Effects of short-term treatment with progesterone superimposed on 11 or 17 days of norgestomet treatment on the interval to oestrus and fertility in Bos indicus heifers

Animal Reproduction Science 51 Ž1998. 169–183

Effects of short-term treatment with progesterone superimposed on 11 or 17 days of norgestomet treatment on the interval to oestrus and fertility in Bos indicus heifers J. Cavalieri ) , J.E. Kinder 1, G. De’ath, L.A. Fitzpatrick Australian Institute of Tropical Veterinary and Animal Science, James Cook UniÕersity of North Queensland, TownsÕille, Queensland 4811, Australia Accepted 30 January 1998

Abstract The aims of this study were to determine: Ž1. if short-term treatment of Bos indicus heifers with progesterone ŽP4 . while implanted with a s.c. norgestomet implant for 17 days would influence the time interval to oestrus and increase fertility of the synchronised oestrus, and Ž2. whether the response to treatment with P4 would differ between heifers treated with a norgestomet implant for 17 vs. 11 days when short-term treatment with P4 is applied 3 days prior to implant removal. B. indicus heifers at two separate sites ŽA and B. were allocated to three groups at each site. Heifers in two groups ŽNG and NGP4 groups. were given a single s.c. norgestomet implant on the first day of treatment Žday 0. while heifers in a third group ŽNGP4 PG group. were implanted on day 6. A single P4 releasing Controlled Internal Drug Release device ŽCIDR. was inserted on day 14 in heifers in the NGP4 and NGP4 PG groups and was removed 23.5 " 0.07 h later Žday 15.. Heifers in the NGP4 PG group were administered an analogue of prostaglandin F2 a ŽPGF2 a . at the time of CIDR removal to regress corpora lutea. Implants were removed from all heifers on the same day Žday 17. and a 400 IU of equine chorionic gonadotrophin ŽECG. was administered s.c. Animals were artificially inseminated 11.1 " 0.17 h after detection of oestrus, using frozen semen from one bull at site A and one of five bulls at site B. Inseminations were carried out by one of two technicians. Treatment with P4 delayed oestrus and reduced the synchrony of oestrus at site A Žhours to oestrus " SD: NG group, 39.0 " 13.7; NGP4 group, 66.3 " 24.4; NGP4 PG group, 58.9 " 20.5 h; P - 0.05. but not at site B Ž41.4 " 15.2, 42.5 " 10.1,

) Corresponding author. Present address: North Carolina State University, College of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606, USA. 1 Present address: Department of Animal Science, University of Nebraska, Lincoln, NE 68583-0908, USA.

0378-4320r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 0 6 4 - 5

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45.4 " 10.3 h; P ) 0.05.. Pregnancy rates 6 weeks after insemination were found to be significantly associated with bull Ž P - 0.001., treatment group Ž P s 0.013. and insemination technician Ž P s 0.033.. Pregnancy rates were greater in the heifers in the NGP4 group than heifers in the NG group w50.3% Ž78r155. vs. 36.4% Ž60r165.; odds ratio s 1.83, 95% CI s 1.14 to 2.96x and similar between heifers in the NGP4 and NGP4 PG groups w50.3% Ž78r155. vs. 51.1% Ž63r117.; odds ratio s 1.06, 95% CI s 0.67 to 1.69x. It was concluded that acute treatment with P4 can improve pregnancy rates in B. indicus heifers treated for 17 days with norgestomet implants. Reducing the duration of norgestomet treatment to 11 days and administration of PGF2 a at the time of ending treatment with a CIDR device resulted in no differences in fertility, mean intervals to oestrus or synchrony of oestrus. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Progesterone; Oestradiol; Oestrous synchronisation; Follicle; Cattle-endocrinology

1. Introduction Progestogens are commonly used to synchronise oestrus in cattle by synchronising the end of the progestational phase of the oestrous cycle ŽOdde, 1990; Wright and Malmo, 1992.. Prolonged treatments with progestogens Ž) 14 days., in concentrations normally used commercially to synchronise oestrus, result in precise synchrony of oestrus but reduced fertility at the synchronised oestrus ŽDe Bois and Bierschwal, 1970; Zimbelman et al., 1970.. Shorter periods of progestogen treatment Ž- 14 days. require the use of a luteolytic treatment and fertility has been improved in some ŽRoche, 1974, 1976; Smith et al., 1984. but not all studies, with lower fertility being reported in animals treated late in the oestrous cycle ŽBeal et al., 1988; Brink and Kiracofe, 1988; Patterson et al., 1989.. The reduction in fertility associated with the use of progestogens, in concentrations normally used to synchronise oestrus, has been attributed to abnormal oocyte development ŽKinder et al., 1996.. Doses of progestogens that are used commercially to synchronise oestrus in the absence of a functional corpus luteum ŽCL. result in a greater frequency of LH pulses than typically occur when concentrations of progesterone ŽP4 . typically present during mid-dioestrus Ž) 3 ngrml. are maintained ŽRoberson et al., 1989; Kojima et al., 1992.. This results in a prolonged period of ovarian follicular dominance of the preovulatory ovarian follicle and prolonged greater circulating concentrations of E 2 ŽSavio et al., 1993a.. These effects have been associated with ovulation of persistent ovarian follicles, abnormal oocytes ŽMihm et al., 1994b; Revah and Butler, 1996., abnormal embryonic development ŽAhmad et al., 1995. and reduced fertility ŽStock and Fortune, 1993; Savio et al., 1993b; Mihm et al., 1994a; Cooperative Regional Research Project, 1996.. Some studies, however, indicate that other factors such as alteration in the uterine environment or reduced sperm transport and survival may also contribute to the reduced fertility that can be associated with the use of progestogens ŽQuinlivan and Robinson, 1969; Lauderdale and Ericsson, 1970; Butcher and Pope, 1979.. It is, therefore, currently not known if the reduction in fertility associated with prolonged treatments with progestogens is entirely due to effects on oocyterearly embryonic development or in part due to an adverse effect of prolonged progestogen treatment on other aspects of reproductive function, such as the uterine environment.

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Acute treatment with P4 causes atresia of dominant ovarian follicles ŽAnderson and Day, 1994, Rajamahendran and Manikkam, 1994. and improves fertility ŽAnderson and Day, 1994. in Bos taurus cattle treated with progestogens to synchronise oestrus. In a recent study in which B. indicus heifers were treated with a norgestomet implant for 17 days and a single CIDR for 24 h 3 days before implant removal ŽCavalieri et al., 1998., treatment resulted in ovulation of an ovarian follicle Žmean age " SD: 5.6 " 1.8 days. that had experienced a short duration of follicular dominance Žmean duration of dominance" SD: 2.4 " 1.5 days.. Mihm et al. Ž1994b. reported that fertility was not reduced when the duration of dominance of the ovulatory follicle was F 4 days. It is not known, however, if the effect of acute treatment with P4 on fertility would differ with the length of the period of progestogen treatment. The aims of the present study were, first, to determine if treatment of heifers with P4 for 24 h 3 days before the end of a long term period of progestogen treatment would influence time interval to oestrus and increase fertility at the synchronised oestrus. Our second aim was to determine if intervals to oestrus and fertility would differ when acute treatment with P4 was applied 3 days before ending a short compared to a long period of progestogen treatment. 2. Materials and methods 2.1. Experimental site and animals This experiment was conducted at two commercial beef properties, Carmila Glen Žlatitude 21855X S; longitude 149825X E; site A. located 500 km south of Townsville in the coastal region of the central north Queensland wet tropics, and Powlathanga Station Žlatitude 20832X S; longitude 145859X E; site B., located 130 km southwest of Townsville in a sub-coastal, open eucalypt woodland within the semi-arid tropics of north Queensland. The climate at the two experimental sites is characterised by a distinct hot, wet summer period Žwet season. and a warm dry winter period Ždry season.. Brahman heifers Žsite A: 7r8 to 15r16 B. indicus, site B: 1r2 to 7r8 B. indicus . about 18 to 28 months of age, at each site, were weighed and condition scored Ž1 s emaciated, 9 s grossly overweight; Holroyd, 1978.. Heifers were sorted in ascending bodyweight and each heifer in each successive group of three heifers were allocated

Table 1 MeanŽ"SEM. body weight and condition score Ž1semaciated, 9s grossly overweight. of heifers in each treatment group at each site Group

Site A

1 ŽNG. 2 ŽNGP4 . 3 ŽNGP4 PG. Total a

vs. b,

c

vs.

d

Site B

n

Weight Žkg.

CS

n

Weight Žkg.

CS

71 74 71 209

345"3 343"3 344"3 344"2 a

5.4"0.04 5.4"0.05 5.4"0.05 5.4"0.03 c

116 112 133 358

254"3 251"3 256"3 254"2 b

4.3"0.05 4.4"0.05 4.3"0.06 4.3"0.03 d

P - 0.001.

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randomly to one of three treatment groups. The number of heifers in each of the three treatment groups at each experimental site and the mean Ž"SEM. body weight and condition score of the heifers at each site are given in Table 1. Heifers at both sites were located on pasture and on a rising plane of nutrition. 2.2. Treatments to synchronise oestrus Heifers in two groups ŽNG and NGP4 groups. were given a single s.c. silicone implant containing 3 mg of the synthetic progestogen norgestomet ŽCrestar; Intervet ŽAust.., Castle Hill, NSW. on the first day of the treatment period Žday 0. while heifers in a third group ŽNGP4 PG group. were implanted on day 6 ŽFig. 1.. A single P4 releasing intravaginal Controlled Internal Drug Release device type B ŽCIDR; CIDR-B, Riverina Artificial Breeders, Albury, NSW. was inserted intravaginally on day 14 in the heifers in the NGP4 and NGP4 PG groups and was removed 23.5 " 0.07 h Žmean " SEM. later Žday 15.. Heifers in the NGP4 PG group were also treated with 15 mg of an analogue of prostaglandin F2 a ŽPGF2 a ; Prosolvin, Intervet. i.m. at the time of CIDR withdrawal to regress corpora lutea. Implants were removed from all heifers on the same day Žday 17. and a s.c. injection of 400 IU of equine chorionic gonadotrophin ŽECG; Folligon, Intervet. was given to facilitate the induction of oestrus in heifers that may have been anoestrous at the commencement of treatment. 2.3. Detection of oestrus, artificial insemination and pregnancy diagnosis Animals were fitted with heatmount detectors ŽKamar w , Kamar Inc., Steamboat Springs, CO, USA. at the time of implant removal to aid detection of oestrus and

Fig. 1. Diagrammatic representation of the treatment protocol. The open rectangles represent a single s.c. norgestomet implant inserted from days 0 to 17 ŽNG and NGP4 groups. or from days 6 to 17 ŽNGP4 PG group.. A CIDR device Žshaded rectangle. was inserted on day 14 in heifers in the NGP4 and NGP4 PG groups and removed on day 15. An injection of an analogue of PGF2 a was administered to the NGP4 PG treated heifers at the time of CIDR removal Žday 15.. Implants were removed from all heifers on day 17 and 400 IU of ECG was injected s.c.

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observations for oestrous behaviour were undertaken twice daily Ž0800, 1700 h. for 6 days. Animals with activated heatmount detectors were separated and artificially inseminated 11.1 " 0.17 h Žmean " SEM. after oestrus was first detected, using frozen semen from a single ejaculate from one bull at site A Žbull 6. and one of five bulls at site B Žbulls 1 to 5.. Allocation of bulls to heifers at Site B was determined by the cattle owner but was done without knowledge of the treatment groups that heifers were allocated to. Thawed semen samples from all six bulls were assessed for motility under light microscopy. Semen quality was also assessed for bulls 1, 2, 4, 5 and 6 using a Hamilton Thorn Motility Analyser ŽHamilton-Thorn Research, Danvers, MA, USA.. Insufficient frozen semen was available from bull 3 for assessment of semen other than by visual assessment. Pregnancy was diagnosed by transrectal ultrasonography ŽAloka 210 Dx: 7.5 MHz probe. 6 weeks after insemination. 2.4. Blood sampling and radioimmunoassays Blood was collected by coccygeal venepuncture into evacuated tubes ŽVacutainer, Becton-Dickinson, Rutherford, NJ, USA. containing lithium heparin on the day of implant insertion and removal from heifers at site A only. Plasma was separated and stored frozen Žy208C. until assayed for P4 and 17b-oestradiol ŽE 2 .. Concentrations of plasma P4 were measured by radioimmunoassay in unextracted plasma samples using a modification of the Danazol method ŽMcGinley and Casey, 1979. described by Jolly Ž1992.. The sensitivity of the assay Ž90% of zero-binding. was 0.05 ngrml and intra- and interassay coefficients of variation were 10.0% and 11.3%. Concentrations of E 2 in plasma were determined by RIA using antiserum to E 2 ŽLilly lot a022367. provided by Dr. N.R. Mason ŽLilly Research Laboratories, Indianapolis, IN., the procedure for which has been previously reported ŽKojima et al., 1992.. The sensitivity of this assay Ž90% of zero binding. was 0.04 pgrml. Intra- and interassay coefficients of variation for E 2 assays were 16.4% and 13.4%, respectively. 2.5. Statistical analyses Statistical analyses, excluding bootstrapping, were performed using the statistical program SPSS for Windows 1993 ŽSPSS, IL, USA.. Logistic regression analysis was used to assess the potential association of insemination technician, bull, treatment group, body weight category with the proportion of heifers that were artificially inseminated that were diagnosed as pregnant 6 weeks after insemination Žpregnancy rate.. Interaction terms based upon their potential biological validity were selected and added to the model. The interaction terms included in the model were treatment group by weight; treatment group by technician; treatment group by bull; technician by weight and bull by weight. Heifers were grouped into four body weight categories, - 240 kg, 240–279 kg, 280–319 kg, ) 320 kg. Since only one bull was used on site A and 5 bulls were used on site B, differences in sites reflected differences in bulls. For this reason site was not included as a factor in the model. Terms were eliminated from the model using step-wise regression with tests based upon the likelihood-ratio test ŽCollett, 1991. using a level of significance of 0.05. As terms were removed from the model differences in

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model Chi-square values were used to calculate the Chi-square values and corresponding probability values for explanatory variables. The odds ratios ŽORs. of the pregnancy rate of the NG group contrasted with the NGP4 group and the NGP4 PG contrasted with the NGP4 group were calculated together with approximated 95% confidence intervals. Comparisons of the NG group with the NGP4 group was used to address the first aim of the study, that is whether short-term treatment with P4 would influence fertility when using a 17-day treatment with norgestomet. Comparison of the NGP4 group with the NGP4 PG group was used to address the second aim of the study, whether the response to treatment with P4 would differ between heifers treated with a norgestomet implant for 17 vs. 11 days when short-term treatment with P4 is applied 3 days prior to implant removal. Comparisons of the mean body weight and condition score of heifers between the two experimental sites were conducted using a Student’s t-test. One-way ANOVAs were used to compare concentrations of P4 and E 2 in plasma at the time of implant insertion and removal among the three treatment groups at site A. A logarithmic transformation of concentrations of E 2 in plasma Žbase 10. was used to stabilise variances across groups. When ANOVAs indicated significant differences Ž P - 0.05. between groups, Tukey’s HSD was used as the means separation procedure ŽZar, 1984.. Logistic regression analysis was used to compare the proportion of heifers in each treatment group with concentrations of P4 in plasma that were greater than 1.0 ngrml. Due to the non-normal and highly variable distribution of the data estimates of location Žmean. and spread Žstandard deviation. for implant removal to oestrus intervals for each treatment group and site were obtained using the bootstrap technique ŽEfron and Tibshirani, 1993.. Bias corrected and accelerated estimates were used to calculate both estimates and confidence intervals ŽEfron and Tibshirani, 1993. using the bootstrap library provided in Statlab ŽS-Plus, StatSci Division, MathSoft, Seattle, WA, 1995.. For the purposes of comparisons of means and standard deviations among treatment groups, non-overlapping 90% confidence intervals represent a significant difference at approximately the 1% level of significance.

3. Results Implants were lost from one heifer in the NG group and four heifers in the NGP4 group at site A and two heifers in the NG group and one heifer in the NGP4 group at site B. One heifer at site A ŽNGP4 group. and one heifer at site B ŽNGP4 PG group. were missing CIDRs on the day of CIDR removal. One heifer at site A ŽNG group. and one heifer from each of the treatment groups at site B also could not be located for recording of data following the removal of implants. Data from these animals were excluded from analyses. 3.1. Concentrations of progesterone No differences in mean concentrations of P4 in plasma were detected among groups at site A at the start Ž4.31 " 0.63, 4.51 " 0.41, 5.43 " 0.61 ngrml, mean " SEM for the NG, NGP4 and NGP4 PG groups, respectively; FŽ2193. s 1.16, P s 0.315. or end of

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treatment Ž0.83 " 0.33, 0.61 " 0.07, 0.58 " 0.05 ngrml for the NG, NGP4 and NGP4 PG groups, respectively; FŽ2204. s 0.47, P s 0.625.. The proportion of heifers at site A with plasma concentrations of P4 ) 1.0 ngrml at the time of implant removal was - 16% for heifers in each of the treatment groups Ž11.8%, 13.0%, 15.7%, for the NG, NGP4 and 2 NGP4 PG groups, respectively, xŽ2. s 0.48, P s 0.788.. 3.2. Concentrations of 17b-oestradiol Concentrations of E 2 in plasma of heifers at site A at the time of implant insertion did not differ between treatment groups Ž FŽ2196. s 1.35, P s 0.261. but by the time of implant removal concentrations of E 2 in plasma were greater in the heifers in the NG group compared with the heifers in the NGP4 and NGP4 PG groups ŽFig. 2; FŽ2203. s 86.09, P - 0.001.. 3.3. InterÕals to and synchrony of oestrus The patterns of onset of oestrus for each group at the two sites are depicted in Fig. 3. Oestrus was delayed in the two groups of heifers treated with P4 compared to the control heifers at site A Ž P - 0.05., however, the mean interval to oestrus was similar among all 3 treatment groups at site B Ž P ) 0.05; Table 2.. Mean time intervals to the detection of

Fig. 2. Concentrations of E 2 in plasma at the start ŽI. and end ŽB. of norgestomet treatment in heifers in the three treatment groups at site A. Note logarithmic Žbase 10. scale. Mean"SEM. Different characters above bars indicate significant differences between groups at the time of blood collection Ž P - 0.05, Tukey’s HSD test..

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Fig. 3. Distribution of onset of oestrus of heifers in groups 1 Ž — ., 2 Ž PPP ., and 3 Ž — . at site A Ža. and B Žb. following removal of norgestomet implants.

oestrus were also less in the heifers in the NGP4 and NGP4 PG groups at site B compared to heifers in these same treatment groups at site A ŽTable 2.. Greater synchrony of oestrus was also observed in heifers treated with P4 at site B compared to heifers administered the same treatments at site A ŽTable 2.. 3.4. Pregnancy rates Factors found to be associated with pregnancy six weeks after insemination were bull 2 2 Ž xŽ5. s 62.3, P - 0.001., treatment group Ž xŽ2. s 8.66, P s 0.013. and insemination 2 Ž . technician xŽ1. s 4.54, P s 0.033 . The pregnancy rates obtained by the two insemination technicians were, 53.2% Ž149r280. and 36.8% Ž81r220. for technician 1 and 2,

Variable

Site A .a

Interval to oestrus Žh.Ž90% CI SD of interval to oestrusŽ90% CI. a PR Ž%. a b

B

NG group

NGP4 group

NGP4 PG group

NG group

NGP4 group

NGP4 PG group

39.0 Ž36.4, 41.9. 13.7 Ž11.4, 18.1. 47.8 Ž32r67. y

66.3 Ž61.1, 71.7. 24.4 Ž22.1, 27.5. 60.3 Ž35r58. y

58.9 Ž55.11, 63.7. 20.5 Ž17.3, 25.1. 58.7 Ž37r63. y

41.4 Ž39.1, 44.4. 15.2 Ž12.5, 18.9. 28.6 Ž28r98. 36.8 b Ž25r68.

42.5 Ž40.9, 44.4. 10.1 Ž8.2, 13.9. 44.3 Ž43r97. 57.4 b Ž39r68.

45.4 Ž44.0, 47.1. 10.3 Ž8.4, 13.2. 47.0 Ž55r117. 57.3 b Ž51r89.

Non-overlapping 90% confidence intervals ŽCI. differ Ž P - 0.05.. Excluding heifers inseminated to bull 1.

J. CaÕalieri et al.r Animal Reproduction Science 51 (1998) 169–183

Table 2 Mean and standard deviation ŽSD. of intervals to oestrus and pregnancy rates ŽPR. for each treatment group at experimental sites A and B

177

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Table 3 Pregnancy rate obtained using semen from six bulls and estimates of semen quality as assessed with a light microscope ŽLM. and computerised semen analyser ŽCSA. Variable

Bull 1

2

3

4

5

6

PR Ž%.

12.6 Ž11r87. 30 42

61.1 Ž33r54. 40 62

40.9 Ž9r22. 40 y

44.0 Ž51r116. 35 44

66.7 Ž22r33. 40 40

55.3 Ž104r188. 35 59

Semen quality a

LM CSA

a

Numerator s% number of live sperm displaying progressively normal motility. Semen from bull 3 was not available for analysis by a CSA.

respectively. Pregnancy rates of heifers inseminated with semen from sire 1, were 69 to 81% lower than pregnancy rates of heifers inseminated with semen from the other four bulls at site B. However, visual assessment of semen quality and assessment of semen by a computerised semen analyser indicated that semen from bull 1 was of adequate quality ŽJ. Cooper, pers. comm... Pregnancy rates obtained with the six bulls used and the results of motility estimates using semen from each of the bulls at site B are shown in Table 3. The odds ratio for pregnancy for heifers treated with norgestomet for 17 days indicated that heifers that were also treated with P4 were more likely to be pregnant 6 weeks after insemination than heifers treated with norgestomet only Žodds ratio: 1.86; 95% CI s 1.14 to 2.96, for the NG compared to NGP4 treated heifers.. The likelihood of pregnancy was, however, similar among the NGP4 and NGP4 PG treated heifers Žodds ratio s 1.06; 95% CI s 0.67 to 1.69.. 4. Discussion The results of this study indicate that short-term treatment with P4 reduced concentrations of E 2 in plasma at the end of a period of norgestomet treatment and improved pregnancy rates in B. indicus heifers treated with a norgestomet implant for 17 days. The findings agree with those of Anderson and Day Ž1994. who administered 200 mg of P4 in an oil base i.m. to cattle, on day 12 of a 14-day treatment with MGA. Pregnancy rates were increased when the CL was absent on day 12. The improvement in fertility observed in the present study was most likely due to short-term treatment with P4 reducing LH secretion and causing atresia of dominant ovarian follicles with subsequent emergence and ovulation of a new ovarian follicle ŽAnderson and Day, 1994; Rajamahendran and Manikkam, 1994; Cavalieri et al., 1998.. Reducing the duration of norgestomet treatment to 11 days with a short-term treatment with P4 3 days before implant removal and administration of PGF2 a at the time of ending exogenous treatment with P4 ŽNGP4 PG group. did not significantly alter pregnancy rates or mean circulating concentrations of E 2 at the time of implant removal compared to the 17-day treatment with norgestomet and treatment with P4 3 days before implant removal ŽNGP4 group.. A possible effect of PGF2 a on fertility in the NGP4 PG treated heifers that may be independent of the duration of norgestomet treatment cannot

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be ignored, however, most studies conducted in cattle have concluded that administration of PGF2 a does not adversely affect fertility ŽMacmillan and Day, 1982; McIntosh et al., 1984.. The use of PGF2 a in this study was deemed necessary in the NGP4 PG treated heifers to ensure that luteolysis occurred in all heifers by the time of implant removal. The results of this study indicate that length of the period of progestogen treatment per se is unlikely to be a critical factor in causing a reduction in fertility following the use of progestogens. This agrees with the findings of McMillan et al. Ž1993. who transferred single frozen-thawed embryos to B. taurus heifers in which oestrus had been synchronised with either a 10- or 19-day CIDR treatment and found no differences in pregnancy rates between the two groups. These authors concluded that an extended period of treatment with P4 did not detrimentally influence either luteal or uterine function. Recently, Wehrman et al. Ž1996. obtained normal pregnancy rates when embryos were transferred 7 days after the detection of behavioural oestrus to cows that ovulated either persistent ovarian follicles or ovarian follicles with a normal duration of follicular dominance. This indicated that the uterine environment was not compromised in animals that ovulated persistent follicles. The use of prolonged periods of progestogen treatment could enable greater flexibility in the design of oestrous synchronising treatments and remove the need and added cost of using luteolytic treatments. In the present study, the use of a 17-day progestogen treatment resulted in less than 16% of heifers having plasma concentrations of P4 ) 1.0 ngrml at the time of implant removal which is comparable to what was achieved in B. indicus cattle when a 10-day period of progestogen treatment was used that incorporated a luteolytic treatment ŽCavalieri and Fitzpatrick, 1995.. If animals are treated with progestogens but treatment is commenced late in the oestrous cycle, declining concentrations of endogenous P4 can lead to the development of persistent follicles ŽSirois and Fortune, 1990; Custer et al., 1994. and reduced fertility ŽBeal et al., 1988; Brink and Kiracofe, 1988; Patterson et al., 1989.. The use of acute treatments with P4 , however, should improve fertility in animals when an oestrous synchronising treatment is initiated late in the oestrous cycle by ensuring the ovulation of a newly emerged dominant follicle. The timing in the onset of oestrus in heifers treated with a CIDR device for 24 h at site A is similar to our previous findings in B. indicus heifers that were similarly treated ŽCavalieri et al., 1998.. In this previous study, a delay in the timing of ovulation was observed in the heifers treated with a CIDR device 3 days before implant removal compared to heifers treated with a norgestomet implant only for 17 days. Treatment with a CIDR device for 24 h was also associated with atresia of dominant ovarian follicles and a delayed emergence of the ovulatory follicle, which lead to a delay in the timing of ovulation. The differences observed in the timing and synchrony of oestrus between sites in the NGP4 and NGP4 PG treated heifers may have been due to differences in body weight at the time treatments began or to other at present unknown causes. Intervals to oestrus in B. indicus heifers treated with a combination of norgestomet and oestradiol to synchronise oestrus have previously been shown to be influenced by ovarian cyclic status with heifers that were anoestrus at time of commencement of treatment being detected in oestrus earlier than heifers that were not anoestrus ŽCavalieri and Fitzpatrick, 1995.. In

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that study B. indicus heifers weighing 344 and 254 kg had a probability of ovarian cyclicity of ) 90% and - 40%, respectively. This would indicate that in the present study the proportion of heifers undergoing oestrous cycles at experimental site A were likely to be greater than at Site B. The shorter intervals from implant removal to oestrus and the greater synchrony of oestrus in the heifers treated at site B compared to site A might, therefore, be due to a greater proportion of heifers at site B being anoestrus at the time of implant insertion compared to heifers at site A. Other reasons for differences in the timing in the onset of oestrus between heifers at the two experimental sites might include that a shorter interval of time between emergence of the ovulatory follicle and ovulation occurred in heifers of lighter body mass. B. indicus heifers of smaller body mass have been shown to ovulate smaller follicles compared to heifers of greater body mass ŽRhodes et al., 1995.. It maybe that the time interval between emergence of the ovulatory follicle and ovulation is less with ovulatory follicles of smaller diameter. Heifers at site A were composed of a slightly greater percentage of B. indicus genotype than some of the heifers at site B and this may have been another factor that may have lead to differences occurring between sites. However, parentage records were not available for heifers at site B and so investigation of the potential association of genotype on intervals to oestrus could not be assessed. Although the exact reasons for differences in the timing and synchrony of ovulation among heifers at the two different sites cannot be discerned from this study, further investigation is needed to investigate the causes of such differences as it carries important implications for the recommended timing of insemination following treatments that synchronise oestrus. Treatment at site B appeared to result in a more synchronised oestrus that had potential for adequate fertility to result following fixed-time insemination. The precision of synchrony of oestrus at site A, however, was unacceptable for fixed-time insemination. Treating heifers with a norgestomet implant for 17 days and administering P4 for 24 h, 7 days before implant removal results in a precise time of ovulation without development of a persistent dominant ovarian follicle ŽCavalieri et al., 1998.. By increasing the interval between treatment with P4 and implant removal to 7 days, tight synchrony of oestrus without compromising fertility might, therefore, be achieved in B. indicus heifers which may differ in, for example, body weight. Effects of inseminator on fertility have been previously reported ŽUwland, 1983; Senger et al., 1984. and has been attributed to differences in semen placement ŽKing and MacPherson, 1965.. The significant effect of sire on fertility observed in the present study demonstrates that both objective and subjective means of assessing frozen–thawed bovine semen for AI programs may not always accurately assess the suitability of semen for AI. Variable correlations exist between tests of semen quality and fertility ŽBuckner and Willett, 1954; Linford et al., 1976. and the need for the development of tests which accurately estimate the fertility of bovine semen in vivo are needed.

5. Conclusion Short-term treatment with P4 3 days before the end of a 17-day period of norgestomet treatment can improve pregnancy rates in B. indicus heifers treated with a norgestomet

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implant for 17 days. Reducing the duration of norgestomet treatment to 11 days with administration of PGF2 a at the time of ending the short-term treatment with P4 did not significantly alter the time intervals to oestrus or fertility compared to the 17-day treatment protocol that included a short-term treatment with P4 . The effect of treatment on time intervals to oestrus and the synchrony of oestrus may, however, vary among cattle that differ in body weight. This study highlights that acute treatment with P4 can be used to counteract the reduction in fertility that can occur following a prolonged treatment with norgestomet. However, a longer interval between treatment with P4 and the end of norgestomet treatment will be needed if precise synchrony of oestrus is to be achieved in heifers of differing body weights and condition.

Acknowledgements The authors thank P. and A. David of the Carmila Glen Pastoral and B. and M. Rea of Powlathanga Station for providing animals and facilities for conducting this study. Assistance with the management of animals was kindly provided by B., C., and S. Coleman, J. Cowan, B. Gourley, J. Measom, J. Palpratt and W. Rea. We thank Dr. J. Britt for critical review of this manuscript, Dr. N.R. Mason for providing antiserum to E 2 and Intervet ŽAust.. for providing Crestar w and Prosolvinw . The assistance of C. Coleman and C. Toombs with hormone assays is gratefully acknowledged. Funding was provided by Intervet ŽAust.., James Cook University Meritorious Research grant and the Meat Research Corporation.

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