and milk yield of dah-y cows under heat stress. J Dairy Sci 1998; 81:390-402.

9. Ar~chiga CF, V(tzquez-Flores S, Ort~z O, Hern(mdez-Cer6n J, Porras A, McDowell LR, Hansen PJ. Effect of injection of l~-carotene or vitamin E and selenium on fertility of lactating dairy cows. Theriogenology 1998; 50:65-76.

10. Badinga L, Thatcher WW, Diaz T, Drost M, Wolfenson D. Effect of environmental heat stress on follicular development and steroidogenesis in lactating Holstein cows. Theriogenology 1993; 39:797-810.

11. Badinga L, Collier ILl, Thatcher WW, Wilcox CJ. Effects of climatic and management factors on conception rate of dairy cattle in subtropical environment. J Dairy Sci 1985; 68:78-85.

12. Berman A, Folman Y, Kaim M, Mamen M, Herz Z, Wolfenson D, Arieli A, Graber Y. Upper critical temperatures and forced ventilation effects for high-yielding dairy cows in a subtropical environment. J Dairy Sci 1985; 68:1488-1495.

13. Biggers BG, Geisert RD, Wettemann RP, Buchanan DS. Effect of heat stress on early embryonic development in the beef cow. J Anita Sci 1987; 64:1512-1518.

14. Burfening PJ, Ulberg LC. Embryonic survival subsequent to culture of rabbit spermatozoa at 38* and 40"C. J Reprod Fertil 1968;15:87-92.

15. Chandolla RK, Reinertsen EM, Hansen PJ. Short communication: Lack of breed differences in responses of bovine spermatozoa to heat shock. J Dairy Sci 1999: 82:2617-2619.

16. Cole JA, Hansen PJ. Effects of administration of recombinant bovine somatotropin on the responses of lactating and nonlactating cows to heat stress. J Am Vet Med Assn 1993; 203:113-117.

17. de la Sota RL, Burke JM, Risco CA, Moreira F, DeLorenzo MA, Thatcher WW. Evaluation of timed insemination during summer heat stress in lactating dairy cattle. Theriogenology 1998; 49:761-770.

18. Drost M, Ambrose JD, Thatcher MJ, Cantrell CK, Wolsdorf KE, Hasler IF, Thatcher WW. Conception rates after artificial insemination or embryo transfer in lactating dairy cows

during summer in Florida. Theriogenology 1999; 52:1161-1167.

Theriogenology 101

19. Ealy AD, Ar6chiga CF, Bray DR, Risco CA, Hansen PJ. Effectiveness of short-term cooling and vitamin E for alleviation of infertility induced by heat stress in dairy cows. J Dairy Sci 1994; 77:3601-3607.

20. Ealy AD, Drost M, Barros CM, Hansen PJ. Thermoprotection of preimplantation bovine embryos from heat shock by glutathione and taurine. Cell Biol Int Rept 1992; 16:125-131.

21. Ealy AD, Drost M, Hansen PJ. Developmental changes in embryonic resistance to adverse effects of mammal heat stress in cows. J Dairy Sci 1993; 76:2899-2905.

22. Edwards JL, Hansen PJ. Elevated temperature increases heat shock protein 70 in bovine two- cellembryos and compromises function of maturing oocytes. Biol Reprod 1996; 55:340-346.

23. Edwards JL, Hansen PL Differential responses of bovine oocytes and preimplantation embryos to heat shock. Mol Reprod Dev 1997; 46:138-145.

27. Elvinger F, Natzke RP, Hansen PJ. Interactions of heat stress and bovine somatotropin affecting physiology and immunology of lactating cows. J Dairy Sci 1992; 75:449-462.

28. Finch VA. Body temperature in beef cattle: its control and relevance to production in the tropics. J Anim. Sci 1986; 62:531-542.

29. Gordon I, Boland MP, McGovern H, Lynn G. Effect of season on superovulatory responses and embryo quality in Holstein cattle in Saudi Arabia. Theriogenology 1987; 27:231 (abstr).

30. Hammond AC, Olson TA, Chase CC JR, Bowers EJ, Randel RD, Murphy CN, Vogt DW, Tewolde A. Heat tolerance in two tropically adapted Bos taurus breeds, Senepol and Romosinuano, compared with Brahman, Angus and Hereford cattle in Florida. J Anim Sci 1996; 74:295-303.

31. Hammond AC, Chase CC JR, Bowers EJ, Olson TA, Randel RD. Heat tolerance in Tuli-, Senepol-, and Brahman-sired F1 Angus heifers in Florida. J Anita Sci 1998; 76:1568-1577.

32. Hansen PJ. Effects of environment on bovine reproduction. In: Current Therapy in Large Animal Theriogenology, Youngquist RS, ed, WB Saunders, Philadelphia, 1997; pp 403-415.

33. I-Iansen PJ, Ar6chiga CF. Strategies for managing reproduction in the heat-stressed dairy cow. J Dairy Sci, 1999; 82 (SuppL 2):36-50.

34. Harmon R J, Lu M, TrammeU DS, Smith BA, Spain JN, Spiers D. Influence of heat stress and calving on antioxidant activity in bovine blood. J Dairy Sci 1997; 80 (SuppL 1):264 (abstr).

35. Howarth B, Alliston CW, Ulberg LC. Importance of uterine environment on rabbit sperm prior to fertilization. J Anita Sci 1965; 24: 1027-1032.

36. Huber Jr , Higginbotham G, Gomez-Alarcon RA, Taylor RB, Chen KH, Chan KS, Wu Z. Heat stress interactions with protein, supplemental fat, and fungal cultures. J Dairy Sci 1994; 77:2080-2090.

37. Hyttel P, Fair T, Callesen H, Greve T. Oocyte growth, capacitation and final maturation in cattle. Theriogenology 1997; 47:23-32.

38. Kamwanja LA, Chase CC Jr, Gutlerrez JA, Guerriero V Jr, Olson TA, Hammond AC, Hansen PJ. Responses of bovine lymphocytes to heat shock as modified by breed and antioxidant status. J Anirn Sci 1994;74:438-44A..

39. King VL, Denise SIC, Armstrong DV, Torabi M, Wiersma F. Effects of a hot climate on the performance of first lactation Holstein cows grouped by coat color. J Dairy Sci 1988; 71:1093-1096.

40. Mihm M, Curran N, Hyttel P, Knight PG, Boland MP, Roche JF. Effect of dominant follicle persistence on follicular fluid oestradiol and inhibin and on oocyte maturation in heifers. J Reprod Fertil 1999; 116: 293-304.

41. Monterroso VII, Drury KC, Ealy AD, Howell JL, Hansen PJ. Effect of heat shock on function of frozen/thawed bull spermatozoa. Theriogenology 1995;44:947-961.

42. Monty DE, Racowsky C. In vitro evaluation of early embryo viability and development in summer heat-stressed, superovulated dairy cows. Theriogenology 1987; 28:451-465.

102 Theriogenology

43. Moreira F, Badinga L, Burnley C, Thatcher WW. Effects of bovine somatotropin on embryo transfer in lactating dairy cows. Theriogenology 2001; 55: in press (abstr).

44. Moreira F, Riseo C, Pires MFA, Ambrose JD, Drost M, DeLorenzo M, Thatcher WW. Effect of body condition on reproductive efficiency of lactating dairy cows receiving a timed insemination. Theriogenology 2000; 53:1305-1319.

45. Nebel R L, Jobst SM, Dransfield MBG, Pandolfl SM, Bailey TL. 1997. Use of radio frequency data communication system, HeatWatch®, to describe behavioral estrus in dairy cattle. J Dairy Sci 1979; 80 (Suppl 1):179 (abstr).

46. Olson TA, Hammond AC, Chase CC Jr. Evidence for the existence of a major gene influencing hair length and heat tolerance in Senepol cattle. J Anita Sci 1997; 75 (Suppl. 1): 147 (abstr).

47. Paula-Lopes, FF, Chase CC, Jr, AI-Katanani YM, Krininger CE, III, Rivera RM, Tekin S, Majewski AC, Ocon OM, Olson TA, Hansen PJ. Breed differences in resistance of bovine preimplantation embryos to heat shock. Theriogenology 2001; 55:436 (abstr).

48. Putney D J, Drost M, Thatcher WW. Embryonic development in superovulated dairy cattle exposed to elevated ambient temperature between days 1 to 7 post insemination. Theriogenology 1988; 30:195-209.

49. Putney D J, Drost M, Thatcher WW. Influence of summer heat stress on pregnancy rates of lactating dairy cattle following embryo transfer or artificial insemination. Theriogenology 1989; 31:765-778.

50. Putney DJ, Mullins S, Thatcher WW, Drost M, Gross TS. Embryonic development in superovulated dairy cattle exposed to elevated ambient temperatures between the onset of estrus and insemination. Anim Reprod Sci 1989; 19:37-51.

51. Putney D J, Thatcher WW, Drost M, Wright JM, DeLorenzo MA. Influence of environmental temperature on reproductive performance of bovine embryo donors and recipients in the southwest region of the United States. Theriogenology 1988; 30: 905-922.

52. Rivera RM, AI-Katanani YM, Paula-Lopes FF, Hansen PJ. Short communication: Seasonal effects on development of bovine embryos produced by in vitro fertilization in a hot environment. J Dairy Sci 2000; 83:305-307.

53. Rivera RaM and Hansen PJ. Development of cultured bovine embryos after exposure to increased temperatures in the physiological range. Reproduction 2001; in press.

54. Rocha A, Randel RD, Broussard JR, Lim JM, Blair RM, Roussel JD, Godke R_A, Hansel W. High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos indicus cows. Theriogenology 1998; 49:657-665.

55. Rutledge J J, Monson RL, Northey DL, Leibfried-Rutledge ML. Seasonality of cattle embryo production in a temperate region. Theriogenology 1999; 51:330 (abstr).

56. Rutledge JJ. Use of embryo transfer and IVF to bypass effects of heat stress. Theriogenology 2001; 55:105-111.

57. Sakurada S, Hales JRS. A role for gastrointestinal endotoxins in enhancement of heat tolerance by physical fitness. J Appl Physiol 1998; 84:207-214.

58. Sartori R, Sartor-Bergfelt R, Mertens SA, Guenther JN, Parrish JJ, Wiltbank MC. Early embryonic development during summer in lactating dairy cows and nulliparous heifers. Biol Reprod 2000; 62 (Suppl 1): 155 (abstr).

59. Spain JN, Spiers DE. Effect of niacin supplementation on milk production and thermoregulatory responses of dairy cows. J Dairy Sci 1997; 80 (Suppl. 1): 153 (abstr).

60. Thatcher WW, Moreira F, Santos JEP, Mattos RC, Lopes FL, Panearci SM, Risco CA. Effects of hormonal treatments on reproductive performance and embryo production. Theriogenology 2001; 55:75-89.

Theriogenology 103

61. Trout JP, McDowell LR, Hansen PJ. Characteristics of the estrous cycle and antioxidant status of lactating Holstein cows exposed to heat stress. J Dairy Sci 1998; 81:1244-1250.

62. Udompraset P, Williamson NB. Seasonal influences on conception efficiency in Minnesota dairy herds. Theriogenology 1987; 28:323-336.

63. Vasconcelos JL, Silcox RW, Rosa GJ, Pursley JR, Wiltbank MC. Synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows. Theriogenology 1999; 52:1067- 1078.

64. West JW, Mullinix BG, Sandifer TG. Effects of physiologic responses of lactating Holstein and Jersey cows during hot, humid weather. J Dairy Sci 1991;74:840-851.

65. Wilson SJ, Kirby CJ, Koenigsfield AT, Keisler DH, Lucy MC. Effects of controlled heat stress on ovarian function of dairy cattle. 2. Heifers. J Dairy Sci 1998;81:2132-2138.

66. Wilson S J, Marion RS, Spain JN, Spiers DE, Keisler DH, Lucy MC. Effects of controlled heat stress on ovarian function of dairy cattle. 1. Cows. J Dairy Sci 1998; 81: 2139-2144.

67. Wolfenson D, Thatcher WW, Badinga L, Savio JD, Meidan R, Lew B J, Braw-Tal R, Berman A. Effect of heat stress on follicular development during the estrous cycle in lactating dairy cattle. Biol Reprod 1995; 52:106-1113.

68. Wolfenson, D, Lew B J, Thatcher WW, Graber Y, Meidan R. Seasonal and acute heat stress effects on steroid production by dominant follicles in cows. Anim Reprod Sci 1997; 47:9- 19.