PHYSIOLOGY AND MANAGEMENT Effect of Gonadotropin-Releasing Hormone at Estrus on Subsequent Luteal Function and Fertility in Lactating Holsteins During

Heat Stress'
G. ULLAH,*32*3 W. F U Q U A Y , * I ~ ~ ~ J. T. KEAWKHONG,*I~~~ B. L. CLARK,*36 D. E. POGUE,t and E. J. MURPHEYS
*Mississippi State University, Mississippi State 39762 tNorlh Mississippi Branch Experiment Station, Mississippi Agricultural and Forestry Experiment Station, Holly Spring 38635 *Coastal Plain Branch Experiment Station, Mississippi Agricultural and Forestry Experiment Station, Newton 39345

ABSTRACT

The experiment used lactating Holstein cows ( n = 9 4 ) from three herds in Mississippi. The experiment was conducted during late summer when temperatures were hot enough to cause a measurable stress response in cows in order to determine the effect of GnRH administration during estrus on fertility and subsequent luteal function of cows under these conditions. The mean ranges for ambient temperature, relative humidity, and temperature-humidity index during the study were 21.4 to 32.8"C, 67 to 95%, and 21.6 to 29.6, respectively. After injection of PGF2,y for synchronization of estrus, cows were alternately injected with 100 pg of either GnRH or saline (control) at detection of estrus, followed by AI 10 to 12 h later. From 14 treated cows and 14 control cows, blood samples were drawn by venipuncture just prior to treatment and at 5-d intervals until 30 d after treatment. Serum progesterone concentrations were determined by radioimmunoassay. Mean progesterone concentrations were higher for the cows treated with GnRH than for the controls. The pregnancy rate from first AI was 28.6% for all treated cows and 17.7% for control cows. On d 20, 42.8% of the treated cows and 57.1% of the control cows exhibited progesterone concentrations that were typical of pregnancy. When

pregnancy was diagnosed in these cows after d 45, pregnancy rates had decreased significantly for controls but not for cows given GnRH at estrus, suggesting greater embryonic survival in treated cows. We concluded that GnRH treatment enhanced secretion of luteal progesterone and embryo survival. ( Key words: heat stress, gonadotropin-releasing hormone, progesterone, embryo survival)
INTR0DUCTlON

Received September 21, 1995. Accepted April 8, 1996. 'Research was supported by the hlississippi Agricultural and Forestry Experiment Station through Project Number MIS-3150, contributing to Cooperative Regional Project W-173. Publication Number 5-8816. 2Present address: Peshawar Cant, Livestock Research and Development Farm, Surezai, Peshawar, Pakistan. "Department of Animal and Dairy Sciences. "Reprint requests. "resent address: Faculty of Agriculture, Rajamangala, PO Box 72, Tungsong, Nakornsrithammarajt, Thailand. Wollege of Veterinary Medicine.
1996 J Dairy Sci 79:19%1953

Summer heat stress impairs reproductive performance in cattle, especially lactating dairy cows (2, 6). When dairy cattle breeds of European origin are moved to environments of high temperature and humidity, stress responses to summer conditions are especially severe, and diurnal patterns of rectal temperature seldom return to the thermoneutral zone (12, 3 0 ). The primary reproductive responses to heat stress include reduced intensity of estrus (10, 12, 30) and lower fertility (9, 25). Although elevated core temperature is a factor that contributes to these reproductive responses ( 2 71, recent research ( 3 0 has shown that the magnitude of the preovulatory surge of LH might be reduced, as well as luteal progesterone secretion (11, 12, 30). A subnormal preovulatory surge of LH could result in reduced luteal function. The effect on fertility of suppression of luteal function that has been induced by heat stress is unknown. Injection of GnRH causes a predictable release of LH and a significant increase in serum progesterone ( 1 6 ) . Treatment with GnRH during the periestrus period has improved fertility of low fertility cows requiring repeated breeding ( 5 , 23) but has not resulted in predictable increases in fertility when cows were treated at first AI 17). In an economics study, Weaver et al. ( 2 8 ) indicated that GnRH treatment during first or second AI was more likely to be

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beneficial for herds with low fertility ( S45% conception rates) than for herds with conception rates >60%. liosenberg et al. ( 2 1 ) reported an improved responsle when repeat breeder cows were injected with GnRH near the start of detected estrus followed by AI 4 to 30 h later than in similarly treated cows that were inseminated <3 h after detected estrus. Given the combination of a suppressed preovulatory surge of LH (30 1, reduced secretion of luteal progesterone (11, 121, and lower fertility ( 9 , 25) of dairy cows inseminated during summer, GnRH treatment could potentially improve fertility in cows inseminated during the summer. The objective of this study was to determine the effect of GnRH treatment at estrus on subsequent function of the corpus luteum and fertility during the summer.
MATERIALS AND METHODS Experimental Plan

Progesterone Radioimmunoassay

Serum progesterone concentrations were determined by using a progesterone radioimmunoassay kit (DSL 3400; Diagnostic Systems Lab, Webster, TX) for the quantitative measurement of progesterone in serum. The kit was validated by linearity of dilution in our laboratory before use. The crossreactivity of the progesterone antiserum, as stated in the company literature provided with the kit, was 100, 5.5, 4.4, and 2.7% against progesterone, 5a-pregnan-3-20-dione, 20a-dihydroprogesterone7 and ll-deoxycortisol, respectively. The crossreactivity against other compounds was <1%. The mean sensitivity of the assay was 0.17 ng/ml. The interassay and intraassay coefficients of variation for progesterone were 8.6 and 7.4%, respectively.
Statistical Analysis

From three herds at Mississippi experiment stations located at Mississippi State University and at Holly Springs and Newton, Mississippi, 94 lactating Holstein cows were used in an experiment conducted during late summer (August t o early September of 1992 and 1993). Estrus was synchronized with a single injection of PGFzo,(LutalyseB;The Upjohn Co., Inc., Kalamazoo, MI), and observations were made during early morning and late afternoon for the next 5 d. Within a single location, alternate cows were injected with either 100 pg of GnRH (Factrel@;Ft. n Dodge Laboratories, Ft. Dodge, IA; = 49) or physiological saline ( n = 45) at observation of estrus and were inseminated 10 to 12 h later. Pregnancy was determined by palpation of the uterus and its contents after d 45. In 28 of these cows (14 treated with GnRH and 14 controls), blood samples were collected from the coccygeal vein a t observation of estrus and at 5-d intervals until d 30 to monitor progesterone concentrations. After centrifugation, serum samples were transferred to labeled vials and maintained at -20C until analysis. Rectal temperatures were measured with an electronic digital thermometer (M2 16; GLA Agricultural Electronics, San Luis Obispo, CA) at observation of estrus in all cows and at time of bleeding in 28 cows. Ambient temperature and relative humidity in the barn were recorded continuously from 10 d before initiation of the experiment until 30 d postbreeding using a hygrothermograph (model H 311; Weathermeasure Corp., Sacramento, CA). The temperaturehumidity index was calculated for use as a comfort index ( 1 4 ) .

No significant year or location differences were detected; therefore, data were combined. Values for plasma concentration of progesterone were analyzed by the GLM procedure of SAS ( 2 2 ) for analysis of variance as a split-plot design of time within treatments; cows within treatment was the error term. Least squares means were separated with a t test using the PDIFF option of the GLM procedure. Oneway analysis of variance and least significant difference tests were applied to analyze and to compare the mean progesterone concentrations of different categories of cows. Rectal temperature was analyzed by using analysis of variance procedures. Differences in pregnancy rate were determined using a series of chi-square tests.
RESULTS AND DISCUSSION Comfort Measures

During the experiment, the mean daily ambient temperature was 27.2"C, and the diurnal range was 21.4 to 32.8"C. Mean daily relative humidity was 80.9%, and the diurnal range was 67 to 95%. The mean temperature-humidity index was 25.6, and the diurnal range was 21.6 t o 29.6 (Figure 1). Therefore, whether based on temperature and humidity ( 1 3 ) or on the temperature-humidity index, the ambient conditions never dropped into the comfort zone for cows (14, 15). The mean daily values of 27.2"C ambient temperature and 80.9% relative humidity were above the 233C and 80% values that have been cited ( 18) as causing a measurable stress response by lactating cows. This degree of stress was reflected in rectal
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Fertility

First AI conception rates, as determined by rectal palpation after d 45, were 28.6 and 17.9% ( P > 0.05) for the treated and control cows, respectively. For the 28 cows that were monitored for progesterone, 42.8% of the cows treated with GnRH and 57.1% of the control cows were determined to be pregnant based on high serum progesterone concentrations (> 1 ng/ml) on d 20 after estrus and AI. When palpated for pregnancy after d 45, respective pregnancy rates were 65 2 4 6 8 10 12 14 16 18 20 22 24 28.6 and 14.396, representing a change ( P < 0.05) in the control cows but not in the treated cows (Figure Time of Day (h) 3). These results are consistent with those of Mee et Figure 1. Mean diurnal ambient temperature ( A), relative hu- al. (161, whose study with repeat breeder cows resulted in 53 and 50% pregnancy rates based on midity ( 0 ) . and temperature-humidity index ( e). progesterone concentrations 20 to 30 d or 20 to 40 d after AI, but 43 and 14% when based on palpation 42 to 56 d after AI for cows treated with GnRH and temperatures, which were above the normal range saline, respectively. In contrast, BonDurant et al. ( 5 ) reported for cattle ( 3 ) . For both treated cows and reported no difference in pregnancy rate when repeat control cows, the mean rectal temperature was 39.1 f breeder cows were treated with GnRH or saline durO.l"C, and the range across the experimental period ing the summer. Progesterone concentrations were was 38.8 f 0.1 to 39.4 f 0.1"C. not monitored during this study. Because rectal temperatures did not differ between Progesterone the treatment groups and because the concentration of luteal progesterone was increased by GnRH treatSerum progesterone concentrations were higher ( P ment, results of our experiment support the hypothe< 0.05) for cows treated with GnRH than for control sis that the suppression of corpus luteum function cows on d 10 (5.0 f 0.9 vs. 3.4 f 0.7 ng/ml, respecthat has been induced by extreme heat is a factor that tively) and d 15 (5.7 k 1.1 vs. 3.7 f 0.9 ng/ml, compromises embryo survival. Jerseys, which are respectively) for the luteal phase following treatment more tolerant of heat stress than are Holsteins, as and on d 25 (4.6 1.2 vs. 2.9 f 0.9 ng/ml, respectively) and d 30 (6.1 0.9 vs. 2.2 k 0.6 ng/ml, was indicated by higher fertility during the summer (2, 24), also have higher concentrations of luteal respectively). Overall treatment means were 5.0 k 0.9 and 3.4 k 0.7 ng/ml, respectively (Figure 2). The higher concentrations of progesterone in the treated cows before d 20 were likely due to the stimulatory effects of GnRH ( 1 6 ) . Our report is the first to observe that this stimulatory effect occurred in the cor8 pus luteum, the function of which was suppressed by heat stress ( 111. The mechanism for this stimulatory effect in the corpus luteum is unknown, but Mee et al. ( 1 6 ) reported that GnRH increased the proportion of large luteal cells in corpora lutea of repeat breeder dairy cows removed 10 d after estrus and GnRH treatment. The higher concentration of progesterone in the cows treated with GnRH on d 25 and 30 after treatment could have been due to the higher proportion of pregnant cows in the group or perhaps to the 0 5 10 15 20 25 30 lingering effects of GnRH on the corpus luteum during early gestation ( 16). Mean concentrations of seBlood Collection (d) rum progesterone were higher ( P < 0.05) for cows treated with GnRH than for control cows whether or Figure 2. Mean serum concentrations of progesterone in cows not they were diagnosed as being pregnant. n treated with GnRH ( m ; n = 14) or saline (0; = 14) ( P i0.05).
+_ _+

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60 70 50 40

III-

ab

an
GnRH

42.86

n
Saline

bc 57.14

Figure 3. Pregnancy rate (percentage) on the basis of rectal palpation ( w ) , progesterone concentrations ( U ) , or both in cows treated with GnRH ( n = 1 4 ) or saline ( n = 14). Different letters ( a , b ) indicate a difference ( P < 0.05).

progesterone and lower rectal temperatures ( 7 , 12). Artificial cooling of dairy cows during the summer, which results in higher fertility (20, 26, 29), also results in higher luteal progesterone (301, lower rectal temperatures (20, 29, 30), and improved body condition ( 8 ) , all of which could affect fertility. Elevated core body temperature in the periestrus period has a negative effect on embryo quality (4, 1 9 ) and embryo survival (1, 27). Although based on limited observations, results of the present study suggest tha.t suppressed secretion of luteal progesterone is a factor in embryo survival as well.
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gGwazdauskas, F. C., C. J . Wilcox, and W. W. Thatcher. 1975. Environmental and management factors affecting conception rate in a subtropical environment. J. Dairy Sci. 58:88. lOHer, E., D. Wolfenson, I. Flamenbaum, Y. Folman, M. Kaim. and A. Berman. 1988. Thermal, productive, and reproductive remonses of hieh vieldine cows exDosed t o short-term cooling in summer. J. Dii, Sci. f1:1085. IlHowell. J. L.. J. W. Fuouav. and A. E. Smith. 1994. Corpus luteum growth and f u n c h i in lactating Holstein cows during spring and summer seasons. J. Dairy Sci. 77:735. 12Imtiaz Hussain, S. M., J . W. Fuquay, and M. Younas. 1992. Estrous cyclicity in nonlactating and lactating Holsteins and Jerseys in a Pakistani summer. J . Dairy Sci. 75:2968. 13 Johnson, D. E. 1986. Climatic stress and production efficiency. Page 17 in Limiting the Effects of Stress on Cattle. Res. Bull. 512, Western Regional Res. Publ. No. 009. Utah Agric. Exp. Stn., Logan. 14Lutgens, F. K., and E. J . Tarbuck. 1982. The Atmosphere: An Introduction to Meteorology. Prentice-Hall, Inc., Englewood Cliffs, NJ. 15 Maunder. W. J . 1970. The Value of Weather. Methuen and Co.. Ltd., London, England. 16 Mee. 0. M.. J . S.Stevenson. B. M. Alexander, and R. G. Sasser. 1993. Administration of GnRH at estrus influences pregnancy rates, serum concentrations of LH, FSH, Estradiol-170, pregnancy-specific protein B. and progesterone, proportion of luteal cell types, and in vitro production of progesterone in dairy cows. J . Anim. Sci. 71:185. 17 Mee, 0. M., J . S. Stevenson, and R. K. Scoby. 1990. Influence of gonadotropin-releasing hormone and timing of insemination relative to estrus on pregnancy rates of dairy cattle a t first insemination. J . Dairy Sci. 73:1500. 18 Nickerson, S . C. 1987. Mastitis management under hot, humid conditions. Page 32 in Proc. Dairy Herd Management Conf., Macon, GA., Univ. Georgia, Athens. 19Putney, D. J., S.Mullins, W. W. Thatcher, M. Drost, and T. S . Gross. 1989. Embryonic development in superovulated dairy cattle exposed to elevated ambient temperature. Anim. Reprod. Sci. 19:37. 20Roman-Ponce, H., W. W. Thatcher, and C. J. Wilcox. 1981. Hormonal relationships and physiological responses of lactating dairy cows to a shade management system in a subtropical environment. Theriogenology 16:139. 21 Rosenberg, M., S.Y. Chun, M. Kaim, Z. Herz, and Y. Folman. 1991. The effect of GnRH administration t o dairy cows during estrus on plasma LH and conception in relation to the time of treatment and insemination. Anim. Reprod. Sci. 24:13. 22 SAS/STAT@Users Guide. Release 6.04. 1990. SAS Inst., Inc., Cary, NC. 23 Stevenson, J . S., E. P. Call, R. K. Scoby, and A. P. Phatak. 1990. Double insemination and gonadotropin-releasing hormone treatment of repeat breeding dairy cattle. J. Dairy Sci. 73:1766. 24 Stott, G. H. 1961. Female and breed associated with seasonal fertility variation in dairy cattle. J. Dairy Sci. 44:1698. 25 Stott, G. H., and R. J . Williams. 1962. Causes of low breeding efficiency in dairy cattle associated with seasonal high temperatures. J. Dairy Sci. 45:1369. 26Thatcher, W. W., F. C. Gwazdauskas, C. J. Wilcox, J. Toms, H. H. Head, D. E. Bufington, and W.B. Fredriksson. 1974. Milking performance and reproductive efficiency of dairy cows in an environmentally controlled structure. J . Dairy Sci. 57:304. 27 Ulberg, L. C., and P. J. Burfening. 1967. Embryo death resulting from adverse environment on spermatozoa or ova. J. Anim. Sci. 26571. 28 Weaver, L. D., C. A. Daley, and W. J. Goodger. 1988. Economic modeling of the use of gonadotropin-releasing hormone a t insemination to improve fertility in dairy cows. JAWMA ( J . Am. Vet. Med. Assoc.) 12:1714. 29 Wolfenson, D., I. Flamenbaum, and A. Berman. 1988. Hyperthermia and body energy store effects on estrous behavior, conceDtion rate. and corDus luteum function in dairy cows. J . Dairy Sci. 71:3497. 30Younas, M., J . W. Fuquay, A. E. Smith, and A. B. Moore. 1993. Estrous and endocrine responses of lactating Holsteins to forced ventilation during summer. J. Dairy Sci. 76:430.
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