A. H. Souza

Follicle deviation and ovulatory capacity in Bos indicus heifers

The objectives of Experiment 1 were to determine the interval from ovulation to deviation, and diameter of the dominant follicle (DF) and largest subordinate follicle (SF) at deviation in Nelore (Bos indicus) heifers by two methods (observed and calculated). Heifers (n = 12) were examined ultrasonographically every 12 h from ovulation (Day 0) to Day 5. The time of deviation and diameter of the DF and largest SF at deviation did not differ (P>0.05) between observed and calculated methods. Overall, deviation occurred 2.5+/-0.2 d (mean +/- S.E.M.) after ovulation, and diameters for DF and largest SF at deviation were 6.2+/-0.2 and 5.9 +/- 0.2 mm, respectively. Experiment 2 was designed to determine the size at which the DF acquires ovulatory capacity in B. indicus heifers. Twenty-nine heifers were monitored every 24 h by ultrasonography, from ovulation until the DF reached diameters of 7.0-8.4 mm (n=9), 8.5-10.0 mm (n=10), or >10.0 mm (n=10). At that time, heifers were treated with 25 mg of pLH and monitored by ultrasonography every 12 h for 48 h. Ovulation occurred in 3 of 9, 8 of 10, and 9 of 10 heifers, respectively (P<0.05). In summary, there was no significant difference between observed and calculated methods of determining the beginning of follicle deviation. Deviation occurred 2.5 d after ovulation when the DF reached 6.2 mm, and ovulatory capacity was acquired by DF as small as 7.0 mm.

Timing of insemination and fertility in dairy and beef cattle receiving timed artificial insemination using sex-sorted sperm

The objective was to evaluate the effects of timing of insemination and type of semen in cattle subjected to timed artificial insemination (TAI). In Experiment 1, 420 cyclic Jersey heifers were bred at either 54 or 60 h after P4-device removal, using either sex-sorted (2.1 × 10(6) sperm/straw) or non-sorted sperm (20 × 10(6) sperm/straw) from three sires (2 × 2 factorial design). There was an interaction (P = 0.06) between time of AI and type of semen on pregnancy per AI (P/AI, at 30 to 42 d after TAI); it was greater when sex-sorted sperm (P < 0.01) was used at 60 h (31.4%; 32/102) than at 54 h (16.2%; 17/105). In contrast, altering the timing of AI did not affect conception results with non-sorted sperm (54 h = 50.5%; 51/101 versus 60 h = 51.8%; 58/112; P = 0.95). There was an effect of sire (P < 0.01) on P/AI, but no interaction between sire and time of AI (P = 0.88). In Experiment 2, 389 suckled Bos indicus beef cows were enrolled in the same treatment groups used in Experiment 1. Sex-sorted sperm resulted in lower P/AI (41.8%; 82/196; P = 0.05) than non-sorted sperm (51.8%; 100/193). In addition, there was a tendency for greater P/AI (P = 0.11) when TAI was performed 60 h (50.8%; 99/195) versus 54 h (42.8%; 83/194) after removing the progestin implant. In Experiment 3, 339 suckled B. indicus cows were randomly assigned to receive TAI with sex-sorted sperm at 36, 48, or 60 h after P4 device removal. Ultrasonographic examinations were performed twice daily in all cows to confirm ovulation. On average, ovulation occurred 71.8 ± 7.8 h after P4 removal, and greater P/AI was achieved when insemination was performed closer to ovulation. The P/AI was greatest (37.9%) for TAI performed between 0 and 12 h before ovulation, whereas P/AI was significantly less for TAI performed between 12.1 and 24 h (19.4%) or >24 h (5.8%) before ovulation. In conclusion, sex-sorted sperm resulted in a lesser P/AI than non-sorted sperm following TAI. However, improvements in P/AI with delayed time of AI were possible (Experiments 1 and 3), and seemed achievable when breeding at 60 h following progestin implant removal, compared to the standard 54 h normally used in TAI protocols.

Ovarian follicular growth suppression by long-term treatment with a GnRH agonist and impact on small follicle number, oocyte yield, and in vitro embryo production in Zebu beef cows

The aim of the present study was to evaluate small follicle number, oocyte yield, and inxa0vitro embryo production (IVEP) in Zebu beef cows treated long term with a GnRH agonist to suppress ovarian follicular growth. Nelore (Bos indicus) cows (nxa0=xa020) showing regular estrous cycles were randomly assigned to one of two groups: control (nxa0=xa010, placebo ear implant without a GnRH agonist); GnRH agonist (nxa0=xa010, GnRH agonist ear implant containing 9.4-mg deslorelin). All cows underwent an ovum pick-up (OPU) session 14xa0days (Day 14) before the start of treatments (Day 0) followed by seven OPU-IVEP procedures at 30-day intervals (Days 0, 30, 60, 90, 120, 150, and 180). Semen from a single batch of a previously tested bull was used for all the IVEP. Cows treated with agonist reported a decrease over time in the proportion of animals with a (CL; Pxa0≤xa00.05) and large follicles (>10xa0mm, Pxa0≤xa00.05). These cows had a lesser number of mediumxa0+xa0large follicles (>5xa0mm; 1.74xa0±xa00.5 vs. 4.13xa0±xa00.5; Pxa0≤xa00.05), greater number of small follicles (2-5xa0mm; 44.3xa0±xa02.8 vs. 30.8xa0±xa01.8; Pxa0≤xa00.05), greater yield of cumulus-oocyte complexes (COCs; 21.0xa0±xa02.3 vs. 15.6xa0±xa01.9; Pxa0≤xa00.05), greater proportion of COCs cultured (79.2 vs. 73.9%; Pxa0≤xa00.05), COCs cleaved (10.6xa0±xa01.5 vs. 6.8xa0±xa01.1, Pxa0≤xa00.05), and cleaved rate (52.8 vs. 44.3%; Pxa0≤xa00.05) compared with control cows. The number (3.4xa0±xa00.7 vs. 3.0xa0±xa00.6; Pxa0>xa00.05) and proportion (16.5 vs. 19.1%; Pxa0>xa00.05) of blastocysts produced were similar between agonist and control cows, respectively. The study has shown that Zebu beef cows treated long term with a GnRH agonist had follicular growth restricted to small follicles. This did not compromise the ability of oocytes to undergo IVF and embryonic development.

Beef donor cows with high number of retrieved COC produce more in vitro embryos compared with cows with low number of COC after repeated ovum pick-up sessions

The aim of the present study was to evaluate whether repeated ovum pick-up (OPU) procedures may affect the efficiency of OPU programs for inxa0vitro embryo production (IVEP) in Bos indicus (Nelore) donors. In addition, the repeatability (r) efficiency of IVEP was also assessed. Data available were from 432 OPU-IVEP sessions that were performed at random stages of the estrous cycle in 36 cycling, nonlactating Nelore donors. Semen from three Nelore bulls was used for the IVF. Donors were submitted to 12 consecutive OPU procedures, with an interval of approximately 30xa0days between sessions. Data were analyzed as repeated measures using the GLIMMIX procedure of SAS 9.3. Cows yielding ≥15 COCs were defined as high COCs and cows with less than 15 COCs were defined as low COCs donors. The number of COCs retrieved decreased over time in donors classified with high COCs and remained fairly steady in cows with low COCs at the beginning of the program (Psession*COC retrieved groupsxa0=xa00.02). Moreover, the number of COCs retrieved (Pxa0<xa00.0001), and number of blastocysts produced per OPU (Pxa0=xa00.001) was greater for the high COCs donors compared with the low COCs category, and these results were consistent across OPU sessions. Interestingly, there was no effect of COCs category on the rate of blastocyst development (Pxa0=xa00.83). In addition, number of blastocysts produced were not affected by repeated OPU (Pxa0=xa00.37) and interactions between time and COC category (Pxa0=xa00.72). Similarly, blastocyst rate was not affected by repeated OPU (Pxa0=xa00.21) and interactions between time and COC category (Pxa0=xa00.58). Despite of COC category of the donor cows, repeatability was high for the number of COCs retrieved (rxa0=xa00.81), number of blastocysts produced per OPU (rxa0=xa00.79), and blastocyst rate (0.69). In conclusion, overall numbers of COCs decreased over time in donors classified as having high COCs. However, cumulative amounts of produced blastocysts were greater in donors with high COCs. More importantly, high repeatability was observed in terms of IVEP efficiency. Therefore, IVEP programs can be significantly improved with more aggressive selection toward donors with greater numbers of COCs.

Impact of phase of the estrous cycle and season on LH surge profile and fertility in dairy cows treated with different GnRH analogs (gonadorelin vs. buserelin)

Our aim was to assess the GnRH-induced LH surge profile in dairy cows receiving two GnRH products (gonadorelin vs buserelin) given at proestrus or diestrus phase and to investigate whether season could alter LH surge profile in dairy cows. In Experiment 1, dairy cows at 108.2xa0±xa02.3 DIM, producing 41.5xa0±xa00.3xa0kg/day were randomized to receive, during proestrus and diestrus: Ovarelin® i.m. (OVA; nxa0=xa056; 100xa0mg of gonadorelin diacetate tetrahydrate; Ceva Animal Health, France) or Receptal® i.m. (REC; nxa0=xa052; 10 mcg of buserelin diacetate; MSD, Germany). In Experiment 1, blood samples were collected at hour 0 (just before GnRH treatment) at 30min, 1xa0h and then hourly until 5xa0h post-GnRH. In Experiment 2, cows were synchronized with a modified G-6-G protocol and randomized to receive either OVA or REC throughout the synchronization program. In Experiment 1, peak LH concentrations (ng/mL) were not affected by type of GnRH (OVAxa0=xa06.2xa0±xa00.4 vs RECxa0=xa06.7xa0±xa00.4; Pxa0=xa00.37) or season (Coolxa0=xa06.8xa0±xa00.4 vs Warmxa0=xa06.1xa0±xa00.4; Pxa0=xa00.22), and there were no interactions between GnRH type and phase of the estrous cycle or season. Interestingly, the area under the curve (AUC) of LH release (ng/ml*time) was significantly lower during warmer months (Coolxa0=xa020.3xa0±xa01.2 vs Warmxa0=xa016.9xa0±xa01.1; Pxa0=xa00.04). As expected, LH peak was affected by phase of the cycle (proestrusxa0=xa08.2xa0±xa00.4 vs diestrusxa0=xa04.7xa0±xa00.4; Pxa0<xa00.01). Ovarelin caused LH concentrations to increase faster, reaching highest concentration sooner (h) than REC (1.5xa0±xa00.1 vs 2.3xa0±xa00.1; Pxa0<xa00.01). As a result, cows receiving OVA had greater circulating LH concentrations (ng/mL) at 1xa0h after GnRH treatment than cows receiving REC (Pxa0<xa00.01). In contrast, cows treated with REC had longer (Pxa0=xa00.01) intervals from peak until return to nadir. In Experiment 2, pregnancy per AI (P/AI) was similar for cows receiving either GnRH product during the synchronization protocol, with no detectable interactions between GnRH type and season. In conclusion, phase of the estrous cycle had a great impact on the GnRH-induced LH surge profile and cows during warm season had reduced AUC. Additionally, type of GnRH did not influence LH surge profile and P/AI in synchronized cows. Regardless of GnRH type, strategies to avoid heat stress and excessively high levels of circulating P4 near the time of GnRH treatment might help improve LH release profile in dairy cows, and ultimately increase P/AI.

The effect of circulating progesterone on magnitude of the GnRH-induced LH surge: Are there any differences between Bos indicus and Bos taurus heifers?

This study evaluated the effects of differing circulating progesterone (P4) levels on the luteinizing hormone (LH) surge profile following treatment with gonadotropin releasing hormone (GnRH) in B.xa0indicus (Nelore, nxa0=xa013) and B.xa0taurus (Holstein, nxa0=xa016) heifers. All heifers were synchronized with a hormonal protocol to induce either a Low or High circulating P4 environment at the time of GnRH treatment. Heifers were randomly assigned to a 2xa0×xa02 factorial design composed by two genetic groups (B.xa0indicus and B.xa0taurus) and two levels of circulating P4 concentrations (Low or High). Blood samples were collected every 30xa0min fromxa0-30 to 210xa0min and at 270xa0min after GnRH treatment. As expected, mean P4 concentration was greater for cows in the High than in the Low P4 group (Pxa0=xa00.0008) and in Bos indicus than in Bos taurus heifers (Pxa0=xa00.06). Despite genetic group, the area under the curve of LH release was greater in the Low-P4 than in High-P4 concentration group (Pxa0<xa00.0001). Interestingly, it appears that High P4 concentrations had a more pronounced effect on LH peak in B.xa0indicus than in B.xa0taurus heifers, as indicated by the interaction (Pxa0=xa00.01) between genetic group and P4 levels. In conclusion, circulating P4 concentration have a great impact on the GnRH-induced LH surge of both B.xa0indicus and B.xa0taurus heifers, but it does not explain the much lower LH peak in B.xa0indicus with low circulating P4. Thus, more studies are essential to uncover some of the underlying physiological factors other than circulating P4 that are limiting LH release following a GnRH treatment in B.xa0indicus cattle.

Factors that interfere with oocyte quality for in vitro production of cattle embryos: effects of different developmental & reproductive stages

The success of IVP is ultimately dependent on the number and quality of the cumulus-oocyte complexes (COC) harvested during the OPU procedure. Several factors appear to be critical to oocyte quality including follicle size, environment factors such as heatstress, genetic background, age and lactation status of donor animals, all having a remarkable influence on the results of IVP. The aim of this review is to highlight some critical areas that can help veterinary practitioners to enhance OPU efficiency and successfully implement IVP into their routine practice. Focus will be given to recent findings in the literature and underlying physiological aspects that may be interfering with the quality of oocytes addressed to IVP in cattle at younger ages (calves and prepubertal heifers), pregnant vs nonpregnant status, and possible interactions with lactation and days postpartum during OPU.

387 EFFECT OF NUMBER OF INSEMINATIONS ON EMBRYO PRODUCTION IN SUPERSTIMULATED NELORE (BOS INDICUS) COWS SUBJECTED TO FIXED-TIME ARTIFICIAL INSEMINATION

In embryo transfer programs, 2 inseminations per superstimulated donor are usually recommended. Based on studies of follicular dynamics in Bos indicus donors (Baruselli et al. 2006 Theriogenology 65, 77–88), we tested the hypothesis that a single fixed-time AI (FTAI) at 16 h after pituitary luteinizing hormone (pLH) treatment is as efficacious as 2 FTAI at 12 and 24 h after the pLH treatment. Suckling Nelore cows (n = 10), 60 ± 12 days postpartum, were equally distributed in 2 experimental groups: G-1TAI vs. G-2TAI, in a crossover design. All animals received an intravaginal progesterone device (DIB; Syntex S.A., Buenos Aires, Argentina) and 2 mg of estradiol benzoate (IM, Ric Be; Syntex) at random stages of the estrous cycle (Day 0). Superstimulatory treatments started on Day 4 with pFSH (133 mg; Folltropin-V; Bioniche Animal Health Canada, Inc., Belleville, Ontario, Canada) in 8 decreasing doses, given IM 12 h apart. On Day 6, all cows received 2 (AM and PM) treatments of 0.15 mg of d-cloprostenol (Prolise; Syntex). The DIB devices were removed 36 h after the first PGF2± injection. The pLH treatment (25 mg IM; Lutropin-V; Bioniche) was given 48 h after the first PGF2± treatment (12 h after the last pFSH treatment). In the G-2TAI group, all cows received 2 inseminations (12 and 24 h after the pLH). Cows in the G-1TAI group received a single insemination (16 h after the pLH). All inseminations were done with the same batch of semen from a single bull. Ultrasound examinations (PIE Medical Scanner 200; Pie Medical Equipment, Maastricht, The Netherlands) were performed every 12 h from 0 to 72 h after pLH treatment to evaluate the number of ovulatory follicles ( 8 mm at the time of pLH administration (16.2 ± 1.4 vs. 14.8 ± 1.2; P > 0.05), ovulation rate (63.8 ± 3.8% vs. 64.2 ± 4.3%; P > 0.05), interval from the first to the last ovulation (32.4 ± 1.8 vs. 33.6 ± 1.6 h; P > 0.05), total ova/embryos (8.2 ± 0.9 vs. 7.2 ± 0.8; P > 0.05), Grade 1 embryos (2.0 ± 0.5 vs. 2.3 ± 0.4; P > 0.05), transferable embryos (Grades 1, 2, and 3) (4.3 ± 0.7 vs. 4.2 ± 0.6; P > 0.05), freezable embryos (Grades 1 and 2) (2.9 ± 0.6 vs. 2.8 ± 0.4; P > 0.05), unfertilized ova (0.6 ± 0.2 vs. 0.8 ± 0.2; P > 0.05), and degenerated embryos (3.3 ± 0.9 vs. 2.2 ± 0.3; P > 0.05). These results suggest no significant differences in embryo production between superstimulated Nelore donors inseminated once or twice by fixed time following administration of pLH. This work was supported by Tecnopec, Brazil.