Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

A retrospective analysis of embryo production records from 2013 to 2017 was carried out to evaluate the in vivo and in vitro production (IVP) of embryos in donors of the Bonsmara breed (i.e. tropically adapted Bos taurus). Only donors with production records of both in vivo and in vitro embryos during the same period were used. A total of 127 superovulations and ova/embryo collections of 19 donors were evaluated. The donors were superstimulated with the following protocol: on Day 0 they received a device with 1g of progesterone (DIB, Zoetis, Argentina), 50mg of rogesterone (Progestar, Zoetis), and 5mg of oestradiol-17β (17ßOestradiol, Rio de Janeiro, Argentina) or 2mg of oestradiol benzoate (Gonadiol, Zoetis) intramuscularly (IM) at the same time. Superstimulatory treatments were initiated on the morning of Day 4 with Folltropin-V (Vetoquinol, France; total dose=240 to 340mg IM) in twice-daily decreasing doses over 4 days. All donors received 2 IM injections of 500µg of cloprostenol (Ciclase DL, Zoetis) on the morning and afternoon of Day 6 and; the intravaginal devices were removed on the morning of Day 7 and 100µg of Gonadorelin (gonadotropin-releasing hormone, Gonasyn gdr, Zoetis) was given on the morning of Day 8. Donors were inseminated using semen from 9 Bonsmara bulls, 12 and 24h after gonadotropin-releasing hormone. On Day 15, ova/embryos were collected and classified according to IETS standards. A total of 89 follicular aspirations (ovum pickup) of 19 donors for IVP were evaluated. The ovum pickups were performed at random stages of oestrous cycle, without superstimulation or other hormone treatments. A total of 1109 viable oocytes (12.5±0.9 per ovum pickup) were collected and matured for 24h in 100-µL drops of maturation medium (TCM-199, supplemented with hormones) under mineral oil and incubated at 38.5°C in 5.5% CO2 and humidity at saturation. Fertilization was performed using 3 Bonsmara bulls that were also used for in vivo embryo production. Viable sperm were obtained using the percoll gradient technique (45-90%). The sperm pellet was dissolved in TL-Sperm, centrifuged, and then diluted to a final concentration of 1.5×106 sperm/mL. Zygotes were stripped and placed in drops of 100µL of SOF medium supplemented with 0.4% BSA under oil at 38.8°C, 5.5% CO2, 7% O2, and humidity at saturation for 7 days. The culture medium was renewed on Days 3 and 5. The data were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA), a P-value <0.05 was considered significant. The mean (±standard error of the means) number of CL, ova/embryos collected, fertilized ova, and transferable embryos were 12.9±0.6, 8.8±0.6, 6.6±0.5, and 4.7±0.4, respectively. A total of 662 oocytes (66.3±2.4%) cleaved 48h post-IVF. On Day 7, an average of 4.4±0.3 embryos were produced. No differences were detected in the number of transferable embryos produced in vivo v. those produced in vitro. Furthermore, no significant differences were found between the techniques or bulls on the proportion of embryos produced in relation to the ova/embryos or oocytes obtained (in vivo 51.5±3.2% v. in vitro 42.9±2.5%). In conclusion, the in vivo and in vitro production of embryos are both effective alternatives to increase the number of offspring from valuable Bonsmara donors.

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<0.01) number of fertilized oocytes, but there were no differences in the other end points evaluated (Table 1). In Exp. 2, no differences were found between FSH+800 eCG and FSH+600 eCG groups in any of the parameters evaluated. In conclusion, the replacement of the last 4 injections of FSH by a single dose of eCG decreases the number of treatments required in a superovulation program without negatively affecting the production of transferable embryos. Table 1.Embryo production (means±s.e.m.) in Bonsmara donors treated with FSH or FSH+eCG

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 (Novormon, 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 u003c 0.01) in recipients showing oestrus (5.6%, 7/124) than in those not showing oestrus (66.7%, 6/9). Similarly, fetal losses between 60 days and calving tended to be lower (P u003c 0.06) in recipients showing oestrus (20.5%, 24/117) than in those not showing oestrus (66.7%; 2/3). Calving rates were higher in recipients showing oestrus (29.3%, 93/317) than in those not showing oestrus (2.9%, 1/35; P u003c 0.01). In conclusion, although both synchronization treatments performed similarly, recipients not showing oestrus after progesterone device removal had lower conception rates and higher pregnancy losses than those showing oestrus at the expected time after treatment.

Embryokines are molecules secreted by the mother that regulate embryonic development. Among these molecules in cattle are colony stimulating factor 2 (CSF2) and dickkopf-related protein 1 (DKK1). Here, we evaluated actions of CSF2 and DKK1 alone or in combination on characteristics of embryos produced in vitro in the presence of serum. A total of 70 beef cows from 4 farms were subjected to oocyte retrieval on 1 to 4 occasions. Within each farm, donors were randomly allocated to 1 of 4 treatment groups (vehicle, CSF2, DKK1, CSF2 + DKK1). Embryos from a given donor were always exposed to the same treatment. Treatments were added to the culture medium on d 5 after insemination, and blastocyst stage embryos were transferred to recipient females 2 d later. Treatment did not affect the percent of oocytes or cleaved embryos that developed to the blastocyst stage or the percent of recipients that became pregnant after embryo transfer. However, calves derived from embryos treated with DKK1 were smaller at birth, regardless of CSF2 treatment. Results indicate no effects of addition of CSF2 or DKK1 to culture of embryos produced in vitro with serum-containing medium on development to the blastocyst stage or competence to establish pregnancy after transfer to recipients. The fact that embryos cultured with DKK1 resulted in calves with reduced birth weight illustrates the potential ability of this embryokine to program postnatal phenotype. Results support the concept that properties of the offspring can be programmed as early as the preimplantation period.

An experiment was designed to evaluate whether expression of oestrus in recipients synchronized with progesterone devices and oestradiol affects pregnancy rates to a fixed-time embryo transfer (FTET). A secondary objective was to determine whether administration of gonadotropin-releasing hormone (GnRH) to those recipients not showing oestrus by 48 h after device removal had an effect on pregnancy rates. Mature, non-lactating beef recipients (Bonsmara, Brangus, and Braford; n = 729), with a corpus luteum (CL) or a follicle ≥8 mm in diameter detected by ultrasonography (Mindray DP 30, Shenzhen, China) and body condition score 2 to 4 (1 to 5 scale) were synchronized in 7 replicates. On Day 0, recipients received a progesterone device (DIB 0.5 g, Zoetis, Buenos Aires, Argentina) and 2 mg of oestradiol benzoate (Gonadiol, Zoetis). On Day 8, DIB were removed and recipients received 400 IU eCG (Novormon 5000, Zoetis) plus 0.5 mg of oestradiol cypionate (Cipiosyn, Zoetis) and 500 μg of cloprostenol (Cyclase, Zoetis). In addition, all cows were tail-painted on the sacrococcygeal area (CeloTest, Biotay, Buenos Aires, Argentina) at DIB removal and were observed to determine the expression of oestrus, according to the percentage of paint loss 48 h later. The animals that showed oestrus (paint loss >30%) were recorded, and those with paint loss ≤30% (not in oestrus) were randomly allocated to receive 100 μg of gonadorelin (GnRH; Gonasyn, Zoetis) or no treatment. On Day 17, all recipients were examined by ultrasonography and those with a CL ≥18 mm (G1), ≥16 and <18 mm (G2), or ≥14 and <16 mm (G3) in diameter received in vivo-produced frozen-thawed embryos by direct transfer or fresh in vitro-produced embryos. Pregnancy was diagnosed by ultrasonography at 23 days after FTET, and data were analysed by logistic regression. There were no differences between replicates, in vivo- and in vitro-produced embryos, or operators (P > 0.2). However, the proportion of recipients transferred and pregnant (P/FTET) was higher (P < 0.05) for those that showed oestrus at 48 h after DIB removal (422/454, 93.0% and 227/471, 48.2%) than those that did not show oestrus (109/211, 51.7% and 45/120, 37.5%). Within the recipients not showing oestrus, P/FTET was significantly higher (P < 0.05) in those that received GnRH (34/74, 45.9%) than in those that did not receive GnRH (12/46, 26.1%). There was a significant interaction (P < 0.05) between CL diameter and expression of oestrus. When recipients had CL ≥18 mm in diameter (G1), P/FTET did not differ (oestrus: 164/338, 48.5% v. no oestrus 30/65, 46.2%); however, when CL diameter was <18 mm and ≥14 mm (G2 and G3), P/FTET was higher (P < 0.05) in those that showed oestrus (37/85, 43.5%) than in those that did not show oestrus (11/43, 25.6%). Finally, P/FTET was higher (P < 0.05) in recipients with a CL (268/573, 46.8%) on Day 0 than in those with follicles >8 mm in diameter (4/18, 22.2%). In conclusion, the expression of oestrus significantly affected the percentage of recipients selected for embryo transfer and P/FTET. Furthermore, treatment of recipients not showing oestrus by 48 h after DIB removal with GnRH at that time increased P/FTET.

A retrospective analysis evaluated pregnancy rates and embryo losses with in vitro-produced embryos in a commercial embryo transfer program on 15 different beef farms. Recipients were beef cows and heifers (n = 1841) that were synchronized with 5 different protocols and transferred at a fixed-time (FTET). Recipients were examined by ultrasonography on Day 0, and those with a corpus luteum (CL) or a follicle ≥8 mm in diameter and with body condition score 2 to 4 (1 to 5 scale) were synchronized. The synchronization treatments were as follows. (T1) Recipients received an intravaginal device with 0.5 g of progesterone plus 2 mg of oestradiol benzoate on Day 0; device removal, plus 500 μg of cloprostenol (prostaglandin F2α), 400 IU of eCG, and 0.5 mg of oestradiol cypionate on Day 8; and FTET on Day 17. (T2) This treatment was similar to T1 but 1 mg of oestradiol cypionate was injected at device removal instead of 0.5 mg of oestradiol cypionate. (T3) This treatment was similar to T1 except that animals were tail-painted on Day 8 and observed on Day 10. Those with the tail-paint intact on Day 10 received 100 μg of gonadorelin (gonadotropin-releasing hormone) and all recipients were FTET on Day 17. (T4) Recipients received a progesterone device on Day 0; device removal, prostaglandin F2α, and eCG on Day 5; gonadotropin-releasing hormone on Day 8; and FTET on Day 15. (T5) Recipients received a progesterone device and 2 mg of oestradiol benzoate on Day 0; device removal, prostaglandin F2α, and eCG on Day 6; gonadotropin-releasing hormone on Day 9; and FTET on Day 16. On the day of FTET all recipients with CL ≥18 mm in diameter (G1), ≥16 and <18 mm in diameter (G2), and ≥14 mm and <16 mm in diameter (G3) received in vitro-produced fresh embryos. Pregnancy was diagnosed by ultrasonography at 30 and 60 days of gestation, and data were analysed by logistic regression. The overall proportion of recipients synchronized that were FTET was 80.8% (1487/1841), with a 30-day pregnancy rate to FTET (P/FTET) of 45.6% (678/1487) and the rate of 30- to 60-day embryo/fetal loses on the 528 recipients that were re-checked at 60 days was 12.8% (68/528). There were no significant differences in P/FTET among operators, animal category, time of the year, embryo stage, or body condition score; however, there was a significant effect of farm (P < 0.001) and CL diameter (P < 0.05), but no interaction between CL diameter and farm or treatment (P > 0.1). Recipients with G1 (443/953, 46%) and G2 (221/462, 47%) CL had higher pregnancy rates than those with G3 CL (23/71, 32%). There was a significant effect of synchronization treatment on the proportion of recipients transferred and on P/FTET (P < 0.01) that was highly influenced by farm (farm by treatment interaction P < 0.01). The proportions of recipients selected for embryo transfer were as follows: T1: 386/486, 79.4%; T2: 233/331, 70.3%; T3: 342/377, 90.7%; T4: 126/160, 78.7%; and T5: 400/487, 82.1%. The P/FTET were as follows: T1: 190/386, 49.2%; T2: 96/233, 41.2%; T3: 175/342, 51.1%; T4: 49/126, 38.8%; and T5: 168/400, 42.0%. Although 30- to 60-day embryo/fetal losses were not influenced by synchronization treatments, they were highly influenced by farm (P < 0.001) and ranged from 0 to 34.5%. In conclusion, P/FTET in a commercial program with beef in vitro-produced embryos was influenced by factors related to the recipient (CL diameter) and the environment (farm), whereas embryo/fetal losses were influenced by farm but not treatment or recipient factors.

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 <16 mm, ≥16 and <18 mm, or ≥18 mm; P = 0.46), number of corpus luteum (1 or ≥2; P = 0.26), and category of recipient (cow or heifer; P = 0.21). However, there were significant effects of farm (P = 0.01) and body condition score (BCS; P = 0.01). Cows with BCS ≥4.5 (1 to 5 scale) resulted in lower pregnancy rates (4/20, 20.0%) than those with BCS 2 (74/225, 32.9%), 2.5 (502/1434, 35.0%), 3 (570/1467, 38.9%), 3.5 (193/532, 36.3%), and 4 (44/118, 37.3%). 2) Factors related to the synchronization treatment; there were no significant differences between recipients receiving eCG at device removal (84/209, 40.2%) or 3 days before device removal (874/2291, 38.1%; P = 0.35). However, recipients synchronized with P4 devices and ECP had higher (P = 0.01) pregnancy rates (232/483, 48.0%) than those treated with EB (679/1888, 36.0%) or gonadotropin-releasing hormone (47/129, 36.4%). 3) Factors related to the embryo transfer technique; day of the recipient’s oestrous cycle (P = 0.36), stage of embryo transferred (IETS stages 6 or 7; P = 0.62), and operator (P = 0.57) did not affect pregnancy rates. However transfers made in the anterior third of the uterine horn resulted in higher (649/1545, 42.0%) pregnancy rates than those in the mid-third (845/2511, 33.6%) or in the distal third (6/35, 17.1%; P = 0.01). It was concluded that factors related to the recipient and the environment (farm and BCS), the synchronization treatment (ECP), and the embryo transfer technique (site of deposition) affect pregnancy rates in recipients of embryos produced in vitro and transferred at a fixed time.

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 < 0.01) in Bos indicus × Bos taurus (19.3 ± 0.4 and 5.3 ± 0.2, respectively) and Bos indicus (15.8 ± 1.4 and 6.8 ± 0.9, respectively) than in beef (11.6 ± 0.5 and 3.0 ± 0.2, respectively) and dairy (8.0 ± 0.2 and 1.6 ± 0.1, respectively) Bos taurus donors. Cleavage rates were higher (P < 0.01) in Bos indicus (72%) than in the other breeds (57% for Bos indicus × Bos taurus and dairy Bos taurus and 54% for beef). Transferable embryo rates were higher (P < 0.01) in Bos indicus (41%) and Bos indicus × Bos taurus (30%) than in beef Bos taurus (26%). Dairy Bos taurus had the lowest (P < 0.01) embryo rates of all breeds (21%). In dairy Bos taurus, cleavage rates, the number of embryos produced, and transferable embryo production rates were higher (P < 0.01) when conventional semen was used (62%, 2.8 ± 0.15, and 27%, respectively) compared to sexed-selected semen (55%, 1.3 ± 0.1, and 19%, respectively). With regards to season, the number of viable COC was highest (P < 0.01) in the spring (14.3 ± 0.5), lowest in the summer (11.3 ± 1.0), and intermediate in the fall (12.2 ± 1.2) and winter (13.7 ± 1.2), which did not differ. Although not affected significantly by season, the number of embryos produced was numerically lower in the summer (2.8 ± 0.4) than in the spring (4.2 ± 0.2), winter (4.5 ± 0.5), or fall (4.6 ± 0.5). In conclusion, in vitro embryo production was directly influenced by breed and season. Bos indicus influenced cattle and the spring season were preferable for commercial IVP programs that did not include superstimulation.

An experiment was designed to evaluate in vitro embryo production following the use of frozen-thawed conventional or Y-sexed semen from a single Brangus and a single Braford bull of proven fertility. Semen was obtained by splitting the same ejaculate to be frozen directly or sex-sorted and then frozen. Oocytes were obtained from 69 ultrasound-guided follicle aspiration (ovum pickup) sessions performed at random stages of the oestrous cycle without superstimulation in 24 Brangus and 10 Braford cows and heifers. Viable oocytes (n = 1120) were matured in TCM-199 medium with NaHCO3 and supplemented with 1% fetal bovine serum. Frozen-thawed sperm from the Brangus and Braford bulls were selected with Percoll for IVF, capacitated in Fert Medium, and used at a final concentration of sperm per milliliter for conventional (non-sexed) semen and 2 × 106 sperm mL–1 for Y-sexed semen. After 16 h (sexed) or 18 h (conventional) of co-incubation with oocytes in Fert Medium, presumptive zygotes were denuded and cultured in SOF supplemented with 0.4% BSA under oil at 37°C, 5% CO2, and saturated humidity for 7 days. The total number of oocytes matured and fertilized from the Brangus donors was 538 and 318 for conventional and sexed semen, respectively. The total numbers of oocytes matured and fertilized from the Braford donors were 139 and 125 for conventional and sexed semen, respectively. Data were compared by ANOVA for mixed models, using breed and type of semen as fixed variables and cow (i.d.) as a random variable. Cleavage and blastocyst rates were first transformed by square root and then analysed by ANOVA for mixed models. Mean (± s.e.m.) number of total viable oocytes collected, cleaved zygotes, and blastocysts produced per ovum pickup session did not differ (P = 0.18) between breeds (Brangus: 17.1 ± 1.6, 10.0 ± 0.9, and 6.2 ± 0.7 v. Braford: 13.9 ± 2.8, 7.6 ± 1.5, and 4.0 ± 0.8), and there was no breed × semen interaction on the mean number of cleaved zygotes and blastocysts produced. However, the mean (± s.e.m.) number of cleaved zygotes and blastocysts produced was significantly higher (P < 0.05) when the oocytes were fertilized with conventional semen (10.7 ± 1.2 and 6.5 ± 0.8) than with sexed semen (7.7 ± 0.7 and 4.3 ± 0.6). The mean cleavage rate was also significantly higher (P < 0.05) when the oocytes were fertilized with conventional semen (76.8 ± 3.9) than with sexed semen (54.1 ± 4.2). Blastocyst rate tended to be higher (P = 0.1) with conventional semen (40.5 ± 3.3) than with sexed semen (33.6 ± 4.2). Although in vitro production may be the preferred alternative for the production of embryos of a known sex, the number of blastocysts produced might be reduced as compared with the use of non-sexed semen from the same bull.

An experiment was designed to evaluate in vivo and in vitro embryo production following the use of frozen–thawed conventional or Y-sexed semen from a Brangus bull with known high fertility. For in vivo embryo production, Brangus heifers (n = 12) were superovulated twice in a crossover design and inseminated with sexed or conventional semen. On Day 0, all heifers received an intravaginal progesterone device (DIB 1 g, Syntex S.A., Buenos Aires, Argentina) and 2.5 mg oestradiol benzoate and 50 mg progesterone (Progestar, Syntex S.A.) by intramuscular injection (IM). On Day 4, heifers were superstimulated with 200 mg of NIH-FSH-P1 Folltropin-V (Bioniche Animal Health, Belleville, Ontario, Canada) in twice-daily decreasing doses over 4 days. In the a.m. and p.m. of Day 6, all heifers received PGF2a (Ciclase, Syntex) and DIBs were removed in the p.m.. In the a.m. of Day 8, heifers received 100 μg de Gonadolerin (Gonasyn, Syntex S.A.) and were randomly allocated to receive either one straw of conventional semen (24 × 106 sperm per dose) 12 and 24 h later or two straws of sexed semen (2.4 × 106 sperm per dose) 18 and 24 h after GnRH. Ova/embryos were collected nonsurgically on Day 15 and evaluated following IETS recommendations. Means were compared by t-test. Mean ( ± s.e.m.) number of ova/embryos, fertilized ova, and transferable embryos were 14.8 ± 2.7, 9.4 ± 1.8, and 7.1 ± 1.7 v. 16.8 ± 3.1, 9.9 ± 2.5, and 8.1 ± 2.0 for donors inseminated with conventional or sexed semen, respectively (P > 0.6). For in vitro production, oocytes were obtained from 50 ultrasound-guided follicle aspiration (OPU) sessions that was performed at random stages of the oestrous cycle and without superstimulation in 22 Brangus cows and heifers. Oocytes were classified and matured in TCM-199 medium with NaHCO3 and supplemented with 1% fetal bovine serum. Semen samples from the same bull used for in vivo embryo production were selected using Percoll and capacitated in Fert medium and used at a final concentration of sperm/mL for nonsexed semen and 2 × 106 sperm mL–1 for sexed semen. After 16 h (sexed) or 18 h (conventional) in Fert medium, zygotes were denuded and cultured in SOF supplemented with 0.4% BSA under oil at 37°C, 5% CO2 and saturated humidity for 7 days. The total number of oocytes matured and fertilized was 528 and 318 for conventional and sexed semen, respectively. Means were compared by t-test and proportions by chi-squared test. Mean (± s.e.m.) number of cleaved zygotes and blastocysts produced per OPU session did not differ between conventional (11.0 ± 1.4 and 7.1 ± 1.0) and sexed (8.7 ± 0.8 and 4.9 ± 0.7; P > 0.2) semen. However, the proportion of cleaved zygotes and blastocysts produced were significantly higher (P < 0.05) with conventional semen (61.2%; 329/538 and 39.4%; 212/538) than with sexed semen (54.4%; 173/318 and 30.8%; 98/318), respectively. In conclusion, comparable number of embryos can be obtained in vivo with sexed or conventional semen from a bull with proven high fertility. However, the proportion of blastocysts produced in vitro is likely to be reduced following the use of sexed as compared with conventional semen from the same bull.

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.

Two experiments were designed to evaluate the superovulatory response of Brangus and Bonsmara donor cows to different dosages of Folltropin®-V (Bioniche Animal Health Inc., Belleville, Ontario, Canada) given by a single i.m. injection or twice-daily i.m. injections. In Experiment 1, Brangus cows (n = 12) were superstimulated by 6 treatments (2 × 3 factorial) in a crossover design (i.e. all cows received the 6 treatments and all treatments were represented on each day). On Day 0, cows received 5 mg of estradiol-17β plus 50 mg of progesterone and a Cue-Mate® (Bioniche Animal Health Inc.). On Day 4, cows were superstimulated with 300, 260, or 200 mg of NIH-FSH-P1 Folltropin®-V (Bioniche Animal Health Inc.) in twice-daily decreasing doses over 4 days or diluted in a slow release formulation (SRF; Bioniche Animal Health) and given in a single i.m. injection. The single injection was prepared by diluting the Folltropin®-V lyophilized powder in 1 mL of saline followed by mixing with 9 mL of the SRF in the syringe immediately before administration. In the am and pm of Day 6, all cows received PGF2, and Cue-Mates® were removed in the pm. Cows received 12.5 mg of porcine LH (Lutropin®-V; Bioniche Animal Health Inc.) in the am of Day 8 and were inseminated 12 and 24 h later. Ova/embryos were collected on Day 15 and data were analyzed by ANOVA. There was no effect of treatment (i.e. single v. twice-daily injections; P > 0.2) nor a treatment by dosage interaction (P < 0.6) on the mean (± SEM) number of total ova/embryos or transferable embryos (13.1 ± 1.9 and 7.5 ± 1.2 v. 15.5 ± 1.7 and 7.6 ± 1.0 for single v. twice-daily injections, respectively). The total number of ova/embryos did not differ among Folltropin®-V dosages (15.0 ± 2.3, 15.7 ± 2.0, and 12.1 ± 2.5 for 300, 260, and 200 mg, respectively; P > 0.4). However, the number of transferable embryos tended (P < 0.09) to be higher in donors receiving 260 mg (9.5 ± 1.6) than 200 mg (5.2 ± 0.8), with 300 mg (7.9 ± 1.5) intermediate. In Experiment 2, Bonsmara cows (n = 16) were superstimulated by 4 treatments (2 × 2 factorial) in a crossover design similarly to Experiment 1, except that 2 dosages of Folltropin®-V (200 and 300 mg) were evaluated. There were no significant effects of dosage of Folltropin®-V (P > 0.9), treatment (P > 0.3), or interaction (P < 0.4) on embryo production. The total number of ova/embryos and transferable embryos were 11.9 ± 2.0 and 7.2 ± 1.1 v. 11.1 ± 1.1 and 7.6 ± 0.7 for single and twice-daily injections, respectively, and 11.9 ± 1.9 and 7.6 ± 1.0 v. 11.1 ± 1.3 and 7.2 ± 0.8 for 300 and 200 mg of Folltropin®-V, respectively. Superstimulation of Brangus and Bonsmara cows with a single i.m. injection of Folltropin®-V diluted in a SRF resulted in comparable embryo production to twice-daily administration of Folltropin®-V over 4 days. While 260 mg seems to be the most appropriate dosage for Brangus donors, 200 mg seems to be adequate for Bonsmara donors.

Two experiments were designed to determine the superovulatory response of Angus donors treated with a single injection of Folltropin®-V diluted in a slow release formulation (SRF; Bioniche Animal Health Inc., Belleville, Ontario, Canada). Experiment 1 was designed to compare superovulatory response in Angus donor cows (n = 29 per group) treated with a single i.m. injection of Folltropin®-V diluted in SRF with cows treated using the traditional twice-daily i.m. injection treatment schedule. On Day 0, all cows received 5 mg of estradiol-17β plus 50 mg of progesterone and a Cue-Mate® (Bioniche Animal Health). On Day 4, cows were superstimulated with 400 mg of NIH-FSH-P1 Folltropin®-V in twice-daily decreasing doses over 4 days or in a single i.m. injection in the neck. The single injection was prepared by diluting the Folltropin®-V lyophilized powder in 1 mL of saline for injection and mixed with 9 mL of the SRF in the syringe immediately before administration. In the am and pm of Day 6, all cows received PGF2 and Cue-Mates® were removed in the pm. In the am of Day 8, cows received 12.5 mg of porcine LH (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 recommendations. Means were compared between groups by t-test Mean (±SEM) number of ova/embryos and transferable embryos were 13.7 ± 2.1 and 7.1 ± 1.3 v. 12.3 ± 1.5 and 5.3 ± 0.8 for donors treated with the single v. twice-daily injections, respectively (P > 0.4). Experiment 2 was designed to confirm the results of Experiment 1 and to compare the effect of different dosages of Folltropin®-V on embryo production in Angus cows. Cows (n = 23) were superstimulated by 6 treatment protocols (2 × 3 factorial) in a crossover design (i.e. all cows received the 6 treatments and all treatments were represented on each collection day). Cows received the same treatments as cows in Experiment 1 except that the dosages of Folltropin®-V used were 200, 300, or 400 mg. Statistical analysis (ANOVA) revealed a significant effect of dosage of Folltropin®-V on embryo production, but there was no effect of treatment (i.e. single v. twice-daily injections; P > 0.2), nor was there a treatment by dosage interaction (P > 0.7). Mean (± SEM) number of ova/embryos and transferable embryos were 10.3 ± 0.9 and 5.4 ± 0.6 v. 11.3 ± 0.6 and 5.6 ± 0.5 for donors treated with the single or twice-daily injections, respectively (P > 0.2). Furthermore, the mean number of ova/embryos and transferable embryos were higher in cows treated with 400 mg (13.9 ± 1.1 and 6.5 ± 0.7) and 300 mg (12.0 ± 1.0 and 6.1 ± 0.7) of Folltropin®-V than those treated with 200 mg (6.6 ± 0.7 and 4.0 ± 0.5). In summary, superstimulation of Angus donor cows with a single i.m. injection of Folltropin®-V diluted in an SRF resulted in comparable embryo production to the traditional twice-daily i.m. administration of Folltropin®-V over 4 days. Although response did not differ between 300 and 400 mg, results suggest that 300 mg of Folltropin®-V given i.m. by a single or twice-daily injections is the most appropriate dose for Angus donor cows.

Background: Successful bovine embryo transfer programs require the use of simple superovulation protocols and high numbers of transferable embryos. The control of follicular wave emergence and ovulation have facilitated donor management, but the most commonly used treatment, estradiol, 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 GnRH or LH, but efficacy in groups of randomly cycling animals is variable. Review: An alternative treatment to control follicular wave emergence 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 arising at the time of the GnRH-induced ovulation did not differ from that achieved 4 days after treatment with estradiol benzoate and progesterone. To further simplify superovulation, FSH has been diluted in a slow-release formulation (SRF) and administered as a single or a split intramuscular injection. Although, a single intramuscular injection of Folltropin-V in SRF was highly efficacious in the induction of superovulation in a variety of breeds of beef cattle, it was difficult to mix with Folltropin-V. However, in a subsequent series of experiments it was shown that reducing the initial concentration of SRF to 25% and administering the Folltropin-V as two intramuscular injections 48 hours apart (called split-single administration) facilitated the dilution of Folltropin-V with the SRF and resulted in a superovulatory response that did not differ from controls. Conclusion: The incorporation of GnRH-based protocol to control follicular dynamics and ovulation have the advantage of being able to schedule the treatments quickly and without the need for detecting estrus in donor cows. The singlesplit intramuscular injection of Folltropin-V in 25% SRF has the potential to reduce labor and handling and may be useful when handling stress is an impediment to success. These treatments are practical and easy to perform by the farm staff, facilitating the widespread application of embryo transfer technologies.

Although we have previously shown that ovarian superstimulation during the first follicular wave resulted in a successful response (Carballo Guerrero D et al. 2009 Reprod. Fertil. 21, 242), the current protocol needs to be optimized in order to be used in the field. Therefore, an experiment was designed to simplify this treatment and to compare it with the traditional superstimulation protocol using progesterone and estradiol. Simmental cows (n = 14) were subjected to 3 superstimulation treatments (2 first wave groups and 1 control group) in a crossover design (i.e. all cows received the 3 treatments and all treatments were represented on each collection day). Cows in Group 1 received a progesterone-releasing device (Cue-Mate®, Bioniche Animal Health, Belleville, Ontario, Canada) along with 0.150 mg of D + cloprostenol (PGF; Bioprost-D®, Biotay, Buenos Aires, Argentina) at random stages of the estrous cycle. A second PGF was injected 5 days after Cue-Mate® insertion, followed by GnRH (0.050 mg of lecirelin; Biosin-OV®, Biotay) 36 h later (i.e. 7 days after Cue-Mate® insertion). Based on previous studies, ovulation was expected to occur 30 to 36 h later. Therefore, superstimulation treatments were initiated 36 h after GnRH (Day 0), with a total dose of 400 mg NIH-FSH-P1 of Folltropin®-V (Bioniche Animal Health) in twice-daily decreasing doses over 4 days. Prostaglandin was administered with the last 2 Folltropin®-V injections and Cue-Mate® devices were removed with the last Folltropin®-V injection. Cows received 12.5 mg of porcine LH (Lutropin®-V, Bioniche Animal Health) 24 h after Cue-Mate® removal and were AI 12 and 24 h later. Ova/embryos were collected 7 days after porcine LH and evaluated following IETS recommendations. Cows in Group 2 were treated similarly to those in the Group 1, except they did not receive the second PGF injection 5 days after Cue-Mate® insertion (thus eliminating the need to handle animals on that day). Finally, cows in Group 3 [estradiol benzoate (EB)+P4 control group] received a Cue-Mate® plus 2.5 mg of EB (Bioestradiol®, Biotay) and 50 mg of progesterone (P4; Lab., Rio de Janeiro, Argentina) at random stages of their estrous cycle. Superstimulation treatments were initiated 4 days later (Day 0) following the same protocol used in Group 1. Data were transformed to square root and analyzed by ANOVA. Mean (± SEM) numbers of ova/embryos collected, fertilized ova, and transferable embryos did not differ among groups (12.9 ± 2.0, 9.8 ± 1.7, and 6.6 ± 1.2; 11.5 ± 1.7, 9.3 ± 1.5, and 7.7 ± 1.6; and 14.5 ± 2.8, 9.4 ± 2.3, and 6.8 ± 1.7 for Groups 1, 2, and 3, respectively). In conclusion, data demonstrated that superstimulation during the first follicular wave can be successfully used in groups of randomly cycling donors without the need for estrus detection or estradiol to synchronize follicular wave emergence. The protocol is easy to follow and embryo production is comparable to that of the estradiol and progesterone protocol.

Latex, a polyisoprene (PI) hydrophobic elastomer, was evaluated in vitro and in vivo as a matrix for intravaginal steroid hormone delivery. Matrices containing hormone were prepared by swelling latex in chloroform that contained soluble progesterone (P4). In vitro studies demonstrate that P4 release from PI follows a zero order model during at least 100 h and depends on initial load up to 10 mg cm−2. The release of P4 from a PI matrix was found to be two times faster than from a polydimethylsiloxane (PDMS) matrix. FT-IR and X-ray powder diffraction analysis of P4 polymorphs show that when nucleated in PDMS, the hormone crystallizes only in α-form while in latex, crystallizes as a mixture of α- and β-form. In vivo studies show that devices with a PI matrix containing 0.5 g of P4 are effective to reach plasma levels above 1 ng ml−1 that are needed to synchronize estrous in cattle. Altogether, the results show that PI, a vulcanized polymer with a carbon–carbon backbone, can be used as a new matrix for the intravaginal administration of progesterone with improved release profile than silicone and that the matrix can influence the crystalline state of the hormone.