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There is evidence that the deleterious effects of heat stress (HS) on fertility are less pronounced in Bos indicus than in B. taurus breeds, due primarily to differences in their thermoregulatory capacity. In the present work, the resistance to heat stress of Nelore embryos (B. indicus) was compared to either a breed not adapted (Angus; B. taurus) or adapted to high temperatures (Bonsmara; 5/8 B. indicus 3/8 B. taurus). In Experiments (Exp.) 1 (Nelore vs. Angus) and 2 (Nelore vs. Bonsmara), oocytes obtained by ovum pickup OPU (during autumn) were matured (TCM-199), fertilized, and cultured (SOFaaci) in vitro. Ninety-six hours post-insemination (hpi), embryos with more than 16 cells were randomly allocated in two main groups: Group Control (embryos were maintained at 39C all of the time) and Group HS (embryos were maintained at 41C during 12 h and afterwards returned to 39C). Blastocyst rates were determined on the 7th day of culture. In Exp. 1, 294 oocytes from Nelore and 144 from Angus cows had a cleavage rate of 67.9 and 59.4?, respectively. Ninety-six-hpi embryos (>16 cells) were distributed in four groups: Nelore Control (n ? 97), Nelore HS (n ? 95), Angus Control (n ? 34) and Angus HS (n ? 25). The blastocyst rates were 39/97 (40.2?), 23/95 (24.2?), 19/34 (55.9?), and e 8/25 (32.0?), respectively. The difference in rate of blatocyst formation caused by heat stress on Nelore (16.0?) and Angus (23.9?) was not significantly different (P < 0.05), and suggests, from oocytes obtained by OPU, that Nelore embryos may be more tolerant to HS than Angus embryos. However, it is necessary to increase the number of blastocysts per group in order to better characterize the effects of heat stress on these embryos. In Exp. 2, 294 oocytes from Nelore and 101 from Bonsmara cows had a cleavage rate of 41.2 and 51.2?, respectively. Ninety-six-hpi embryos (>16 cells) were distributed in four groups: Nelore Control (n ? 44), Nelore HS (n ? 49), Bonsmara Control (n ? 22), and Bonsmara HS (n ? 22). The blastocyst rates were 35/44 (79.5?), 30/49 (61.2?), 10/22 (45.5?), and 6/22 (27.3?), respectively. In spite of the fact that Bonsmara embryos had a lower blastocyst rate as compared to Nelore, the decline on blastocyst rate caused by HS was very similar in Nelore (18.3?) and Bonsmara embryos (18.2?). Additional OPU are underway to test the hypothesis that thermotolerance of Nelore embryos is similar to that in embryos from a breed adapted to high temperatures (Bonsmara), and superior to embryos from a non adapted breed (Angus).

The objective of this study was to characterize the pattern of gene expression of LH receptor (LHR) transcripts from bovine antral follicles from 5 to 14 mm in diameter (theca and granulosa cells, TC and GC, respectively) in crossbreed cattle and from cultured GC. From ovaries collected in abattoir (Bos indicusB. taurus cattle), antral follicles were dissected, and samples of TC and GC were obtained for total RNA extraction (Trizol protocol). Steroid concentrations in the follicular fluid were determined by RIA. Samples of GC cultured for 6 days (obtained from B. taurus follicles), were treated with FSH (0 (control), 1, or 10 ng) and processed for total RNA extraction. Total RNA (1 g) was utilized in the RT reaction (SuperScript III; Invitrogen, Brasil Ltd., So Paulo, Brazil). The PCR conditions were 29 cycles of 95C, 60C, and 70C (1 min each) for denaturation, annealing, and extension (PTC200 thermocycler; MJ Research, Biozym, Landgraaf, The Netherlands). Gene expression of LHR was measured by semiquantitative RT-PCR with specific primers to amplify the fragment from exon 9 to exon 11 (LHRBC; full-length amplicon with 1240 bp). To investigate a putative site of alternative splicing on exon 3 specific primers were utilized to amplify a fragment containing the exons 2 to 9 (LHRA). As an internal control of the PCR, GAPDH expression was used. After sequencing, four isoforms were detected from the LHRBC fragment two with deletion of exon 10 (M2 and M4) and two with deletion of part of exon 11 (M3 and M4) as well as the fragment without deletions (M1). There was no correlation (P > 0.05; Spearman correlation) between LHRBC isoform expression and steroid hormones or follicular diameter (TC). However, estradiol and progesterone concentrations (r > 0.51 and P < 0.01) and follicular diameter (r > 0.82 and P < 0.01) were correlated with expression of the four LHRBC isoforms (GC). Expression of isoforms from fragment LHRBC was observed in GC from follicles with a diameter of 7 mm. In six follicles with a diameter of 7 mm, only one (16.7%) expressed LHR in GC, whereas most of the follicles e 8 mm expressed LHR (87.5%, 21/24). No LHR isoforms were detected in GC from follicles (n = 7) d 6 mm in diameter. From LHRA fragment amplification, an alternative transcript with a deletion of 75 bp, homologous to the rat exon 3, was detected by sequencing. In cultured GC (without FSH treatment), only a weak LHR expression (in vitro control) was observed when compared with the in vivo control (TC sample). The treatment with FSH (1 or 10 ng) was effective to induce LHR expression in cultured GC, however, with a different pattern of expression (lower M1/M2 ratio) when compared to in vivo GC samples (0.8 0.14 vs. 3.5 0.66; mean SEM, P < 0.01, unpaired t-test). It is concluded that, in GC from follicles with a diameter e 7 mm, the gene expression of the LHR was positively correlated with follicular diameter and with estradiol and progesterone concentration in the follicular fluid. Treatment with FSH was effective in inducing LHR expression in cultured GC, however, with a different pattern than in the in vivo control. Additionally, sites of alternative splicing were detected in exons 3, 10, and 11 from the bovine LHR gene. This work was supported by a Fellowship from FAPESP.

Reports indicate that either temporary calf removal or equine chorionic gonadotropin administration can increase the efficiency (pregnancy rate) of hormonal treatments with progestins during postpartum anestrus. This experiment evaluated effects of TCR and/or eCG administration in a protocol with progesterone that is frequently used for fixed-time artificial insemination (FTAI) in cows during postpartum anestrus. The protocols were tested at three farms in lactating Nelore cows (40 to 70 days post-partum, n = 361) with body condition scores from 2.5 to 3.0 (0- to 5-point scale). At a random stage of the estrous cycle (Day 0), animals received a basic PEPE (progesterone-estrogen-prostaglandin-estrogen) protocol with insertion of an intravaginal device with 1.0 g progesterone (DIB, Syntex, Buenos Aires, Argentina) and an i.m. injection of 2.5 mg of estradiol benzoate (EB, Estrogin, Farmavet, So Paulo, Brazil). Eight days later (Day 8) cows were treated i.m. with 150 mg D-cloprostenol (PGF2 Prolise, ARSA S.R.L., Buenos Aires, Argentina), and the DIB was removed. Twenty-four hours after DIB removal, cows received 1.0 mg EB i.m. and 30 to 36 h later all animals were FTAIed without estrus detection. Cows were allocated randomly to four groups: PEPE, PEPE/TCR, PEPE/eCG, and PEPE/TCR/eCG. In Group PEPE/TCR, calves were removed temporarily for 54 h (from DIB removal until FTAI). In Group PEPE/eCG, animals received PEPE treatment plus one dose i.m. of 300 UI eCG (Novormon, Syntex) following PGF2 administration (Day 8). In Group PEPE/TCR/eCG, animals were treated as in protocol PEPE/TCR plus eCG on D8. All animals were examined by ultrasonography (Aloka SSD 500, 7.5 MHz probe) 10 days before and at the beginning of hormonal treatment in order to detect anestrous cows (absence of corpus lutcum in both exams). Pregnancy diagnosis was performed by ultrasonography 30 days after FTAI. The data were analyzed by logistic regression. The following variables were considered in the model and did not affect pregnancy rates: farms, inseminators, and semen (sire). A total of 75% of the animals were in anestrus (absence of CL) and the pregnancy rates were similar (P > 0.05) among the four groups: PEPE (43/85 = 50.6%), PEPE/TCR (42/98 = 42.9%), PEPE/eCG (41/88 = 46%), and PEPE/TCR/eCG (39/90 = 43.3%). The results indicate that in Nelore cows, in postpartum anestrus and good body condition, TCR and/or eCG administration do not improve the efficiency (pregnancy rate) of the PEPE protocol.This work was supported by FAPESP. V.G.P. and A.F.S. received fellowships from CNPq, Brazil.

Postpartum anestrous cows are usually treated with progesterone/progestagen protocols in order to induce ovulation and sustain pregnancy. In the present work, the efficiency of a protocol (modified GPE), in which temporary calf removal (TCR) and/or administration of eCG, replaced the use of progesterone/progestagens, was evaluated in anestrous animals. Anestrous Nelore cows (40 to 60 days postpartum, n = 22) were randomly allocated to two groups: GPE/eCG and TCR/GPE/eCG. At a random stage of the estrous cycle (D0), animals from Group GPE/eCG were treated with GnRH (50 g, licereline, i.m., Gestran Plus; Tecnopec, Sao Paulo, Brazil) and seven days later (D7) they received PGF2 (150 mcg, d-cloprostenol, i.m., Prolise) and eCG (300 UI, i.m., Novormon). On D8, estradiol benzoate (EB, 1 mg, Estrogin; Tecnopec) was administered, and 30 to 36 h afterwards all cows were inseminated at fixed-time (FTAI), without estrus detection. The animals from group TCR/GPE/eCG received the same treatment described above, but with temporary calf removal (during 48 h) before beginning hormonal treatments. The ovaries were examined by ultrasonography (Aloka SSD 500, 7.5 MHz probe) before (D-12, D-2), during (D0, D7, D8), and after (D9, D10, D11, D17, D60) hormonal treatments. The absence of CL, during the ultrasonography performed before starting the treatments, was the criterion used to classify the cows as in postpartum anestrus. Administration of GnRH (D0) induced ovulation (determined by the presence of CL on D7) in 6 of 11 cows (54.5%) from Group GPE/eCG and TCR/GPE/eCG. In relation to synchronization of ovulation after EB administration, in Group GPE/eCG, two cows (18.1%) ovulated until 12 h after FTAI, and four (36.3%) 12 h afterwards (24 h after FTAI). In Group TCR/GPE/eCG, one cow (9%) ovulated approximately 6 h before FTAI, two (18.1%) 12 h after FTAI, and two (18.1%) 12 h afterwards. Preovulatory follicles had a diameter of 11.0 1.3 and 11.5 2.6 mm in Groups GPE/eCG and TCR/GPE/eCG, respectively. Pregnancy rates, determined by ultrasonograpy 40 to 46 days after FTAI, were 27.2% (3/11) and 45.4% (5/11), respectively. The results indicate that association of TCR with protocol GPE/eCG did not improve synchronization of ovulation or pregnancy rates (P > 0.005). However, increase in pregnancy rates was observed in another experiment with a large number of animals (Pinheiro et al. 2005 Reprod. Fert. Dev. 17, 161, abstr), in which cows from Group TCR/GPE/eCG had higher pregnancy rates (51.5%; 34/66) when compared to animals from Group GPE/eCG (28.3%, 21/74; P < 0.05).This work was supported by FAPESP and by fellowships for A. F. Souza and V. G. Pinheiro from CNPq, Brazil.