Abstract. The aim of the present study was to investigate the effect of short-term progesterone (P4) supplementation during the early metoestrous period on circulating P4 concentrations and conceptus development in cattle. The oestrous cycles of cross-bred beef heifers were synchronised using a 7-day P4-releasing intravaginal device (PRIDÒ Delta; 1.55 g P4) treatment with administration of a prostaglandin F 2a analogue (Enzaprost; CEVA Sante Animale) the day before PRIDÒ Delta removal. Only those heifers recorded in standing oestrus (Day 0) were used. In Experiment 1, heifers were randomly assigned to one of five groups: (1) control: no treatment; (2) placebo: insertion of a blank device (no P4) from Day 3 to Day 7; (3) insertion of a PRIDÒ Delta from Day 3 to Day 7; (4) insertion of a PRIDÒ Delta from Day 3 to Day 5; or (5) insertion of a PRIDÒ Delta from Day 5 to Day 7. In vitro-produced blastocysts were transferred to each heifer in Groups 2-5 on Day 7 (n ¼ 10 blastocysts per heifer) and conceptuses were recovered when heifers were killed on Day 14. Based on the outcome of Experiment 1, in Experiment 2 heifers were artificially inseminated at oestrus and randomly assigned to one of three treatment groups: (1) placebo; (2) PRID from Day 3 to Day 5; or (3) PRID from Day 3 to Day 7. All heifers were killed on Day 16 and recovered conceptuses were incubated in synthetic oviducal fluid medium for 24 h; spent media and uterine flushes were analysed for interferon-t (IFNT). In both experiments, daily blood samples were taken to determined serum P4 concentrations. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute, Cary, NC, USA). Insertion of a PRID resulted in an increase (P , 0.05) in serum P4 that declined following removal. In Experiment 1, P4 supplementation from Day 3 to Day 5 (17.0 AE 1.4 mm) or Day 3 to Day 7 (11.3 AE 2.3 mm) increased conceptus length compared with placebo (2.1 AE 1.8 mm). Serum P4 was significantly lower from Day 9 to Day 14 (P , 0.05) and the weight of the Day 14 corpus luteum (CL) was lower in the PRID Day 3-7 group than the placebo or control groups. In Experiment 2, supplementation from Day 3 to Day 5 (94.0 AE 18.8 mm) or Day 3 to Day 7 (143.6 AE 20.6 mm) increased conceptus length on Day 16 compared with placebo (50.3 AE 17.4 mm). Serum P4 was significantly lower in the two supplemented groups following PRID removal compared with placebo (P , 0.05) and was associated with a lower CL weight in the Day 3-7 group. Conceptus length was strongly correlated with the IFNT concentration in the uterine flush (r ¼ 0.58; P ¼ 0.011) and spent culture medium (r ¼ 0.68; P , 0.002). The findings of the present study highlight the somewhat paradoxical effects of P4 supplementation when given in the early metoestrous period in terms of its positive effect on conceptus development and its potentially negative effects on CL lifespan.

The selection of competent oocytes for in vitro maturation is still a major problem during bovine in vitro embryo production. Markers for in vitro cytoplasmic maturation, based on the organization of cortical granule and mitochondria, are lacking. We examined the pre-selection of immature bovine oocytes by brilliant cresyl blue stain (BCB test) based on glucose-6-phosphate dehydrogenase (G6PDH) activity during oocyte development. Oocytes were recovered from ovarian follicles exposed to 26 μM BCB stain and classified according to the aspect of their cytoplasm: BCB(+) (oocytes with blue cytoplasm) and BCB(-) (unstained cytoplasm) and then in vitro matured into a conventional in vitro maturation (IVM) medium and standard procedure. In Experiment 1, nuclear maturation was determined by polar body identification, while cytoplasmic maturation was based on cortical granule (CG) migration (peripheral) and mitochondria distribution (central). Evidence of polar body, cortical granule migration and of centrally located mitochondria was significantly (p < 0.05) higher in BCB(+) oocytes than in BCB(-) (polar body present: 65% vs 20%; peripheral CG: 72% vs. 14%; and central mitochondria: 85% vs. 19%, respectively). In Experiment 2, the efficiency pre-selection of bovine oocytes by BCB on embryo development in vitro was assessed. Cleavage rates were similar (75%) among control, BCB(+) and BCB(-) groups, while blastocyst rates on D7 were (p < 0.05) higher (35%) in BCB(+) vs BCB(-) (10%) or control (28%). We showed that the BCB test is efficient to identify competent immature bovine oocytes to undergo synchronous nuclear and cytoplasmic in vitro maturation thus yielding higher in vitro embryo development to blastocyst stage.

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