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M. Mihm, A. Baguisi, M. P. Boland, and J. F. Roche

After luteolysis, subluteal concentrations of progesterone or treatment with a synthetic progestagen result in an extended period of dominance (persistence) of the dominant follicle in cattle. Two experiments studied (1) the relationship between the duration of dominance of the ovulatory follicle and pregnancy rate and (2) the ability of a persistent dominant follicle to ovulate and form a normal functioning corpus luteum. In Expt 1, beef heifers were either untreated (n = 30) or given a synthetic progestagen implant (3 mg norgestomet) for 12 days starting on day 16 of their cycle (n = 32). The mean duration of dominance of the ovulatory follicle differed (P < 0.05) between treated and control heifers (10.8 ± 1.2 and 3.3 ± 0.8 days, respectively) and 20 of 26 control and 7 of 30 treated heifers were diagnosed pregnant 28 days after artificial insemination (P < 0.01). In Expt 2, on the first day of dominance of the second dominant follicle, heifers received either a PGF analogue alone (controls; n = 18), or prostaglandin and a norgestomet implant for 6 (T6; n = 19) or 10 days (T10; n = 20). Increases in the duration of dominance of the second dominant follicle (controls, 4.1 ± 0.2 days; T6, 8.6 ± 0.2 days; T10, 12.1 ± 0.2 days; P < 0.05) resulted in a decrease in pregnancy rate (controls, 14 of 16; T6, 11 of 19; T10, 0 of 13; P ≤ 0.05). Progesterone concentrations on days 7 and 12 and the area of luteal tissue on day 12 after artificial insemination were not different (P > 0.05) between treatments. It is concluded that (1) treatment with a synthetic progestagen towards the end of the luteal phase causes a variable extension of the period of dominance of the ovulatory follicle with a significant reduction in pregnancy rate, (2) the persistent dominant follicle can ovulate and form a functional corpus luteum, and (3) the pregnancy rate is sequentially decreased as the duration of dominance increases from 4 to 8 days, and is further significantly reduced if the duration of dominance exceeds 10 days.

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M Mihm, P J Baker, L M Fleming, A M Monteiro, and P J O'Shaughnessy

This study was designed to identify genes that regulate the transition from FSH- to LH-dependent development in the bovine dominant follicle (DF). Serial analysis of gene expression (SAGE) was used to compare the transcriptome of granulosa cells isolated from the most oestrogenic growing cohort follicle (COH), the newly selected DF and its largest subordinate follicle (SF) which is destined for atresia. Follicle diameter, follicular fluid oestradiol (E) and E:progesterone ratio confirmed follicle identity. Results show that there are 93 transcript species differentially expressed in DF granulosa cells, but only 8 of these encode proteins known to be involved in DF development. Most characterised transcripts upregulated in the DF are from tissue development genes that regulate cell differentiation, proliferation, apoptosis, signalling and tissue remodelling. Semiquantitative real-time PCR analysis confirmed seven genes with upregulated (P≤0.05) mRNA expression in DF compared with both COH and SF granulosa cells. Thus, the new genes identified by SAGE and real-time PCR, which show enhanced mRNA expression in the DF, may regulate proliferation (cyclin D2; CCND2), prevention of apoptosis or DNA damage (growth arrest and DNA damage-inducible, β; GADD45B), RNA synthesis (splicing factor, arginine/serine rich 9; SFRS9) and unknown processes associated with enhanced steroidogenesis (ovary-specific acidic protein; DQ004742) in granulosa cells of DF at the onset of LH-dependent development. Further studies are required to show whether the expression of identified genes is dysregulated when abnormalities occur during DF selection or subsequent development.

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M. Mihm, N. Curran, P. Hyttel, P. G. Knight, M. P. Boland, and J. F. Roche

The aim of the present study was to characterize in detail the cytoplasmic and nuclear morphology of cattle oocytes recovered from follicles that are dominant for more than 9 days (with low fertility after ovulation), and to relate morphological changes to intrafollicular markers of follicle health. Beef heifers received prostaglandin F and a synthetic progestagen (3 mg Norgestomet) for 2 or 10 days on the first day of dominance of the second dominant follicle (DF2) of the oestrous cycle, to give a 4 day (n = 19; N2) or 12 day (n = 21; N10) duration of dominance of the dominant follicle at ovariectomy 18 h after implant removal and before the predicted gonadotrophin surge. Ultrasound scanning determined emergence of a new wave of follicles in five N10 heifers the day before (n = 1) or day of ovariectomy (n = 4) (N10-NonDom). Dominant follicles from the remaining N10 heifers (N10-Dom) were larger (P < 0.05) on the day of ovariectomy (17.8 ± 0.6 mm) than those from N2 heifers (13.6 ± 0.4 mm). The oestradiol:progesterone ratio of follicular fluid from N10-Dom heifers was reduced (21.7 ± 3.1 versus 34.1 ± 4.4; P < 0.05), while inhibin A (as measured by immunoradiometric assay) was increased (12.7 ± 1.0 versus 9.0 ± 0.7 μg ml−1; P < 0.05) compared with N2 heifers. Eleven of twelve N2 oocytes demonstrated nuclear activation without germinal vesicle breakdown, while seven of eight N10-Dom oocytes had undergone germinal vesicle breakdown and had progressed to metaphase I (6/8) or II (1/8). In contrast to N2 oocytes, N10-Dom oocytes showed a larger perivitelline space containing more cumulus cell process endings, vacuoles, irregular vesicles, and more mitochrondia and lipid droplets throughout the ooplasm, yet the degree of cumulus cell expansion and atresia was similar. Thus, final oocyte maturation leading to metaphase I is initiated in most dominant follicles with a dominance period of > 9 days before the gonadotrophin surge and is associated with a reduction in dominant follicle health. However, ovulatory ability is maintained and will lead to the ovulation of aged oocytes, markedly reducing subsequent pregnancy rates.

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EJ Austin, M Mihm, AC Evans, JL Ireland, JJ Ireland, and JF Roche

Antral follicle development in cattle is initially FSH dependent and then LH dependent. The aim of the present study was to determine the effects of oestradiol- and progesterone-induced suppression of FSH and LH on growth and differentiation of first wave follicles. Cyclic heifers (n = 45, n = 6-10 per group) received the following i.m. injections or treatments beginning 30 h after oestrus: (i) saline (controls); (ii) 0.75 mg oestradiol benzoate (ODB); (iii) insertion of a progesterone-releasing intravaginal device (PRID) for 42 h (progesterone); (iv) 0.75 mg oestradiol benzoate plus PRID (ODB plus progesterone); (v) 0.75 mg ODB plus injection of 1 mg Ovagen(TM) at 33, 39 and 45 h after onset of oestrus (ODB plus FSH). In Expt 1, follicle development was monitored by ovarian ultrasonography once a day. In Expt 2, heifers were ovariectomized. Emergence of the first follicle wave and dominant follicle selection were delayed in ODB plus progesterone-treated heifers compared with controls. Interval to nadir FSH concentration was shorter in ODB-, progesterone- and ODB plus progesterone-treated heifers compared with controls. Frequency of LH pulses was unaffected in ODB- or ODB plus FSH-treated heifers, decreased in progesterone-treated heifers and further decreased in ODB plus progesterone-treated heifers. Intrafollicular oestradiol concentrations were lower in the largest follicle from ODB plus progesterone-treated heifers compared with control (66 h) heifers, but follicle diameter and concentrations of insulin-like growth factor binding proteins (IGFBPs) and inhibin forms were unaffected. Treatment with ODB decreased follicular oestradiol concentration in smaller follicles in the cohort. It is concluded that growing cohort follicles are uniformly responsive to increased FSH concentration but differentially responsive to suppressed FSH and LH release, which is consistent with an LH-mediated survival advantage of the largest follicle in the cohort before cessation of the growth of remaining follicles in the cohort occurs.