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Follicular dynamics and superovulatory response in heifers
REP~hON SCIENCE
ELSEVIER
Animal Reproduction Science 43 ( 19%) 183- 190
Follicular dynamics and superovulatory response in heifers S.P. Singh “b”, P.J. Broadbent a’*, J.S.M. Hutchinson b, R.G. Watt a, D.F. Dolman a a Scottish Agricultural College, School of Agriculture, 581 King Street, Aberdeen AB9 IUD, UK b University of Aberdeen, School of Agriculture, 581 King Street, Aberdeen AB9 1UD. UK
Accepted I December 1995
Abstract The study examined whether the response of heifers to exogenous gonadotrophin superovulatory treatment could be predicted from a knowledge of previous antral follicular dynamics. During a pretreatment monitoring phase, of 24 normal oestrous cycles (20.1 + 0.33 days long) observed in 17 heifers, one, 15 and seven cycles showed one, two and three antral follicular waves respectively, as measured by ultrasonography. The subsequent ovulatory response (number of corpora lutea) to ovine FSH stimulation, after a CIDR-B/oestradiol benzoate/prostaglandin analogue cycle synchronisation regime, was not correlated with either oestrous cycle length or follicle wave number during the monitoring phase or with the number of follicles observed at the start of FSH treatment, but was related to the number of follicles observed during the monitoring
phase (r = 0.47, P < 0.05). In conclusion, the present results show that the outcome of FSH superovulatory stimulation in heifers cannot be predicted from a knowledge of prior follicular dynamics. Keywords: Heifers; Superovulatoly response; Follicular waves; Ultrasound
1. Introduction Ovarian status at the time that gonadotrophin treatment commences appears to be a major determinant of a superovulatory response. This is demonstrated by the differences in response observed when gonadotrophin treatment is initiated at different stages of the
* Corresponding author. Tel: 44 1224 480291; fax: 44 1224 276717. ’Present address: Prabhat Nagar, Meerut 250006, UP, India.
0378-4320/%/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)01480-7
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Reproduction
Science 43 (1996) 183-190
oestrous cycle with higher responses obtained when treatment begins in mid- rather than early or late cycle (Sreenan and Gosling, 1977; Lemer et al., 1986; Goulding et al., 1990; Macmillan et al., 1994). At a more detailed level, the number of antral follicles present when gonadotrophin treatment begins has been shown to be positively related to the outcome of superovulation (Monniaux et al., 1983; Romero et al., 1991). The presence of a dominant follicle at this time, however, resulted in a reduced response (Guilbault et al., 1991, Huhtinen et al., 1992) although Gray et al. (1992) did not observe an inhibitory effect due to the presence of a regressing dominant follicle. Follicular development and the dominant follicle can be controlled by ablation of follicles (Ko et al., 1991; Adams et al., 1993) or hormonal treatments (Sirois and Fortune, 1990; Adams et al., 1992a; Bo et al., 1993). Ablation of the dominant follicle, or its natural absence, resulted in improved superovulatory responses (Hahn, 1992; Bungartz and Niemann, 1994). Similar improvements were observed following hormonal suppression of the dominant follicle and emergence of a new wave of follicles (Bo et al., 1995). The use of transrectal ultrasound imaging of the ovaries (Pierson and Ginther, 1984; Pierson and Ginther, 1988) provides a means of evaluating follicular dynamics in a frequent and non-invasive manner. This technique has been used to confirm the wave-like pattern of follicular development in cattle (Pierson and Ginther, 1988; Savio et al., 1988; Sirois and Fortune, 1988). It has also been used to monitor the dominant follicle and its control by ablation or endocrine treatment (Ko et al., 1991; Armstrong, 1993; Sawyer et al., 1992; Sawyer et al., 1995; Bo et al., 1995). The purpose of the present study was to examine the proposition that the outcome of gonadotrophin treatment could be predicted from a knowledge of the follicular dynamics exhibited by the animal.
2. Materials and methods There were two phases to the study. In the first phase, of 7 weeks duration, the natural oestrous cycles of the animals were characterised in terms of their length and follicular dynamics using a combination of observation, plasma progesterone concentration and ovarian ultrasonography. In the second or superovulation phase, the oestrous cycles of the animals were controlled by synchronisation followed by superovulation and embryo recovery. The data were examined for correlations between features of the oestrous cycle or follicular dynamics, in either the monitoring or superovulatory phases, and the subsequent superovulatory response. 2.1. Animals The study was carried out using 18 purebred Simmental nulliparous heifers. The live weights and condition scores (Lowman et al., 1976) at the beginning and end of the study (mean + SEM) were 553 + 6.8 and 563 k 7.7 kg; and 3.35 + 0.1 and 3.46 + 0.1, respectively. The animals were housed in straw-bedded pens in groups of nine (4.7 mz per head) during the monitoring phase and groups of six (7.8 m2 per head) during the
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superovulation phase. Their daily diet during the study was 3 kg concentrates (1: 1:1 of sugar beet pulp, distillers’ dark grains and barley) plus hay and straw ad libitum, supplemented with 100 g mineral and vitamin mixture. 2.2. Ovarian ultrasonography Transrectal ultrasonography was performed using a 5 MHz linear probe (Aloka 210 DXII; BCF, UK) and all follicles 2 2 mm diameter plus the presence of corpora lutea were recorded separately for each ovary. Observations were made three times per week (Monday, Wednesday, Friday) during the monitoring phase. During the superovulation phase, ultrasonography was performed once when intravaginal devices were inserted; on two occasions prior to, and once at the start of, gonadotrophin treatment. The growth and regression of the follicles on each ovary were identified by a modified non-identity method (Ginther, 1993). The modification was that changes in the size of the largest (dominant) and second largest (subdominant) follicle by 1 mm followed by a regaining of the original diameter were not considered as the start of a new follicle because observations were not carried out daily. Follicular waves were identified by inspection of graphical presentations of the data. The number of follicles per wave was counted on the day of divergence i.e. the day on which the largest subordinate follicle ceased to grow or began to regress (Ginther, 1993). 2.3. Observation of oestrus Oestrous behaviour observed during the course of monitoring and handling the animals was recorded. Formal observations of oestrus (Broadbent et al., 1991) were carried out at the reference oestrus prior to superovulation over 5 days and at the superovulated oestrus for 3 days. The occurrence of oestrus and the length of the oestrous cycle during the monitoring phase was determined by observation of oestrus or inspection of the profiles for follicular dynamics and plasma progesterone concentrations. 2.4. Plasma progesterone Blood samples were taken by jugular venepuncture, plasma prepared for assay and stored at - 20°C three times per week during the monitoring phase, at oestrous synchronisation and at reference oestrus observations; at insertions or removals of intravaginal devices; daily during gonadotrophin treatment; and on the days of artificial insemination and embryo recovery. Progesterone was assayed using an enzyme-linked immunosorbant assay kit (Ridgeway Science, UK). 2.5. Superovulation and embryo recovery The heifers were synchronised for a reference oesbus by the intravaginal insertion of a CIDR-B (SmithKline Beecham, UK) and injection of 10 mg oestradiol benzoate im (Intervet, UK) on Day 1 of the programme. Prostaglandin F2 o: analogue (15 mg
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luprostiol; Intervet, UK) was administered im on Day 10 and the intravaginal device removed on Day 12 of this programme. The superovulatory protocol began on Day 7 + 1 after the reference oestrus (Sawyer et al., 1995) with the insertion of a second CIDR-B plus 10 mg oestradiol benzoate im. Gonadotrophin treatment began 9 days later and a total of 9.0 mg NIADDK-oFSH-17 equivalent ovine pituitary follicle stimulating hormone (ICP, New Zealand) was administered twice daily over 4 days in the pattern 2 (1.80 + 1.35 + 0.90 + 0.45) mg. On the third day of FSH treatment 30 mg luprostiol was administered im and the CIDR-B was removed 24 h later. Artificial insemination was performed at fixed-times, 32 and 48 h after CIDR-B removal, using frozen-thawed semen. Embryos and ova were recovered, non-surgically, 7 f 0.5 days after the first insemination. They were classified and graded as excellent, good, moderate, poor, degenerate and non-fertile. For the purposes of statistical analysis the viable embryos were re-classified on a numerical scale where 1 is excellent plus good; 2 is moderate; and 3 is poor. 2.6. Sratistical analysis Data are presented as means f SEM. The significance of differences between categories of data was tested by C-test. Linear relationships were tested by means of correlation analysis.
3. Results During the monitoring phase 24 oestrous cycles, 20.1 f 0.3 days long, were identified in 17 heifers. One heifer had a persistent corpus luteum and did not exhibit oestrus but five follicular waves were identified in this period. The average number of follicular waves, excluding this animal, was 2.3 + 0.1 per cycle. Two follicular waves were observed in 15 (65.2%; 19.9 f 0.3 days), three in 7 (30.5%; 21.3 + 2.0 days) and one in 1 (4.3%; 17 days) of the oestrous cycles. The number of follicular waves could not be determined for one of the oestrous cycles.
Table 1 Number of follicles 2 2 mm in different categories (mean f SEM) observed during each wave of the normal oestrous cycle and at initiation of gonadotrophin treatment on Day 16 f 1 after the reference oestrus Category of follicle
2to5mm 6to9mm 210mm Total
Number of follicles Mean per wave
At gonadotrophin
10.6f0.94 2.4kO.31 1.3kO.14 15.0k0.72
13.2f0.73 1.9 f 0.24 0.2 f 0.09 15.3 *0.67
stimulation
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Table 1 shows the total number of follicles and their distribution by size on average per wave in the normal oestrous cycle and at initiation of gonadotrophin treatment. The total number of follicles was similar in both cases but at the start of gonadotrophin stimulation there were more 2 to 5 mm follicles (10.6 vs. 13.2; P < 0.05) and fewer follicles 2 10 mm (1.3 vs. 0.2; P < 0.001). The number of corpora lutea palpated at embryo recovery was 16.3 + 1.l and a recovery rate of 82.3% produced a total of 13.4 f 1.7 ova plus embryos. Of these, there were 5.8 + 1.0 usable (Grades 1, 2 and 3) embryos and 4.1 rt 0.7 Grade 1 embryos. There was no significant relationship between the length of oestrous cycle (r = 0.15, P > 0.05) observed in the monitoring phase and number of corpora lutea at embryo recovery. The number of corpora lutea was similar for those animals which had two or three follicular waves during their normal oestrous cycles. The number of ovulations was not correlated (r = 0.08, P > 0.05) with the number of follicles present at the beginning of FSH treatment but was related to the number of follicles observed during the natural oestrous cycle (r = 0.47, P < 0.05). Three of the 18 heifers had a large follicle (2 10 mm) present at the time that gonadotrophin treatment commenced. There were no differences in ovulation rate between these animals and the remaining 15 heifers (15.3 + 2.7 vs. 16.5 f 1.2) but the heifers with a large follicle had fewer ova plus embryos recovered (10.0 &-4.7 vs. 14.1 f 1.9), usable embryos (4.0 IL-2.6 vs. 6.1 + 1.O) and grade 1 embryos (3.0 k 2.1 vs. 4.3 + 0.8). These differences were not significant.
4. Discussion The length of the oestrous cycle observed for pure Simmental heifers in this study was similar to that reported by Cardenas et al. (1991) for animals of the same breed and parity. Furthermore, the difference in oestrous cycle length between heifers exhibiting two or three follicular waves during the normal oestrous cycle was similar to that for other studies with heifers (Ginther et al., 1989). The maximum size of the dominant follicle (12.4 + 0.02 mm), however, was small relative to some other studies (approximately 16 mm; Pierson and Ginther, 1987; Ginther, 1993). Murphy et al. (1991) found that the dominant follicle was smaller in animals fed on a low plane compared with those on a moderate or high plane of nutrition. The diet of the heifers in the present study would equate with the moderate plane of Murphy et al. (1991) and the maximum diameter of the dominant follicle was similar in both studies (12.4 vs. 13.2 mm). The correlation of number of follicles during the normal oestrous cycles of the monitoring phase with the superovulatory response was unexpected but suggests that this information could be used as an indicator of the follicle population that can be stimulated in an animal provided that it continues to receive the same nutrition and management. The lack of any correlation between follicle number at the start of gonadotrophin treatment and subsequent ovulation rate indicates that one examination may not identify which follicles are capable of responding to gonadotrophin stimulation. The data suggest that some follicles 2 2 mm diameter may have been atretric and probably luteinised subsequently, rather than ovulated (Purwantara et al., 1993). Further-
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more, it is probable that some follicles < 2 mm were capable of responding satisfactorily to gonadotrophin treatment and that not all follicles 2 2 mm were identified. This finding contrasts with that of Romero et al. (1991) who, however, observed a larger range of follicle numbers at the initiation of FSH stimulation and worked with more animals. The feature of ovarian status at initiation of gonadotrophin treatment which tended to be related to superovulatory response was the presence or absence of a large (2 10 mm) follicle. The number of animals in each category meant that the differences observed were not statistically significant. Furthermore, the frequency of ultrasound examination and the use of the non-identity method meant that a clear determination of whether the largest follicles were in the growing, plateau or regressing phase could not be made. The results, however, are in line with the observations by others (Guilbault et al., 1991; Huhtinen et al,, 1992) that superovulatory response is lower when stimulation begins in the presence of a dominant follicle; and that response is higher following ablation of the dominant follicle (Hahn, 1992; Bungartz and Niemamr, 1994) or its control by hormonal treatment (Bo et al., 1993; Bo et al., 1995). Gonadotrophin treatment was initiated in the presence of exogenous progesterone, previously administered with oestradiol benzoate, on Day 7 (oestrus is Day 0) of a synchronised oestrous cycle. This protocol had previously been shown to improve superovulatory response (Sawyer et al., 1992; Sawyer et al., 1995) due to the suppressive effects of exogenous progesterone on follicular development (Adams et al., 1992b; Savio et al., 1993) and the induction of atresia, particularly of the dominant follicle, by oestradiol (Bo et al., 1991; Sawyer et al., 1992; Sawyer et al., 1995). The use of oestradiol 17/3 in the presence of exogenous progesterone has been shown to synchronise the emergence of a new follicular wave (Adams et al., 1994; Bo et al., 1994) and initiation of stimulatory treatments on the day before or the day of wave emergence to produce a higher superovulatory response (Adams et al., 1994; Nasser et al., 1993). These authors used oestradiol 17p in preference to oestradiol valerate because the effect of the latter, used in the absence of exogenous progesterone, depended on the stage of the oestrous cycle at which it was administered (Bo et al., 1993). It was suggested that oestradiol valerate did not suppress the dominant follicle completely and the long half-life of this substance resulted in the asynchronous emergence of follicular waves. The half life of the benzoate ester may be expected to be longer than that of oestradiol 17p but not as long as oestradiol valerate and its effects to be intermediate between those of these two compounds. This could explain why three of the 18 heifers in the present study had a follicle 2 10 mm at the start of FSH treatment. The superovulatory responses in this experiment were, however, good for maiden heifers and in a subsequent report (Broadbent et al., 1995) we have shown that the protocol of Sawyer et al. (1992) is equally efficacious to that of Adams et al. (1994). The implication may be that oestradiol benzoate administered simultaneously with exogenous progesterone not only suppresses the dominant follicle but may synchronise the emergence of a new follicular wave with the start of gonadotrophin treatment when the protocol used in the present study is followed. In conclusion, the present results show that the outcome of FSH superovulatory stimulation in heifers cannot be predicted from a knowledge of prior follicular dynamics.
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Acknowledgements SAC receives financial support from the Scottish Office Agriculture, Environment and Fisheries Department.
References Adams, G.P., Bo, G.A., Martinez, M., Caccia, M., Tribulo, H., Pierson, R.A. and Mapletoft, R.J., 1994. The effect of estradiol- 17p and progestogen treatment on superovulatory response in beef cows. Theriogenology, 41: 153. (Abstr.). Adams, G.P., Kot, K., Smith, C.A. and Ginther, O.J., 1993. Effect of the dominant follicle on regression of its subordinates in heifers. Can. J. Anim. Sci., 73: 267-275. Adams, G.P., Matteri, R.L. and Ginther, O.J., 1992a. Effect of progesterone on ovarian follicles, emergence of follicular waves and circulating follicle-stimulating hormone in heifers. J. Reprod. Fertil., 96: 627-640. Adams, G.P., Matteri, R.L., Kastelic, J.P., Ko, J.C.H. and Ginther, O.J., 1992b. Association between surges of follicle stimulating hormone and the emergence of follicular waves in heifers. J. Reprod. Fertil., 94: 177-188. Armstrong, D.T., 1993. Recent advances in superovulation of cattle. Theriogenology, 39: 7-24. Bo, G.A., Pierson, R.A. and Mapletoft, R.J., 1991. The effect of oestradiol valerate on follicular dynamics and superovulatory response in cows with Synchro-Mate B implants. Theriogenology. 36: 169- 183. Bo, G.A., Adams, G.P., Nasser, L.F., Pierson, R.A. and Mapletoft, R.J., 1993. Effect of oestradiol valerate on ovarian follicles, emergence of follicular waves and circulating gonadotrophins in heifers. ‘lheriogenology, 40: 225-239. Bo, G.A., Caccia, M., Martinez, M., Adams, G.P., Pierson, R.A. and Mapletoft, R.J., 1994. The use of oestradiol-17fi and progestogen treatment for the control of follicular wave emergence in beef cattle. Theriogenology, 41: 16.5.(Abstr.). Bo, G.A., Adams, G.P., Pierson, R.A. and Mapletoft, R.J., 1995. Exogenous control of follicular wave emergence in cattle. Theriogenology, 43: 3 l-40. Broadbent, P.J., Stewart, M. and Dolman, D.F., 1991. Recipient management and embryo transfer. Theriogenology, 35: 125- 139. Broadbent, P.J., Gebbie, F.E., Dolman, D.F., Watt, R.G., King, M.E. and Higgins, L.C., 1995. Superovulatory responses in cattle pre-treated with estradiol and progestogen. Theriogenology, 43: 176. (Abstr.). Bungartz, L. and Niemann, H., 1994. Assessment of the presence of a dominant follicle and selection of dairy cows suitable for superovulation by a single ultrasound examination. J. Reprod. Fertil., 101: 583-591. Cardenas, H., Padilla, A., Alvarado, E., Vivanco, W. and Berardinelli, J.G., 1991. Natural and prostaglandin F (PG)-synchronised oestrous cycle in Brown Swiss and Simmental heifers in the Highlands of Peru. Anim. Reprod. Sci., 26: 211-217. Ginther, O.J., Knopf, L. and Kastelic, J.P., 1989. Temporal association among ovarian events in cattle during oestrous cycles with two and three follicular waves. J. Reprod. Fertil., 87: 223-230. Ginther, O.J., 1993. A method for characterising ultrasonically-derived follicular data in heifers. Theriogenology, 39: 363-371. Goulding, D., Williams, D.H., Duffy, 0.. Boland, M.P. and Roche, J.F., 1990. Superovulation in heifers given FSH initiated either at Day 2 or Day 10 of the oestrous cycle. Theriogenology, 34: 767-778. Gray, B.W., Cartee. R.E., Stringfellow, D.A., Riddell, M.G., Riddell, K.P. and Wright, J.C., 1992. The effects of FSH priming and dominant follicular regression on the superovulatory response of cattle. ‘Iheriogenology, 37: 631-639. Guilbault, L.A., Grasso, F., Lussier, J.G., Rouilher, P. and Matton, P., 1991. Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle. J. Reprod. Fertil., 91: 81-89. Hahn, J., 1992. Attempts to explain and reduce variability of superovulation. Theriogenology, 38: 269-275. Huhtinen, M., Rainio, V., Aalto, J., Bmdbacka, P. and Maki-Tanila, A., 1992. Increased ovarian responses in the absence of a dominant follicle in superovulated cows. Theriogenology, 37: 457-463.
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Ko, J.C.H., Kastelic, J.P., Del Campo, M.R. and Ginther, O.J., 1991. Effects of a dominant follicle on ovarian dynamics during the oestrous cycle in heifers. J. Reprod. Fertil., 91: 511-519. Lemer, S.P., Thayne, W.V., Baker, R.D., Henschen, T., Meredith, S., Inskeep, E.K., Dailey, R.A., Lewis, P.E. and Butcher, R.L., 1986. Age, dose of FSH and other factors affecting superovulation in Holstein cows. J. Anim. Sci., 63: 176-183. Lowman, B.G., Scott, N.A. and Somerville, S.H., 1976. Condition scoring of cattle. Revised edition. Bulletin, East of Scotland College of Agriculture, Number 6. Macmillan, K.L., Taufa, V.K. and Hayman, D.L., 1994. Superovulation responses to ovine FSH administered during late dioestrus to cows fitted with an intravaginal progesterone device. Theriogenology, 41: 243. (Abstr.). Momtiaux, D., Chupin, D. and Saumande, J., 1983. Superovulatory responses of cattle. Theriogenology, 19: 55-81. Murphy, M.G., Enright, W.J., Crowe, M.A., McConnell, K., Spicer, L.J., Boland, M.P. and Roche, J.F., 1991. Effect of dietary intake on pattern of growth of dominant follicles during the oestrous cycle in beef heifers. J. Reprod. Fertil., 92: 333-338. Nasser, L.F., Adams, G.P., Bo, G.A. and Mapletoft, R.J., 1993. Ovarian superovulatory response relative to follicular wave emergence in heifers. Theriogenology, 40: 713-724. Pierson, R.A. and Ginther, O.J., 1984. Ultrasonography of the bovine ovary. Theriogenology, 21: 495-504. Pierson, R.A. and Ginther, O.J., 1987. Follicular populations during the oestrous cycle in heifers I. Influence of day. Anim. Reprod. Sci., 14: 165-176. Pierson, R.A. and Ginther, O.J., 1988. Ultrasonic imaging of the ovaries and uterus in cattle. Theriogenology, 29: 21-37. Punvantara, B., Schmidt, M., Greve, T. and Callensen, H., 1993. Follicular dynamics prior to and during superovulation in heifers. Theriogenology, 40: 913-921. Romero, A., Albert, J., Brink, 2. and Siedel, G.E., 1991. Numbers of small follicles in ovaries affect superovulation response in cattle. ‘Iberiogenology, 35: 265. (Abstr.). Savio, J.D., Keenan, L., Boland, M.P. and Roche, J.F., 1988. Pattern, of growth of dominant follicles during the oestrous cycle of heifers. J. Reprod. Fertil., 83: 663-671. Savio, J.D., Thatcher, W.W., Badinga, L., de la Sota, R.L. and Wolfenson, D.. 1993. Regulation of dominant follicle turnover during the oestrous cycle in cows. J. Reprod. Fertil., 97: 197-203. Sawyer, G.J., Dolman, D.F. and Broadbent, P.J., 1992. Responses to oestrous synchronisation and superovulation in cattle monitored by ultrasonography. Theriogenology, 37: 290. (Abstr.1. Sawyer, G.J., Broadbent, P.J. and Dolman, D.F., 1995. Ultrasound-monitored ovarian responses in normal and superovulated cattle given exogenous progesterone at different stages of the cycle. Anim. Reprod. Sci., 38: 187-201. Sirois, J. and Fortune, J.E., 1988. Ovarian follicular dynamics during the oestrous cycle in heifers monitored by real-time ultrasonography. Biol. Reprod., 39: 308-317. Sirois, J. and Fortune, J.E., 1990. Lengthening the bovine estrous cycle with low levels of exogenous progesterone: a model for studying ovarian follicular dominance. Endocrinology, 127: 916-925. Smenan, J.M. and Gosling, J.P., 1977. The effect of cycle stage and plasma progesterone level on the induction of multiple ovulations in heifers. J. Reprcd. Fertil., 50: 366-369.
ELSEVIER
Animal Reproduction Science 43 ( 19%) 183- 190
Follicular dynamics and superovulatory response in heifers S.P. Singh “b”, P.J. Broadbent a’*, J.S.M. Hutchinson b, R.G. Watt a, D.F. Dolman a a Scottish Agricultural College, School of Agriculture, 581 King Street, Aberdeen AB9 IUD, UK b University of Aberdeen, School of Agriculture, 581 King Street, Aberdeen AB9 1UD. UK
Accepted I December 1995
Abstract The study examined whether the response of heifers to exogenous gonadotrophin superovulatory treatment could be predicted from a knowledge of previous antral follicular dynamics. During a pretreatment monitoring phase, of 24 normal oestrous cycles (20.1 + 0.33 days long) observed in 17 heifers, one, 15 and seven cycles showed one, two and three antral follicular waves respectively, as measured by ultrasonography. The subsequent ovulatory response (number of corpora lutea) to ovine FSH stimulation, after a CIDR-B/oestradiol benzoate/prostaglandin analogue cycle synchronisation regime, was not correlated with either oestrous cycle length or follicle wave number during the monitoring phase or with the number of follicles observed at the start of FSH treatment, but was related to the number of follicles observed during the monitoring
phase (r = 0.47, P < 0.05). In conclusion, the present results show that the outcome of FSH superovulatory stimulation in heifers cannot be predicted from a knowledge of prior follicular dynamics. Keywords: Heifers; Superovulatoly response; Follicular waves; Ultrasound
1. Introduction Ovarian status at the time that gonadotrophin treatment commences appears to be a major determinant of a superovulatory response. This is demonstrated by the differences in response observed when gonadotrophin treatment is initiated at different stages of the
* Corresponding author. Tel: 44 1224 480291; fax: 44 1224 276717. ’Present address: Prabhat Nagar, Meerut 250006, UP, India.
0378-4320/%/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)01480-7
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oestrous cycle with higher responses obtained when treatment begins in mid- rather than early or late cycle (Sreenan and Gosling, 1977; Lemer et al., 1986; Goulding et al., 1990; Macmillan et al., 1994). At a more detailed level, the number of antral follicles present when gonadotrophin treatment begins has been shown to be positively related to the outcome of superovulation (Monniaux et al., 1983; Romero et al., 1991). The presence of a dominant follicle at this time, however, resulted in a reduced response (Guilbault et al., 1991, Huhtinen et al., 1992) although Gray et al. (1992) did not observe an inhibitory effect due to the presence of a regressing dominant follicle. Follicular development and the dominant follicle can be controlled by ablation of follicles (Ko et al., 1991; Adams et al., 1993) or hormonal treatments (Sirois and Fortune, 1990; Adams et al., 1992a; Bo et al., 1993). Ablation of the dominant follicle, or its natural absence, resulted in improved superovulatory responses (Hahn, 1992; Bungartz and Niemann, 1994). Similar improvements were observed following hormonal suppression of the dominant follicle and emergence of a new wave of follicles (Bo et al., 1995). The use of transrectal ultrasound imaging of the ovaries (Pierson and Ginther, 1984; Pierson and Ginther, 1988) provides a means of evaluating follicular dynamics in a frequent and non-invasive manner. This technique has been used to confirm the wave-like pattern of follicular development in cattle (Pierson and Ginther, 1988; Savio et al., 1988; Sirois and Fortune, 1988). It has also been used to monitor the dominant follicle and its control by ablation or endocrine treatment (Ko et al., 1991; Armstrong, 1993; Sawyer et al., 1992; Sawyer et al., 1995; Bo et al., 1995). The purpose of the present study was to examine the proposition that the outcome of gonadotrophin treatment could be predicted from a knowledge of the follicular dynamics exhibited by the animal.
2. Materials and methods There were two phases to the study. In the first phase, of 7 weeks duration, the natural oestrous cycles of the animals were characterised in terms of their length and follicular dynamics using a combination of observation, plasma progesterone concentration and ovarian ultrasonography. In the second or superovulation phase, the oestrous cycles of the animals were controlled by synchronisation followed by superovulation and embryo recovery. The data were examined for correlations between features of the oestrous cycle or follicular dynamics, in either the monitoring or superovulatory phases, and the subsequent superovulatory response. 2.1. Animals The study was carried out using 18 purebred Simmental nulliparous heifers. The live weights and condition scores (Lowman et al., 1976) at the beginning and end of the study (mean + SEM) were 553 + 6.8 and 563 k 7.7 kg; and 3.35 + 0.1 and 3.46 + 0.1, respectively. The animals were housed in straw-bedded pens in groups of nine (4.7 mz per head) during the monitoring phase and groups of six (7.8 m2 per head) during the
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superovulation phase. Their daily diet during the study was 3 kg concentrates (1: 1:1 of sugar beet pulp, distillers’ dark grains and barley) plus hay and straw ad libitum, supplemented with 100 g mineral and vitamin mixture. 2.2. Ovarian ultrasonography Transrectal ultrasonography was performed using a 5 MHz linear probe (Aloka 210 DXII; BCF, UK) and all follicles 2 2 mm diameter plus the presence of corpora lutea were recorded separately for each ovary. Observations were made three times per week (Monday, Wednesday, Friday) during the monitoring phase. During the superovulation phase, ultrasonography was performed once when intravaginal devices were inserted; on two occasions prior to, and once at the start of, gonadotrophin treatment. The growth and regression of the follicles on each ovary were identified by a modified non-identity method (Ginther, 1993). The modification was that changes in the size of the largest (dominant) and second largest (subdominant) follicle by 1 mm followed by a regaining of the original diameter were not considered as the start of a new follicle because observations were not carried out daily. Follicular waves were identified by inspection of graphical presentations of the data. The number of follicles per wave was counted on the day of divergence i.e. the day on which the largest subordinate follicle ceased to grow or began to regress (Ginther, 1993). 2.3. Observation of oestrus Oestrous behaviour observed during the course of monitoring and handling the animals was recorded. Formal observations of oestrus (Broadbent et al., 1991) were carried out at the reference oestrus prior to superovulation over 5 days and at the superovulated oestrus for 3 days. The occurrence of oestrus and the length of the oestrous cycle during the monitoring phase was determined by observation of oestrus or inspection of the profiles for follicular dynamics and plasma progesterone concentrations. 2.4. Plasma progesterone Blood samples were taken by jugular venepuncture, plasma prepared for assay and stored at - 20°C three times per week during the monitoring phase, at oestrous synchronisation and at reference oestrus observations; at insertions or removals of intravaginal devices; daily during gonadotrophin treatment; and on the days of artificial insemination and embryo recovery. Progesterone was assayed using an enzyme-linked immunosorbant assay kit (Ridgeway Science, UK). 2.5. Superovulation and embryo recovery The heifers were synchronised for a reference oesbus by the intravaginal insertion of a CIDR-B (SmithKline Beecham, UK) and injection of 10 mg oestradiol benzoate im (Intervet, UK) on Day 1 of the programme. Prostaglandin F2 o: analogue (15 mg
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luprostiol; Intervet, UK) was administered im on Day 10 and the intravaginal device removed on Day 12 of this programme. The superovulatory protocol began on Day 7 + 1 after the reference oestrus (Sawyer et al., 1995) with the insertion of a second CIDR-B plus 10 mg oestradiol benzoate im. Gonadotrophin treatment began 9 days later and a total of 9.0 mg NIADDK-oFSH-17 equivalent ovine pituitary follicle stimulating hormone (ICP, New Zealand) was administered twice daily over 4 days in the pattern 2 (1.80 + 1.35 + 0.90 + 0.45) mg. On the third day of FSH treatment 30 mg luprostiol was administered im and the CIDR-B was removed 24 h later. Artificial insemination was performed at fixed-times, 32 and 48 h after CIDR-B removal, using frozen-thawed semen. Embryos and ova were recovered, non-surgically, 7 f 0.5 days after the first insemination. They were classified and graded as excellent, good, moderate, poor, degenerate and non-fertile. For the purposes of statistical analysis the viable embryos were re-classified on a numerical scale where 1 is excellent plus good; 2 is moderate; and 3 is poor. 2.6. Sratistical analysis Data are presented as means f SEM. The significance of differences between categories of data was tested by C-test. Linear relationships were tested by means of correlation analysis.
3. Results During the monitoring phase 24 oestrous cycles, 20.1 f 0.3 days long, were identified in 17 heifers. One heifer had a persistent corpus luteum and did not exhibit oestrus but five follicular waves were identified in this period. The average number of follicular waves, excluding this animal, was 2.3 + 0.1 per cycle. Two follicular waves were observed in 15 (65.2%; 19.9 f 0.3 days), three in 7 (30.5%; 21.3 + 2.0 days) and one in 1 (4.3%; 17 days) of the oestrous cycles. The number of follicular waves could not be determined for one of the oestrous cycles.
Table 1 Number of follicles 2 2 mm in different categories (mean f SEM) observed during each wave of the normal oestrous cycle and at initiation of gonadotrophin treatment on Day 16 f 1 after the reference oestrus Category of follicle
2to5mm 6to9mm 210mm Total
Number of follicles Mean per wave
At gonadotrophin
10.6f0.94 2.4kO.31 1.3kO.14 15.0k0.72
13.2f0.73 1.9 f 0.24 0.2 f 0.09 15.3 *0.67
stimulation
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Table 1 shows the total number of follicles and their distribution by size on average per wave in the normal oestrous cycle and at initiation of gonadotrophin treatment. The total number of follicles was similar in both cases but at the start of gonadotrophin stimulation there were more 2 to 5 mm follicles (10.6 vs. 13.2; P < 0.05) and fewer follicles 2 10 mm (1.3 vs. 0.2; P < 0.001). The number of corpora lutea palpated at embryo recovery was 16.3 + 1.l and a recovery rate of 82.3% produced a total of 13.4 f 1.7 ova plus embryos. Of these, there were 5.8 + 1.0 usable (Grades 1, 2 and 3) embryos and 4.1 rt 0.7 Grade 1 embryos. There was no significant relationship between the length of oestrous cycle (r = 0.15, P > 0.05) observed in the monitoring phase and number of corpora lutea at embryo recovery. The number of corpora lutea was similar for those animals which had two or three follicular waves during their normal oestrous cycles. The number of ovulations was not correlated (r = 0.08, P > 0.05) with the number of follicles present at the beginning of FSH treatment but was related to the number of follicles observed during the natural oestrous cycle (r = 0.47, P < 0.05). Three of the 18 heifers had a large follicle (2 10 mm) present at the time that gonadotrophin treatment commenced. There were no differences in ovulation rate between these animals and the remaining 15 heifers (15.3 + 2.7 vs. 16.5 f 1.2) but the heifers with a large follicle had fewer ova plus embryos recovered (10.0 &-4.7 vs. 14.1 f 1.9), usable embryos (4.0 IL-2.6 vs. 6.1 + 1.O) and grade 1 embryos (3.0 k 2.1 vs. 4.3 + 0.8). These differences were not significant.
4. Discussion The length of the oestrous cycle observed for pure Simmental heifers in this study was similar to that reported by Cardenas et al. (1991) for animals of the same breed and parity. Furthermore, the difference in oestrous cycle length between heifers exhibiting two or three follicular waves during the normal oestrous cycle was similar to that for other studies with heifers (Ginther et al., 1989). The maximum size of the dominant follicle (12.4 + 0.02 mm), however, was small relative to some other studies (approximately 16 mm; Pierson and Ginther, 1987; Ginther, 1993). Murphy et al. (1991) found that the dominant follicle was smaller in animals fed on a low plane compared with those on a moderate or high plane of nutrition. The diet of the heifers in the present study would equate with the moderate plane of Murphy et al. (1991) and the maximum diameter of the dominant follicle was similar in both studies (12.4 vs. 13.2 mm). The correlation of number of follicles during the normal oestrous cycles of the monitoring phase with the superovulatory response was unexpected but suggests that this information could be used as an indicator of the follicle population that can be stimulated in an animal provided that it continues to receive the same nutrition and management. The lack of any correlation between follicle number at the start of gonadotrophin treatment and subsequent ovulation rate indicates that one examination may not identify which follicles are capable of responding to gonadotrophin stimulation. The data suggest that some follicles 2 2 mm diameter may have been atretric and probably luteinised subsequently, rather than ovulated (Purwantara et al., 1993). Further-
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more, it is probable that some follicles < 2 mm were capable of responding satisfactorily to gonadotrophin treatment and that not all follicles 2 2 mm were identified. This finding contrasts with that of Romero et al. (1991) who, however, observed a larger range of follicle numbers at the initiation of FSH stimulation and worked with more animals. The feature of ovarian status at initiation of gonadotrophin treatment which tended to be related to superovulatory response was the presence or absence of a large (2 10 mm) follicle. The number of animals in each category meant that the differences observed were not statistically significant. Furthermore, the frequency of ultrasound examination and the use of the non-identity method meant that a clear determination of whether the largest follicles were in the growing, plateau or regressing phase could not be made. The results, however, are in line with the observations by others (Guilbault et al., 1991; Huhtinen et al,, 1992) that superovulatory response is lower when stimulation begins in the presence of a dominant follicle; and that response is higher following ablation of the dominant follicle (Hahn, 1992; Bungartz and Niemamr, 1994) or its control by hormonal treatment (Bo et al., 1993; Bo et al., 1995). Gonadotrophin treatment was initiated in the presence of exogenous progesterone, previously administered with oestradiol benzoate, on Day 7 (oestrus is Day 0) of a synchronised oestrous cycle. This protocol had previously been shown to improve superovulatory response (Sawyer et al., 1992; Sawyer et al., 1995) due to the suppressive effects of exogenous progesterone on follicular development (Adams et al., 1992b; Savio et al., 1993) and the induction of atresia, particularly of the dominant follicle, by oestradiol (Bo et al., 1991; Sawyer et al., 1992; Sawyer et al., 1995). The use of oestradiol 17/3 in the presence of exogenous progesterone has been shown to synchronise the emergence of a new follicular wave (Adams et al., 1994; Bo et al., 1994) and initiation of stimulatory treatments on the day before or the day of wave emergence to produce a higher superovulatory response (Adams et al., 1994; Nasser et al., 1993). These authors used oestradiol 17p in preference to oestradiol valerate because the effect of the latter, used in the absence of exogenous progesterone, depended on the stage of the oestrous cycle at which it was administered (Bo et al., 1993). It was suggested that oestradiol valerate did not suppress the dominant follicle completely and the long half-life of this substance resulted in the asynchronous emergence of follicular waves. The half life of the benzoate ester may be expected to be longer than that of oestradiol 17p but not as long as oestradiol valerate and its effects to be intermediate between those of these two compounds. This could explain why three of the 18 heifers in the present study had a follicle 2 10 mm at the start of FSH treatment. The superovulatory responses in this experiment were, however, good for maiden heifers and in a subsequent report (Broadbent et al., 1995) we have shown that the protocol of Sawyer et al. (1992) is equally efficacious to that of Adams et al. (1994). The implication may be that oestradiol benzoate administered simultaneously with exogenous progesterone not only suppresses the dominant follicle but may synchronise the emergence of a new follicular wave with the start of gonadotrophin treatment when the protocol used in the present study is followed. In conclusion, the present results show that the outcome of FSH superovulatory stimulation in heifers cannot be predicted from a knowledge of prior follicular dynamics.
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Acknowledgements SAC receives financial support from the Scottish Office Agriculture, Environment and Fisheries Department.
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