- Email: [email protected]
Effect of hCG administration on day 7 of the estrous cycle on follicular dynamics and cycle length in cows
ELSEVIER
EFFECT OF hCG ADMINISTRATION ON DAY 7 OF THE ESTROUS CYCLE ON FOLLICULAR DYNAMICS AND CYCLE LENGTH IN COWS P.C. Sianangama and R Rajamahendran Department of Animal Science The University of British Columbia Vancouver, British Columbia, Canada V6T lZ4 Received for publication: December 6, 19% Accepted: September II, 1995 ABSTRACT TheuseofhCGincattleatbreedingoratdifferenttimesafterbreedinghasbeen associated with extension in estrous cycle length among cows that do not become pregnant. The objective of this study was to determine whether the increase in estrous cycle length observed in hCG-treated cows that fail to become pregnant is due to changes in ovarian follicular dynamics. Twelve no&ted lactating cows were randomly assigned either to receive hCG on Day 7 of the cycle (Day 0 = day of es&us, n=6) or to serve as controls (n=6). Ultrasound scanning was conducted daily from Day 0 until the onset of the next ovulation to monitor follicular and corpus luteum (CL) dynamics. Blood samples were collected for progestemne analysis at each ultrasound session. Ovulation of the Day 7 follicle occurred in all 6 hCG-treated cows. Ihe time of emergence of the second-wave of follicular growth was advanced in hCG-treated cows but was not statistically different (PXl.05) from that of the control cows (10.8 + 0.3 vs 12.7 + 1.4 d). The mean diameter of the second-wave dominant follicle from Days 15 to 18 was not different @0.05) between the treatment groups. However, the second-wave dominant follicle had a slower growth rate (0.8 vs 1.3 mm/d) among cows treated with hCG compared with that of the controls. The second-wave dominant follicle was the ovulatory follicle in 5 control cows, but only in 3 hCG-treated cows. The dominant follicle tirn the third wave ovulated in 1 control and in 3 hCG-treated cows. The lifespan of the spontaneous CL and the time to low progesterone levels (~1 n&nl) were not different between the control and hCG-treated cows. Ihese results suggest an altered follicular dynamic but no extension in estrous cycle length when hCG is administered on Day 7 of the cycle in postpartum cows. Key words:
cows, hCG, follicular dynamics, cycle length, corpus luteum, progesterone
Acknowledgements This study was funded by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC). Sianangama was supported by the National Council for Scientific Research (NCSR), Lusaka, Zambia.
Theriogenology 45:583-592, 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 100 10
0093-691W96/$15.00 SSDI 0093-691X(95)00405-X
584
Theriogenology
INTRODUCTION It has been estimated that fertilization mtes in the cow mnge firom 88 to 90% (4), but as shown by pregnancy diagnosis conducted 60 d post breeding, only 60% of these embryos are maintained. One factor that contributes to the loss of embryos is CL function (3). Consequently, gonadotxopic hormones such as hCG have been used to augment the function of the inadequate CL in cattle (6,8,25). Although hCG increased pregnancy rates, results have been variable, ranging f?om a net loss of 11% to a net gain of 13% (5). In a recent study, we demonstrated that the greatest increase in pregnancy rate occurs when hCG is administered on Day 7 after breeding (19). We showed (19) that treatment with hCG on Day 0 (day of e&us), 7 or 14 was associated with an 8,49 and 35% increase in pregnancy rates, respectively. Upon repeating this experiment we showed an even higher response to hCG given on Days 0, 7 and 14 after breeding (24, 57 and 400/4 respectively). However, the beneficial effectsof hCG have been associated with extension of estrous cycle length among cows that either do not conceive or fail to maintain their pregnancies (8, 25). ‘Ihis was confirmed in our study (19), with the cycle extension being dependent on the timing of the hCG treatment. ‘Ihe frequency of extended cycles was higher and the duration of the extension longer among cows treated on Day 14, as compared with cows treated on either Day 0 or 7 after lmxding. Recently, de Los Santos-Valadez and co-workers (4) reported that up to 60% of cows that were treated with hCG but did not become pregnant had extended cycles lasting more than 24.5 d, as compared with 24% of cows that did not receive hCG. A retrospective evaluation of our previous study (18) suggested that al&&ions in follicular dynamics among hCG-treated cows, which were bred 12 h a&r standiig estrus. Administmtion of hCG on Day 0,7 and 14 led to persistence of large follicles in a majority of treated cows and was associated with lack of new waves of follicular growth. Such changes in follicular dynamics may explain extensions in estrous cycle length observed in hCG treated cows. However, cycles in animals that were bred at estrus and treated with hCG at specific times relative to estrus, can also be extended by antiluteolytic factors elaborated by the conceptus during early pregnancy. For instance, the removal of the bovine conceptus f+om the uterus on Day 17 extends the cycle to Day 25 & 1.2 as compared with the control cycle of 2 1 d (IO). Confirmation that the conceptus initiates the observed extension is provided by the fact that intrauterine infbsion of embryonic homogenates between Days 15 and 17 extends both the inter&tus interval and the life span of the CL (10). Taken together, our results (18) and those presented by others (10) suggest that the extension in cycle length could be attributed to either an alteration in follicular dynamics, to factors of embryonic origin or to a combination of both. Ihe objective of this study was to determine whether the increase in estrous cycle length observed in hCG-treated cows that fail to be-comepregnant is due to changes in ovarian follicular dynamics.
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MATERIALSAND METHODS Animal and Materials Regularly cycling postpartum cows, 3 to 5 yr of age, were randomly selected from the dairy herd at Ihe University of British Columbia They were housed in a closed fke-stall barn and received a standard dairy ration of alfalfa cubes and a barleykanol&ased concentrate adjusted to meet requirements for milk pmduction. Human chorionic gonadotropin (hCG, APL) was pwchased horn Ayerst I&oratories (MontreaI, PQ., Canada). Heparmkd vacutainer tubes were purchased ti-om Becton Dickinson (Vacutainer Systems, Rutherford, NJ, USA). Progesterone kit (Coat-A-Count) was purchased from Diagnostic Products Corporation (Los Angeles, CA, USA).
Experimental Procedure At standing estrus (Day 0), 12 cows were randomly assigned to either serve as untreated controls (n=6) or receive a single intramuscular injection of hCG (1000 IU) on Day 7 post e&us (n==6). A real time linear-anay ultrasound scanning device (model LS 300, Tokyo Keiki Company Ltd, Tokyo, Japan) equipped with a 5-MHz tlan&wrwasusedto characterize follicular and CL dynamics. Ultrasonogmphy was conducted as previously described (12) and was modified for this experiment as noted below. Ultrasound scanning was started at Day 0 and conducted daily until the subsequent ovulation was detected. ovulations resulting from tmatment with hCG were verified by observing the diqpearance of one or more of the large follicles present at the time when mxtement was administered. The emergence of an induced CL was characterized by 1uteaItissue appearing on a site previously occupied by a large follicle. The dimensions of spontaneous and induced CL and of observed follicles were obtained using a built-in system of calibrated callipers. Hard copies of the frozen images of the ovary bearing the CL and follicles were obtained using a Mitsubishi Video Copy Processor (Model number P6OU, Mitsubishi Electric Sales America Inc., Cypress, CA, USA) connected to the ultrasound scanner. Jugular blood samples were obtained at each ultrasound session. Blood samples were collected into 8ml hepatkkd vacutainer tubes, and plasma was harvested within 30 min of collection. Plasma samples were stored at -20°C until assayed for progesterone. Plasma progesterone concentrations were determined using a solid-phase radioimmunoassay Coat-ACount kit. This kit had been previously validated in our laboratory for the measurement of progesterone in bovine plasma (11). The inter- and intm-assay coefficients of variation were 7 and 5?4&, respectively, and sensitivity was 0.05 ng/ml. Statistical Analyses Data for follicle, CL diameter and progesterone concentrations were analyzed as a split plot experimental design. The least squares analysis of variance in the General Linear Model Pro&me. (SAS, version 6.09) was used to test for differences behveen control and hCGtreated cows (15). The model included the following effects: treatma cows nested within treatment, day after estrus and the interaction between tmatment and day after e&us. The
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Theriogenology
cowmeansquarewasusedtotest~~tmeansquare;therestweretestedusingthe residual error mean square. Categorical data were analyzed using contingency tables in Chisquare (15) analysis. RESULTS
Administration of hCG on Day 7 resulted in ovulation of the first wave dominant follicle in 5 cows. The sixth cow ovulated both the dominant and the second largest follicle and had 2 spontaneous CL at the time of hCG treatment (Table 1). Control cows had 1 CL each. Least squares means of the spontaneous CL diameter among control cows and cows treated with hCG on Day 7, as well as the diameter of the induced CL are shown in Figure la The diameter of the spontaneous CL between control and hCG-treated cows before hCG administration was not different (PXI.05). Similarly, the diameter of spontaneous CL between the 2 groups did not differ following hCG administration. However, the diameter of the CL showed a significant increase over time, and no interaction of treatment by time (PXl.05) was detected. Accessory CL induced by hCG given on Day 7 were detectable starting on Day 9, and were distinguishable from spontaneous CL by their smaller diameter during the treatment period Concentrations of progesterone increased steadily horn Day 0 to Day 7 in both groups (Figure lb). The mean concentration of progesterone was similar (PXOS) between the 2 groups until Day 16, and thereafter a rapid decline in progesterone was observed in the hCG-treated group. Table 1.
Effect of hCG on Day 7 of the cycle on accessory CL formation, follicular waves, CL regression and ovulation in cows x-eatrnent
Characteristics Cows with accessory CL Number of waves per cycle Time of emergence of Wave 2 (Day) Time to low progesterone level (Day) Time to ovulation of Wave 2 follicles (Day) Estrous cycle length (Days)
hCG 6 2.5 ? 10.8 2 21.8 2 13.0 + 23.3 +
0.2 0.2 0.5 1.8 1.4
Control 0 2.1 + 0.1 12.7 + 1.4 22.3 + 1.0 9.7 + 2.1 22.4 + 1.6
Profiles of the dominant follicle among control cows and cows treated with hCG are presented in Figures 2 and 3. The diameter of the first wave dominant follicle was similar between hCG and control cows (M.05). The Day of emergence of the second wave of follicular growth was advanced, but not significantly, in hCG-treated cows (10.8 + 0.3 vs 12.7 + 1.4 d). The diameter of the second wave dominant follicle from Days 15 to 18 did not differ (FM.05) between the treatment groups. However, the second wave dominant follicle had a slower growth rate (0.8 vs 1.3 &d) among cows treated with hCG compared with the controls. The second wave dominant follicle was the ovulatory follicle in 5 control cows, but only in 3 hCG-treated cows. The second wave dominant follicle among hCG-treated cows took longer to ovulate (36 h) than among control cows (10 h) after progesterone reached a
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.“..“.“.“~“~‘~~““‘~’
4
0
3
6
9
12
14
17
20
23
26
8
0
3
6
9 Number
12
14
of days after
17
20
23
26
estrus
Figure 1. Lease squares means (& SEM) of spontan~us CL diarnete~ (panel a) among control cows (0) and cows txated with hCG on Day 7 (0) as well as the diameter of induced CL among hCG-treated cows (A). Profiles of least squares means for progesterone concentration among control (0) and hCG-treated cows (0) are also presented ( Panel b).
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0
5
10
19
14
24
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20:
!j .d w Q)
14:
z =:
7:
g
0 3:
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10 .’
0
‘.‘..‘..‘..‘..‘..‘..‘..r..“.J 3 6 9 12
15
18
21
24
27
30
Number of days after estrus
Figure 2.
Least squans means (& SEM) of the ckmeter of the dominant follicle of the fkst (W and second (0) wave among the 5 control cows that presented a 2-wave pattern of follicular growth (RUMAa). One cunlrol cow had 3 waves of follicular growth @=I b).
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589
1‘ ’ -. 0
* ‘. 5
. . . ‘. 10
14
19
24
Ovulation
0
Figure 3.
5
10
Number
of days
14
after
19
24
estrus
Least squares means (& SEM) of the diameter of the dominant follicle of the first (0) and second wave (a) among the 3 hCG-treated cows that presented a 2-wave pa&n of follicular growth (Pam9 a). The remainder of the hCG-tnzated cows showed a 3-wave pattern (Panel b).
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basal level of 0.05 @ml. The dominant follicle I?om the third wave ovulated in 1 control and in 3 hCG-treated cows. There was no effect (PXOS) of hCG with regard to the number of Days to regression of the CL (hCG-treated, 18.8 +1.2d, vs control 19.3 + 2.7d). Similarly, them was no difference with regard to time to low progesterone concentration, the interval between emergence and ovulation of the second wave dominant follicle and estrous cycle length (Table 1). DISCUSSION The classical explanation for the extension in estrous cycle length observed following hCG tteatment has been that the functional status of the CL is maintained beyond its normal duration (6,23,25). The present study evaluated the use of hCG in nonbred cows and found no difference in the CL diameter (spontaneous CL) or in the onset of CL regression of control and hCG-treated cows. This finding is consistent with our previous observation (12). Similarly, the Day at which progesterone concentration declined to less than 1.0 ng/ml was also not different between the 2 treatment groups. However, a trend was observed for a more rapid and earlier decline in progesterone among hCG-treated cows. This is in contrast to the progesterone profile observed among cows which were bred and treated with hCG either at the time of breeding or afler 7 or 14 days (19). The earlier decline in progesterone levels observed in this study might be due to several factors. Treatment with hCG in sheep (7) and pigs (24) is associated with an increase in the number of large luteal cells and a concomitant reduction in the number of small luteal cells in spontaneous CL. Recently, this pattern was also observed in the hCG-induced and hCGexposed spontaneous CL of cattle (20). Furthermore, large luteal cells are reported to possess receptors to the lutelysin PGF, (9). This increase in the number of large luteal cells and, therefore, potential increase in the sensitivity to PGF, would explain the premature physiological regression of hCG-induced and hCG-exposed CL observed in the present study. Moreover, if spontaneously formed CL are exposed to hCG during the period of its formation (Days 0 to 3), estrous cycle length and CL lifespan is shorter than normal (2, 3). Although not statistically different, the time of emergence of the second wave of follicular growth was advanced in hCG-treated cows. Such a finding is in agreement with the hypothesis that dominant follicles suppress the emergence of new waves of follicular growth (1, 13). In the present study, removal of the suppressive effect of the dominant follicle, by way of induced ovulations, created a permissive environment, thus allowing a new wave of follicles to emerge early. The second wave dominant follicle was the ovulatory follicle in 5 controls, but only in 3 hCG-treated cows. Also in these 3 cows, the dominant follicle took longer to ovulate following regression of the CL and decline in progesterone to < 0.5 ng/ml. In a recent study, we (22) reported that administration of progesterone early in the cycle caused either regression or delayed ovulation of the first wave dominant follicle. Therefore, regression or delayed ovulation of the second wave dominant follicle observed in the present study could be ascribed to high progesterone or to the carry-over effect of hCG.
591
Theriogenology
Due to the onset of at&a of the second wave follicle in hCG-treated cows, a third wave was required to provide an ovulatory follicle. This need for a third wave may explain a nonsignificant extension in the estrous cycle length observed in hCG-treated cows, and is in agreement with previous reports (16,21) that have demonstrated that 3-wave cycles are associated with an extension in cycle length. Nonetheless, the extension observed in the present study was shorter than when hCG was given later in the luteal phase of the cycle (6, 17, 18, 25). In conclusion, the current study found alteration in follicular dynamics among cows treated with hCG on Day 7 of the cycle. This alteration did not affect the cycle length. Therefore, based on pregnancy data in our previous studies (12, 19) and the cycle length data in the present study, hCG at Day 7 after breeding could be used as a management tool for improving the pregnancy rate in postpartum cows. REFERENCES 1
Adams GP, Kot K Smith CA, Ginther OJ. Effect of dominant follicle on regression of its subordinated follicle in heifa. Can J Anim Sci 1993; 73:267-275. 2 Bat&a PJ, Rexroad Jr CE, Williams WF. Effects of progesterone administered to dairy heifers on sensitivity of corpora lutea to PGF, and on plasma LH concentrations. Theriogenology 1984; 22:47-58. 3 Bulrnan DC, Lamming GE. Milk progesterone levels in relation to conception, repeat breeding and factors influencing a cyclic&y in daii cows. J Reprod Fertil 1978; 54:447458. 4 de Los Santos-Valadez S, Seidel GE Jr, Elsden RP. Effect of hCG on pregnancy rates in embryo transfer recipients. ‘lheriogenology 1982; 17:85 abstr. 5 Diskin MG, Sreenan JM. Progesterone and embryo survival in the cow. In: Diskin MG, Screenan JM teds). Embryonic Mortality in Farm Animals. Dordtrecht, The Netherlands: Martinus Nijhoff Publishers, 1985; 142-158. 6 Eduvie LO, Seguin BE. Corpus luteum function and pregnancy rates in lactating dairy cows given human chorionic gonadotropin at mid die&us. ‘Iheriogenology 1982; 17: 415-422. 7 Farin CE, Moeller CL, Mayan H Gamboni F, Sawyer HR, Niswender GD. Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum. Biol Reprod 1988; 38:413-421. 8 Morris LM, Gonzalez-Padilla E, Niswender GD, Wiltbank JN. Peripheral progesterone levels in pregnant and non-pregnant heifers following use of hCG. Theriogenology 1976; 6:367-378. 9 Niswender GD, Schwall RH, Fitz CE, Sawyer HR Regulation of luteal function in domestic ruminants: new concepts. Ret Prog Horm Res 1985; 41:101-151. 10 Northey DL, French LR Effect of embryo removal and intrauterine infusion of embryonic homogenates on the lifespan of the bovine corpus luteum. J Anim Sci 1980; 501298-302. 11 Rajamahendran R, Taylor C. characterization of ovarian activity in postpartum dairy cows using ultrasound imaging and progesterone profiles. Anim Reprod Sci 1990; 22:171-180.
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12 Rajamahendran R, Sianangama PC. Effect of human chorionic gonadotropin on dominant follicles in cows: f&nation of accessory corpom lutea, progesterone production and pregnancy rates. J Reprod Fertil 1992; 95577-584. 13 Rajamahendmn R, Calder MC. Superovulatory responses in dairy cows following ovulation of the dominant follicle of the first wave with hCG. neriogenology 1993; 40:99-109. 14 Roche J, Boland MP, McGeady TA. Reproductive wastage following artificial imemimtion of heifm. Vet Ret 1981; 109:401-403. 15 SAS User’s Guide: Statistics, Personal Computer, Version 6.3. SAS Institute, Gary, NC 1986. 16 Savio JD, Thatcher WW, Bad&a IU, de la Sota RL, Wolfmson D. Regulation of follicular turnover during the estrous cycle in cows. J Reprod Fertil 1993; 97: 197-203. 17 Seguin BE, Oxender WD, Britt JH. Effect of human chorionic gonadotropin and gonadotropin-releasing hormone on cmpus luteum fimction and estrom cycle duration in dairy heifixs. Am J Vet Res 1977; 38:1153-1156. 18 Sianangama PC, Rajamahemlran R Effect of hCG on follicular dynamics in lactating dairy cattle. Biol Repmd 1991; 44 (Suppl 1): 66 abstr. 19 Sianangama PC, Rajamahendran R Effect of human chorionic gonadotropin administered at specific times following breeding on milk progesterone and pregnancy rates in cows. ‘I&riogenology 1992; 38:85-96. 20 Sianangama PC. Ihe Characteristics of Corpora Lutea Induced by hCG in Cattle. Ph.D. Dissertation, Ihe University of British Columbia, Vancouver, Canada, 1994. 21 Taylor C, Rajamahendran R Follicular dynamics, corpus h&urn growth and regression in lactating dairy cattle. Can J Anim Sci 1991; 71:61-68. 22 Taylor C, Rajamahendran R Effect of mid-l&al phase progesterone levels on the first wave dominant follicle in cattle. Can J Anim Sci 1994; 74:281-285. 23 Wagner JF, Veenhuizen EL, Tonkinson LV, Rathmacker RP. Effect of placental gonadotropin on pregnancy rate in the bovine. J Anim Sci 1973; 36:1129-l 136. 24 Wlesak T. Effect of pregnancy, injection of oe&adiol bemmate or hCG on steroid concentration and release by pig luteal cells. J Reprod Fertil 1989; 86:247-254. 25 Wiltbank JN, Rothlisberger JA, Zimmerman DR Effect of human chorionic gonadotropin on maintenance of the corpus luteum and embryo survival in the cow. J Anim Sci 1961; 20:827-829.
EFFECT OF hCG ADMINISTRATION ON DAY 7 OF THE ESTROUS CYCLE ON FOLLICULAR DYNAMICS AND CYCLE LENGTH IN COWS P.C. Sianangama and R Rajamahendran Department of Animal Science The University of British Columbia Vancouver, British Columbia, Canada V6T lZ4 Received for publication: December 6, 19% Accepted: September II, 1995 ABSTRACT TheuseofhCGincattleatbreedingoratdifferenttimesafterbreedinghasbeen associated with extension in estrous cycle length among cows that do not become pregnant. The objective of this study was to determine whether the increase in estrous cycle length observed in hCG-treated cows that fail to become pregnant is due to changes in ovarian follicular dynamics. Twelve no&ted lactating cows were randomly assigned either to receive hCG on Day 7 of the cycle (Day 0 = day of es&us, n=6) or to serve as controls (n=6). Ultrasound scanning was conducted daily from Day 0 until the onset of the next ovulation to monitor follicular and corpus luteum (CL) dynamics. Blood samples were collected for progestemne analysis at each ultrasound session. Ovulation of the Day 7 follicle occurred in all 6 hCG-treated cows. Ihe time of emergence of the second-wave of follicular growth was advanced in hCG-treated cows but was not statistically different (PXl.05) from that of the control cows (10.8 + 0.3 vs 12.7 + 1.4 d). The mean diameter of the second-wave dominant follicle from Days 15 to 18 was not different @0.05) between the treatment groups. However, the second-wave dominant follicle had a slower growth rate (0.8 vs 1.3 mm/d) among cows treated with hCG compared with that of the controls. The second-wave dominant follicle was the ovulatory follicle in 5 control cows, but only in 3 hCG-treated cows. The dominant follicle tirn the third wave ovulated in 1 control and in 3 hCG-treated cows. The lifespan of the spontaneous CL and the time to low progesterone levels (~1 n&nl) were not different between the control and hCG-treated cows. Ihese results suggest an altered follicular dynamic but no extension in estrous cycle length when hCG is administered on Day 7 of the cycle in postpartum cows. Key words:
cows, hCG, follicular dynamics, cycle length, corpus luteum, progesterone
Acknowledgements This study was funded by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC). Sianangama was supported by the National Council for Scientific Research (NCSR), Lusaka, Zambia.
Theriogenology 45:583-592, 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 100 10
0093-691W96/$15.00 SSDI 0093-691X(95)00405-X
584
Theriogenology
INTRODUCTION It has been estimated that fertilization mtes in the cow mnge firom 88 to 90% (4), but as shown by pregnancy diagnosis conducted 60 d post breeding, only 60% of these embryos are maintained. One factor that contributes to the loss of embryos is CL function (3). Consequently, gonadotxopic hormones such as hCG have been used to augment the function of the inadequate CL in cattle (6,8,25). Although hCG increased pregnancy rates, results have been variable, ranging f?om a net loss of 11% to a net gain of 13% (5). In a recent study, we demonstrated that the greatest increase in pregnancy rate occurs when hCG is administered on Day 7 after breeding (19). We showed (19) that treatment with hCG on Day 0 (day of e&us), 7 or 14 was associated with an 8,49 and 35% increase in pregnancy rates, respectively. Upon repeating this experiment we showed an even higher response to hCG given on Days 0, 7 and 14 after breeding (24, 57 and 400/4 respectively). However, the beneficial effectsof hCG have been associated with extension of estrous cycle length among cows that either do not conceive or fail to maintain their pregnancies (8, 25). ‘Ihis was confirmed in our study (19), with the cycle extension being dependent on the timing of the hCG treatment. ‘Ihe frequency of extended cycles was higher and the duration of the extension longer among cows treated on Day 14, as compared with cows treated on either Day 0 or 7 after lmxding. Recently, de Los Santos-Valadez and co-workers (4) reported that up to 60% of cows that were treated with hCG but did not become pregnant had extended cycles lasting more than 24.5 d, as compared with 24% of cows that did not receive hCG. A retrospective evaluation of our previous study (18) suggested that al&&ions in follicular dynamics among hCG-treated cows, which were bred 12 h a&r standiig estrus. Administmtion of hCG on Day 0,7 and 14 led to persistence of large follicles in a majority of treated cows and was associated with lack of new waves of follicular growth. Such changes in follicular dynamics may explain extensions in estrous cycle length observed in hCG treated cows. However, cycles in animals that were bred at estrus and treated with hCG at specific times relative to estrus, can also be extended by antiluteolytic factors elaborated by the conceptus during early pregnancy. For instance, the removal of the bovine conceptus f+om the uterus on Day 17 extends the cycle to Day 25 & 1.2 as compared with the control cycle of 2 1 d (IO). Confirmation that the conceptus initiates the observed extension is provided by the fact that intrauterine infbsion of embryonic homogenates between Days 15 and 17 extends both the inter&tus interval and the life span of the CL (10). Taken together, our results (18) and those presented by others (10) suggest that the extension in cycle length could be attributed to either an alteration in follicular dynamics, to factors of embryonic origin or to a combination of both. Ihe objective of this study was to determine whether the increase in estrous cycle length observed in hCG-treated cows that fail to be-comepregnant is due to changes in ovarian follicular dynamics.
Theriogenology
585
MATERIALSAND METHODS Animal and Materials Regularly cycling postpartum cows, 3 to 5 yr of age, were randomly selected from the dairy herd at Ihe University of British Columbia They were housed in a closed fke-stall barn and received a standard dairy ration of alfalfa cubes and a barleykanol&ased concentrate adjusted to meet requirements for milk pmduction. Human chorionic gonadotropin (hCG, APL) was pwchased horn Ayerst I&oratories (MontreaI, PQ., Canada). Heparmkd vacutainer tubes were purchased ti-om Becton Dickinson (Vacutainer Systems, Rutherford, NJ, USA). Progesterone kit (Coat-A-Count) was purchased from Diagnostic Products Corporation (Los Angeles, CA, USA).
Experimental Procedure At standing estrus (Day 0), 12 cows were randomly assigned to either serve as untreated controls (n=6) or receive a single intramuscular injection of hCG (1000 IU) on Day 7 post e&us (n==6). A real time linear-anay ultrasound scanning device (model LS 300, Tokyo Keiki Company Ltd, Tokyo, Japan) equipped with a 5-MHz tlan&wrwasusedto characterize follicular and CL dynamics. Ultrasonogmphy was conducted as previously described (12) and was modified for this experiment as noted below. Ultrasound scanning was started at Day 0 and conducted daily until the subsequent ovulation was detected. ovulations resulting from tmatment with hCG were verified by observing the diqpearance of one or more of the large follicles present at the time when mxtement was administered. The emergence of an induced CL was characterized by 1uteaItissue appearing on a site previously occupied by a large follicle. The dimensions of spontaneous and induced CL and of observed follicles were obtained using a built-in system of calibrated callipers. Hard copies of the frozen images of the ovary bearing the CL and follicles were obtained using a Mitsubishi Video Copy Processor (Model number P6OU, Mitsubishi Electric Sales America Inc., Cypress, CA, USA) connected to the ultrasound scanner. Jugular blood samples were obtained at each ultrasound session. Blood samples were collected into 8ml hepatkkd vacutainer tubes, and plasma was harvested within 30 min of collection. Plasma samples were stored at -20°C until assayed for progesterone. Plasma progesterone concentrations were determined using a solid-phase radioimmunoassay Coat-ACount kit. This kit had been previously validated in our laboratory for the measurement of progesterone in bovine plasma (11). The inter- and intm-assay coefficients of variation were 7 and 5?4&, respectively, and sensitivity was 0.05 ng/ml. Statistical Analyses Data for follicle, CL diameter and progesterone concentrations were analyzed as a split plot experimental design. The least squares analysis of variance in the General Linear Model Pro&me. (SAS, version 6.09) was used to test for differences behveen control and hCGtreated cows (15). The model included the following effects: treatma cows nested within treatment, day after estrus and the interaction between tmatment and day after e&us. The
586
Theriogenology
cowmeansquarewasusedtotest~~tmeansquare;therestweretestedusingthe residual error mean square. Categorical data were analyzed using contingency tables in Chisquare (15) analysis. RESULTS
Administration of hCG on Day 7 resulted in ovulation of the first wave dominant follicle in 5 cows. The sixth cow ovulated both the dominant and the second largest follicle and had 2 spontaneous CL at the time of hCG treatment (Table 1). Control cows had 1 CL each. Least squares means of the spontaneous CL diameter among control cows and cows treated with hCG on Day 7, as well as the diameter of the induced CL are shown in Figure la The diameter of the spontaneous CL between control and hCG-treated cows before hCG administration was not different (PXI.05). Similarly, the diameter of spontaneous CL between the 2 groups did not differ following hCG administration. However, the diameter of the CL showed a significant increase over time, and no interaction of treatment by time (PXl.05) was detected. Accessory CL induced by hCG given on Day 7 were detectable starting on Day 9, and were distinguishable from spontaneous CL by their smaller diameter during the treatment period Concentrations of progesterone increased steadily horn Day 0 to Day 7 in both groups (Figure lb). The mean concentration of progesterone was similar (PXOS) between the 2 groups until Day 16, and thereafter a rapid decline in progesterone was observed in the hCG-treated group. Table 1.
Effect of hCG on Day 7 of the cycle on accessory CL formation, follicular waves, CL regression and ovulation in cows x-eatrnent
Characteristics Cows with accessory CL Number of waves per cycle Time of emergence of Wave 2 (Day) Time to low progesterone level (Day) Time to ovulation of Wave 2 follicles (Day) Estrous cycle length (Days)
hCG 6 2.5 ? 10.8 2 21.8 2 13.0 + 23.3 +
0.2 0.2 0.5 1.8 1.4
Control 0 2.1 + 0.1 12.7 + 1.4 22.3 + 1.0 9.7 + 2.1 22.4 + 1.6
Profiles of the dominant follicle among control cows and cows treated with hCG are presented in Figures 2 and 3. The diameter of the first wave dominant follicle was similar between hCG and control cows (M.05). The Day of emergence of the second wave of follicular growth was advanced, but not significantly, in hCG-treated cows (10.8 + 0.3 vs 12.7 + 1.4 d). The diameter of the second wave dominant follicle from Days 15 to 18 did not differ (FM.05) between the treatment groups. However, the second wave dominant follicle had a slower growth rate (0.8 vs 1.3 &d) among cows treated with hCG compared with the controls. The second wave dominant follicle was the ovulatory follicle in 5 control cows, but only in 3 hCG-treated cows. The second wave dominant follicle among hCG-treated cows took longer to ovulate (36 h) than among control cows (10 h) after progesterone reached a
Theriogenology
587
.“..“.“.“~“~‘~~““‘~’
4
0
3
6
9
12
14
17
20
23
26
8
0
3
6
9 Number
12
14
of days after
17
20
23
26
estrus
Figure 1. Lease squares means (& SEM) of spontan~us CL diarnete~ (panel a) among control cows (0) and cows txated with hCG on Day 7 (0) as well as the diameter of induced CL among hCG-treated cows (A). Profiles of least squares means for progesterone concentration among control (0) and hCG-treated cows (0) are also presented ( Panel b).
588
Theriogenology
0
5
10
19
14
24
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20:
!j .d w Q)
14:
z =:
7:
g
0 3:
17:
10 .’
0
‘.‘..‘..‘..‘..‘..‘..‘..r..“.J 3 6 9 12
15
18
21
24
27
30
Number of days after estrus
Figure 2.
Least squans means (& SEM) of the ckmeter of the dominant follicle of the fkst (W and second (0) wave among the 5 control cows that presented a 2-wave pattern of follicular growth (RUMAa). One cunlrol cow had 3 waves of follicular growth @=I b).
Theriogenology
589
1‘ ’ -. 0
* ‘. 5
. . . ‘. 10
14
19
24
Ovulation
0
Figure 3.
5
10
Number
of days
14
after
19
24
estrus
Least squares means (& SEM) of the diameter of the dominant follicle of the first (0) and second wave (a) among the 3 hCG-treated cows that presented a 2-wave pa&n of follicular growth (Pam9 a). The remainder of the hCG-tnzated cows showed a 3-wave pattern (Panel b).
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Theriogenology
basal level of 0.05 @ml. The dominant follicle I?om the third wave ovulated in 1 control and in 3 hCG-treated cows. There was no effect (PXOS) of hCG with regard to the number of Days to regression of the CL (hCG-treated, 18.8 +1.2d, vs control 19.3 + 2.7d). Similarly, them was no difference with regard to time to low progesterone concentration, the interval between emergence and ovulation of the second wave dominant follicle and estrous cycle length (Table 1). DISCUSSION The classical explanation for the extension in estrous cycle length observed following hCG tteatment has been that the functional status of the CL is maintained beyond its normal duration (6,23,25). The present study evaluated the use of hCG in nonbred cows and found no difference in the CL diameter (spontaneous CL) or in the onset of CL regression of control and hCG-treated cows. This finding is consistent with our previous observation (12). Similarly, the Day at which progesterone concentration declined to less than 1.0 ng/ml was also not different between the 2 treatment groups. However, a trend was observed for a more rapid and earlier decline in progesterone among hCG-treated cows. This is in contrast to the progesterone profile observed among cows which were bred and treated with hCG either at the time of breeding or afler 7 or 14 days (19). The earlier decline in progesterone levels observed in this study might be due to several factors. Treatment with hCG in sheep (7) and pigs (24) is associated with an increase in the number of large luteal cells and a concomitant reduction in the number of small luteal cells in spontaneous CL. Recently, this pattern was also observed in the hCG-induced and hCGexposed spontaneous CL of cattle (20). Furthermore, large luteal cells are reported to possess receptors to the lutelysin PGF, (9). This increase in the number of large luteal cells and, therefore, potential increase in the sensitivity to PGF, would explain the premature physiological regression of hCG-induced and hCG-exposed CL observed in the present study. Moreover, if spontaneously formed CL are exposed to hCG during the period of its formation (Days 0 to 3), estrous cycle length and CL lifespan is shorter than normal (2, 3). Although not statistically different, the time of emergence of the second wave of follicular growth was advanced in hCG-treated cows. Such a finding is in agreement with the hypothesis that dominant follicles suppress the emergence of new waves of follicular growth (1, 13). In the present study, removal of the suppressive effect of the dominant follicle, by way of induced ovulations, created a permissive environment, thus allowing a new wave of follicles to emerge early. The second wave dominant follicle was the ovulatory follicle in 5 controls, but only in 3 hCG-treated cows. Also in these 3 cows, the dominant follicle took longer to ovulate following regression of the CL and decline in progesterone to < 0.5 ng/ml. In a recent study, we (22) reported that administration of progesterone early in the cycle caused either regression or delayed ovulation of the first wave dominant follicle. Therefore, regression or delayed ovulation of the second wave dominant follicle observed in the present study could be ascribed to high progesterone or to the carry-over effect of hCG.
591
Theriogenology
Due to the onset of at&a of the second wave follicle in hCG-treated cows, a third wave was required to provide an ovulatory follicle. This need for a third wave may explain a nonsignificant extension in the estrous cycle length observed in hCG-treated cows, and is in agreement with previous reports (16,21) that have demonstrated that 3-wave cycles are associated with an extension in cycle length. Nonetheless, the extension observed in the present study was shorter than when hCG was given later in the luteal phase of the cycle (6, 17, 18, 25). In conclusion, the current study found alteration in follicular dynamics among cows treated with hCG on Day 7 of the cycle. This alteration did not affect the cycle length. Therefore, based on pregnancy data in our previous studies (12, 19) and the cycle length data in the present study, hCG at Day 7 after breeding could be used as a management tool for improving the pregnancy rate in postpartum cows. REFERENCES 1
Adams GP, Kot K Smith CA, Ginther OJ. Effect of dominant follicle on regression of its subordinated follicle in heifa. Can J Anim Sci 1993; 73:267-275. 2 Bat&a PJ, Rexroad Jr CE, Williams WF. Effects of progesterone administered to dairy heifers on sensitivity of corpora lutea to PGF, and on plasma LH concentrations. Theriogenology 1984; 22:47-58. 3 Bulrnan DC, Lamming GE. Milk progesterone levels in relation to conception, repeat breeding and factors influencing a cyclic&y in daii cows. J Reprod Fertil 1978; 54:447458. 4 de Los Santos-Valadez S, Seidel GE Jr, Elsden RP. Effect of hCG on pregnancy rates in embryo transfer recipients. ‘lheriogenology 1982; 17:85 abstr. 5 Diskin MG, Sreenan JM. Progesterone and embryo survival in the cow. In: Diskin MG, Screenan JM teds). Embryonic Mortality in Farm Animals. Dordtrecht, The Netherlands: Martinus Nijhoff Publishers, 1985; 142-158. 6 Eduvie LO, Seguin BE. Corpus luteum function and pregnancy rates in lactating dairy cows given human chorionic gonadotropin at mid die&us. ‘Iheriogenology 1982; 17: 415-422. 7 Farin CE, Moeller CL, Mayan H Gamboni F, Sawyer HR, Niswender GD. Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum. Biol Reprod 1988; 38:413-421. 8 Morris LM, Gonzalez-Padilla E, Niswender GD, Wiltbank JN. Peripheral progesterone levels in pregnant and non-pregnant heifers following use of hCG. Theriogenology 1976; 6:367-378. 9 Niswender GD, Schwall RH, Fitz CE, Sawyer HR Regulation of luteal function in domestic ruminants: new concepts. Ret Prog Horm Res 1985; 41:101-151. 10 Northey DL, French LR Effect of embryo removal and intrauterine infusion of embryonic homogenates on the lifespan of the bovine corpus luteum. J Anim Sci 1980; 501298-302. 11 Rajamahendran R, Taylor C. characterization of ovarian activity in postpartum dairy cows using ultrasound imaging and progesterone profiles. Anim Reprod Sci 1990; 22:171-180.
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12 Rajamahendran R, Sianangama PC. Effect of human chorionic gonadotropin on dominant follicles in cows: f&nation of accessory corpom lutea, progesterone production and pregnancy rates. J Reprod Fertil 1992; 95577-584. 13 Rajamahendmn R, Calder MC. Superovulatory responses in dairy cows following ovulation of the dominant follicle of the first wave with hCG. neriogenology 1993; 40:99-109. 14 Roche J, Boland MP, McGeady TA. Reproductive wastage following artificial imemimtion of heifm. Vet Ret 1981; 109:401-403. 15 SAS User’s Guide: Statistics, Personal Computer, Version 6.3. SAS Institute, Gary, NC 1986. 16 Savio JD, Thatcher WW, Bad&a IU, de la Sota RL, Wolfmson D. Regulation of follicular turnover during the estrous cycle in cows. J Reprod Fertil 1993; 97: 197-203. 17 Seguin BE, Oxender WD, Britt JH. Effect of human chorionic gonadotropin and gonadotropin-releasing hormone on cmpus luteum fimction and estrom cycle duration in dairy heifixs. Am J Vet Res 1977; 38:1153-1156. 18 Sianangama PC, Rajamahemlran R Effect of hCG on follicular dynamics in lactating dairy cattle. Biol Repmd 1991; 44 (Suppl 1): 66 abstr. 19 Sianangama PC, Rajamahendran R Effect of human chorionic gonadotropin administered at specific times following breeding on milk progesterone and pregnancy rates in cows. ‘I&riogenology 1992; 38:85-96. 20 Sianangama PC. Ihe Characteristics of Corpora Lutea Induced by hCG in Cattle. Ph.D. Dissertation, Ihe University of British Columbia, Vancouver, Canada, 1994. 21 Taylor C, Rajamahendran R Follicular dynamics, corpus h&urn growth and regression in lactating dairy cattle. Can J Anim Sci 1991; 71:61-68. 22 Taylor C, Rajamahendran R Effect of mid-l&al phase progesterone levels on the first wave dominant follicle in cattle. Can J Anim Sci 1994; 74:281-285. 23 Wagner JF, Veenhuizen EL, Tonkinson LV, Rathmacker RP. Effect of placental gonadotropin on pregnancy rate in the bovine. J Anim Sci 1973; 36:1129-l 136. 24 Wlesak T. Effect of pregnancy, injection of oe&adiol bemmate or hCG on steroid concentration and release by pig luteal cells. J Reprod Fertil 1989; 86:247-254. 25 Wiltbank JN, Rothlisberger JA, Zimmerman DR Effect of human chorionic gonadotropin on maintenance of the corpus luteum and embryo survival in the cow. J Anim Sci 1961; 20:827-829.