The effectiveness of low-dose of eCG in timed AI Nelore (Bos indicus) heifers.

This study evaluated the efficacy of the administration of different doses of equine chorionic gonadotropin (eCG; 0 IU, 200 IU, or 300 IU) at the time of the progesterone device removal in 2-year-old Nelore (Bos indicus) heifers synchronized for fixed-timed artificial insemination (FTAI). On day 0 (D0), a total of 398 heifers received 2 mg of oestradiol benzoate i.m., 0.53 mg of cloprostenol i.m., and an eight-day previously used (second use) intravaginal device containing 1 g of progesterone (P4). Eight days later (D8), simultaneous with the P4 device removal, 0.5 mg of oestradiol cypionate i.m. and 0.53 mg of cloprostenol i.m. were administered. At the same time, heifers were randomly assigned to receive one of the following treatments: G-0 IU (n = 141; no eCG treatment), G-200 IU (n = 132; treated with 200 IU of eCG), and G-300 IU (n = 125; treated with 300 IU of eCG). FTAI was performed 48 h after the P4 device removal (D10). Ultrasonographic evaluations were performed at D0, D10, and D17. Heifers were scanned to measure the size of the largest follicle (LF), the presence, number, and size of the corpus luteum (CL), and the ovulation rate. Subsequently, at D40, the heifers underwent scanning to determine the pregnancy rate and identify any twin pregnancies. Additionally, at D70, scans were performed to assess pregnancy loss (PG). Data were analysed by orthogonal contrasts [C1 (eCG effect): control x (200 IU + 300 IU) and C2 (eCG dose effect): 200 IU × 300 IU]. On D0, CL presence was similar between the groups [G-0 IU = 65.2% (92/141), G-200 IU = 55.3% (73/132), and G-300 IU = 63.2% (79/125); p = .16]. No interactions between the presence of CL on D0 and eCG treatment were found for any of the variables (p > .05). The diameter of the LF at FTAI (D10) was not influenced by eCG treatment (p = .22) or eCG dose (p = .18). However, treatment with eCG increased the diameter of the CL at D17 (G-0 IU = 15.7 ± 0.3 mmb , G-200 IU = 16.6 ± 0.2 mma , and G-300 IU = 16.6 ± 0.3 mma ; p = .001), regardless of the dose used (p = .94). The ovulation rate was higher in heifers treated with eCG [G-0 IU = 79.4%b (112/141), G-200 IU = 90.2%a (119/132), and G-300 IU = 93.6%a (117/125); p = .002], but there was no effect of eCG dose (p = .36). Pregnancy per AI (P/AI) on D40 [G-0 IU = 32.6%b (46/141), G-200 IU = 42.4%a (56/132), and G-300 IU = 42.4%a (53/125); P = 0.05] and D70 [G-0 IU = 29.1%b (41/141), G-200 IU = 40.9%a (54/132), and G-300 IU = 40.8%a (51/125); p = .02] were higher on heifers that received eCG; however, no dose effect was observed for P/AI on D40 (p = .89) nor D70 (p = .98). Pregnancy loss between D40 and D70 tended to reduce (p = .07) in eCG-treated heifers without dose effect (p = .91). Heifers with CL at D0 presented a greater follicle diameter (LF) on D10 (With CL = 11.2 ± 0.2 mm and Without CL = 10.2 ± 0.2 mm; p = .05), CL diameter on D17 (With CL = 15.8 ± 0.03 mm and Without CL = 11.8 ± 0.6 mm; p = .01), and ovulation rate [With CL = 95.5% (233/244) and Without CL = 74.7% (115/154); p = .01]. However, no difference in pregnancy rate at D40 (p = .52) and D70 (p = .84) was found. In conclusion, eCG treatment increases ovulation and pregnancy rates of heifers submitted to a FTAI protocol. Furthermore, eCG treatment increases the diameter of the CL after FTAI and reduces pregnancy losses. No dose effect was observed, suggesting Nelore (Bos indicus) heifers respond to 200 IU of eCG treatment for FTAI.

Exposure to progesterone before an ovulation synchronization protocol increases the follicular diameter and fertility of multiparous suckled Bos taurus cows.

The objective of this study was to evaluate the effect of administering injectable progesterone (P4i) before a timed artificial insemination (TAI) protocol on the follicular growth, ovulation, and pregnancy rate of Bos taurus suckled cows. The effect of P4i administration before the TAI on the pregnancy rate (P/AI) was evaluated in 576 suckled Bos taurus cows at 30-90 days postpartum. In addition, the effect of P4i administration before TAI on follicular dynamics was evaluated in subgroup of 401 suckled Bos taurus cows. On Day -10 (D-10), cows were divided into two experimental groups (Control and P4i). In this moment, P4i cows received i.m. 150 mg of injectable long-action progesterone. After that, both experimental groups received a synchronization protocol (Day 0; D0) that consisted of administration i.m. of 2 mg of estradiol benzoate and a progesterone intravaginal insert on D0. On Day 8 (D8), the progesterone insert was removed, and the cows received 500 µg of cloprostenol, 400 IU of eCG, and 1 mg of estradiol cypionate. TAI was performed 48 h after the removal of the progesterone insert. The ultrasound exams were performed in a subgroup of cows on Days 0, 8, 10 and 12 to evaluate the diameter of the largest follicle, rate of follicular growth and risks of single and double ovulation. The pregnancy diagnosis was performed 30 days after TAI in all cows to determine the pregnancy rate. The diameter of the largest follicle, on D10 (P = 0.84), rate of follicular growth (P = 0.14), ovulation rate (P = 0.40) and double ovulation rates (P = 0.23) did not differ between experimental groups. The pregnancy rate was greater in the P4i group [Control 46.2 % (133/288) vs. P4i 55.6 % (160/288); P = 0.03]. The diameter of the largest follicles (LF) on D0 (Control 11.6 ± 0.2 vs. P4i 13.3 ± 0.3) was greater (P = 0.01) in the P4i group. In conclusion, injectable progesterone before the ovulation synchronization protocol increased the diameter of the largest follicle on the D0 and the pregnancy rate in multiparous Bos taurus suckled beef cows.

Use of injectable progesterone at the beginning of the TAI protocol is not necessary in super-early resynchronization started 14 days after artificial insemination in Bos indicus beef heifers.

In a protocol for the resynchronization of ovulation starting 14 days (D14) after timed artificial insemination, the effects of short-action injectable progesterone (P4i) administration and the length of treatment with an intravaginal progesterone (P4) device on follicular dynamics, the conception rate (P/AI) and the percentage of false positives were evaluated in 1065 Nelore heifers previously timed-inseminated. On D14, P4 devices were inserted, and heifers allocated, based on a 2 × 2 factorial design, to receive (P4i) or not 50 mg of P4i (NoP4i), and to remove the P4 device after 6 (6Day) or 8 days (8Day). At the time of P4 device removal (D20 and D22), non-pregnancy diagnosis was performed using vascularization of the corpus luteum (VCL) evaluated. At this time, non-pregnant heifers received 150 µg of D-cloprostenol, 0.6 mg of estradiol cypionate and 300IU of eCG. TAI was performed 48 h after P4 device removal. For these heifers, ultrasound examinations were performed at P4 device removal and at TAI to evaluate follicular dynamics and at 30 days after TAI to evaluate the P/AI. Pregnant heifers based on VCL were evaluated using B-mode ultrasonography 10 days after Doppler ultrasound to evaluate the percentage of false positives. Statistical analyses were performed using the GLIMMIX procedure of SAS. There were no interaction effects between P4i and duration of treatment with a P4 device. The P/AI was diagnosed by Doppler, 1st TAI, 2nd TAI, total and percentage of false positives did not differ between heifers receiving or not P4i. Similarly, duration of treatment with a P4 device did not influence the P/AI by Doppler, 1st TAI, 2nd TAI and total. However, the percentage of false positives diagnoses was higher in 6Day heifers (P = 0.01). The diameter of largest follicle at P4 device removal was greater in the 8Day heifers (P = 0.001), and at TAI was higher in the P4i-treated heifers (P = 0.03). Additionally, the percentage of false positives diagnoses was lower in heifers that ovulated to the 1st TAI protocol (P = 0.001). In conclusion, for resynchronization 14 days after TAI, it is not necessary to inject P4i at the beginning of the protocol. In addition, P4 device removal after 6 instead of 8 days increases the percentage of false positives because of the earlier diagnosis (20 days after TAI), but does not interfere in P/AI of resynchronization protocol. Furthermore, the percentage of false positives is higher in heifers that did not ovulate to 1st TAI protocol.

Effects of eCG and FSH in timed artificial insemination treatment regimens on estrous expression and pregnancy rates in primiparous and multiparous Bos indicus cows.

Effects were evaluated in Bos indicus cows of eCG and FSH on follicular growth, estrous expression, and pregnancy per artificial insemination (P/AI) as a result of fixed-time artificial insemination (TAI). In Experiment 1, extent of timing-of-ovulation synchronization among cows was evaluated after imposing an estrogen/progesterone-based treatment regimen. At progesterone device removal (D8), cows were administered: eCG, or FSH or served as untreated Controls. In Experiment 2, percentage of cows P/AI was evaluated when the Experiment 1-treatment regimen was imposed. On D10, all cows were artificially inseminated. In Experiment 3, cows were assigned to two treatment groups (Control and eCG) on D8 to evaluate percentage of cows P/AI and estrous expression. In Experiment 1, follicular dynamics were similar among treatment groups. In Experiment 2, follicular growth was greater (P = 0.0001) with the eCG treatment. There was an interaction of treatment × parity (P = 0.007) on percentage of cows P/AI. There was a greater percentage of primiparous cows P/AI in the eCG-treated than Control and FSH-treated cows. There was a greater percentage of eCG-treated multiparous cows pregnant as a result of TAI than Control cows. There was an interaction of treatment × parity (P = 0.005) on P/AI in Experiment 3, in which the eCG effect was more pronounced in primiparous cows. Treatment with FSH, therefore, was not as effective as eCG in stimulation of follicular growth or enhancing percentage of cows pregnant as a result of TAI. Physiological effects of eCG, however, were also more evident in primiparous cows.

Nuclear maturation kinetics and in vitro fertilization of immature bovine oocytes injected into pre-ovulatory follicles.

The maturation kinetics and in vitro fertilization of immature bovine oocytes injected by the intra-follicular oocyte injection (IFOT) technique into pre-ovulatory follicles of previously synchronized cows were evaluated. In Experiment 1, grade I, II and III cumulus-oocyte complexes (COCs) were randomly distributed to one of three Groups: Matvitro22 (COCs matured in vitro for 22 h), MatFol20 and MatFol28 (COCs matured in vivo after being injected into a pre-ovulatory follicle of previously synchronized cows for 19.8 ± 0.1 h and 28.3 ± 0.1 h, respectively). Cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at the time of IFOT in the MatFol20 Group or 10 h after IFOT in the MatFol28 Group. MatFol20 and MatFol28 COCs were aspirated approximately 20 h after the LH injection for nuclear maturation kinetics and recovery rate assessment. In Experiment 2, grade I, II, and III COCs were randomly distributed into two Groups: Matvitro22 Group, COCs were matured and fertilized in vitro, and MatFol20 Group, COCs were matured as in the MatFol20 Group in Experiment 1, but COCs were fertilized in vitro. Putative zygotes were classified as fertilized, unfertilized or polyspermic. In Experiment 1, the recovery rate was lower (P < 0.001) in the MatFol20 Group (52.9%, 91/172) compared with MatFol28 (72.9%, 113/155). Rate of oocytes in germinal vesicle stage, metaphase I, anaphase I and telophase I were similar among Groups. However, oocytes matured in vivo for 28.3 h had lower rate of metaphase II (P = 0.001) and greater rates of degenerated (P = 0.001) and parthenogenetically activated (P = 0.001) oocytes. In experiment 2, the rates of polyspermy and degenerated were similar between Groups. However, the rate of fertilized oocytes was greater (P = 0.05) in oocytes in the MatFol20 Group. It is concluded that oocyte in vivo maturation for 19.8 h after IFOT does not compromise the nuclear maturation kinetics and increases in vitro fertilization rates. However, the extra 10 h of intra-follicular incubation time decreased oocyte viability.

Effect of induction of ovulation with estradiol benzoate at P4 device removal on ovulation rate and fertility in Bos indicus cows submitted to a TAI protocol.

This study aimed to minimize the number of times cattle need to be confined during protocols for TAI in beef cows treated for induction of ovulation with EB at the time of P4 device removal (P4r). In Experiment 1, cows were treated with P4 plus EB (Day 0; AM) and were allocated to one of three groups at P4r: EB8.5, EB at P4r on Day 8.5 (PM; three confinements); EB9, EB 24 h after P4r on Day 8 (AM; four confinements) and EC8, EC at P4r on Day 8 (AM; positive control; three confinements). At P4r, cows were treated with PGF2a plus eCG. Ultrasonography was performed from D8 to D12. The interval from P4r to ovulation was less in the EB8.5 compared to EB9 and EC8 group. There was no difference in the ovulation rate between groups. The variability of ovulation was greater in the EB8.5 and EC8 compared to EB9 group. In Experiment 2, cows of EC8 and EB9 groups were submitted to TAI 48 to 52 h (AM) or 54 to 58 h (PM) after P4r (D10). Cows of the EB8.5 group were submitted to TAI 38 to 42 h (AM) or 44 to 48 h (PM) after P4r (D10). There was no interaction between treatments and timing of AI and no treatment effect and timing of AI on P/AI. In conclusion, the delay compared to what typically occurs by 10 h of P4r concomitant with EB administration (Day 8.5) reduced the frequency of animal confinement for the TAI protocol without affecting the reproductive efficiency and the flexibility to perform the TAI in suckled beef cows.

Subclinical mastitis interferes with ovulation, oocyte and granulosa cell quality in dairy cows.

The objective was to evaluate the effect of mastitis by somatic cell count (SCC) on follicular growth, ovulation, oocytes and cumulus cells quality and on the concentration and size of exosomes in follicular fluid of dairy cows. In the study, crossbred cows (Bos taurus - Holstein x Bos indicus - Gir) were classified for analysis as Control (SCC<200.000 cells/mL) and Mastitis (SCC>400.000 cells/mL) groups. In experiment 1 (follicular dynamics), cows (n = 57) were submitted to ultrasound evaluations every 24 h, from progesterone-releasing-intravaginal-device (PRID) removal (D8) until 48 h later (D10). Thereafter, evaluations were performed every 12 h, until ovulation or up to 96 h after PRID removal. In experiment 2 (oocyte, cumulus complexes, and follicular fluid evaluation), cows (n = 26) were submitted to follicular aspiration (OPU) for oocyte quality and cumulus cells transcript evaluation. The amount of cumulus complexes transcripts (BCL2, BAX, PI3K, PTEN, FOXO3) was determined by Real-Time Polymerase Chain Reaction. Moreover, seven days after the OPU session, the dominant follicle was aspirated. Exosomes were isolated from the follicular fluid for evaluation of particle size and concentration. Ovulation rate [Control 77.4% (24/31) and Mastitis 57.7% (15/26); P = 0.09] and viable oocytes rate [Control 59.1% (130/220) and Mastitis 41.9% (125/298); P = 0.01] were higher in Control animals. Additionally, there was a greater number of degenerate oocytes [Control 6.7 ±â€¯1.2 and Mastitis 13.3 ±â€¯5.5; (P = 0.001)] in subclinical mastitis cows. There was greater abundance (P = 0.003) of BAX cumulus cell transcripts and exosome mean (P = 0.03) and mode (P = 0.02) was smaller in subclinical mastitis cows. In conclusion, ovulation rate, oocyte quality, and exosome diameter were smaller in cows with SCC>400.000 cells/mL.

Presynchronization by induction of a largest follicle using a progesterone device in GnRH-based-ovulation synchronization protocol in crossbred dairy cows.

The objective was to compare the fertility of dairy cows using a presynchronization protocol by induction of a largest follicle using a progesterone intravaginal device prior to an Ovsynch protocol (P4synch) with the Double-Ovsynch in lactating dairy cows. Lactating Bos indicus x Bos taurus crossbred cows (n = 440) were randomly allocated to one of two treatments: (I) Double-Ov (n = 228), GnRH (D-17), PGF2α 7 days later (D-10) and GnRH 3 days later (D-7) followed by an Ovsynch protocol 7 days later (GnRH on D0, PGF on D7, GnRH on D9); (II) P4synch (n = 212), insertion of a sustained release progesterone intravaginal device (D-10), 10 days later (D0) an Ovsynch protocol was initiated (GnRH on D0, PGF on D7, GnRH on D9) with progesterone device withdrawal on Day 7. All cows were artificially inseminated (TAI) 16 h after the second GnRH of the Ovsynch protocol and pregnancy diagnosis was performed by transrectal ultrasonography 30 and 60 days after TAI. A subset of cows (n = 52 for Double-Ov and n = 50 for P4synch) ultrasonography was performed on days 0, 7, 9 and 24 of the experimental period. There were no differences among treatments on presynchronization rate [presence of a follicle>12 mm on D0, Double-Ov 94.2% (49/52) and P4synch 92.0% (46/50); P = 0.66], follicular diameter on the 1st GnRH (Double-Ov 17.2 ±â€¯0.7 mm e P4synch 18.6 ±â€¯0.9 mm; P = 0.28), ovulation rate to the 1st GnRH [Double-Ov 86.3% (44/51) and P4synch 81.2% (39/48); P = 0.50], synchronization rate [presence of a follicle>12 mm on D9; Double-Ov 84.6% (44/52) and P4synch 86.0% (43/50); P = 0.84], follicular diameter on the 2nd GnRH (Double-Ov 17.5 ±â€¯0.6 mm and P4synch 18.0 ±â€¯0.5 mm; P = 0.48), ovulation rate to the 2nd GnRH [Double-Ov 90.9% (40/44) and P4synch 86.0% (37/43); P = 0.48] and CL diameter on D24 (Double-Ov 27.9 ±â€¯0.7 mm and P4synch 29.4 ±â€¯0.9 mm; P = 0.19). Corpus luteum presence on D0 was different (P = 0.03) among treatments [Double-Ov 57.7% (30/52) and P4synch 36.0% (18/50)]. There was no difference (P = 0.85) among the pregnancy per AI on day 30 [Double-Ov 39.0% (89/228) and P4synch 40.1% (85/212)], on day 60 [Double-Ov 34.8% (79/227) and P4synch 38.7% (82/212); P = 0.41] and pregnancy loss [Double-Ov 10.2% (9/88) and P4synch 3.5% (3/85); P = 0.08]. The presynchronization by induction of a largest follicle using a sustained release progesterone device prior to Ovsynch yielded similar results compared with the Double Ovsynch protocol on follicular development patterns and on the fertility of lactating dairy cows.

Exposure to progesterone previous to the protocol of ovulation synchronization increases the follicular diameter and the fertility of suckled Bos indicus cows.

The objective was to evaluate the effect of injectable progesterone previous to the timed artificial insemination (TAI) protocol on follicular growth, ovulation and pregnancy rate of suckled Bos indicus cows. In experiment 1, 10 days before the beginning to TAI protocol (D-10), 431 suckled-anestrus Nelore cows (249 multiparous and 182 primiparous), were allocated to one of three treatments groups (control, P4i and P4iGnRH). At this moment, cows in the P4i and P4iGnRH group received 150 mg of injectable progesterone intramuscularly (Sincrogest injetável®, Ouro Fino, Brazil). On Day 0 (D0), all cows were synchronized using an estradiol/progesterone-based TAI protocol. Simultaneously, in the P4iGnRH group, cows received 10 µg of Busereline intramuscularly (Sincroforte®, Ouro Fino, Brazil). Ultrasound exams were performed to evaluate the diameter of the largest follicle (D0, D8 and D10), ovulation rate and diameter of the corpus luteum (D24). In experiment 2, 681 suckled Nelore cows (356 multiparous and 325 primiparous) were synchronized using an estradiol/progesterone-based TAI protocol and received treatments similar to experiment 1. TAI was performed 48 h after removal of the progesterone (P4) device. Pregnancy diagnosis was 30 d after TAI. In experiment 3, blood samples were collected to evaluated the progesterone concentration for 168 h after administration of injectable progesterone intramuscularly. Statistical analyses were performed by GLIMMIX procedure of SAS. In experiment 1, the diameter of the largest follicle (LF) on D10 (P = 0.21), follicular growth rate (P = 0.34) and ovulation rate (P = 0.62) were similar among experimental groups. However, there was difference among groups for the LF on D0 [Control (10.9 ±â€¯0.2 mm)b, P4i (12.7 ±â€¯0.3 mm)a and P4iGnRH (12.6 ±â€¯0.3 mm)a; P = 0.001], LF on D8 [Control (9.7 ±â€¯0.2 mm)b, P4i (10.4 ±â€¯0.2 mm)a and P4iGnRH (9.9 ±â€¯0.2 mm)ab; P = 0.05], presence of the CL on D8 [Control 0% (0/136)b, P4i 0% (0/140)band P4iGnRH 26.4% (38/144)a; P = 0.001], diameter of the CL on D24 [Control (19.7 ±â€¯0.4 mm)ab, P4i (20.1 ±â€¯0.4 mm)a and P4iGnRH (18.5 ±â€¯0.4 mm)b; P = 0.001] and pregnancy rate [Control 35.0% (78/223)b, P4i 45.9% (105/229)a and P4iGnRH 40.6% (93/229)ab; P = 0.01]. The circulating concentration of P4 remained above 1.5 ng/mL until 168 h after the P4i treatment. In conclusion, the injectable progesterone previous to the TAI protocol increased diameter of the LF on D0 and D8 without interfering on the ovulation rate. Furthermore, such exposure increases the pregnancy rate in suckled Nelore cows.

Synchronization treatments previous to natural breeding anticipate and improve the pregnancy rate of postpartum primiparous beef cows.

Timed AI has become a potential tool to bypass postpartum acyclicity, yet only a small percentage of the world bovine herd is inseminated. Most females are still subjected to bull mating; therefore, the frequent occurrence of postpartum anestrus may compromise their reproductive efficiency. Thus, the aim of this study was to develop an approach that allows the early conception of postpartum primiparous beef cows that are exposed to natural breeding (NB). For this purpose, 350 primiparous Nelore cows 35-65 d postpartum were allocated into three groups: Control (no hormonal treatment), TNB (hormonal protocol for timed-NB without equine chorionic gonadotropin; eCG) and TNB + eCG (hormonal protocol for TNB with eCG) groups. The protocol for TNB consisted of the insertion of a 1-g progesterone device and the intramuscular (IM) administration of 2 mg estradiol benzoate on D-9 (nine days before bull exposure), followed by device removal and the administration of 1 mg estradiol cypionate IM on D0. An additional 300 IU of eCG was given only to TNB + eCG cows. All cows were exposed to bull mating from D0 to D105. Pregnancy was checked by ultrasonography every 50 d, gestational age was estimated, and the number of new gestations every 21-d cycle (P21, P42, P63, P84 and P105) was predicted. Control cows had lower pregnancy at P21 (5.7%c, 7/123) than TNB (30.4%b, 35/115) and TNB + eCG (51.8%a, 58/112; P = 0.001) cows. Pregnancy rate increased across P42, P63 and P84 (P = 0.001) but remained higher for TNB + eCG cows. Regarding time to conception, the TNB + eCG cows achieved a greater than 50% pregnancy rate within the first days after bull exposure, while the TNB and Control cows took more than 40 d and 90 d to achieve 50% pregnancy rates, respectively. At the end of the breeding season (BS), TNB + eCG cows had a 21% higher pregnancy rate than the Control cows and a 16% higher rate than the TNB cows. The probability of conceiving increased 1.5-fold for cows treated with TNB (P = 0.0079) and 2.2-fold for cows additionally treated with eCG (P < 0.0001). The average interval between the onset of the BS and conception was reduced (P < 0.0001) for the TNB + eCG cows (26.5 ± 3.8c) compared to the TNB (35.7 ±â€¯4.1b) and Control (64.7 ±â€¯3.9a days) cows. Thus, the use of TNB, especially when associated with eCG, efficiently improved the early conception of postpartum primiparous beef cows after exposure to NB. The increased number of cows conceiving early in the BS is crucial to improve reproductive efficiency by reducing the interval between parturitions and improving the number of pregnant cows at the end of BS, thus resulting in greater farm income.

Molecular and endocrine factors involved in future dominant follicle dynamics during the induction of luteolysis in Bos indicus cows.

The growth profiles of the future dominant follicle (DF) and subordinate follicle (SF) and the gene expression of the granulosa cells during luteolysis induction in Bos indicus cows were evaluated. Forty cows were synchronized with a progesterone and estradiol based protocol. After synchronization, cows with a corpus luteum (CL) were evaluated by ultrasonography every 12 h, beginning at eight days post ovulation. Cows identified with a follicle of at least 6.0 mm in diameter in the second wave were split into two groups (BD-before follicular deviation and AD-after follicular deviation. In the BD group cows received 500 µg of cloprostenol (a synthetic analogue of prostaglandin F2α) when the DF reached a mean diameter of 7.0 mm (6.5-7.5 mm). In the AD group, cows received 500 µg of cloprostenol when the DF reached a mean diameter of 8.0 mm (7.5-8.5 mm). Cows in both groups were submitted to aspiration of the DF at 96 and 72 h after prostaglandin was given. Follicular aspirations were performed to quantify IGF1R, LHR and PAPPA transcripts in the granulosa cells. The diameter of the DF at the moment of prostaglandin administration (P = 0.001) and the growth rate of the SF (P = 0.05) were greater in the AD group. There was greater abundance of LHR transcripts in BD cows (P = 0.04). The remaining variables tested were similar between the experimental groups (P > 0.05). In conclusion, the induction of luteolysis before follicular deviation does not interfere with dominant follicle dynamics. However, it causes granulosa cell LHR down regulation.

Although it induces synchronized ovulation, hCG reduces the fertility of Santa Ines ewes submitted to TAI / Embora induza a ovulação sincronizada, hCG reduz a fertilidade de ovelhas Santa Inês submetidas à IATF

The aim of this study was to evaluate hCG treatment on ovarian response and on pregnancy rate using a 9-day oestrus synchronization protocol in Santa Ines ewes. On a random oestrus cycle day, ewes received an intravaginal progesterone device (Primer-PR®, Tecnopec, Brazil). Nine days later (Day 9), 30µg of d-cloprostenol (Prolise®, Syntex, Argentina) and 250IU of eCG (Folligon®, Intervet, Brazil) were administered and the progesterone device was removed. This moment, the ewes were randomly assigned on two groups: Control Group and hCG Group. In the hCG Group, the ewes received 500IU of hCG (Vetecor®, Hertape-Calier, Spain) 24h after device removal. In the Control Group, the ewes did not receive any ovulation inductor. Control and hCG Groups ewes were inseminated 60h and 48h after device removal, respectively. There was no difference between the groups regarding the first ovulatory follicle diameter and the second ovulatory follicle. hCG Group ewes had shorter interval between device removal and ovulation (Control: 79.9±15.4h and hCG: 54.7±4.9h; P=0.001) and more synchronized ovulations. However, the treatment with hCG decreased the pregnancy rate after TAI (P=0,009). In conclusion, hCG administration improves ovulatory synchronisation, but causes a decrease in the pregnancy rate.(AU)

Avaliou-se o tratamento com hCG na resposta ovariana e na taxa de prenhez utilizando protocolo de sincronização do estro de nove dias em ovelhas Santa Inês. As ovelhas receberam um dispositivo intravaginal de progesterona em fase aleatória do ciclo (dia zero= D0). No momento da remoção do dispositivo (D9), as fêmeas receberam 30µg de d-cloprostenol (Prolise®, Syntex, Argentina) e 250UI de eCG (Folligon®, Intervet, Brasil). Nesse momento, as ovelhas foram aleatoriamente distribuídas em dois grupos de tratamento: controle sem indução de ovulação e tratamento com 500UI hCG para indução de ovulação. As ovelhas dos grupos controle e hCG foram inseminadas 60h e 48h após a remoção do dispositivo, respectivamente. Não houve diferença entre os grupos para o diâmetro do primeiro e do segundo folículo pré-ovulatório. As avelhas do grupo hCG apresentaram menor intervalo entre a remoção do dispositivo e a ovulação (grupo controle: 79.9±15.4h e grupo hCG: 54.7±4.9h; P=0.001) e maior sincronização das ovulações. No entanto, o tratamento com hCG diminuiu a taxa de prenhez após a IATF (P=0,009). Conclui-se que, apesar de a administração de hCG aumentar a sincronização da ovulação, reduz a taxa de prenhez.(AU)

Ovulation synchronization with estradiol benzoate or GnRH in a timed artificial insemination protocol in buffalo cows and heifers during the nonbreeding season.

The aim of this study was to compare estradiol benzoate (EB) and GnRH for the induction of ovulation in a TAI protocol in buffalo during the nonbreeding season. In experiment 1, 141 buffaloes (56 cows and 85 heifers) received an intravaginal P4 device (1.0 g) plus EB (2.0-mg, intramuscular [im]) at random stage of the estrous cycle (Day 0). On Day 9, the P4 device was removed, and buffaloes were given PGF2α (0.53-mg im sodium cloprostenol) plus eCG (400-IU im). Buffaloes were then randomly allocated to one of three groups and treated as follows: EB24 (n = 47), EB (1.0 mg im) 24 hours after P4 device removal; EB36 (n = 50), EB 36 hours after P4 device removal; GnRH48 (n = 44), GnRH (10 µg im buserelin acetate) 48 hours after P4 device removal. Ultrasound examinations were performed on Day 0 to ascertain ovarian follicular status, Day 9 to measure follicular diameter, and from Day 11 to Day 14 (every 12 hours for 60 hours) to establish the time of ovulation. There were no significant differences between EB24, EB36, and GnRH48 for diameter of the ovulatory follicle (13.1 ± 0.3, 13.7 ± 0.3, and 13.7 ± 0.3 mm; P = 0.26) and ovulation rate (78.7%, 82.0%, and 84.1%; P = 0.93). When compared with heifers, cows had a greater diameter of the dominant follicle on Day 9 (10.3 ± 0.3 and 8.6 ± 0.2 mm; P = 0.0001), diameter of the ovulatory follicle (14.1 ± 0.3 and 13.1 ± 0.2 mm; P = 0.01), ovulation rate (91.1% and 75.3%; P = 0.02), and interval from P4 device removal to ovulation (76.3 ± 1.3 and 72.5 ± 1.4 hours; P = 0.05). In experiment 2, 511 buffaloes (354 cows and 157 heifers) were assigned to the same treatments described in experiment 1 (EB24, n = 168; EB36, n = 172; and GnRH48, n = 171), and all animals were submitted to timed artificial insemination (TAI) 64 hours after P4 device removal. Pregnancy diagnosis was undertaken 30 days after TAI. There were no significant differences between EB24, EB36, and GnRH48 for pregnancy rate (45.2%, 43.0%, and 49.7%; P = 0.46), and the pregnancy rate did not differ (P = 0.31) for cows (47.5%) and heifers (42.7%). The findings from the two experiments indicated that EB (24 or 36 hours) and GnRH (48 hours) induce comparable follicular responses, ovulation, and pregnancy rates in buffalo cows and heifers. Although there were some differences in the follicular responses between cows and heifers, the pregnancy rate to TAI was nonetheless similar.

Effects of eCG are more pronounced in primiparous than multiparous Bos indicus cows submitted to a timed artificial insemination protocol.

The effects of eCG on follicular growth, ovulation, and pregnancy per artificial insemination (P/AI) in multiparous and primiparous Bos indicus beef cows submitted to timed artificial insemination (TAI) were evaluated in three experiments. In experiments 1 (follicular responses; n = 64), 2 (follicular growth and ovulation rate; n = 662), and 3 (P/AI; n = 2092), cows submitted to TAI were assigned to receive one of two treatments on Day 8 of the synchronization protocol: control (no additional treatment) or eCG (300-IU of eCG intramuscularly). In experiment 1, largest follicle (LF) diameter on Day 8 (P = 0.56) and the interval from progesterone (P4)-device removal to ovulation (P = 0.79) did not differ between treatments. However, the maximum diameter of the LF (P = 0.05) and ovulation rate (P = 0.03) were greater in cows that received eCG. In experiment 2, the diameter of the LF on Day 10, follicular growth, and ovulation rate were greater in eCG-treated cows (P < 0.01). However, CL diameter was similar between treatments (P = 0.11). In experiment 3, there was a treatment-by-parity interaction (P = 0.003) on P/AI, such that treatment with eCG was more effective in primiparous cows. In conclusion, eCG treatment resulted in increased final follicular growth, ovulation rate, and fertility in B indicus cows submitted to TAI protocols, especially in primiparous cows.

Comparative efficacy of exogenous eCG and progesterone on endogenous progesterone and pregnancy in Holstein cows submitted to timed artificial insemination.

The objectives were to evaluate the effects of the administration of either eCG or progesterone (P4) alone or combined on endogenous P4 concentrations and pregnancy per AI in lactating dairy cows. Cows received a P4-releasing intravaginal device (PRID) and estradiol benzoate on D-8. The PRID was removed and a PGF2α injection was given on D-3. An estradiol cypionate was given on D-2 and TAI was performed on D0. On D-2, cows were randomly allocated to treatments in a 2×2 factorial design: Control-saline solution on the D-2 and D+3 (n=104), eCG - 400IU eCG on D-2 (n=93), P4 - 600mg of P4 on D+3 (n=106), and eCG+P4 - 400IU eCG on D-2 and 600mg of P4 on D+3 (n=95). Blood samples were collected on days three, four, and thirteen and pregnancy diagnoses were performed at 32 and 46 days after AI. There was no interaction between eCG and P4 injection. Cows treated with eCG and with P4 injection had higher serum P4 on Day +4. On Day +13 serum P4 was lower in eCG-untreated primiparous cows (Interaction eCG×parity). Cows with serum P4<4.57ng/mL on Day +13 had lower probability to be pregnant on day 32. P/AI on days 32 and 46 and embryonic losses were not influenced by eCG and P4 injection. In conclusion, the addition of 400IU of eCG on D-2 and/or 600mg of P4 on D+3 to the present TAI protocol did not increase P/AI.

Effects of a high-energy diet on oocyte quality and in vitro embryo production in Bos indicus and Bos taurus cows.

The effects of different dietary energy levels [100 and 170% for maintenance (M) and high energy (1.7M), respectively] on metabolic, endocrine, and reproductive parameters were evaluated in nonlactating Bos indicus (Gir; n=14) and Bos taurus (Holstein; n=14) cows submitted to ultrasound-guided ovum pick-up followed by in vitro embryo production. The oocyte donor cows were housed in a tiestall system and fed twice daily (0800 and 1600 h). Twenty-one days before the beginning of the experiment, the animals were fed with a maintenance diet for adaptation followed by the experimental diets (M and 1.7M), and each cow underwent 9 ovum pick-up procedures 14 d apart. The recovered oocytes were cultured in vitro for 7 d. We measured glucose and insulin concentrations and performed glucose tolerance tests and the relative quantification of transcripts (PRDX1, HSP70.1, GLUT1, GLUT5, IGF1R, and IGF2R) from the oocytes recovered at the end of the experimental period. No interactions were observed between the effects of genetic groups and dietary energy level on the qualitative (viable oocytes, quality grade, and oocyte quality index) and quantitative (oocytes recovered) oocyte variables. There were no effects of dietary energy level on the qualitative and quantitative oocyte variables. However, Bos indicus cows had greater numbers of recovered structures, viable oocytes, and A and B oocyte grades as well as better oocyte quality index scores and lower DNA fragmentation rates compared with Bos taurus donors. In vitro embryo production (cleavage and blastocyst rates and number of embryos) was similar between diets, but the 1.7M diet reduced in vitro embryo production in Bos indicus cows after 60 d of treatment. Moreover, Bos indicus cows on the 1.7M diet showed lower transcript abundance for the HSP70.1, GLUT1, IGF1R, and IGF2R genes. All cows fed 1.7M diets had greater glucose and insulin concentrations and greater insulin resistance according to the glucose tolerance test. In conclusion, increasing dietary energy did not interfere with oocyte numbers and quality, but the 1.7M diet reduced in vitro embryo production in Bos indicus cows after 60 d of treatment. Finally, Bos indicus cows had greater oocyte quality, greater numbers of viable oocytes and greater in vitro embryo yield than Bos taurus.

Effect of circulating progesterone concentration during synchronization for fixed-time artificial insemination on ovulation and fertility in Bos indicus (Nelore) beef cows.

Four experiments were designed to evaluate the effect of different circulating progesterone (P4) concentrations during a synchronization of ovulation protocol for the timed artificial insemination (TAI) of Bos indicus (Nelore) beef cattle. In the first trial, 13 ovariectomized Nelore heifers were randomly allocated into one of three groups using new P4 devices (New; 1.0 g P4), previously used P4 devices for 8 days (Used1x), and previously used P4 devices for 16 days (Used2x), in a crossover experimental design. The circulating P4 concentrations during the P4 device treatment were lower for Used1x (2.3 ± 0.1 ng/mL) and Used2x (2.0 ± 0.1 ng/mL) than those for New (3.8 ± 0.2 ng/mL; P = 0.001). In the second trial, the ovarian follicular dynamics of 60 anestrous cows were evaluated after the cows received the treatments described previously (New [n = 20], Used1x [n = 20], and Used2x [n = 20]). During the insertion of the P4 device, the cows were administered 2.0-mg estradiol benzoate. Eight days later, the P4 device was removed, and the cows were administered 0.53-mg sodium cloprostenol, 300 IU eCG, and 1-mg estradiol cypionate. There were no differences among the groups during the interval from P4 device removal to ovulation (73.7 ± 2.9 vs. 69.8 ± 2.4 vs. 68.4 ± 2.3 hours) or regarding the ovulation rate (70.0% vs. 80.0% vs. 85.0%). However, the maximum diameter of the largest follicle was greater (P = 0.06) in the Used2x (15.3 ± 0.4 mm) than that of New (13.5 ± 0.8 mm) and Used1x (14.9 ± 0.5 mm). In experiment 3, 443 anestrous cows were randomly assigned into one of the three treatments (New [n = 144] vs. Used1x [n = 167] vs. Used2x [n = 132]) and received a TAI 48 hours after the P4 device removal. The diameter of the largest follicle during the device removal (10.7 ± 0.3 vs. 11.2 ± 0.2 vs. 11.3 ± 0.3 mm) and the 30-day pregnancy rates (51.4% vs. 53.9% vs. 43.2%) did not differ among the experimental groups. In experiment 4, a field trial with 593 B indicus (Nelore) cows was conducted to evaluate the pregnancy per AI using different levels of P4 in a TAI protocol (New [n = 189] vs. Used1x [n = 203] vs. Used2x [n = 201]). The pregnancy per AI was similar between the treatment groups (63.5% vs. 57.6% vs. 62.7%). In conclusion, the low circulating P4 concentrations that were released from a used P4 device efficiently controlled the ovarian follicular growth and exhibited no detrimental effects on the pregnancy rates of the B indicus (Nelore) beef cattle.

A comparison of two different esters of estradiol for the induction of ovulation in an estradiol plus progestin-based timed artificial insemination protocol for suckled Bos indicus beef cows.

The abilities of two different estradiol esters to induce ovulation in a timed AI (TAI) synchronization protocol in suckled Bos indicus cows were evaluated. In Experiment 1 (synchrony of ovulation), 31 cows were submitted to an estradiol/progestin-based synchronization protocol (Day 0) and randomly assigned to one of three treatments at the time of progestin removal on Day 8: 0.5 or 1.0mg of estradiol cypionate (EC) at that time or 1.0mg of estradiol benzoate (EB) 24h later (Day 9). To determine the timing of ovulation, ultrasound examinations were performed every 12h from ear implant removal to 96 h after the removal. Orthogonal comparisons were performed to determine the effects of estradiol ester and the effects of the dose of EC on reproductive parameters. Although neither the E2 ester (P = 0.83) nor the dose of EC (P = 0.55) affected the ovulation rate, the interval from progestin removal to ovulation was longer (P=0.04) in EC-treated cows (1.0mg EC = 71.1 ± 3.6 and 0.5mg EC = 78.0 ± 3.5) than EB-treated cows (EB = 66.0 ± 2.3) was detected. Ovulation in 0.5-mg-EC-treated cows was less synchronous than that in 1.0-mg-EC-treated cows (distribution curves compared using kurtosis). In Experiment 2 (pregnancy per AI; P/AI), 660 cows at two different locations received the same synchronization protocol (n = 361 at Farm A and n = 299 at Farm B) and were treated with estradiol esters as in Experiment 1 [0.5mg EC (n = 220) or 1.0mg EC (n = 219) at the time of progestin removal or 1.0mg EB (n = 221) 24h later]. The cows were inseminated 54 to 56 h after progestin removal. As applied in the Experiment 1, orthogonal comparisons were performed to evaluate the effect of estradiol ester and the dose of EC on P/AI. Although the type of estradiol ester used did not affect the P/AI (P = 0.57; EB - 43.0% vs. EC - 44.6%), the P/AI was higher (P=0.03) in cows treated with 1.0mg EC (55.7%) than in those treated with 0.5mg EC (38.6%). In summary, the administration of 0.5mg EC at the time of progestin removal altered the distribution of ovulation and resulted in a lower P/AI when compared with the use of 1.0mg EC in suckled B. indicus cows. However, the P/AI following the administration of 1.0mg EC at the time of progestin removal did not differ from that after the administration of 1.0mg EB 24h later.

Donor category and seasonal climate associated with embryo production and survival in multiple ovulation and embryo transfer programs in Holstein cattle.

The present study investigated the effect of Holstein donor category (cows vs. heifers) and climate variation (hot vs. cooler season) on the efficiency of in vivo embryo production programs as well as embryo survival after transferred to Holstein recipient cows. A total of 1562 multiple ovulation (MO) procedures (cows: n = 609, and heifers: n = 953) and 4076 embryo transfers (ETs) performed in two dairy herds were evaluated. Donor cows had greater number of CLs (10.6 ± 0.6 vs. 7.5 ± 0.4; P < 0.0001) and ova/embryos recovered (7.6 ± 0.6 vs. 4.6 ± 0.4; P < 0.0001) compared with donor heifers. However, fertilization rate (47.9 vs. 82.4%; P < 0.0001) and proportion of transferable embryos (31.5 vs. 67.4%; P < 0.0001) were lower in donor cows than heifers, respectively. Regardless of donor category, the proportion of freezable embryos was less (P < 0.001) during hot season than in cooler season (21.4 vs. 32.8%). However, greater decline in the proportion of freezable embryos during the hot season was observed in cows (21.7 vs. 10.7%) compared with heifers (46.2 vs. 38.1%; P = 0.01). In contrast, the season on which the embryo was produced (hot or cool) did not affect pregnancy rate on Day 31 (30.5 vs. 31.7%; P = 0.45) and 45 (25.3 vs. 25.1%; P = 0.64) of pregnancy. Regardless of the season in which the embryos were produced, embryonic survival after transferring embryos retrieved from donor cows was greater on Days 31 (36.0 vs. 30.7%; P = 0.001) and 45 (28.3 vs. 23.1%; P = 0.001) of pregnancy when compared with embryos from donor heifers. In conclusion, MO embryo production efficiency decreased during the hot seasons both in cows and heifers; however, the decline was more pronounced in donor cows. Regardless of the embryo source, similar pregnancy rate was observed in the recipient that received embryos produced during the hot and cooler seasons. Curiously, embryos originating from donor cows had higher embryonic survival when transferred to recipient cows than embryos originating from heifers.

Different circulating progesterone concentrations during synchronization of ovulation protocol did not affect ovarian follicular and pregnancy responses in seasonal anestrous buffalo cows.

Three experiments were designed to evaluate the effect of different circulating progesterone (P4) concentrations during synchronization of ovulation protocol for timed artificial insemination of seasonal anestrous buffalo cows. In the first trial, ovariectomized cows were randomly allocated into one of three groups: using new P4 devices (G-New; n = 8), using devices previously used for 9 days (G-Used1x; n = 8), and using devices previously used for 18 days (G-Used2x; n = 8). The P4 device was maintained for 9 days, and the circulating P4 concentration was measured daily. The circulating P4 concentrations during the P4 device treatment were the lowest for G-Used2x (1.10 ± 0.04 ng/mL), intermediate for G-Used1x (1.52 ± 0.05 ng/mL), and the highest for G-New (2.47 ± 0.07 ng/mL; P = 0.001). In the second trial, 31 anestrous cows had their ovarian follicular dynamics evaluated after receiving the treatments described previously (G-New [n = 10], G-Used1x [n = 11], and G-Used2x [n = 10]). At insertion of the P4 device, cows were administered 2.0 mg of estradiol benzoate. Nine days later, the P4 device was removed and cows were administered 0.53 mg of cloprostenol sodium plus 400 IU of eCG. Forty-eight hours after P4 device removal, 10 µg of buserelin acetate was administered. There were no differences among the groups (G-New vs. G-Used1x vs. G-Used2x) in diameter of the largest follicle at P4 device removal (9.0 ± 0.8 vs. 10.1 ± 0.9 vs. 8.6 ± 0.8 mm; P = 0.35), in interval from P4 device removal to ovulation (77.1 ± 4.5 vs. 76.5 ± 4.7 vs. 74.0 ± 4.4 hours; P = 0.31), or in ovulation rate (80.0% vs. 81.8% vs. 60.0%; P = 0.51). In experiment 3, 350 anestrous cows were randomly assigned into one of the three treatments described previously (G-New, n = 111; G-Used1x, n = 121; G-Used2x, n = 118) and received a timed artificial insemination for 16 hours after buserelin treatment. The 30-day pregnancy rates did not differ among groups (55.9% vs. 55.4% vs. 48.3%; P = 0.39). Thus, the low circulating P4 concentrations released from a used P4 device efficiently control the ovarian follicular growth and had no detrimental effect on the pregnancy rates of the seasonal anestrous buffalo cows.