Fábio Morotti

Pregnancy rate and birth rate of calves from a large-scale IVF program using reverse-sorted semen in Bos indicus, Bos indicus-taurus, and Bos taurus cattle.

Obtaining sexed sperm from previously frozen doses (reverse-sorted semen [RSS]) provides an important advantage because of the possibility of using the semen of bulls with desired genetic attributes that have died or have become infertile but from whom frozen semen is available. We report the efficiency of RSS on the pregnancy rate and birth rate of calves in a large-scale program using ovum pick-up and in vitro embryo production (IVEP) from Bos indicus, Bos indicus-taurus, and Bos taurus cattle. From 645 ovum pick-up procedures (Holstein, Gir, and Nelore), 9438 viable oocytes were recovered. A dose of frozen semen (Holstein, Nelore, Brahman, Gir, and Braford) was thawed, and the sperm were sex-sorted and cooled for use in IVF. Additionally, IVF with sperm from three Holstein bulls with freeze-thawed, sex-sorted (RSS) or sex-sorted, freeze-thawed (control) was tested. A total of 2729 embryos were produced, exhibiting a mean blastocyst rate of 29%. Heifers and cows selected for adequate body condition, estrus, and health received 2404 embryos, and 60 days later, a 41% average pregnancy rate was observed. A total of 966 calves were born, and 910 were of a predetermined sex, with an average of 94% accuracy in determining the sex. Despite the lower blastocyst rate with freeze-thawed, sex-sorted semen compared with sex-sorted semen, (P < 0.05), the pregnancy rate (bull I, 45% vs. 40%; II, 35% vs. 50%; and III, 47% vs. 48% for RSS and control, respectively; P > 0.05) and sex-sorted efficiency (bull I, 93% vs. 98%; II, 96% vs. 94%; and III, 96% vs. 97% for RSS and control, respectively; P > 0.05) were similar for each of the three bulls regardless of the sperm type used in the IVF. The sexing of previously frozen semen, associated with IVEP, produces viable embryos with a pregnancy rate of up to 40%, and calves of the desired sex are born even if the paternal bull has acquired some infertility, died, or is located a long distance from the sexing laboratory. Furthermore, these data show the feasibility of the process even when used in a large-scale IVEP program.

Antral Follicle Populations and Embryo Production – In Vitro and In Vivo – of Bos indicus–taurus Donors from Weaning to Yearling Ages

Interest in indicus-taurus cattle has been increasing, as these animals are likely to present the best characteristics of Zebu and European bovine breeds. The aim of this study was to compare the embryo production of indicus-taurus donors with high vs low antral follicle counts obtained by ovum pickup/in vitro production (OPU/IVP) and superovulation (SOV)/embryo collection. Braford females at weaning age (3/8 Nelore × 5/8 Hereford, n = 137, 9 ± 1 month old) were subjected to six serial ovarian ultrasonographs and were assigned to two groups according to the number of antral follicles ≥ 3 mm as follows: G-High antral follicular count (AFC, n = 20, mean ≥ 40 follicles) and G-Low AFC (n = 20, mean ≤ 10 follicles). When the females (n = 40) reached 24 months of age, they were subjected to both OPU/IVP and SOV/embryo collection. The average number of follicles remained highly stable throughout all of the ultrasound evaluations (range 0.90-0.92). The mean number of COCs recovered (36.90 ± 13.68 vs 5.80 ± 3.40) was higher (p < 0.05) for females with high AFC, resulting in higher (p < 0.05) numbers of total embryos among females with high vs low AFC (6.10 ± 4.51 vs 0.55 ± 0.83). The mean number of embryos per collection was also higher (p < 0.05) for G-High vs G-Low (6.95 ± 5.34 vs 1.9 ± 2.13). We conclude that a single ultrasound performed at pre-pubertal ages to count antral follicles can be used as a predictor of embryo production following IVP and SOV/embryo collection in indicus-taurus females.

Asynchronous embryo transfer as a tool to understand embryo–uterine interaction in cattle: is a large conceptus a good thing?

The aim was to examine the effect of embryo-uterine synchrony on conceptus elongation and pregnancy rate in cattle. In Study 1, crossbred beef heifers each received 10 Day-7 in vitro-produced blastocysts on either Day 5, 7 or 9 after oestrus. A proportion of Day 5 recipients were supplemented with progesterone, via a progesterone-releasing intravaginal device from Days 3-5 plus either 750IU equine chorionic gonadotrophin or 3000IU human chorionic gonadotrophin on Day 3. At embryo age Day 14, all heifers were slaughtered and the uterus was flushed. Fewer recipients yielded conceptuses (P<0.05) and fewer conceptuses were recovered (P<0.05) following transfer on Day 5 compared with Day 7 or 9. Supplementation with progesterone resulted in short cycles in approximately 50% of recipients. Mean conceptus length was greater (P<0.05) following transfer to an advanced uterus. In Study 2, overall pregnancy rate following the fresh transfer of a single in vitro-produced blastocyst was 43.5% (2065/4749). Transfer of a Day 7 embryo to a synchronous Day-7 uterus resulted in a pregnancy rate of 47.3%. Transfer to a Day-5 (40.8%) or a Day-8 (41.3%) uterus moderately impacted pregnancy rate (P<0.01) while transfer to a uterus 2 days in advance (Day-9, 24.4%) or 3 days behind (Day-4, 27.0%) reduced (P<0.001) pregnancy rate compared with synchronous transfers. In conclusion, transfer of an embryo into an advanced uterus results in an acceleration of conceptus development, but does not result in greater pregnancy rates.

High numbers of antral follicles are positively associated with in vitro embryo production but not the conception rate for FTAI in Nelore cattle.

The objective was to compare the conception rates for FTAI and in vitro embryo production between Nelore cows with different antral follicle counts (AFC=number of follicles ≤3mm in diameter in the ovaries). Nelore cows (n=547) were subjected to ovulation synchronization. Randomly during the estrous cycle (D0), cows received an intravaginal device containing 1.9g P4 (CIDR(®)) and 2mg BE (Estrogin(®)), IM. When the device was removed (D8), the cows received 500μg PGF2α (Ciosin(®)), 300IU eCG (Novormon(®)) and 1mg EC (ECP(®)), IM. All cows were inseminated 48h after P4 device removal. Antral follicles ≥3mm were counted using an intravaginal microconvex transducer (D0), and the cows were assigned to high (G-High, ≥25 follicles, n=183), intermediate (G-Intermediate, 16-20 follicles, n=183) or low AFC groups (G-Low, ≤10 follicles, n=181). In another experiment, COCs were retrieved by OPU from Nelore cows (n=66), which were assigned to groups according to oocyte production: G-High (n=22, ≥40 oocytes), G-Intermediate (n=25, 18-25 oocytes) or G-Low (n=19, ≤7 oocytes). All COCs from the same cow were cultured individually (maximum of 25 COCs per drop) and then in vitro fertilized using thawed frozen sperm (2×10(8)/dose) from a Nelore sire of known fertility. The data were analyzed using a Kruskal-Wallis and a Chi-square test (P≤0.05). There was no difference in the conception rates after FTAI between Nelore cows with high, intermediate or low AFC (51.9 vs. 48.6 vs. 58.6%). The number of viable embryos was 18.4±6.7 (G-High), 6.1±3.6 (G-Intermediate) and 0.6±0.7 (G-Low; P<0.05). Therefore, AFC had no influence on the conception rates for FTAI; however, Nelore cows with high oocyte production exhibited better in vitro embryo production.

Resynchronization of estrous cycle with eCG and temporary calf removal in lactating Bos indicus cows

The aim of this study was to compare four methods of estrus resynchronization performed 23 days after timed artificial insemination (TAI) plus estrus observation in Bos indicus cows. Eight hundred fourteen lactating Nelore cows were submitted to TAI and then randomly assigned to one of the five following treatments: R23 (resynchronization without eCG), R23/200 (resynchronization with 200 IU of eCG), R23/300 (resynchronization with 300 IU of eCG), R23/TCR (resynchronization with temporary calf removal [TCR]), and a control group, with estrus observation followed by AI (with no resynchronization). Treatment consisted of a progesterone device plus administration of estradiol benzoate on Day 0; on Day 8, the device was removed and cloprostenol was applied, together with estradiol cypionate. Also on Day 8, either eCG was administered or TCR was performed in the resynchronized groups, except for R23. The females were inseminated 48 hours after device removal or TCR (33 days after the first TAI). The control group was kept under estrus observation from 18 to 23 days after the first TAI and was inseminated 12 hours after detection of estrus. The first pregnancy evaluation was performed using ultrasound examination 31 days after the first TAI. After 30 days of the resynchronization, a second pregnancy evaluation was performed and the animals in the R23/300 and R23/TCR groups achieved the highest conception rates, 76.6% and 74.0%, respectively (P < 0.05). There were no differences between the conception rates of the animals in the R23/200 (63.3%), R23 (61.3%), and control (54.3%) groups (P > 0.05). These results suggest that estrus resynchronization at 23 days after TAI can effectively improve the conception rate of lactating Bos indicus cows in a short time period. Furthermore, resynchronization with 300 IU of eCG or with TCR provided the best results.

Dynamics of follicular growth and progesterone concentrations in cyclic and anestrous suckling Nelore cows (Bos indicus) treated with progesterone, equine chorionic gonadotropin, or temporary calf removal

The objective of this study was to investigate the effects of eCG and temporary calf removal (TCR) associated with progesterone (P4) treatment on the dynamics of follicular growth, CL size, and P4 concentrations in cyclic (n = 36) and anestrous (n = 30) Nelore cows. Cyclic (C) and anestrous (A) cows were divided into three groups. The control group received 2 mg of estradiol benzoate via intramuscular (IM) injection and an intravaginal device containing 1.9 g of P4 on Day 0. On Day 8, the device was removed, and the animals received 12.5 mg of dinoprost tromethamine IM. After 24 hours, the animals received 1 mg of estradiol benzoate IM. In the eCG group, cows received the same treatment described for the control group but also received 400 UI of eCG at the time of device removal. In the TCR group, calves were separated from the cows for 56 hours after device removal. Ultrasound exams were performed every 24 hours after device removal until the time of ovulation and 12 days after ovulation to measure the size of the CL. On the same day as the CL measurement, blood was collected to determine the plasma P4 level. Statistical analyses were performed with a significance level of P ≤ 0.05. In cyclic cows, the presence of the CL at the beginning of protocol resulted in a smaller follicle diameter at the time of device removal (7.4 ± 0.3 mm in cows with CL vs. 8.9 ± 0.4 mm in cows without CL; P = 0.03). All cows ovulated within 72 hours after device removal. Anestrous cows treated with eCG or TCR showed follicle diameter at fixed-timed artificial insemination (A-eCG 10.2 ± 0.3 and A-TCR 10.3 ± 0.5 mm) and follicular growth rate (A-eCG 1.5 ± 0.2 and A-TCR 1.3 ± 0.1 mm/day) similar to cyclic cows (C-eCG 11.0 ± 0.6 and C-TCR 12.0 ± 0.5 mm) and (C-eCG 1.4 ± 0.2 and C-TCR 1.6 ± 0.2 mm/day, respectively; P ≤ 0.05). Despite the similarities in CL size, the average P4 concentration was higher in the A-TCR (9.6 ± 1.4 ng/mL) than in the A-control (4.0 ± 1.0 ng/mL) and C-TCR (4.4 ± 1.0 ng/mL) groups (P < 0.05). From these results, we conclude that eCG treatment and TCR improved the fertility of anestrous cows by providing follicular growth rates and size of dominant follicles similar to cyclic cows. Additionally, TCR increases the plasma concentrations of P4 in anestrous cows.

Influence of category-heifers, primiparous and multiparous lactating cows-in a large-scale resynchronization fixed-time artificial insemination program

This study was conducted to evaluate the influence of category (heifers, primiparous or multiparous cows) on pregnancy rates in a large scale resynchronization ovulation program. Nelore heifers (n = 903), primiparous lactating cows (n = 338) and multiparous lactating cows (n = 1,223) were synchronized using a conventional protocol of estradiol/P4-based fixed-time artificial insemination (FTAI). Thirty days after ultrasonography, females who failed the first FTAI were resynchronized with the same hormonal protocol prior to a second FTAI. The pregnancy status of each cohort was evaluated by ultrasonography 30 days after each FTAI. The average conception rate after the first FTAI and resynchronization was 80.5%. Heifers had a higher conception rate (85%) than primiparous (76%) or multiparous cows (78%; p = 0.0001). The conception rate after the first FTAI was similar among heifers (57%), primiparous cows (51%) and multiparous cows (56%; p = 0.193). After the second FTAI, heifers exhibited a higher conception rate (66%) than primiparous or multiparous cows (51%; p = 0.0001). These results demonstrate the feasibility of resynchronization in large beef herds for providing consistent pregnancy rates in a short period of time. We also demonstrated that ovulation resynchronization 30 days after FTAI is particularly effective for heifers, providing a conception rate of up to 66%.

Histophilus somni -associated syndromes in sheep from Southern Brazil

Histophilus somni is a Gram-negative bacterium that is associated with a disease complex (termed histophilosis) that can produce several clinical syndromes predominantly in cattle, but also in sheep. Histophilosis is well described in North America, Canada, and in some European countries. In Brazil, histophilosis has been described in cattle with respiratory, reproductive, and systemic disease, with only one case described in sheep. This report describes the occurrence of Histophilus somni-associated disease in sheep from Southern Brazil. Eight sheep with different clinical manifestations from five farms were investigated by a combination of pathological and molecular diagnostic methods to identify additional cases of histophilosis in sheep from Brazil. The principal pathological lesions were thrombotic meningoencephalitis, fibrinous bronchopneumonia, pulmonary abscesses, and necrotizing myocarditis. The main clinical syndromes associated with H. somni were thrombotic meningoencephalitis (n = 4), septicemia (n = 4), bronchopneumonia (n = 4), and myocarditis (n = 3). H. somni DNA was amplified from multiple tissues of all sheep with clinical syndromes of histophilosis; sequencing confirmed the PCR results. Further, PCR assays to detect Pasteurella multocida and Mannheimia haemolytica were negative. These findings confirmed the participation of H. somni in the clinical syndromes investigated during this study, and adds to the previous report of histophilosis in sheep from Brazil.

Ovarian follicular dynamics and conception rate in Bos indicus cows with different antral follicle counts subjected to timed artificial insemination

Two experiments in Nelore cows subjected to timed artificial insemination (TAI) were designed to compare the influence of the antral follicle count (AFC/AFCs) on (1) ovarian follicular dynamics and (2) conception rates. First, multiparous cows with high (≥45 follicles; n=43) or low (≤15 follicles; n=32) AFCs were selected to undergo a TAI protocol to monitor ovarian follicular dynamics. Second, the AFCs of 962 cows also subjected to TAI were determined and classified as groups of high (G-high; ≥45 follicles; n=194), intermediate (G-intermediate; ≥20≤40 follicles, n=397) or low (G-low; ≤15 follicles; n=243) AFCs. In study I, the ovarian measurements (diameter, perimeter and area) were greater (P≤0.05) and there was a greater consistency in number of antral follicles during the period of synchronization for TAI in the high than low group. Effects of the AFC and ultrasonic evaluation interval on the follicular diameter were observed (P≤0.05); however, there was no interaction (P>0.05). Dominant follicles had greater diameters (P≤0.05) in the G-low than in the G-high at D4 (7.3±2.2 vs. 6.2±1.4mm, respectively, P=0.06), D8 (11.2±1.8 vs. 9.5±1.8mm, respectively), D9 (12.3±1.7 vs. 10.6±1.7mm, respectively), and D10 (13.4±1.3 vs. 12.2±1.8mm, respectively), as well as greater estimated diameters of ovulatory follicles (14.4±1.5 vs. 13.4±2.1mm, respectively, P=0.08). In study II, the cows with fewer AFCs had greater (P≤0.05) conception rates (61.7%) than the cows with the intermediate (52.9%) and greater (49.5%) AFCs. Nelore cows with fewer AFCs subjected to synchronization of time of ovulation had a larger follicular diameter and a greater conception rate than the groups with intermediate and a greater AFCs.

The impact of sanity on the fertility of cows submitted to reproductive biotechnics

The productivity in the herds is directly associated with reproduction. Thus, in order to achieve a better efficiency, the most varied reproductive biotechniques have been applied to the plants. Several factors have a direct influence on reproductive performance, including genetics, nutrition, zootechnical management, and sanitation. However, the sanity of the herd through the use of prophylactic measures against infectious agents is often neglected. In this context, particular attention should be paid to health programs, since 37-50% of gestational losses in the herd are consequences of infectious diseases of the reproductive tract caused by viral, bacterial or protozoan agents. Thus, this review will discuss the main advantages obtained with the use of reproductive biotechniques to increase fertility and the epidemiological aspects related to the diseases that cause reproductive failures in cows. Finally, we will also explain the importance of implementing sanitary programs with adequate management and vaccination of animals, to minimize economic losses related to reproductive diseases and, consequently, obtain higher pregnancy rates in the use of reproduction biotechnologies.