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Three experiments were designed to evaluate the effect of the length of insertion of a intravaginal progesterone (P4) device on follicular and corpus luteum (CL) diameter, time of ovulation, and pregnancy rates following fixed-time AI (P/FTAI) in Bos indicus cows treated with an oestradiol/P4-based treatment with a lengthened proestrus, named J-Synch. Multiparous suckled Bos indicus beef cows that were 35 to 60 days postpartum were used in Experiments 1 and 2, whereas non-lactating cows were used in Experiment 3. All cows had a CL or a follicle of at least 8 mm in diameter and a body condition score between 2.5 and 4 (1 to 5 scale) at the time of treatment. On Day 0, all cows received 2 mg of oestradiol benzoate (EB; Sincrodiol, Ourofino, Brazil) and a previously used intravaginal device (1 g of P4; Sincrogest, Ourofino). The P4 device was removed on Day 6 in the J-Synch 6-day group and on Day 7 in the J-Synch 7-day and Control groups. All cows received 500 µg of cloprostenol (Sincrocio, Ourofino) and 400 IU of equine chorionic gonadotrophin (SincroeCG, Ourofino) at the time of P4 device removal, and cows in the Control group also received 0.5 mg of oestradiol cypionate (ECP, SincroCP, Ourofino) at the same time. In Experiment 1, cows (n = 20 per group) were examined every 12 h, from P4 device removal to determine the time of ovulation and 7 days after ovulation to measure the CL. In Experiment 2, all cows (n = 200 per group) were tail-painted for oestrous detection. Cows with = 50% of the tail-paint rubbed-off by 72 h (J-Synch groups) or 48 h (Control group) after P4 device removal, were FTAI at that time. Cows not designated as showing oestrus at that time received 10 µg Buserelin (GnRH, Sincroforte, Ourofino) and were FTAI 8 h later. In Experiment 3, P/FTAI was compared in cows that received the J-Synch 7-day treatment (n = 265) or the 7-day Control treatment (n = 250). Data were analysed using the GLM mixed procedure (InfoStat). In Experiment 1, the diameter of the ovulatory follicle was larger (P

An experiment was designed to evaluate the effect of the length of insertion of a progesterone (P4)-releasing device on follicular and luteal characteristics and pregnancy rates to fixed-time AI (P/FTAI) in Bos indicus cows treated with oestradiol/P4-based treatments with a prolonged pro-oestrus. Multiparous Bos indicus suckled beef cows (n = 659), 60-90 days postpartum, with a corpus luteum (CL) or at least one follicle ≥8 mm in diameter and with a body condition score between 2 and 3.5 (1-to-5 scale) were synchronised using three treatments. On Day 0, all cows received 2 mg of oestradiol benzoate (Sincrodiol, Ourofino) and an intravaginal device with 1 g of P4 (Sincrogest, Ourofino). The P4 device was removed on Day 6 in cows in the J-Synch 6 group (n = 195) and on Day 7 in cows in the J-Synch 7 group (n = 262) and conventional group (n = 202). All cows received 500 μg of cloprostenol (Sincrocio, Ourofino) and 400 IU of equine chorionic gonadotrophin (SincroeCG 6000UI, Ourofino) at the time of P4 device removal. Cows in the conventional treatment group also received 0.5 mg of oestradiol cypionate (SincroCP, Ourofino) at the same time. In addition, all cows were tail-painted for oestrous detection (CeloTest, Biotay). Cows that had lost ≥50% of the tail paint 62-66 h (J-Synch groups) or 48-52 h (conventional group) after device removal were FTAI at that time. Cows not showing oestrus by 62-66 h (J-Synch groups) or 48-52 h (conventional group) received 10 μg of GnRH (Sincroforte, Ourofino) at the same time and were FTAI 8 h later. A subset of 20 cows per group was also examined using ultrasonography (Mindray DP50 Vet) every 12 h from the time of device removal to determine the time of ovulation, 6 days after ovulation to determine CL diameter, and 30 days after FTAI to determine P/AI. Data were analysed using the GLM Mixed procedure (InfoStat). The results are shown in Table 1. The diameter of the dominant preovulatory follicle was larger and the interval from device removal to ovulation was longer in cows in the J-Synch groups compared with those in the conventional group (P

An experiment was designed to evaluate pregnancy rates per AI (P/AI) in suckled beef cows synchronised with a progesterone (P4)/oestradiol-based protocol and AI with non-sexed (conventional) semen and with a sexed-sorted semen with 65% X-bearing sperm and 35% Y-bearing sperm that was named SuperConventionalTM. Angus and Hereford suckled cows (n=558), 60-90 days postpartum, with a body condition score of 2 to 3.5 (1-to-5 scale) and 48% (269/558) with a corpus luteum, were randomly allocated to be AI with non-sexed (conventional) or SuperConventional semen. The experiment was performed on 3 farms using straws with sexed-sorted SuperConventional semen containing 6 or 8 million sperm and non-sorted conventional semen with 25 million sperm per straw. Semen was from split ejaculates from two Angus bulls. All cows received a P4 device with 0.6g of P4 (Pluselar, Calier) and 2mg of oestradiol benzoate (Calier) on Day 0. All P4 devices were removed on Day 8, and all cows received 400IU of equine chorionic gonadotrophin (Vetegon, Calier), 150µg of d+cloprostenol (Veteglan, Calier), and 1mg of oestradiol cipionate (Calier) at the same time. All cows were also tail-painted and observed for signs of oestrus. Cows with >30% of the tail paint rubbed off by 48-50h after P4 device removal were AI at that time with either SuperConventional or conventional semen. Those not showing oestrus by 48-50h received 10µg of buserelin (Pluserelina, Calier) at that time and were AI with either SuperConventional or conventional semen 50-52h after P4 device removal. Pregnancy was diagnosed using ultrasonography 60 days after AI, and all pregnancies were sexed to determine the proportion of female pregnancies. Data were analysed using GLM for binary data. The P/AI were different between cows showing or not showing oestrus (P

An experiment was designed to evaluate the effect of the length of insertion of a progesterone (P4)-releasing device and the length of pro-oestrus on follicular and luteal characteristics and pregnancy rates to AI (P/AI) in Bos indicus heifers treated with oestradiol/P4-based treatments. Bos indicus beef heifers (n=374), 22-26 months of age, with a corpus luteum (CL) or at least one follicle ≥8mm in diameter and with a body condition score between 2.5 and 3.5 (1-to-5 scale) were synchronised using three treatments for fixed-time AI (FTAI). On Day 0, all heifers received 2mg of oestradiol benzoate (Sincrodiol, Ourofino) and an intravaginal device with 1g of P4 (Sincrogest, Ourofino). The P4 device was removed on Day 6 in heifers in the J-Synch 6 group (n=120) and on Day 7 in heifers in the J-Synch 7 group (n=105) and conventional group (n=165). All heifers received 500μg of cloprostenol (Sincrocio, Ourofino) and 300IU of equine chorionic gonadotrophin (SincroeCG 6000UI, Ourofino) at the time of P4 device removal. Furthermore, heifers in the conventional treatment group received 0.5mg of oestradiol cypionate (SincroCP, Ourofino) at the same time. In addition, all heifers were tail-painted for oestrus detection (CeloTest, Biotay). Heifers that had lost ≥50% of the tail paint by 70-74h (J-Synch groups) or 48-52h (conventional group) after device removal were FTAI at that time. Heifers not showing oestrus by 70-74h (J-Synch groups) or 48-52h (conventional group) received 10μg of gonadotrophin-releasing hormone (Sincroforte, Ourofino) at the same time and were FTAI 8h later. All heifers were also examined using ultrasonography (Mindray DP50 Vet) every 12h from the time of device removal to determine the time of ovulation, 6 days after ovulation to determine the diameter of the CL, and 28 days after FTAI for P/AI determination. Data were analysed using the MLGM procedure (InfoStat) for normal data families (follicular dynamics) and binary data family (P/AI). The results are shown in Table 1. The diameter of the dominant preovulatory follicle and the CL did not differ among groups (P>0.12). However, the interval from device removal to ovulation was longer in heifers in the J-Synch groups than in heifers in the conventional group (P

The objective of the present study was to evaluate the superovulatory response and embryo production in beef donors using 8 twice-daily injections of FSH or an alternative protocol in which the last 4 FSH injections were replaced with a single injection of eCG. In Exp. 1, 12 mature Bonsmara donor cows, with a body condition score between 3 and 4 (1 to 5 scale) were superstimulated twice every 46 days in a crossover design (i.e. in each experiment all cows received 2 treatments and the 2 treatments were equally represented in each replicate). On Day 0 a.m., all donors received an intravaginal device with 1.2g of progesterone (Diprogest 1200®, Zoovet, Santa Fe, Argentina), along with 50mg of progesterone i.m (Progestar®, Zoetis, Buenos Aires, Argentina) and 5mg of oestradiol-17β (17βOestradiol®, Rio de Janeiro, Argentina) IM. On Day 4 a.m., the superstimulatory treatments were initiated and donors in the control group received 8 applications of FSH (Folltropin-V, Vetoquinol, Lure, France), IM (total dose: 300mg NIH-FSH-P1) in a twice-daily decreasing dosage schedule over 4 days (i.e. 60, 60, 50, 50, 30, 30, 10, and 10mg, respectively). Donors in the FSH+800 eCG group received only the first 4 applications of FSH (i.e. 60, 60, 50 and 50mg, respectively) and on Day 6 a.m. they received 800IU of eCG (Novormón®, Zoetis) IM in a single dose. All donors received 500μg of cloprostenol (Ciclase DL®, Zoetis) IM on Day 6 a.m. and p.m., and the intravaginal devices were removed on Day 7 a.m. All cows also received 100μg of gonadorelin acetate (GnRH, Gonasyn gdr, Zoetis) on Day 8 a.m. and were inseminated with frozen-thawed semen from 2 bulls 12 and 24h later. On Day 15, ova/embryos were collected and evaluated according to the IETS standards. The data were analysed by GLMM (Infostat, 2018). In Exp 2., 18 Bonsmara donors with similar conditions as those in Exp. 1 were superovulated twice in a crossover design. Cows in both groups received similar treatments to those in the FSH+eCG treatment of Exp. 1, except that the total dosage of FSH was 200mg (i.e. 60, 60, 40, and 40mg, respectively) and the eCG given on Day 6 a.m. was either 600IU (group 1) or 800IU (group 2). In Exp 1., the FSH (control) group had a higher (P

An experiment was designed to evaluate pregnancy rates (P/AI) in suckled beef cows synchronized with a progesterone (P4)/oestradiol-based protocol (J-Synch) and inseminated (AI) with sexed or nonsexed (conventional) semen. Angus or Angus crossbred cows (n=877), 60 to 90 days postpartum, with a body condition score of 2.5 to 3.5 (scale of 1 to 5) and a corpus luteum or a follicle >8mm in diameter detected by ultrasonography, were randomly allocated to be AI with sexed-sorted or nonsexed (conventional) semen. The experiment was performed in 4 farms, and the sexed-sorted (SexedUltra™ 4M, with 4 million sperm; ST Genetics, Navasota, TX, USA) and conventional (25 million sperm) semen were from split ejaculates from 5 Angus bulls. All cows received a P4 device (DIB 0.5, Zoetis, Villa Adelina, Argentina) and 2mg of oestradiol benzoate (Gonadiol, Zoetis) on Day 0. On Day 6, cows received 400IU of eCG (Novormon 5000, Zoetis) and 500µg of cloprostenol (Ciclase DL, Zoetis) and DIB was removed. All cows were also tail painted and observed for signs of oestrus (i.e. >30% of the tail paint rubbed off) at 60, 72, and 84h after DIB removal. Cows with the tail paint rubbed off by 60 or 72h after DIB removal were artificially inseminated at 72h with either sexed or conventional semen. Those not showing oestrus by 72h received 100µg of gonadorelin acetate (Gonasyn GDR, Zoetis) at that time and were artificially inseminated with either sexed-sorted or conventional semen 12h later (i.e. 84h). Pregnancy was diagnosed by ultrasonography 30 days after AI. Data were analysed by general linear model for binary data with a logit link. Overall, 76.4% (678/877) of cows showed oestrus 60 to 84h after DIB removal, and there were differences in P/AI between sexed-sorted and conventional semen (P

The objective of this study was to evaluate the effect of synchronization treatment and oestrus expression on conception rates and pregnancy losses in recipients receiving in vitro-produced (IVP) embryos. Crossbred beef cows (n = 407) with a corpus luteum (CL) determined by ultrasonography and body condition between 2.5 and 4.5 (1 to 5 scale) received a progesterone-releasing device (DIB 0.5 g, Zoetis, Argentina) and 2 mg of oestradiol benzoate (EB, Gonadiol, Zoetis) on Day 0 and were randomly allocated to 1 of 2 treatments. Recipients in the Conventional group (n = 201) had their devices removed and received 500 µg of cloprostenol (PGF, Ciclase, Zoetis), 0.5 mg of oestradiol cypionate (ECP, Cipiosyn, Zoetis), and 400 IU of eCG (Novormón, Zoetis) on Day 8. Recipients in the J-Synch group (n = 206) had their devices removed and received PGF and eCG on Day 6. All recipients were tail-painted at device removal to determine the presence of oestrus on Day 10 (a.m.) in the Conventional group and on Day 9 (a.m.) in the J-Synch group. All recipients not in oestrus in both groups received 100 µg of gonadorelin (GnRH, Ovurelin, Bayer, New Zealand) at that time. In addition, the paint was observed again in the p.m. and it was recorded. Recipients were examined by ultrasonography 7 days after oestrus or GnRH treatment (Day 16 in the J-Synch group and Day 17 in the Conventional group) and those with a CL = 16 mm in diameter received an IVP fresh embryo non-surgically. Pregnancy was determined by ultrasonography 23 and 53 days after embryo transfer and calving rates were recorded. Data were analysed using generalized linear models for binary data and a logit link (InfoStat, https://www.infostat.com.ar/). The proportion of recipients transferred was higher (P 0.11), but they were lower (P

The adaptation of Bos indicus cattle to tropical and subtropical environments has led to their widespread distribution around the world. Although artificial insemination (AI) is one of the best alternatives to introduce new genetics into Bos indicus herds, the peculiarity of their temperament and the tendency to show short oestrus (many of them during the night) greatly affects the effectiveness of genetic improvement programs. Therefore, the most useful alternative to increase the number of females that are inseminated is the use of protocols that allow for AI without the need for oestrus detection, usually called fixed-time AI (FTAI). Besides, the development of protocols to advance the resumption of cyclicity during the early postpartum period has a great impact on beef production and will allow for the inclusion of a significantly larger population of animals into genetic improvement programs. Fixed-time AI protocols using progestin devices, oestradiol and eCG have resulted in consistent pregnancy rates in suckled Bos indicus and Bos indicus x Bos taurus cows. Furthermore, fertility in the successive cycles and the overall pregnancy rates at the end of the breeding season, have been shown to be improved by the use of progestin devices at the beginning of the breeding season. In summary, exogenous control of luteal and follicular development has facilitated the application of assisted reproductive technologies in Bos indicus-influenced cattle, by offering the possibility of planning programs without the necessity of oestrus detection and may provide the opportunity to improve reproductive performance of beef cattle in tropical climates.

We have previously shown that expression of oestrus significantly influences the time of ovulation and pregnancy rates to timed-AI (P/TAI) in beef cattle synchronized with progesterone devices and oestradiol (Bó et al. 2016 Theriogenology 86, 388–396). Furthermore, delaying TAI from 48 h to 54–58 h after device removal improved P/TAI of those animals not showing oestrus by 48 h. The objective of the present study was to determine whether the administration of GnRH to those animals not showing oestrus by 48 h after device removal had an impact on P/TAI. Non-lactating and suckled beef cows and heifers (Bonsmara, Brangus, and Braford; n = 868), with a corpus luteum (CL) or a follicle ≥8 mm in diameter detected by ultrasonography (Mindray DP 30 Vet, 7.5 MHz, China) and body condition score 2 to 4 (1 to 5 scale) were synchronized in 3 replicates. On Day 0, animals received a progesterone device (DIB 0.5 g, Zoetis, Argentina) and 2 mg of oestradiol benzoate (Von Franken, Argentina). On Day 8, DIB were removed and cows received 0.150 mg of D+cloprostenol (Río de Janeiro, Argentina) plus 0.5 mg oestradiol cypionate (Cipiosyn, Zoetis). In addition, all cows were tail-painted on the sacrococcygeal area (CeloTest, Biotay, Argentina) at DIB removal and were observed to determine the expression of oestrus, according to the percentage of paint loss (PL) 48 h later. The animals that showed oestrus (PL > 30%) were recorded and TAI at that time while those with PL ≤ 30% (not in oestrus) were randomly allocated to receive 100 μg of gonadorelin (GnRH; Gonasyn, Zoetis) or no treatment and TAI between 54 to 58 h after DIB removal. Pregnancy was diagnosed by ultrasonography at 45 days after TAI, and data were analysed by logistic regression. There were no significant differences among replicates, inseminators, semen source or animal class (dry cows, suckled cows or heifers; P > 0.11). However, animals showing oestrus by 48 h after DIB removal had higher P/TAI (371/666; 55.7; P

A retrospective analysis of factors that affect pregnancy rates from 4214 fresh in vitro-produced (IVP) embryos that were transferred at a fixed-time (FTET) in 20 different farms. Recipients were all cycling cows or heifers that were synchronized with 1 of 3 treatments: 1) treatments with progesterone (P4) devices and 2 mg of oestradiol benzoate (EB) on Day 0 (day of insertion) and 24 h after device removal (Day 8); 2) treatments with P4 devices and EB on Day 0, but with 0.5 mg of oestradiol cypionate (ECP) at device removal (Day 8); or 3) treatments with P4 devices and GnRH on Day 0 and a second GnRH 60 h after device removal (Day 5). Cows in all treatment groups also received 500 µg of cloprostenol (prostaglandin F2α) at the time of P4 device removal and 400 IU of eCG either at device removal or 3 days before device removal. All embryos were transferred 7 or 8 days after the expected time of oestrus (24 h after EB, 48 h after ECP or at the time of the second GNRH for each synchronization treatment, respectively). On the day of embryo transfer, recipients were examined by ultrasonography and those with corpus luteum >14 mm in diameter received a fresh, IVP embryo in the uterine horn ipsilateral to the corpus luteum. Pregnancy rates were determined by ultrasonography 35 days after FTET. Data were analysed by logistic regression. Independent variables were classified into the following three categories. 1) Factors related to the recipient and the environment; there were no significant differences in pregnancy rates for corpus luteum diameter (≥14 and

A retrospective analysis of in vitro production (IVP) data was done to determine the influence of breed and season on the production of viable oocytes and embryos. Cumulus‐oocyte complexes (COC) were obtained from 1946 ultrasound-guided follicle aspiration (ovum pickup) sessions performed at random stages of the oestrous cycle without superstimulation in Bos taurus and Bos indicus donors in commercial IVP in Argentina. Frozen-thawed conventional semen was used in beef cattle and conventional (n = 139) and sexed-selected (n = 481) semen in dairy cattle. The COC were classified, matured in B-199 medium, fertilized in IVF-SOF medium (Day 0), and cultured in SOF medium supplemented with 0.4% BSA under oil at 38.8°C, 5.5% CO2, and saturated humidity for 7 days. The number of viable COC and transferable embryos in each breed and season were compared by ANOVA and means were compared by Fisher’s Least Significant Difference test. Proportions were first transformed by arcsin and then analysed by ANOVA. To simplify the interpretation of the results, breeds were grouped as follows: dairy Bos taurus (Holstein, n = 620), beef Bos taurus (Angus and Bonsmara, n = 229), Bos taurus × Bos indicus (Brangus and Braford, n = 1045), and Bos indicus (Brahman, n = 52). There was no interaction between breed and season for any of the end points analysed (P > 0.1). Mean (± standard error of the mean) numbers of viable COC and transferable embryos were higher (P

Superovulation protocols have improved greatly since the early days of bovine embryo transfer when purified gonadotrophins were not available, follicular wave dynamics were unknown physiological phenomena and prostaglandins were not available. Although superstimulatory protocols in cattle are normally initiated mid-cycle, elective control of follicular wave emergence and ovulation have had a great impact on the application of on-farm embryo transfer. However, the most common treatment for the synchronisation of follicular wave emergence involves the use of oestradiol, which cannot be used in many parts of the world. Therefore, the need for alternative treatments has driven recent research. An approach that has shown promise is to initiate follicle-stimulating hormone (FSH) treatments at the time of the emergence of the new follicular wave following ovulation induced by gonadotrophin-releasing hormone. Alternatively, it has been shown that it may be possible to ignore follicular wave status and, by extending the treatment protocol, induce subordinate follicles to superovulate. Finally, the short half-life of pituitary FSH necessitates twice-daily treatments, which are time-consuming, stressful and subject to error. Recent treatment protocols have permitted superstimulation with a single FSH treatment or two treatments 48 h apart, reducing the need for animal handling during gonadotrophin treatments.

Although embryo transfer technology has been used commercially in cattle for many years, the inefficiency of oestrus detection, especially in recipients, has limited the widespread application of this technology. The most useful alternative to increase the number of recipients utilised in an embryo transfer program is the use of protocols that allow for embryo transfer without the need for oestrus detection, usually called fixed-time embryo transfer (FTET). Most current FTET protocols are based on progestin-releasing devices combined with oestradiol or GnRH, which control and synchronise follicular wave dynamics and ovulation. Conception rates to a single FTET have been reported to be similar to those after detection of oestrus, but pregnancy rates are higher because these treatments have increased the proportion of recipients that receive an embryo. Recent changes to treatments for FTET, such as the administration of eCG, have resulted in increased pregnancy rates and provide opportunities to make these treatments easier to perform on farm.

An oestradiol-based protocol (named J-Synch) has been developed recently with the aim of providing for a longer proestrus, enhancing the development of the dominant follicle before ovulation, and increasing pregnancy rates following fixed-time AI (FTAI) in heifers (de la Matta and Bo 2012 Taurus 55, 17–23). Two experiments were performed in Uruguay with 1953 Angus × Hereford crossbred heifers to compare pregnancy rates obtained with the J-Synch protocol with the conventional 7-day oestradiol-based protocol. A secondary objective was to determine the effects of the time of FTAI and the addition of eCG at the time progestin device removal on pregnancy rates. In Experiment 1, 966 heifers received a DIB device (0.5 g of progesterone, Syntex, Buenos Aires, Argentina) plus 2 mg of oestradiol benzoate IM (Syntex) on Day 0. Heifers in the conventional treatment group (n = 485) received cloprostenol (500 μg, Ciclase DL, Syntex) and oestradiol cypionate (0.5 mg, Cipiosyn, Syntex) IM and had their DIB removed on Day 7 a.m. Heifers in this group were then subdivided to be FTAI on Day 9 a.m. or p.m. (i.e. 48 or 56 h after DIB removal). Heifers in the J-Synch treatment group (n = 481) received cloprostenol IM and DIB removal on Day 6 p.m. and received gonadotropin-releasing hormone (100 μg gonadorelin acetate, Gonasyn Gdr, Syntex) on Day 9 a.m.; heifers were then FTAI on Day 9 a.m. or p.m. (i.e. 60 or 72 h after DIB removal). All heifers in this experiment were also treated with 300 IU of eCG (Novormon, Syntex) at DIB removal. In Experiment 2, 987 heifers were treated with the J-Synch protocol as described in Experiment 1. At device removal (Day 6 p.m.), heifers were divided to receive (n = 488) or not (n = 499) 300 IU of eCG IM at that time and were further subdivided to receive gonadotropin-releasing hormone and FTAI on Day 9 a.m. or p.m. (i.e. 60 or 72 h after DIB removal). Pregnancy rates were determined by ultrasonography 30 days after FTAI. Data were analysed using logistic regression, and results are shown in Table 1. J-Synch-treated heifers tended (P

An embryo transfer program in cows relies on the superovulatory (SPO) response after receiving a hormonal treatment. Anti-Müllerian hormone (AMH) has been proposed as an endocrine marker associated to superovulation; however, the SPO response of donors has also been influenced by the environment. Thus, objective herein was to evaluate effects of serum AMH levels on number of ovarian follicles (NOF), corpora lutea (CL), and transferable embryos (TE) in superovulated donor cows managed under summer or winter conditions in northwest Mexico. Fifty-two cows in a random stage of the oestrous cycle were used in this study. All cows received an Eazi-Breed CIDR cattle insert (1.384 g of progesterone; Zoetis, USA), and 2.76 mg of oestradiol benzoate (Sigma-Aldrich, St. Louis, MO, USA) plus 50 mg of progesterone (Vetoquinol, Canada) given i.m. to synchronize follicular wave emergence (Day 0). The SPO treatment started on Day 4 with decreasing doses (120, 80, 40, and 20 mg) of Folltropin-V (Vetoquinol, Canada) injected twice daily until Day 7. Artificial insemination was performed on days 8 p.m. and 9 a.m. and ova/embryos were collected on Day 15. Transrectal ultrasonography was done on Days 8 and 15 to determine the number of follicles >9 mm in diameter on Day 8 and CL on Day 15, respectively. Blood samples were collected on the first day of SPO treatment (Day 4) to measure serum AMH concentrations, which served to classify cows as high- (>400; n = 29) and normal-AMH donors (100–400 pg mL–1; n = 23). A mixed effects model was used to analyse SPO response in cattle including level of AMH and season as fixed effects, age as covariate, and sire as random. Level of AMH, season, and their interaction resulted as significant (P 0.05). Results from this study indicated that SPO response in donor cows is associated with serum concentrations of AMH; however, the relationship among these variables appears to be affected in the summer when drought and severe heat stress are common. We concluded that under winter conditions of northwest Mexico, the serum levels of AMH should be considered as an endocrine marker associated with ovarian traits indicative of the SPO response in donor cows.

Although oestradiol has been used successfully to synchronize follicle wave emergence for superovulation, it cannot be used in many countries. Attention has turned to alternatives, including the use of GnRH to induce ovulation of a dominant follicle, which will be followed by emergence of a new follicular wave in 1 to 2 days. However, GnRH synchronizes follicular wave emergence only when it induces ovulation and administration of GnRH at random stages of the oestrous cycle results in ovulation in less than 60% of animals. The objective of the study was to compare superovulatory response and ova/embryo production following synchronization of follicle wave emergence for superovulation with GnRH administered 2 days after insertion of a progestin device, with a protocol in which GnRH is administered 7 days after administration of prostaglandin F2α and a progestin device. Beef donors of various breeds were placed at random into 1 of 2 groups and superstimulated by replicate so that one cow in each group had ova/embryos collected on the same day. Sixty-six superstimulations were performed in this study; 26 were performed in 13 donors that were superstimulated twice in a crossover design, and 40 donors were superstimulated once (i.e. 20 donors in each treatment group). Cows in group 1 received CIDR devices (Zoetis Animal Health, USA) on Day –2 and 100 μg of GnRH (Cystorelin, Merial USA) on Day 0; FSH treatments were initiated on Day 2 with 288 mg of Folltropin-V (Vetoquinol, Canada) given in twice-daily decreasing doses for 4 days. Prostaglandin F2α (PGF; 35 mg dinoprost, Lutalyse, Zoetis) was given with the last 2 injections of Folltropin-V and CIDR were removed with the last Folltropin-V administration (i.e. Day 6). Donors received a second GnRH at the onset of oestrus and were AI 8 and 20 h later. Donors that were still in standing oestrus at the second AI were AI again at 30 h. Ova/embryo collections were done on Day 14 and embryos were classified according to the IETS manual. Cows in group 2 received an injection of PGF and a CIDR on Day –7 and 100 μg of GnRH on Day 0; FSH treatments were initiated on Day 2 and the remainder of the treatment protocol was as in group 1. Data (total ova/embryos collected and transferable embryos) were analysed by ANOVA for mixed models, using treatment as a fixed variable and cow (i.d.) as a random variable. The group 1 cows produced a mean (± s.e.m.) of 18.6 ± 1.9 total ova/embryos of which 12.7 ± 1.5 were of transferable quality (7.2 ± 1.3 grade 1). Cows in group 2 produced a mean (± s.e.m.) of 19.5 ± 1.7 total ova/embryos, of which 14.8 ± 1.5 were of transferable quality (8.9 ± 1.2 grade 1). Although 2 more transferable embryos were obtained in group 2, differences were not significant (P > 0.3). At the same time as this experiment was done, 214 other cows were superstimulated in this practice, yielding an average of 7.9 transferable embryos per donor. Results suggest that both approaches are efficacious for the superstimulation of beef cows.Special thanks to Vetoquinol/Bioniche Animal Health, USA for support.

Our expanding knowledge of the control of follicular wave dynamics during the bovine estrous cycle has resulted in renewed enthusiasm for the prospects of precisely controlling the follicular and luteal dynamics and finely controlling the time of ovulation. Follicular wave development can be controlled mechanically by ultrasound-guided follicle ablation or hormonally by treatments with GnRH or estradiol and progestogen/progesterone in combination. Treatment of cattle with GnRH in combination with prostaglandin F2 alpha (PGF) 7 d later and a second GnRH 48 h after PGF (known as Ovsynch) has resulted in acceptable pregnancy rates after fixed-time AI in lactating dairy cows and in recipients in which embryos were transferred without estrus detection. Alternatively, treatments with estradiol and progestogen/progesterone-releasing devices resulted in synchronous emergence of a new follicular wave and, when a second estradiol treatment was given 24 h after device removal, synchronous ovulation and high pregnancy rates to fixed-time AI. Self-appointed embryo transfer (without estrus detection) using estradiol and progesterone treatments have resulted in pregnancy rates comparable with those obtained with recipients transferred 7 d after estrus. Furthermore, estradiol and progesterone treatments combined with PGF and eCG (given 1 d after the expected time of wave emergence) have resulted in high rates of recipients selected for transfer (84.6%) and an overall pregnancy rate of 48.7% (recipients pregnant/recipients treated). Estradiol and progestogen/progesterone treatments have also been widely used for self-appointed superstimulation protocols with equivalent embryo production to that of donor cows superstimulated using the traditional approach beginning 8 to 12 d after estrus. In summary, exogenous control of luteal and follicular development facilitates the application of assisted reproductive technologies in cattle by offering the possibility of planning the superstimulation of donors and synchronization of recipients at a self-appointed time, without the necessity of estrus detection and without sacrificing results.

Several studies, performed mainly in North America, have shown that the 5-day co-synch timed-AI (TAI) protocol results in similar or higher pregnancy rates than conventional 7-day co-synch protocols in beef cows and beef and dairy heifers. It is hypothesised that the 5-day co-synch protocol reduces the incidence of persistent follicles and provides for a longer proestrus, with increasing oestradiol concentrations due to continuous gonadotropin support for the dominant follicle (Bridges et al. 2008 Theriogenology 69, 843–851). Similar findings have been reported recently following the use of a shortened oestradiol-based protocol that also provides for a longer proestrus (named J-synch; de la Matta and Bo 2012 Taurus 55, 17–23). A study was designed to compare these 2 treatment protocols for synchronization of ovulation that allow for a prolonged proestrus with the conventional oestradiol-based protocol currently used for TAI in South America for heifers. Cycling Holstein heifers that were, on average, 17 months of age, weighting 350 to 380 kg, and with a body condition score of 2.5 to 3.5 (scale of 1 to 5), were randomly allocated to 1 of 3 treatment groups. Heifers in the 7-day estradiol benzoate (EB) group received a progesterone device (DIB 1 g of progesterone; Syntex SA, Buenos Aires, Argentina) and 2 mg EB (oestradiol benzoate, Syntex SA) on Day 0, 500 μg of cloprostenol (prostaglandin F2α; Sincronil, Proagro SA, Argentina) and DIB removal on Day 7, 1 mg of EB on Day 8, and TAI 30 h later (54 h after DIB removal). Heifers in the J-synch group received a DIB and 2 mg of EB on Day 0, PGF and DIB removal on Day 6, and 10 μg of buserelin acetate [gonadotropin-releasing hormone (GnRH); Receptal, Intervet, Millsboro, MD, USA] and TAI on Day 8 (72 h later). Heifers in the 5-day co-synch group received a DIB and GnRH on Day 0, DIB removal and prostaglandin F2α (twice, 12 h apart) on Day 5, and GnRH along with TAI on Day 8 (72 h later). Treatments were coordinated so that DIB were removed in all groups at the same time to decrease a possible time effect of follicle parameters and pregnancy rates. Two experiments were conducted; the first evaluated follicular dynamics and ovulation rates by twice daily ultrasonography (Honda 101 V, 5.0 MHz) in 20 heifers per group and the second determined pregnancy rates to TAI in 172 heifers. The mean (± standard error of the mean) interval from DIB removal to ovulation was shorter in the 7-day EB group (82.7 ± 3.1 h) than in the J-synch (103.8 ± 3.3 h) and 5-day co-synch (96.8 ± 3.3 h) groups (P = 0.01). However, the mean diameter of the dominant preovulatory follicle was smaller (P = 0.01) in the 7-day EB (12.8 ± 0.5 mm) and the J-synch (13.1 ± 0.6 mm) groups than in the 5-day co-synch group (14.9 ± 0.6 mm). Although pregnancy rates in the second experiment were numerically lower in the 7-day EB group (31/58; 53.4%) and 5-day co-synch group (30/55; 54.5%) than in the J-synch group (37/59; 62.7%), differences were not significant (P > 0.3). Preliminary results indicate that the 3 protocols evaluated result in comparable pregnancy rates to TAI in dairy heifers.

Two experiments were designed to evaluate the effect of administering 1 or 2 doses of prostaglandin F2α (PGF) at the time of ova/embryo collection on luteal regression and return to oestrus and ovulation in donor cows. In Experiment 1, 14 Angus cows were blocked at the time of embryo collection by the number of corpora lutea (CL) determined by rectal palpation [6 to 10 CL (n = 6), 11 to 15 CL (n = 4), >15 CL (n = 4)] and were randomly assigned to 2 groups to receive a single injection of 500 µg of sodium cloprostenol (PGF, Ciclase, Syntex S.A., Buenos Aires, Argentina) on the day of embryo collection (Day 0) or a single injection of PGF on Day 0 and a second injection of PGF 24 h later. All cows were bled daily to determine progesterone concentrations by RIA and ultrasonically scanned daily (Falco 100, Pie Medical, Maastricht, the Netherlands) for 19 days after embryo collection, to determine ovulation of a newly recruited dominant follicle. In Experiment 2, 47 superovulated Angus cows were blocked by CL numbers [1 to 4 CL (n = 5), 5 to 10 (n = 11), 11 to 15 (n = 13), >15 CL (n = 18)] and treated as in Experiment 1. In this experiment, cows were bled and scanned on Days 0 (i.e. embryo collection), 4, 7, 10, 13, 16, and 19. The interval from embryo collection to luteal regression (i.e. plasma progesterone 0.5). The interval from treatment to luteal regression was not affected by PGF treatment (1 PGF: 2.0 ± 1.2 days; 2 PGF: 2.4 ± 1.0 days). Furthermore, 6/7 animals (85.7%) were observed in oestrus in both groups, and 6/7 (85.7%) and 4/7 (57.1%) ovulated within 19 days in the 1 PGF and 2 PGF groups, respectively (P > 0.2). The interval to oestrus and ovulation did not differ among groups (7.0 ± 0.7 days and 8.5 ± 0.6 days v. 9.7 ± 0.5 days and 10.0 ± 0.7 days, for 1 PGF and 2 PGF, respectively; P > 0.1). In Experiment 2, the mean number of CL in donor cows did not differ from that in Experiment 1 (13.9 ± 1.1; P > 0.37), and the effect of CL numbers on luteal regression and ovulation was not significant (P > 0.7). Although luteal regression occurred earlier (P = 0.03) in the 2 PGF group (4.1 ± 0.6 days) compared with the 1 PGF group (5.3 ± 2.2 days), no differences were detected between PGF groups in any of the other parameters. The proportion of cows in oestrus and the interval to oestrus was 19/24 (79.2%) and 7.2 ± 0.4 days v. 18/23 (78.3%) and 7.2 ± 0.3 days for cows in the 1 PGF and 2 PGF groups, respectively (P > 0.7). Finally, the proportion of cows ovulating within 19 days after collection and the interval to ovulation were 20/24 (83.3%) and 10.3 ± 0.4 days v. 21/23 (91.3%) and 10.1 ± 0.4 days for cows in the 1 PGF and 2 PGF groups, respectively (P > 0.4). In summary, treatment with PGF at the time on embryo collection results in luteal regression in 4 to 5 days and ovulation of a new dominant follicle in 80 to 90% of the donors within 19 days. The addition of a second PGF treatment did not shorten the interval to oestrus and ovulation.

An experiment was designed to compare pregnancy rates in lactating dairy cows synchronized with a 7-day CIDR-Synch or a 5-day CIDR-Synch program and to determine if the addition of a second prostaglandin F2α (PGF) injection to the 7-day CIDR-Synch program would improve pregnancy rates following fixed-time AI (FTAI). The experiments were performed on 2 dairy farms in Argentina, with year-round calving and a mixed feeding system (35% grazing plus 65% corn silage and grain). Cows (n = 621) were 39.3 ± 6.5 days in milk (DIM, mean ± SD) when they were enrolled in the program, had 2.4 ± 1.5 lactations and a body condition score (BCS) of 3.1 ± 0.2 (range: 2.7 to 4.0). All cows received a pre-synchronization treatment with 2 doses of prostaglandin (PGF, 25 mg of dinoprost, Lutalyse, Pfizer Animal Health, Argentina) 14 days apart, and 11 days after the second PGF (Day 0) received 10 µg of Buserelin (GnRH, Receptal, MSD-Intervet, Argentina) and a CIDR device (1.9 g of progesterone, Pfizer Animal Health). Cows were randomly allocated to 1 of 3 groups. The CIDR devices were removed and PGF was administered to cows in Groups 1 and 2 on Day 7. A second GnRH was given 56 h later and cows experienced FTAI 16 h after gonadotropin-releasing hormone (GnRH) injection (i.e. 72 h after CIDR removal). Cows in Group 2 also received a second PGF injection on the afternoon of Day 7. Cows in Group 3 had the CIDR removed and received 2 PGF injections 12 h apart on Day 5. A second dose of GnRH was given and FTAI was performed at the same time, on Day 8 (i.e. 72 h after CIDR removal). All cows were examined by ultrasonography (Aloka 500V, Aloka, Tokyo, Japan) on the day of the first PGF injection and at CIDR removal to determine the presence and number of corpora lutea (CL), and 30 days after FTAI to determine pregnancy status. Data were analyzed by logistic regression to determine the effects of treatment, parity, days postpartum, milk production, BCS, presence of a CL at enrollment, and number of CL at the time of CIDR removal on pregnancy rates. Overall pregnancy rates did not differ among groups: 32.9% (68/207), 38.2% (78/204), and 38.3% (80/209) for Groups 1, 2, and 3, respectively (P = 0.2). Although the number of CL present at CIDR removal did not significantly affect pregnancy rates (P = 0.4), pregnancy rates in cows with 1 CL in Groups 1 and 2 tended to differ [29.0% (11/38) v. 48.9% (21/43); P 3 had 1.63 (1.16–2.28) times more chance of getting pregnant than those with BCS

An experiment was designed to evaluate the effect of brilliant cresyl blue (BCB) selection of immature oocytes and the addition of sodium hyaluronate (HA) to the vitrification solution on survival rates of bovine oocytes vitrified using solid-phase vitrification. Bovine cumulus–oocyte complexes (COC; n = 716) obtained from slaughterhouse ovaries were used in 6 replicates. Cumulus–oocyte complexes were washed in tissue culture medium 199 (TCM-199) and randomly allocated to 2 groups to be exposed to BCB stain (Sigma Chemical Company, St. Louis, MO, USA) for 90 min as described by Alm et al. (2005 Theriogenology 63, 2194–2205) or (control) maintained in Vigro holding medium (Bioniche Animal Health, Belleville, Canada) for 90 min (n = 220). Cumulus–oocyte complexes in the BCB group were selected based on their response to BCB as BCB+ (colored, n = 248) or BCB– (colorless, n = 248), whereas those in the control group were selected morphologically as described by Rodríguez-González et al. (2002 Theriogenology 57, 1397–1409). Oocytes from both BCB groups and 100 oocytes in the control group were vitrified by solid-phase vitrification as previously described by Rodriguez et al. (2012 Reprod. Fertil. Dev. 24, 132). The remaining 120 oocytes in the control group were not vitrified and were matured, fertilized, and cultured in vitro (in SOFaa in a controlled atmosphere) for 7 days. Vitrified oocytes were exposed to 10% ethylene glycol for 10 min, and 20% ethylene glycol + 0.2-M trehalose for 30 s, and then were subdivided to be exposed to 30% ethylene glycol + 0.5-M trehalose with or without 0.1 mg mL–1 HA (MAP 5, Bioniche Animal Health). Vitrified oocytes were stored in liquid nitrogen for at least one week and then placed directly into a 0.5-M sucrose solution (in TCM 199) at 37°C for 5 min, 0.25 M of sucrose for another 5 min, and finally TCM-199 and matured, fertilized, and cultured. Development rates (i.e. proportion of blastocysts) were examined on Day 7 after fertilization. Proportional data were first transformed by square root and then analyzed by ANOVA to detect the effect of replicate, type of oocyte (BCB+, BCB–, controls), and vitrified with or without HA or not vitrified as main effects, using the software Infostat (UNC, Argentina, 2010). There was a significant effect of oocyte type on blastocyst rate (P

The objectives of this study were to evaluate the reproductive and productive performance of dairy cows with and without puerperal metritis and to evaluate the effectiveness of using a long-acting antibiotic (200 mg mL–1 ceftiofur, Excede, Pfizer Animal Health, New York, NY, USA). Dairy cows in one dairy farm calving from July 2009 to January 2010 were examined between 3 and 14 days postpartum and classified based on vaginal discharge into 3 groups: cows with normal discharge (control); cows with purulent or red-brown vaginal discharge without foul smell, and cows with purulent or red-brown vaginal discharge with foul smell (Sheldon et al. 2006 Theriogenology 65, 1516–1530). Cows in the first 2 groups were not treated, whereas those in the third group were randomly allocated to receive 1 mL/30 kg of body weight of Excede subcutaneously behind the ear or remain untreated. From the 640 cows examined, 372 (58.2%) had normal discharge, 86 (13.4%) had a discharge without foul smell and 182 (28.4%) had a discharge with foul smell. Kaplan-Meier survival curves were obtained to compare pregnancy rates across days postpartum, and differences between curves were tested with the log rank statistic. Proportional hazards regression with PROC PHREG Cox of SAS were done to estimate the risk of pregnancy in terms of treatment, parity (first parity or ≥2 parities), type of delivery (normal or assisted), and milk production. Finally, lactation curves were modeled using SAS PROC NLMIXED to estimate the cumulative milk production at 305 days. Survival curves differed between cows in the control group and those with foul-smell discharge that were not treated (P

Although slow programmable freezing is currently the standard method for bovine embryo cryopreservation, vitrification has become an alternative for in vitro-produced embryos. A study was designed to compare the in vitro survival rates of in vivo- and in vitro-produced bovine embryos with 1 of 2 commercially available methods of cryopreservation: slow freezing and solid surface vitrification. In vivo-produced Grade 1 blastocysts (n = 210) collected from superovulated donor cows 7 days post-insemination and in vitro-produced Grade 1 blastocysts (n = 122) from slaughterhouse oocytes, produced with the procedure described by Chaubal et al. (2007 Theriogenology 67, 719–728) were randomly allocated in 2 groups. Group 1 (slow freezing) embryos were exposed to 1.5 M ethylene glycol (ViGro EG; Bioniche Animal Health USA Inc., Pullman, WA, USA) for 5 min and loaded in 0.25-mL plastic straws. The straws were placed in a Freeze Control CL 5500 freezer (CryoLogic, Victoria, Australia) at –6.5°C, seeded and after 10 min of equilibration, cooled at –0.6°C min–1 until –CE°C, before plunging into liquid nitrogen. Group B (vitrification) embryos were exposed to a AE% EG+0.BEM trehalose solution for A min and then into C0% EG+AM trehalose solution for C0 sec at room temperature to be vitrified using the CVM system (CryoLogic). The CVM used a cryohook and the solutions with the embryos are exposed to a metal solid surface cooled at –AIF°C. The vitrification solution was chosen after a toxicity test in which several EG and trehalose combinations were tested (Rodriguez Villamil et al. Ith IRAC Symposium, Argentina B0AA). After at least 1 wk of storage, embryos in the slow freezing groups were thawed in water bath at C0°C for AB s, placed in holding medium for E min and then cultured in SOF. Vitrified embryos were placed directly in a 0.BE M sucrose solution for E min (at CG°C) and then cultured in SOF medium. Re-expansion and hatching rates were evaluated at BD and GB h, respectively. Data was analyzed by nonparametric tests with type of embryo and cryopreservation procedure as main effects, using the software Infostat (UNC, Argentina, B0A0). In vivo-produced embryos had higher (P

The aim of this study was to compare the effects of 2 different commercially available spermatozoa separation techniques, Isolate® (Irving-Scientific, Santa Ana, CA, USA) and Percoll® (Nutricell, São Paulo, Brazil), on sperm quality and in vitro embryo production using sexed and nonsexed semen. Oocytes (n = 5046) were obtained from slaughterhouse ovaries and fertilized with frozen-thawed sexed or nonsexed semen from the same 4 Holstein bulls. The experiment was done in 10 replicates, with all treatment groups included. Sperm quality (motility, concentration, morphology and membrane integrity) was evaluated and compared before and after sperm selection by the 2 methods. Oocytes were maturated in TCM-199 supplemented with 0.4% of BSA for 24 h in a controlled atmosphere and then selected and randomly allocated into 4 different groups. Group 1: oocytes fertilized with sexed semen selected by Percoll®; Group 2: oocytes fertilized with sexed semen selected by Isolate®; Group 3: oocytes fertilized with nonsexed semen selected by Percoll®; Group 4: oocytes fertilized with nonsexed semen selected by Isolate®. Fertilization was performed in Fert-TALP medium for 18 h under the same conditions as maturation. Presumptive zygotes were cultured for 7 days in SOF medium in a 39°C humidified incubator with 5% CO2, 5% O2 and 90% N2. Cleavage and blastocyst rates were evaluated on Day 2 and 7, respectively, after fertilization. Proportional data were transformed by square root and then analysed by ANOVA, with type of semen and sperm selection method as the main effects. Regardless of the sperm selection technique, sperm motility and percentage of normal sperm increased (P

Commercial embryo transfer programs require frequent superovulation of embryo donors. Although early reports suggested that donor cows require 60 to 90 days to recover from superovulation, recent information suggests that this interval could be reduced to 25 to 30 days. Because donor cows reovulate at variable intervals after embryo collection, some donors do not have an ultrasonically detectable corpus luteum (CL) when frequent superovulation programs are initiated. A retrospective study was performed to evaluate the influence of the presence of a CL at the time of initiating treatments on superovulatory response of beef donors that had been previously collected once and then were collected every 28 to 35 days 2 or 3 times. The data were from 190 collections performed in 48 Angus, 36 in 10 Brangus and 74 in 20 Bonsmara donors. On Day 0, all cows were ultrasonically examined for the presence of a CL (Falco 100 Vet, 8-MHz transducer, Pie Medical, the Netherlands) and were treated with 5 mg of oestradiol-17β and 50 mg of progesterone IM and a progesterone-releasing intravaginal device (Cue-Mate, Bioniche Animal Health, Belleville, Ontario, Canada). On Day 4, donors were superstimulated with Folltropin-V (Bioniche Animal Health), in twice-daily injections over 4 days or diluted in 20 mg mL–1 hyaluronan and given by a single IM injection. Folltropin-V dosages used were 300 or 400 mg (Angus), 260 or 300 mg (Brangus), or 200 or 300 mg (Bonsmara). On Day 6, all cows received 2 doses of PGF2α 12 h apart and Cue-Mates were removed in the p.m. In the a.m. of Day 8, cows received 12.5 mg pLH (Lutropin-V; Bioniche Animal Health) and were inseminated 12 and 24 h later. Ova/embryos were collected nonsurgically on Day 15 and evaluated following IETS guidelines. For each breed, superovulatory response (i.e. mean number of CL) and embryo data were evaluated by ANOVA for mixed models, using CL, FSH dose and treatment as fixed variables and cow identification as a random variable. Forty-six (24.2%) Angus donors, 4 (11.1%) Brangus donors and 5 (6.8%) Bonsmara donors did not have a CL at the time of initiating FSH treatment. There was no significant effect of FSH dose or treatment protocol on superovulatory response and the presence or absence of a CL did not influenced embryo production (P > 0.31). Mean (± SEM) number of CL, ova/embryos and transferable embryos were 13.2 ± 0.5, 12.4 ± 0.7 and 6.1 ± 0.4 in Angus donors with a CL vs 13.0 ± 0.7, 11.5 ± 0.9 and 5.6 ± 0.7 for those without a CL (P > 0.37); 14.8 ± 1.2, 14.2 ± 1.6 and 8.4 ± 1.1 for Brangus donors with a CL vs 15.2 ± 3.1; 11.2 ± 3.3 and 5.7 ± 2.8 for those without a CL (P > 0.34); and 13.2 ± 0.8, 11.2 ± 1.0 and 7.4 ± 0.5 for Bonsmara donors with a CL vs 17.4 ± 3.7, 13.6 ± 6.5 and 9.2 ± 4.3 for those without a CL (P > 0.16). In summary, the presence or absence of a CL did not affect embryo production in donor cows superstimulated every 28 to 35 days using a progesterone-releasing device and FSH. Study was supported by Bioniche Animal Health, Belleville, Ontario, Canada.

An important proportion of embryonic deaths in cattle occur during the critical period of embryo development and maternal recognition of pregnancy. High serum progesterone concentrations during this period are associated to higher embryo survival, and the use of eCG could enhance luteal activity. Two experiments were carried out to evaluate the effect of administering eCG at the time of removal of a progesterone-releasing device and 14 days after fixed-time artificial insemination (FTAI) in beef cattle. Multiparous suckled cows between 60 and 90 days postpartum (n = 297; Exp. 1), and two-year old prepuberal heifers (n = 689; Exp. 2) with a body condition score (BCS) between 3.5 and 5 (1 to 8 scale) were used. At the beginning of the experiments, none of females used had a corpus luteum detected by ultrasonography. Females received an intravaginal device containing 0.5 g progesterone (DIB 0.5, Syntex, Buenos Aires, Argentina) and 2 mg oestradiol benzoate i.m. (Benzoato de Oestradiol, Syntex). Eight days later, 500 μg of cloprostenol (Ciclase DL, Syntex) and 0.5 mg oestradiol cypionate (Cipiosyn, Syntex) were given i.m. and devices were removed; FTAI was performed 52 to 56 h after DIB removal. At the time of device removal, the animals were randomly allocated to 4 groups, to receive 400 IU of eCG i.m. (Novormon, Syntex) or placebo at device removal and/or 14 days after FTAI, in a 2 × 2 factorial design. Control group did not receive eCG. In Exp. 1, nose plates were placed on calves on the day of DIB insertion and left for 10 days in order to prevent suckling. Pregnancy diagnosis was performed by ultrasonography at 30 and 60 days after FTAI. Data were analysed by logistic regression. Pregnancy rate at 30 days after FTAI was affected by treatment (P

We have previously shown that superstimulation of beef donors with a single intramuscular (IM) injection of Folltropin-V (Bioniche Animal Health, Belleville, Ontario, Canada) diluted in a proprietary slow-release formulation (SRF; Bioniche Animal Health) results in in vivo embryo production comparable with the traditional twice-daily IM administration over 4 days (Tribulo et al. 2010 Reprod. Fertil. Dev. 22, 367). Two experiments were designed to evaluate oocyte recovery and in vitro blastocyst production of oocyte donors superstimulated with Folltropin-V, administered in twice-daily injections or diluted in SRF and given by a single injection. In Experiment 1, crossbred beef cows (n = 11) and heifers (n = 5) were randomly allocated to 3 treatment groups in a crossover design (i.e. all animals were treated 3 times, and all treatments were represented in the 3 replicates). All donors had all follicles >8 mm in diameter ablated by ultrasound-guided follicle aspiration on Day 0, received 500 μg of cloprostenol (prostaglandin F2α, Ciclase, Syntex, Argentina), and were treated as follows: Group 1 (multiple FSH): 160 mg of Folltropin-V divided into 4 IM injections administered twice daily (i.e. Days 1 and 2); Group 2 (single FSH): 160 mg of Folltropin-V diluted in 4 mL of 25% SRF and given by a single IM injection in the neck on Day 1; Group 3 (control): no FSH treatment. On Day 4, oocytes were aspirated from all follicles >3 mm in diameter by ultrasound-guided follicular aspiration, classified, matured in TCM-199 supplemented with 0.4% BSA, fertilized in Fert medium, and in vitro cultured in SOF supplemented with 0.4% BSA under oil at 37°C, 5% CO2, 5% O2, 90% N2, and saturated humidity for 7 days. Data were analyzed by ANOVA. The mean (±SEM) numbers of follicles aspirated and oocytes recovered were higher (P

There is continuing need to simplify bovine superovulation protocols without compromising embryo production. The control of follicular wave emergence and ovulation has facilitated donor management, but the most commonly used treatment, oestradiol, cannot be used in many parts of the world and mechanical removal of the dominant follicle is difficult to apply in the field. Other alternatives include gonadotrophin-releasing hormone (GnRH) or LH, but efficacy in groups of randomly cycling animals is variable. Another alternative is to increase the response to GnRH by inducing a persistent follicle and initiating FSH treatments following GnRH-induced ovulation. The number of transferable embryos following superovulation during the first follicular wave did not differ from that achieved 4 days after oestradiol benzoate and progesterone. To further simplify superovulation, FSH has been administered as a single intramuscular injection. Superovulation of beef donors with a single intramuscular injection of Folltropin-V (Bioniche Animal Health, Belleville, ON, Canada) diluted in a slow-release formulation resulted in embryo production comparable to that obtained using the traditional twice-daily protocol. The single intramuscular injection has the potential to reduce labour and handling and may be useful when handling stress is an impediment to success. These alternatives provide ways of facilitating widespread application of embryo transfer technologies.

Numerous studies have shown that it is possible to manipulate follicular and luteal dynamics, thereby eliminating the need for oestrus detection in embryo transfer (ET) programmes. Fixed-time ET (FTET) protocols are based on the use of gonadotrophin-releasing hormone (GnRH) and prostaglandin (PG) F or progesterone/progestogen (P4)-releasing devices and oestradiol. The FTET protocols increases the proportion of recipients transferred, and therefore pregnancy rates, compared with the use of PGF followed by ET 7 days after oestrus. Furthermore, the addition of equine chorionic gonadotrophin (eCG) to the P4 and oestradiol-based FTET protocols results in an even higher proportion of recipients transferred, and thus higher pregnancy rates. The beneficial effect of eCG treatment may be related to increased growth of the dominant follicle and increased plasma P4 concentrations during the subsequent luteal phase. In Bos taurus × Bos indicus recipients, pregnancy rates were positively correlated with the diameter of the corpus luteum (CL) and the number of CL at ET. When repeat-breeder Holstein cows were used as recipients, FTET protocols increased number of recipients transferred and pregnancy rates compared with the traditional PGF-based synchronisation protocols. In conclusion, the use of FTET protocols eliminates the need for the detection of oestrus and results in a greater proportion of recipients transferred and satisfactory pregnancy rates. Thus, FTET optimises the use of recipients, reducing labour and animal handling and facilitating the use of ET.

Two experiments were designed to evaluate the effect of the timing of fixed-time AI (FTAI) in relation to the removal of an intravaginal progesterone-releasing device (1 g of progesterone, DIB, Syntex SA, Buenos Aires, Argentina) on pregnancy rates in Bos indicus × Bos taurus cross-bred heifers. In experiment 1, 285 Bonsmara × zebu cross-bred heifers, between 18 and 24 months of age and with a body condition score (BCS) between 3.0 and 3.5 (1-5 scale) were used. On the day of initiation of treatment (Day 0), the heifers’ ovaries were palpated (92% of them had a CL) and they received a new DIB plus 2 mg of estradiol benzoate (EB; Syntex SA) and 250 μg of cloprostenol (Ciclase DL, Syntex SA). On Day 8, DIB devices were removed and all heifers received 250 μg of Ciclase plus 0.5 mg of estradiol cypionate (ECP; Cipiosyn, Syntex SA). At that time the heifers were randomly divided to receive FTAI between 48 to 49 h, 53 to 54 h, or 58 to 59 h after DIB removal. The heifers underwent FTAI with semen from 4 bulls by 2 inseminators. In experiment 2, 260 heifers from the same group as those used in experiment 1 (87% with a CL) were treated exactly as those in experiment 1, except that previously used DIB was inserted on Day 0. Pregnancy diagnosis was performed 30 days post-fixed-time AI by ultrasonography. The data were analyzed by logistic regression, taking into account the effect of time of FTAI, semen, and inseminator on pregnancy rates. In experiment 1, pregnancy rates were lower (P = 0.04) in the heifers undergoing FTAI between 48 and 49 h after DIB removal (46/95, 48.4%) than those undergoing FTAI 53 to 54 h (61/99, 61.6%) or 58 to 60 h (57/91, 62.6%) after DIB removal. However, no differences in pregnancy rates were found (P = 0.72) in experiment 2 between the 3 treatment groups, with 39/91 (42.9%) for the 48 to 49 h group, 45/89 (50.6%) for the 53 to 54 h group, and 35/89 (43.8%) for the 58 to 59 h group. There was no effect of the semen or inseminator (P > 0.2) in either experiment. We conclude that when Bos indicus × Bos taurus beef heifers are synchronized with new DIB devices and ECP, higher pregnancy rates are obtained in heifers undergoing FTAI late (between 53 to 60 h after DIB removal) than in those undergoing FTAI early (48 to 49 h after DIB removal). However, time of insemination does not apparently affect pregnancy rates when Bos indicus × Bos taurus beef heifers are synchronized with previously used DIB devices and ECP.

An experiment was designed to test the following hypotheses: (1) the administration of 400IU of eCG (Novormón, Syntex SA, Buenos Aires, Argentina) 14 days after fixed-time artificial insemination (FTAI) increases pregnancy rates in cross-breed Zebu × Bonsmara suckled cows synchronized with progesterone releasing devices (DIB 0.5, 0.5 g of progesterone, Syntex SA); and (2) pregnancy rates in cows treated with estradiol or GnRH, combined with a DIB device, are not different. Primiparous suckled cows (n = 260), between 60 and 120 days postpartum (DPP) and with a body condition score between 2.0 and 2.5 (1 to 5 scale) were used. At the beginning of the experiment (Day 0), all cows were examined by rectal palpation (only 11% had a CL), received a DIB device, and were randomly allocated to 1 of 3 treatment groups. Cows in the E2 group received 2 mg of estradiol benzoate (EB) intramuscularly (i.m.) at DIB insertion and those in the two GnRH groups received 100 μg of gonadorelin (GDR Gonasyn, Syntex SA) at the same time. On Day 7, DIB devices were removed and all cows received 500 μg of cloprostenol (Ciclase DL, Syntex SA) plus 400 IU of eCG (i.m.). Cows in the E2 group received 0.5 mg of estradiol cypionate (ECP, Cipiosyn, Syntex SA) and underwent FTAI administered by one technician, using semen from 1 of 2 bulls between 52 and 56 h later. Cows in the GnRH groups received a dose of 100 μg of gonadorelin (full GnRH group) or 50 μg of gonadorelin (1/2 GnRH group) at the time of the FTAI (52 to 56 h after DIB removal). Fourteen days after FTAI cows in the 3 groups were further subdivided (3 × 2 factorial) to receive or not 400 IU of eCG i.m. Pregnancy was determined 30 days after FTAI using real-time ultrasonography. Data were analyzed by logistic regression, taking into account the effect of the synchronization treatment, eCG or noteCG, 14 days after FTAI and sire used. No differences in pregnancy rates were found (P = 0.16) among treatment groups (E2: 39/87, 43.7%, full GnRH: 38/92, 41.3%, and 1/2 GnRH: 25/81, 30.9%). However, there was a significant (P = 0.02) effect of eCG treatment 14 days after FTAI (eCG: 60/127, 47.2% v. no eCG: 41/133, 30.8%). Furthermore, bull A (77/155, 47.1%) resulted in higher pregnancy rate (P = 0.009) than bull B (28/107, 26.2%), but there were no significant interactions between bull and synchronization treatment or eCG administration 14 days after FTAI (P > 0.1). We conclude that the application of 400 IU of eCG 14 days after FTAI increases pregnancy rates in suckled Bos taurus × Bos indicus primiparous cows with fair to poor body condition score synchronized with progesterone releasing devices. Furthermore, the use of estradiol ester or GnRH at DIB insertion and removal did not affect pregnancy rates.

The success of an embryo transfer program is measured by the number of calves born alive by female donor in a given period. The success is influenced by several factors related to the number of ovulations, fertilization rate, and embryo viability (Armstrong D 1993 Theriogenology 39, 7–24). One of the main inconveniences of embryo transfer programs is the variability of superovulatory response to treatments. An experiment was designed to determine the optimal dose of ovine pituitary gland extract Ovagen® (ICPbio, Ltd., Auckland, New Zealand) for inducing superovulation in Aberdeen Angus donor cows. Sixty cycling multiparous donors with a condition score between 3.5 and 4.5 (Scale 1 to 5) were used. All donors received an intravaginal progesterone device DIB (1 g of Progesterone, Syntex, Buenos Aires, Argentina), along with 2 mg of EB (Estradiol Benzoate, Syntex, Buenos Aires, Argentina) and 50 mg of progesterone (Laboratorio Rio de Janeiro, Argentina) on Day 0. Superestimulatory treatment began on Day 4 and donors were randomly assigned to 3 treatment groups according to the total dose of NIADDK-oFSH-Z (Ovagen®) as follows: Group 1, cows received the total dose recommended by the manufacturer (100%* Group), 17.6 mg; Group 2, cows received 75% of the total dose recommended by manufacturer (75% Group), 13.2 mg and Group 3, cows received 50% of the total dose recommended by manufacturer (50% Group), 8.8 mg. All cows received two 150 μg of D+ cloprostenol IM (Ciclase, Syntex) injections on Day 6 given at 12 h interval. DIB was removed on Day 7 a.m. On Day 8 a.m., cows received 0.05 mg of GnRH IM (Gonasyn, Syntex). Fixed time AI was done on Day 8 p.m. and Day 9 a.m. with high quality frozen–thawed semen. On Day 15 embryo collection was performed by non-surgically method and evaluated by developmental stage and quality. The efficiency of superestimulatory response was evaluated by total amount of collected ova-embryos, fertilized ova and embryos Grade 1, 2, and 3, (according to IETS manual). One way AOV test was used to compare variables among groups and results are shown in Table 1. A significant increase in total CL was observed for the groups receiving 75% or 100% of the recommended dose with respect to the group receiving 50% of that dose. There was also a significant greater number of grade 1 embryos for the groups receiving 75% or 100% of the dose than in the group receiving 50% of the recommended dose. There were no differences for any of the evaluated parameters between 75% and 100% dose groups. These results suggest that acceptable superstimulatory responses can be obtained using reduced doses of Ovagen® in Aberdeen Angus donor cows. (*) the percentage makes reference to the total dose recommended by the laboratory of origin. Table 1.Embryo collection results of superstimulated donors with different dose* of NIADDK-oFSH-Z (Ovagen) Research supported by Syntex S.A. and Eolia S.A.

We have previously shown that the addition of eCG improved pregnancy rates following fixed-time artificial insemination (FTAI) in lactating dairy cows treated with estradiol benzoate (EB) and progesterone releasing devices (Veneranda et al. 2006 Reprod. Fertil. Dev. 18, 118). An experiment was designed to compare pregnancy rates in lactating dairy cows treated with progesterone-releasing devices and EB plus eCG with different gonadotropin-releasing hormone (GnRH)-based protocols. Five-hundred lactating dairy cows between 30 and 51 days postpartum (when prostaglandin F (PGF) treatments were given in the Pre-Synch groups), with a milk yield of 29.5 � 7.0 kg per day (range 13.0 to 46.0 kg) and a body condition score (BCS) between 2.5 to 3.5 out of 5 were used. Cows were blocked by days postpartum and randomly assigned to five treatment groups. Cows in the Pre-Synch group received PGF 28 and 14 days prior to the insertion of a DIB device (1 g progesterone, Syntex SA, Buenos Aires, Argentina) and the administration of 50 µg of Lecirelin (GnRH, Ovusin, Syntex SA) IM (Day 0). On Day 7, DIB were removed and cows received PGF (0.5 mg cloprostenol, Ciclase, Syntex SA). On Day 9, cows received a second GnRH treatment and were FTAI 16 h later (60 h after device removal). Cows in the Modified Pre-Synch group were treated similarly except that they received a DIB for 7 days prior to the second PGF injection and no DIB at the time of the first GnRH. Cows in the P4-Synch group were treated as those in the Pre-synch group but did not receive the two doses of PGF 28 and 14 days earlier. Cows in the P4 + EB + eCG groups received a DIB for 8 days, 2 mg of EB at DIB insertion (Day 0), and PGF and 400 IU eCG (Novormon, Syntex SA) IM at DIB removal (Day 8); they were subdivided to receive 1 mg of EB on Day 9 (P4 + EB + eCG + EB group) or GnRH on Day 10 (P4 + EB + eCG + GnRH group). Cows in these latter two groups were also FTAI 60 h after device removal. Pregnancy was determined by rectal palpation 50 days after FTAI and data were analyzed by logistic regression. Pregnancy rates were not affected by BCS, days postpartum, or milk yield (P > 0.5). However, there was a significant group effect due to a higher (P < 0.05) pregnancy rate in the P4 + EB + eCG + EB (49%) and P4 + EB + eCG + GnRH (44%) treatment groups than in those in the Modified Pre-Synch group (30%). Pregnancy rate in the P4-Synch group (39%) was lower (P < 0.05) than those in the P4 + EB + eCG + EB group but not different from the others. Finally, pregnancy rate in Pre-Synch group (41%) was intermediate and not different from the other treatment groups. Results of these experiments suggest that treatments with progesterone-releasing devices, EB, and eCG result in pregnancy rates that are comparable to or better than those obtained in GnRH-based protocols in lactating dairy cows. Further studies are needed to investigate whether eCG could increase pregnancy rates in cows treated with GnRH and progesterone-releasing devices. The authors thank Syntex SA for providing the hormones used in the study.

In Holstein cows, the diameter of the dominant follicle (DF) at the time of follicle deviation is 8.5 mm and the subordinate follicle (SF) is 7.2 mm (Ginther et al. 1996 Biol. Reprod. 55, 1187–1194). However, follicular responsiveness to an ovulatory treatment occurs only with 10.0-mm-diameter follicles (Sartori et al. 2001 Biol. Reprod. 65, 1403–1409). The current study tested the hypothesis that, in Bos indicus (Nelore and crossbred Nelore � Gir) females, the follicular diameters at the time of deviation and ovulation responsiveness are smaller than those in Holstein cows. The experiment was performed in two phases. In the first phase, 12 Nelore heifers were previously synchronized with a protocol using progestagen and estradiol benzoate. After implant removal, all heifers were evaluated by transrectal ultrasonography (Aloka SSD-500, Tokyo, Japan) every 12 h until Day 5 of the estrous cycle (Day 0 = Day of the ovulation) to assess the time of ovulation, the time of follicle deviation, and the follicular diameter at the deviation. In the second phase, 29 Bos indicus heifers (Nelore and crossbred Nelore � Gir) were previously synchronized with the same protocol as cited above. After the ovulations (Day 0), the follicles were evaluated by transrectal ultrasonography every 24 h, until they reached the diameter of 7.0–8.4 mm (n = 9); 8.5–10.0 mm (n = 10); and >10.0 mm (n = 10). In order to assess the ovulatory capacity, all animals were treated with 25 mg of LH (Lutropin-V�; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) at these follicle diameter ranges. After the LH treatment, all animals were monitored by ultrasonography every 12 h for 48 h. ANOVA, Bartlett, and chi-square tests were used in the statistical analyses. In the first phase, the diameters of the DF and SF at the time of follicular deviation (61.9 � 4.9 h after ovulation) were 6.2 � 0.2 and 5.8 � 0.2 mm, respectively. In the second phase, the the average follicular diameters at the time of LH administration in the groups 7.0–8.4 mm, 8.5–10.0 mm, and >10.0 mm were 7.6a � 0.1 mm, 9.6b � 0.1 mm, and 10.9c � 0.2 mm; and their ovulation rates were 33.3%a (3/9), 80.0%b (8/10), and 90.0%b (9/10), respectively (P < 0.05). The interval from LH treatment to ovulation was 38.0 � 4.0 h, 31.5 � 2.7 h, and 30.0 � 2.0 h, respectively (P > 0.05). In conclusion, in Bos indicus heifers, follicle deviation occurred with smaller diameters than previously reported in Bos taurus breeds. In addition, Bos indicus heifers are able to ovulate in response to 25 mg of LH with smaller diameters compared to those of Bos taurus breeds. Moreover, in Bos indicus heifers, ovulatory capacity is acquired by follicles as small as 7.0–8.4 mm, but this responsiveness significantly increases after follicles reach 8.5–10.0 mm. This work was supported by FAPESP (Proc:03/10203-4); Bioniche Animal Health, Inc., Belleville, Ontario, Canada; and Tecnopec, S�o Paulo, Brazil.

An experiment was designed to evaluate alternative treatment protocols for fixed-time embryo transfer (FTET) in recipients in order to reduce cattle handling. Non-lactating, cycling Bos taurus � Bos indicus beef cows (n = 918), with a body condition score (BCS) of 2.5 to 3.5 out of 5, received a progesterone-releasing device (DIB; Syntex, Buenos Aires, Argentina) and 2 mg estradiol benzoate (EB; Syntex) IM on random days of the cycle (Day 0); DIB were removed on Day 8. Cows were placed randomly into 6 treatment groups in a 3 � 2 factorial design. The factors evaluated were time of administration of PGF and eCG [control treatment: PGF Day 5 and eCG Day 5 (Bo et al. 2002 Theriogenology 57, 53–72); PGF Days 0 and 8 and eCG Day 5; PGF Days 0 and 8 and eCG Day 8] and the time of administration of the second injection of EB (at DIB removal or 24 h later). Therefore, cows in the control treatment received 150 �g of d-Cloprostenol (PGF, Ciclase; Syntex) and 400 IU eCG (Novormon; Syntex) on Day 5, whereas cows in the other 2 treatments received 75 �g of PGF on Days 0 and 8 and 400 IU eCG on either Day 5 or Day 8. Each treatment group was further subdivided to receive 1 mg EB IM on either Day 8 (EB Day 8) or Day 9 (EB Day 9). All recipients, whether observed in estrus or not and with more than one CL with luteal area (total CL area minus cavity area) >76 mm2 on the day before embryo transfer, received frozen-thawed embryos (IETS Grade 1) on Day 16 (EB Day 8) or Day 17 (EB Day 9) by direct transfer. Pregnancy was determined by ultrasonography 30 days after FTET. Continuous data were analyzed by ANOVA and pregnancy rates were analyzed by logistic regression. No effects of body condition score or technician were detected (P > 0.1). Although there were no differences in conception rates between recipients treated with EB on Day 8 or Day 9 (168/321, 52.3% vs. 203/350, 58.0%, respectively; P > 0.1), treatment with EB on Day 8 resulted in fewer recipients selected/treated (321/460, 69.7% vs. 350/458, 76.4%; P < 0.05) and pregnant/treated (168/460, 36.5% vs. 203/458, 44.3%; P < 0.05) than 24 h later (EB Day 9), respectively. The mean interval from DIB removal to estrus was shorter (P < 0.05) for recipients in the EB Day 8 (28.1 � 7.2 h) than in the EB Day 9 (43.5 � 9.4 h) group, and the mean (� SEM) CL area was also less in the EB Day 8 group than in the EB Day 9 group (261.7 � 96.7 vs. 293.1 � 114. mm2; P < 0.05). The number of recipients selected/treated was higher in the group receiving PGF on Days 0 and 8 and eCG on Day 8 (243/305, 79.6%) than in the group receiving PGF on Days 0 and 8 and eCG on Day 5 (208/301, 69.1%) or the control group (220/312, 70.5%). Although the luteal area was greater (P < 0.05) in the PGF on Days 0 and 8 and eCG on Day 5 group (304.4 � 127.1 mm2) than in the groups receiving PGF and eCG on Day 5 (274.6 � 98.6 mm2) or PGF on Days 0 and 8 and eCG Day 8 (258.4 � 90.2 mm2), pregnancy rates did not differ between PGF and eCG treatment groups (P > 0.05). It was concluded that it may be possible to reduce animal handling by treating recipients with a half dose of PGF at the time of DIB insertion and removal, when eCG is also administered, without compromising pregnancy rates.

An experiment was designed to evaluate the effect of the interval between thawing to deposition of the embryo into the uterine horn on pregnancy rates of in vivo-produced frozen–thawed embryos in 1.5 M ethylene glycol (direct transfer). Data were collected from 1122 embryo transfers performed in the same farm (Estancia El Mangrullo, Lavalle, Santiago del Estero, Argentina) during the spring and summer of 2004/05 and 2005/06 (6 replicates, ambient temperature between 20 and 40�C). Recipients used in all replicates were non-lactating, cycling, multiparous Bos taurus � Bos indicus crossbred cows with body condition score between 3 and 4 (1 to 5 scale) that were synchronized using fixed-time embryo transfer protocols. Briefly, the synchronization treatments consisted of the insertion of a Crestar ear implant (Intervet, Sao Paulo, Brazil) or a progesterone-releasing device (DIB; Syntex SA, Buenos Aires, Argentina), plus 2 mg of estradiol benzoate (EB; Syntex) intramuscularly (IM) on Day 0, and 400 IU of eCG (Folligon 5000; Intervet, or Novormon 5000; Syntex) IM plus 150 �g d-cloprostenol IM (Preloban; Intervet, or Ciclase; Syntex) on Day 5. Progestin devices were removed on Day 8 and all cows received 1 mg of EB IM on Day 9. All cows were examined by ultrasonography on Day 16 and those with a luteal area >76 mm2 (by calculating the area of the CL minus the area of the cavity) received, on Day 17, frozen–thawed embryos by nonsurgical transfer. All embryos were Grade 1, and all were frozen in 1.5 M ethylene glycol at the Embryo Plus Laboratory (Brits, South Africa). After being stored in liquid nitrogen, the embryos were plunged directly (no air thawing) in a 30�C water bath for 30 s, and then transferred to the recipient cows by either one of two technicians. Based on the interval between thawing and transfer, the transfers were classified as being in one of 3 groups: Group 1: 0.6. These results may be interpreted to suggest that there is no significant effect of time from thawing to transfer (up to 16 min) in direct transfer embryos using Bos taurus � Bos indicus recipients transferred at a fixed time.

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.

It has been shown than it is possible to AI embryo donors at a fixed time (FTAI) by removing the progestin-releasing device at the time of the last FSH treatment and inducing ovulation with gonadotropin-releasing hormone (GnRH) or porcine luteinizing hormone (pLH) at either 12 h (dairy cows: Bos indicus) or 24 h (beef cows: Bos taurus) after device removal (Bo et al. 2006 Theriogenology 65, 89–101). An experiment was designed to determine the best interval from device removal to GnRH or pLH treatment in Angus (beef cow) donors. Red Angus donors (43 cows and 56 heifers) were blocked by parity and randomly assigned to 1 of 4 treatment groups in a 2 × 2 factorial design. On Day 0, all donors received a DIB vaginal device (Syntex, Buenos Aires, Argentina) plus 2.5 mg estradiol-17β and 50 mg progesterone (P4; Laboratorio Rio de Janeiro, Argentina) IM. Superstimulatory treatments were initiated on Day 4, with a total dose of 320 mg (cows) or 200 mg (heifers) NIH-FSH-P1 of Folltropin-V (Bioniche Animal Health Inc., Belleville, Ontario, Canada) in twice daily IM injections over 4 days. All cows received prostaglandin-F treatment (150 µg d-cloprostenol; Bioprost; Biotay, Buenos Aires, Argentina) on Day 6 (AM and PM); DIB devices were removed with the last Folltropin-V treatment on Day 7 PM. Donors received 0.050 mg GnRH (Lecirelina; Gonasyn; Syntex, Buenos Aires, Argentina) or 25 mg Armour pLH (Lutropin-V; Bioniche) 12 h (Day 8 AM) or 24 h (Day 8 PM) later and were FTAI 12 and 24 h after GnRH or pLH. Ova/embryos were collected nonsurgically and classified according to IETS recommendations on Day 15 AM for donors receiving GnRH or pLH on Day 8 AM and on Day 15 PM for those receiving GnRH or pLH on Day 8 PM. Data were analyzed by two-way ANOVA. Although there was no effect of induction agent (GnRH vs. pLH; P > 0.2) and/or interaction between the induction agent and the time of treatment (P > 0.13) on ova/embryo production, treatment with GnRH or pLH on Day 8 AM (i.e. 12 h after DIB removal) resulted in a significantly lower (P < 0.05) number of fertilized ova and Grades 1 and 2 embryos than treatment on Day 8 PM (i.e. 24 h after DIB removal; Table 1). It was concluded that GnRH and pLH were equally efficacious for inducing ovulation in superstimulated donors that were fixed-time AI. Furthermore, a 24-h interval from progestin device removal to GnRH or pLH administration was preferable for Bos taurus beef donors. Table 1. Ova/embryo production in Angus donors treated with GnRH or pLH 12 or 24 h after removal of a progestin-releasing device and FTAI 12 and 24 h later

Various intravaginal progesterone-releasing devices are commercially available and each is impregnated with different amounts of progesterone. An experiment was designed to characterize plasma progesterone release profiles from Cue-Mate in comparison with other commercially available progesterone-releasing devices. Cycling, lactating Holstein cows (n = 60; BCS between 2.0 and 3.0 out of 5) from commercial dairy farms in Argentina were used in 2 replicates. All cows received 2 injections of D+ cloprostenol (PGF, Bioprost-D�; Biotay, Buenos Aires, Argentina) 14 days apart and were randomly assigned to one of four groups to receive intravaginal devices as follows: Cue-Mate (1.56 g progesterone; Bioniche Animal Health, A/Asia Pty, Armidale, Australia; n = 10), DIB (1 g progesterone; Syntex SA, Argentina; n = 10), CIDR with 1.9 g progesterone (Pfizer Salud Animal S.A., Buenos Aires, Argentina; n = 10), or CIDR with 1.38 g progesterone (Pfizer Animal Health, Groton, CT, USA; n = 10). All devices were inserted 24 hours after the second PGF and were left in place for 31 days. Blood samples were taken for progesterone analysis at 6-hour intervals for the first 24 hours, then daily for a week, and every 2 days for an additional 25 days. Plasma progesterone concentrations were measured with a modified human double-antibody RIA kit (DPC Coat-A-Count�; Diagnostic Products Corporation, Los Angeles, CA, USA). Cows were observed twice daily to confirm that vaginal inserts were still in place. Time-series hormone data were analyzed using the MIXED procedure for repeated measures (SAS Institute, Inc., Cary, NC, USA). Main effects of device and time, the highest mean concentration of progesterone, and the mean day on which this occurred were compared by a protected LSD test. There was no evidence of cow discomfort with any of the vaginal devices, and loss rates did not differ. There was an effect of group (P < 0.001) and day (P < 0.001) for progesterone concentrations, but there was no interaction (P = 0.89). Peak concentrations of progesterone (ng mL-1) did not differ among groups (Cue-Mate: 5.0 � 1.1; DIB: 3.9 � 0.6; 1.9-g CIDR: 4.6 � 0.6; 1.38-g CIDR: 3.7 � 0.4; P = 0.51). Mean (� SEM) concentrations of progesterone over the 31-day insertion period differed between 1.9-g CIDR (1.8 � 0.2 ng mL-1) and DIB (1.5 � 0.1 ng mL-1, P < 0.006), with Cue-Mate (1.5 � 0.2 ng mL-1) and 1.38-g CIDR (1.6 � 0.1 ng mL-1) intermediate and not different from either. When data were evaluated from insertion until Day 7, mean plasma progesterone concentrations did not differ among groups (Cue-Mate: 2.2 � 0.2 ng mL-1; DIB: 2.2 � 0.1 ng mL-1; 1.9-g CIDR: 2.6 � 0.2 ng mL-1; 1.38-g CIDR: 2.1 � 0.1 ng mL-1). However, progesterone concentrations from insertion until Day 21 were higher (P < 0.03) in cows with a 1.9-g CIDR (2.1 � 0.1 ng mL-1) than in those with a DIB (1.6 � 0.1 ng mL-1) or a 1.38-g CIDR (1.6 � 0.1 ng mL-1), with Cue-Mate (1.7 � 0.1 ng mL-1) intermediate and not different. Based on progesterone release patterns, it was concluded that Cue-Mate intravaginal devices can be used in protocols for synchronization of estrus and ovulation as with other progesterone-releasing devices.

High temperatures can be harmful to the competence of cumulus–oocyte complexes and to embryo development (Al-Katanani et al. 2002 J. Dairy Sci. 85, 390–396). The aim of this study was to evaluate the effect of maternal heat stress on in vitro embryo yield. Ten multiparous nonlactating Gir (Bos indicus) cows were kept in tie stalls for an adaptive period of 28 days [pre-heat-stress period (PRE-HS)/Days -28 to -1]. Cows were subjected to 2 OPU (ovum pickup) sessions (Days -14 and -7). In the heat-stress period (HS; Days 0 to 28), cows were divided into control (C: n = 5) and heat-stressed (HS: n = 5) groups. During this period, OPU sessions were performed once a week from Days 0 to 28. The C group remained in a thermo-neutral environment, and the HS group was kept in a climatic chamber with controlled temperature and humidity (38�C and 80% during the day and 30�C and 80% during the night). In the post-heat-stress period (POST-HS; Days 28 to 147), all cows returned to thermo-neutral conditions. Then 17 OPU sessions were performed once a week from Days 35 to 147. In all periods, blood samples were collected weekly for progesterone (P4) analysis, and ovarian follicles were counted, measured, and aspirated. The COCs were evaluated and selected for the IVF procedure. Data were analyzed by ANOVA (PROC MIXED of SAS) and a chi-squared test. The luteal phase was defined as the period between 2 samples with P4 below 1.0 ng mL-1. A handling accident caused the exclusion of an HS cow after the sixth session. The C and HS groups were subjected to 125 and 107 OPU sessions, respectively. Means � SEM for the C vs. HS groups, in the PRE-HS, HS, and POST-HS periods, respectively, were visualized follicles: 25.5 � 2.5 vs. 28.5 � 2.8, 24.2 � 1.1 vs. 24.0 � 1.9, and 15.3 � 0.6 vs. 15.8 � 0.8; largest follicle diameter: 12.1 � 1.5 vs. 11.1 � 1.7, 13.3 � 0.8 vs. 13.0 � 0.6, and 11.4 � 0.4b vs. 14.0 � 0.4a; P < 0.05; 2nd largest follicle diameter: 6.2 � 1.3 vs. 6.0 � 1.2, 5.9 � 0.6 vs. 7.1 � 0.8, and 6.3 � 0.3b vs. 8.7 � 0.5a; recovered COCs: 11.2 � 2.8 vs. 14.3 � 2.5, 9.6 � 1.0 vs. 11.0 � 1.3, and 8.6 � 0.7 vs. 7.9 � 0.6; COCs selected for IVF: 69/112 (61.6%)b vs. 108/143 (75.5%)a, 164/241 (68%) vs. 172/265 (64.9%), and 426/712 (59.8%) vs. 305/535 (75.0%); cleavage: 44/59 (74.5%) vs. 87/105 (82.9%), 72/101 (71.3%) vs. 74/121 (61.2%), and 226/317 (71.3%) vs. 159/230 (69.1%); embryos per cow/OPU: 2.1 � 1.1y vs. 4.1 � 1.0x, 0.4 � 0.3 vs. 0.5 � 0.3, and 0.9 � 0.2x vs. 0.4 � 0.1y; P < 0.1; and blastocyst yield: 16/59 (27.1%) vs. 33/105 (31.5%), 11/31 (35.5%) vs. 13/52 (25.0%), and 76/279 (27.2%)a vs. 25/188 (13.3%)b. In conclusion, maternal heat stress increased the percentage of short estrous cycles, decreased the P4 concentrations, and decreased the number of embryos produced by Bos indicus cows, mainly from 28 to 147 days post-heat-stress, showing long-term deleterious effects on blastocyst development.

Three experiments were designed to evaluate the effect of the duration of treatment with progesterone (P4) vaginal devices (1 g of P4, Triu-B; Biog�nesis, Buenos Aires, Argentina) on pregnancy rates in crossbred zebu cattle artificially inseminated at a fixed time (FTAI). In Exp. 1, 371 non-lactating multiparous crossbred zebu cows with a body condition score (BCS) between 2.0 and 3.0 out of 5 were randomly allocated to one of nine treatment groups in a 3 � 3 factorial design. On Day 0, all cows received 2 mg of estradiol benzoate (EB, Bioestrogen; Biog�nesis) i.m. and were divided into groups to receive a new Triu-B device, a once-previously-used Triu-B (2nd-use), or a Triu-B that was previously used twice (3rd-use) and in which three silicone rings (2 cm in length) impregnated with 100 mg P4 each were threaded through the wings of the device. Triu-B devices are currently approved for re-use up to three times. Cows were further subdivided to have their Triu-B devices removed on Days 7, 8, or 9. PGF (150 �g D(+)cloprostenol, Enzaprost-DC; Biog�nesis) was administered i.m. at the time of Triu-B removal, followed by 1 mg EB 24 h later; all cows were FTAI 52 to 56 h after Triu-B removal. In Exp. 2, 417 crossbred zebu heifers, 20 to 26 months of age and BCS between 2.5 and 3.5, were treated similarly to the cows in Exp. 1, except that only new or 3rd-use Triu-B were used. In Exp. 3, 836 crossbred zebu heifers, similar to those used in Exp. 2, were also treated similarly to the cows in Exp. 1, but in this case, Triu-B devices were inserted for 8, 9, or 10 days. Pregnancy rates were determined by ultrasonography 50 to 60 days after FTAI and were analyzed by logistic regression. In Exp. 1, there were no significant effects of duration of treatment [7 days: 69/137 (50.4%), 8 days: 68/139 (48.9%), or 9 days: 41/95 (43.2%); P = 0.28], or type of Triu-B used (P = 0.91; new Triu-B: 47.7%, 2nd-use: 48.9%, or 3rd-use: 47.2%). In Exp. 2, there were no significant effects of duration of treatment [7 days: 76/139 (54.7%), 8 days: 63/143 (44.1%), or 9 days: 73/135 (54.1%); P = 0.98] or type of Triu-B used (new Triu-B: 48.6% and 3rd-use Triu-B: 53.2%; P = 0.33). In Exp. 3, there was an interaction between duration of treatment and pregnancy rates (P < 0.05). Lower pregnancy rates were obtained in 9- and 10-day treatments than in 8-day treatments, but only when previously used Triu-B devices were used. Pregnancy rates were: new Triu-B for 8 days: 42/89 (47.2%), 9 days: 37/90 (41.1%), and 10 days: 41/91 (45.1%); 2nd-use Triu-B for 8 days: 51/103 (49.5%), 9 days: 39/104 (37.5%), and 10 days: 39/109 (35.8%); 3rd-use Triu-B for 8 days: 52/90 (57.4%), 9 days: 31/90 (34.4%), and 10 days: 25/70 (35.7%); P < 0.01. Results demonstrate that treatment of crossbred zebu cattle with P4 devices for 7 to 10 days results in comparable pregnancy rates when new Triu-B devices are used. However, pregnancy rates decreased when previously used Triu-B devices were inserted for more than 8 days.

An experiment was designed to determine plasma progesterone (P4) concentrations in ovariectomized cows treated with either CIDR-B devices (Pfizer Animal Health, Hamilton, New Zealand) impregnated with 1.9 g of P4 or DIB devices (Syntex, Argentina) impregnated with 0.5 or 1.0 g of P4. Fifteen ovariectomized beef cows, weighing 350 to 450 kg and with a body condition score (BCS) 2.5 to 3.0 out of 5 were used. All cows were ovariectomized 30 days before the beginning of the trial and had plasma P4 concentrations 0.13) or a day by treatment interaction (P > 0.16; Table 1). The area under the curve did not differ (P = 0.95) among groups (DIB 0.5: 2236.4 ng, DIB 1.0: 2164.6 ng, and CIDR-B: 2266.8 ng). In all treatments, plasma P4 profiles were characterized by a sudden increase in P4 concentrations within 12 h of device insertion and a gradual decrease during the following several days. It was concluded that the use of CIDR-B devices impregnated with 1.9 g of P4 or DIB devices impregnated with 1.0 or 0.5 g of P4 did not result in different plasma P4 concentrations in ovariectomized cows. Further studies are needed to confirm these results in lactating and dry intact (not ovariectomized) cattle. Table 1. Mean plasma P4 concentrations (ng/mL ± SEM) in ovariectomized cows treated with intravaginal devices containing 0.5 g (DIB 0.5), 1.0 g (DIB 1.0), or 1.9 g (CIDR-B) of P4*

Three experiments were conducted to compare pregnancy rates in cows and heifers treated with intravaginal progesterone (P4)-releasing devices impregnated with either 0.5 or 1.0 g of P4 and inseminated at a fixed time (FTAI). In Experiment 1, 102 postpartum beef cows (Hereford and Angus crossbred with Tuli, 60 to 90 d postpartum) with a body condition score (BCS) of 2.5 to 3.0 (1 to 5 scale) were used. On Day 0, all cows received 2 mg of estradiol benzoate (EB; Syntex, Argentina) i.m. and were randomly assigned to receive an intravaginal progesterone-releasing device with 0.5 g of P4 (DIB 0.5 g; Syntex) or 1.0 g of P4 (DIB 1.0 g; Syntex). On Day 7, DIB devices were removed and all cows received 150 µg D(+)cloprostenol (Ciclase; Syntex) i.m. On Day 8, all cows received 1 mg EB and were FTAI 52 to 56 h after DIB removal. In Experiment 2, 93 Holstein cows that were 90 to 110 days in milk (DIM), producing on average 5000 kg of milk per lactation and with a BCS of 2.5 to 3.0 received the same treatments as those in Experiment 1. In Experiment 3, 100 Holstein heifers, 20 to 22 months old, with a BCS of 2.5 to 3.0, also received the same treatments as those in Experiment 1. Pregnancy rates in all experiments were determined by ultrasonography 35 days after FTAI and compared by chi-square test. No differences in pregnancy rates were detected between lactating beef cows, dairy cows, or dairy heifers treated with DIB devices impregnated with 0.5 or 1.0 g of P4 (Table 1). It was concluded that vaginal P4 releasing devices impregnated with 0.5 g or 1.0 g of P4 results in comparable pregnancy rates in FTAI protocols in beef and dairy cattle. Table 1. Pregnancy rates in postpartum beef cows, Holstein cows, and Holstein heifers treated with DIB devices impregnated with 0.5 or 1.0 g of P4 and subjected to FTAI

Two experiments were designed to investigate the use of eCG in a fixed-time embryo transfer (FTET) protocol for Chinese Yellow crossbred recipients receiving in vitro-produced (IVP) bovine embryos. In Experiment 1, cows were observed for spontaneous estrus (Group 1) or following 500 �g cloprostenol (PGF; Schering-Plough Animal Health, Montreal, Quebec, Canada; Group 2) with nonsurgical embryo transfer 7.5 or 8.0 days later. Cows in Groups 3, 4, and 5 were treated with a CIDR insert (Bioniche Animal Health, Beijing, China), 2 mg estradiol benzoate (EB), and 50 mg progesterone (Sigma-Aldrich, Canada) i.m. on Day 0, PGF on Day 5, CIDR removal on Day 7, and 1 mg EB i.m. 24 h later with nonsurgical transfer of Holstein IVP embryos 8.5 days later, without estrus detection. In addition, cows in Group 3 received no further treatment, whereas cows in Group 4 received 400 IU eCG i.m. (Pregnecol; Bioniche) on Day 5, and cows in Group 5 received eCG on Day 7. Holstein embryos were produced utilizing slaughterhouse ovaries and standard IVF procedures. Expanded blastocysts of quality grade 1 (IETS) were cryopreserved in 10% ethylene glycol and 20% fetal bovine serum using standard procedures. Straws were thawed in a 30�C water bath, and embryos were expelled directly into holding medium and evaluated prior to nonsurgical transfer (NT) ipsilateral to the corpus luteum (CL). Pregnancy was diagnosed ultrasonically 30 d later. Although pregnancy rates, based on numbers of recipients synchronized, numerically favored the administration of eCG on Day 5, there were no significant differences (P = 0.40) among groups (Group 1: 15.4%, n = 52; Group 2: 20.0%, n = 50; Group 3: 19.2%, n = 99; Group 4: 28.1%, n = 96; Group 5: 21.3%, n = 75). In Experiment 2, Chinese Yellow crossbred cattle were synchronized with a CIDR insert plus estradiol and progesterone on Day 0, PGF on Day 5, CIDR removal on Day 7 and estradiol on Day 8, and were randomly assigned to received no further treatment (Group 1; n = 400) or an injection of 400 IU eCG on Day 5 (Group 2; n = 391). Recipients with a detectable CL received a frozen-thawed IVP Holstein embryo by NT 8.5 days after the second injection of estradiol without estrus detection, as in the first experiment. Pregnancy diagnosis was done ultrasonically 30 days later. Although CL size, cow age, and embryo quality, prior to transfer, were recorded, no effects on pregnancy rates were demonstrated (P = 0.30). Pregnancy rates, based on recipients receiving embryos, did not differ (P = 0.5) between groups (Group 1: 21.4%, n = 154; Group 2: 24.5%, n = 290). Overall pregnancy rates (based on the total number of recipients synchronized) were significantly higher (P < 0.001) in Group 2 (eCG; 18.2%) than in Group 1 (no eCG; 8.3%), because of the significantly higher (P < 0.03) percentage of recipients used following treatment with eCG (74.2% vs. 38.5%). Results indicate that the administration of eCG on Day 5 of an 8-d synchronization protocol for FTET of frozen-thawed bovine IVP embryos will improve pregnancy rates in beef recipients, especially those of marginal quality.

Two experiments were designed to compare the effects of eCG treatment and temporary weaning on ovulation and pregnancy rates in postpartum cows. In Experiment 1, 39 lactating multiparous crossbred Bos indicus cows, 60 to 80 d postpartum with a body condition score (BCS) between 2.0 to 2.5 out of 5 were randomly allocated to one of four treatment groups in a 2 � 2 factorial design. On Day 0, all cows received a progesterone (P4) intravaginal device (DIB; Syntex, Argentina) and 2 mg estradiol benzoate (EB) i.m. (Syntex). On Day 8, DIB devices were removed and all cows received 150 �g D(+)cloprostenol i.m. (Ciclase; Syntex) and were randomly divided to receive 400 IU eCG i.m. (Novormon 5000; Syntex) at the same time or no treatment. In addition, half of the cows in each group had their calves weaned temporarily (TW) for 56 h at the time of DIB removal; the other half remained with their calves. All cows received 1 mg EB i.m. on Day 9 and were examined every 8 h by ultrasonography from the time of DIB removal until ovulation. Data were analyzed by ANOVA and chi-square test. The diameter of the dominant follicle on Day 8 (eCG: 7.8 � 0.5 mm vs. no eCG: 8.2 � 0.4 mm, and TW: 7.6 � 0.4 mm vs. no TW 8.4 � 0.5 mm), the proportion of cows that ovulated (eCG: 12/20, 60% vs. no eCG: 9/19, 47%; and TW: 13/20, 65% vs. no TW: 8/19, 42%, and the interval to ovulation (eCG: 72.0 � 1.4 h vs. no eCG: 75.6 � 2.0 h, and TW: 73.8 � 1.6 h vs. no TW: 73.0 � 1.8 h) did not differ among groups (P > 0.05). Although there was no effect of eCG treatment or eCG by TW interaction (P > 0.3) on the size of the preovulatory follicle (eCG: 11.1 � 0.4 mm vs. no eCG: 10.1 � 0.6 mm), it was smaller in cows TW (9.9 � 04 mm), compared to those not TW (11.8 � 0.3 mm; P < 0.05). Nevertheless, the growth rate of the ovulatory follicle was greater (P < 0.02) in cows treated with eCG (1.1 � 0.1 mm/day) than in those not treated with eCG (0.6 � 0.1 mm/day). In Experiment 2 769 lactating multiparous crossbred Bos indicus cows received the same treatments as those in Experiment 1 and were fixed-time inseminated (FTAI) 52 to 56 h after DIB removal. Cows were examined by ultrasonography on Day 0 (22.5% with corpora lutea (CL), 30.0% with follicles >8 mm, and 47.5% with follicles 0.7) by TW (TW: 141/397, 37.2% vs. no TW: 141/390, 36.1%) or the TW by eCG interaction (P > 0.7). However, pregnancy rates were higher (P < 0.05) in cows treated with eCG (154/377, 40.8%) than in those not treated with eCG (128/392, 32.6%). The use of eCG but not TW improved pregnancy rates following FTAI in postpartum Bos indicus cows. Results also suggest that the eCG-related increase in pregnancy rates may be due to the final growth rate of the ovulatory follicle.

An experiment was designed to evaluate the effect of the time of removal of an intravaginal progesterone (P4) releasing device and GnRH treatment on the interval to and the distribution of ovulations in beef cows superstimulated with Folltropin-V (Bioniche Animal Health, Belleville, Ontario, Canada). Red Angus donors (22 cows and 12 heifers) with a body condition score between 2.5 to 3.5 out of 5 were blocked by parity and randomly assigned to one of four treatment groups in a 2 × 2 factorial design. On Day 0, all donors received an i.m. DIB vaginal device (Syntex, Argentina) plus 2.5 mg estradiol-17β and 50 mg P4 (Laboritorio Rio de Janeiro, Argentina). Superstimulatory treatments were initiated on Day 4, with a total dose of 320 mg (cows) or 200 mg (heifers) NIH-FSH-P1 (Folltropin-V; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) in twice daily i.m. injections over 4 days. All cows received PGF treatment (150 μD(+)cloprostenol: Ciclase; Syntex) in the morning and afternoon of Day 6 and were randomly divided to have DIB devices removed at the time of the second PGF treatment (Day 6.5) or 12 h later (Day 7). On Day 8, donors were further subdivided to receive GnRH (0.050 mg Lecirelina, Gonasyn; Syntex) or no further treatment, and all cows were fixed-time AI either 60 and 72 h after the first PGF injection (0 h). All cows and heifers were examined by ultrasonography every 8 h for a period of 120 h commencing at the time of DIB removal, to determine the time and distribution of ovulations. For each cow, mean time of ovulation was used for the statistical analysis. The distributions of ovulations were compared among groups by Levene's test, and then by ANOVA (equal variances) or Friedman two-way non-parametric ANOVA (unequal variances). No significant differences were detected between cows and heifers (P > 0.13) for the parameters evaluated, and data were combined. There was no effect (P > 0.22) of treatment on the number of ovulations (GnRH: 12.6 ± 1.6 vs. no GnRH: 15.1 ± 1.1 and Day 6.5: 13.7 ± 1.3 vs. Day 7: 14.3 ± 1.5), or numbers of transferable embryos (GnRH: 5.1 ± 1.1 vs. no GnRH: 3.1 ± 0.8 and Day 6.5: 3.5 ± 0.8 vs. Day 7: 4.81 ± 0.1). However, there was a significant effect of GnRH treatment and a GnRH-by-time-of-DIB-removal interaction on the distribution of ovulations (P < 0.05; Table 1). The administration of GnRH in a superstimulatory protocol using P4 releasing devices results in earlier and more synchronous ovulations than when GnRH is not used. Table 1. Distribution of ovulations (hours after the first PGF) in superstimulated Red Angus donors treated with progesterone-releasing devices (DIB) for 6.5 or 7 days with and without GnRH treatment on Day 8

Two experiments were designed to evaluate the effect of different treatments for the synchronization of ovulation on pregnancy rates following fixed-time AI (FTAI) of lactating dairy cows. In Experiment 1, 394 Holstein cows that were 61.7 � 13.6 days postpartum (range 35 to 94 days), with a milk yield of 30.7 � 6.8 liters per day (range 12.0 to 52.4 liters) and a body condition score (BCS) between 2.5 to 3.5 out of 5, were used. Cows were blocked by days postpartum and milk yield and randomly assigned to one of four treatment groups. Cows in P4+EB treatment groups received an intravaginal DIB device (1 g P4; Syntex, Argentina) and 2 mg of estradiol benzoate (EB; Syntex) i.m. on Day 0. On Day 8, DIB devices were removed, and cows received PGF (150 �g D+cloprostenol: Ciclase; Syntex), and were subdivided to receive 400 IU eCG (Novormon 5000, Syntex) i.m. or no further treatment at that time. On Day 9, all cows received 1 mg of EB and were FTAI 60 h after DIB removal. Cows in P4-Synch groups received a DIB device and 50 �g of GnRH (Lecirelina, Gonasyn; Syntex) i.m. on Day 0. On Day 7, DIB devices were removed; cows received PGF and were divided to receive 400 IU eCG i.m. or no further treatment. On Day 9, all cows received a second GnRH treatment and were FTAI 60 h after DIB removal. Blood samples were taken on Days -10 and 0 to determine plasma P4 concentrations; 93% of the cows had >1 ng/mL P4 in at least one sample. In Experiment 2, 200 lactating cows from the same farm were treated with the P4+EB+eCG and P4-Synch without eCG treatments with either a DIB or a CIDR-B (1.9 g P4; Pfizer Animal Health, Groton, CT, USA) in a 2 � 2 factorial design. Cows were examined by rectal palpation 50 days after FTAI to determine pregnancy status, and data were analyzed by chi-square Mantel-Haenszel test. There was an EB/GnRH by eCG interaction (P < 0.05) which was attributed to a higher pregnancy rate in the P4+EB+eCG group (44/98; 44.9%) than in the P4+EB without eCG group (30/100; 30.0%) and P4-Synch+eCG group (30/98; 30.6%); the P4-Synch without eCG group had an intermediate pregnancy rate that did not differ from the other treatment groups (37/98; 38.8%). In Experiment 2, no significant differences (P = 0.40) in pregnancy rates were detected between cows treated with DIB (51/100; 51.0%) or CIDR-B (42/100; 42.0%), and there was no difference (P = 0.18) between P4+EB+eCG (52/100; 52.0%) and P4-Synch without eCG (41/100; 41.0%) treated groups. Results suggest that the addition of eCG will improve pregnancy rates following FTAI in lactating dairy cows treated with EB but not in those treated with GnRH at the time of insertion and after removal of a P4 releasing device. Although treatment with P4+EB+eCG resulted in numerically higher pregnancy rates, results were not different from those obtained in the GnRH-based treatment, without the addition of eCG.

Although treatments with progesterone (P4) releasing devices, estradiol benzoate (EB) and eCG have been shown to result in acceptable pregnancy rates after embryo transfer, the treatment requires that the cows be run through the chute at least four times for treatments. An experiment was designed to compare pregnancy rates in cows treated with P4 releasing devices plus EB and eCG, and induced to ovulate with EB given at device removal or 24 h later. Non-lactating Bos taurus × Bos indicus crossbred beef cows (n = 165), with a body condition score between 2.5 to 3.5 (1 to 5 scale), were treated with a P4-device (DIB, Syntex, Argentina) and 2 mg EB i.m. (Syntex), on Day 0 and 400 IU of eCG i.m. (Novormon, Syntex) plus 150 μg D(+)cloprostenol i.m. (Ciclase, Syntex) on Day 5. On Day 8, DIB devices were removed and cows were randomly divided into two groups to receive either 1 mg EB i.m. at the time of DIB removal (EB0) or 24 h later (Day 9; EB24). Recipients were observed for signs of estrus for 48 h after DIB removal. On Day 16, all recipients observed in estrus and with >1 CL or a single CL with an area >256 mm2 were selected to receive fresh embryos on Day 16 (EB0) or Day 17 (EB24). Furthermore, 20 recipients not observed in estrus but with a CL >256 mm2 were randomly selected and transferred. The embryos used were 28 Grade 1, 40 Grade 2, and 24 Grade 3. Ovarian ultrasonography was performed on Day 0, to determine ovarian status (only cows with a CL or a follicle >10 mm and uterine tone were used), on Day 16 to measure CL area, and 60 days after embryo transfer to determine pregnancy status. Quantitative data were analyzed by Student's t-test and qualitative data were analyzed by logistic regression. There were no differences between groups in the mean (± SEM) CL area on Day 16 (EB0: 300.3 ± 12.0 mm2 and EB24: 324.9 ± 11.7 mm2; P = 0.14), the proportion of recipients in estrus (EB0: 38/82, 46.3%, and EB24: 34/83, 41.0%; P = 0.49), the number of recipients with >1 CL or a CL >256 mm2 (EB0: 68/82, 82.9%, and EB24: 72/83, 86.7%; P = 0.49) and the number of recipients pregnant/transferred (EB0: 31/49, 63.4%, and 20/43, 46.5%; P = 0.23). Furthermore, there were no significant effects of embryo quality (P = 0.31) or technician (P = 0.12) on pregnancy rates. The mean interval from DIB removal to estrus was shorter (P = 0.001) for recipients in the EB0 group (22.7 ± 1.0 h) than for those in the EB24 group (37.4 ± 1.2 h). Nevertheless, pregnancy rates did not differ (P = 0.14) between recipients seen in estrus (EB0: 21/38, 55.3%, and EB 24: 16/34, 47.1%) and those not seen in estrus but with a CL >256 mm2 on Day 16 (EB0: 10/11, 90.9%, and EB24 4/9, 44.4%). It was concluded that the two treatments evaluated are equally efficacious for synchronizing Bos taurus × Bos indicus recipients. Furthermore, the use of EB at device removal could reduce the number of trips through the chute without affecting pregnancy rates. Research was supported by Syntex S.A., Estancia El Mangrullo S.A., and Agencia Cordoba Ciencia S.E.

Although treatments with progesterone (P4)-releasing devices and estradiol benzoate (EB) have been extensively used in fixed-time AI (FTAI) programs in beef cattle, pregnancy rates in postpartum Bos indicus cows kept on pasture often have been lower than expected because of poor body condition score (BCS) and a high incidence of anestrus. Temporary weaning and eCG treatment have been shown to increase pregnancy rates in suckled beef cows. Therefore, this experiment was designed to compare the effect of eCG treatment and temporary weaning on pregnancy rates in postpartum cows in fair to poor BCS. Lactating multiparous crossbred Bos indicus cows (n = 393), 60 to 90 d postpartum with a BCS 2.0 (1 to 5 scale) were randomly allocated to 1 of 4 treatment groups, in a 2 × 2 factorial design. At the beginning of the experiment (Day 0), all cows received a P4 intravaginal device (DIB, Syntex, Argentina) and 2 mg EB i.m. (Syntex). On Day 8, DIB devices were removed and all cows received 150 μg D (+) cloprostenol i.m. (Ciclase, Syntex) and were randomly divided to receive 400 IU eCG (Novormon 5000, Syntex) or no treatment at the same time. Furthermore, half of the cows in each treatment group have their calves weaned temporarily, from the time of DIB removal until the end of the FTAI, whereas the other half remained with their calves all the time. Finally, all cows received 1 mg EB i.m. on Day 9 and were FTAI 52 to 56 h after DIB removal. Cows were examined by ultrasonography 42 d after FTAI to determine pregnancy status. Data were analyzed by logistic regression and the effects of treatment and semen used were considered in the model. Ovarian activity was estimated by rectal palpation on Day 0: there were 72/393 (18.2%) cows with a CL, 140/393 (35.6%) cows with palpable medium size follicles, and 181/393 (46.1%) cows with ovaries containing no detectable structures. There was no effect of ovarian status at the time of treatment (P = 0.91) or semen (P = 0.91) on pregnancy rates. Treatment with eCG tended (P = 0.08) to increase pregnancy rates (94/192, 48.9% and 79/201, 39.3% for cows treated or not treated with eCG, respectively). However, there was no effect of weaning or eCG by weaning interaction (P = 0.98) on pregnancy rates (86/191, 45.1% and 87/202, 43.1% for cows that have their calves weaned or not weaned for 56 h, respectively). Results confirm those of previous studies that demonstrated that the use of eCG in a P4/EB/FTAI program improved pregnancy rates in postpartum Bos indicus crossbred cows that were in fair-to-poor BCS. However, no improvement in pregnancy rates was observed after temporary weaning in the present study. Research was supported by Syntex S.A., Estancia El Mangrullo S.A., and Agencia Cordoba Ciencia S.E.

Previous studies have shown that the use of eCG given at the time of removal of a progesterone (P4) releasing device improved pregnancy rates to fixed-time AI (FTAI) in postpartum Bos indicus cows (Bo GA et al. 2004 Reprod. Fertil. Dev. 16, 127). Suckling restriction with the placement of nose tags in calves has also been shown to induce cyclicity and increase pregnancy rates in postpartum Bos indicus cows (Stahringer RC 2003 Taurus 18, 21–23). An experiment was designed to compare the effect of eCG treatment and restricted suckling on pregnancy rates in postpartum cows in fair to poor body condition score (BCS). A secondary objective was to evaluate the effect of restricted suckling on weaning weights. Lactating primiparous crossbred Bos indicus cows (n = 399), 60 to 90 days postpartum with a BCS 2.0 (1 to 5 scale), were randomly allocated to 1 of 4 treatment groups, in a 2 × 2 factorial design. On Day 0, all cows received a P4 intravaginal device (DIB, Syntex, Argentina) and 2 mg estradiol benzoate (EB) i.m. (Syntex). On Day 8, DIB devices were removed and all cows received 150 μg D(+)cloprostenol i.m. (Ciclase, Syntex) and were randomly divided to receive 400 IU eCG (Novormon, Syntex) or no treatment at the same time. On Day 9, all cows received 1 mg EB i.m. and were FTAI 52 to 56 h after DIB removal. Nose tags were placed in half of the calves from Day 0 to the time of FTAI, whereas the other half of the calves remained untreated. All cows were examined by ultrasonography 30 d after FTAI to determine pregnancy status. Pregnancy data were analyzed by logistic regression, and birth and weaning weights were analyzed by Student's t-test. Ovarian activity was estimated by rectal palpation on Day 0: there were 57/399 (14.3%) cows with a CL, 203/399 (50.8%) cows with palpable medium size follicles, and 139/399 (34.8%) cows with ovaries containing no detectable structures. There was no effect of ovarian status at the time of treatment (P = 0.52) or semen (P = 0.89) on pregnancy rates. Suckling restriction increased (P = 0.03) pregnancy rates (91/195, 46.6% and 81/204, 39.7% for cows not suckled or suckled during the treatment, respectively). However, there was no effect of eCG or eCG by weaning interaction (P = 0.82) on pregnancy rates (82/192, 42.7% and 90/207, 43.4% for cows treated or not treated with eCG, respectively). Although mean (±SEM) birth weights were not different (29.4 ± 0.4 vs. 29.1 ± 0.4; P = 0.6), weaning weights were significantly affected (P = 0.001) by the suckling restriction (190.1 ± 1.9 vs. 200.4 ± 2.1 for calves treated or not treated with the nose tags, respectively). Although restricting suckling, by placement of a nose tag from Day 0 to FTAI, may increase pregnancy rates in primiparous Bos indicus cows in fair to poor BCS, the adverse effect of this treatment on weaning weights of the calves has to be considered. Furthermore, eCG did not increase pregnancy rates in the present study, as it has been previously reported. Research was supported by Syntex S.A., Estancia El Mangrullo S.A., and Agencia Cordoba Ciencia S.E.