Fixed-time Artificial Insemination Research Articles

Synchronization of the bovine estrous cycle: Current and emerging opportunities

Controlling the bovine estrous cycle is a necessity if performing fixed-time artificial insemination or embryo transfer. Addition­ally, irrespective of the genetic objectives driving the decision to use reproductive technologies, the use of synchronization protocols often has beneficial effects on the overall reproduc­tive performance and commercial production output of a beef cattle operation. This proceedings paper provides a general review of the conceptual goals and challenges associated with synchronization of estrus, and directs readers to resources on current protocols and emerging opportunities in the field.

Use of long-acting injectable progesterone in fixed-time artificial insemination protocols

Knowledge of the physiology of the estrous cycle in bovine females has been extremely important in monitoring and improving strategies in the use of drugs to control their estrous cycle and follicular development, providing better recruitment, selection, growth, and maturation until ovulation, with strategies to further optimize estrus and ovulation synchronization protocols. The objective of this work is to carry out a bibliographical analysis comparing the use of slow-release progesterone devices in relation to the use of long-acting injectable progesterone, taking into account their advantages, disadvantages and characteristics within a fixed-time artificial insemination program.

242 Comparison of GnRH administration day in the 7&7 Synch versus 8&6 Synch Protocol for fixed-timed artificial insemination in beef heifers

Abstract The research aimed to investigate the impact of reducing the time interval for progesterone exposure on the ovulatory follicle just before performing fixed-timed artificial insemination (FTAI). This was achieved by adjusting the timing of administering a gonadotropin-releasing hormone agonist (GnRH; specifically, 100 µg gonadorelin, known as Cystorelin). The study assessed the effectiveness of two estrous synchronization protocols, the “7 & 7 Synch” and a modified protocol, by examining estrus expression (EE) and the resulting pregnancy rates (PR) following FTAI. The research utilized frozen conventional Angus semen from Select Sires, Inc. (Plain City, OH), and involved 66 Angus-cross heifers from the beef cattle research unit near Fayetteville at the University of Arkansas. To ensure consistency, the heifers were divided into two treatment groups based on their cyclicity status (CYC) and age. CYC status was determined by assessing a reproductive tract score (RTS) 1 wk before the synchronization process. RTS scores ranged from 1 to 5, with RTS 1-3 indicating pre- or peripubertal status and scores of 4 and 5 indicating cycling. Heifers with an RTS of 1 and 2 were excluded from the study before synchronization commenced. Both treatment groups involved the insertion of a controlled internal drug-releasing device (CIDR; specifically, Eazi-breed, containing 1.38 g of progesterone) into the vagina on d -14. Simultaneously, a prostaglandin F2α analogue (PGF; 500 µg cloprostenol sodium, known as Synchsure) was administered. In the first treatment group (7&7), GnRH was administered on d -7, while in the second group (8&6), GnRH was given on d -6. In both cases, PGF was administered on d 0 when the CIDR was removed, precisely 72 ± 1 h before FTAI, along with another dose of GnRH. Estrus expression (EE) was assessed during FTAI by examining Estrotect patches (Rockway Inc., Spring Valley, WI). EE was considered present when a patch had 50% or more of its color rubbed off, and it was deemed absent when less than 50% of the color had worn off. Pregnancy rates (PR) were determined through ultrasound examination on d 55. EE and PR were analyzed by treatment using the GLIMMIX procedure on SAS 9.4 (SAS Institute Inc., Cary, NC). Statistical differences were declared at α ≤ 0.05 and tendencies declared between 0.05 < P ≤ 0.1. Heifers on the 8&6-d protocol had greater rectal temperatures at breeding compared with 7&7-d heifers (P < 0.0001). AI pregnancy rates were greater in heifers on the 8&6-d protocol compared with the heifers on 7&7-d protocol (P = 0.625) with average pregnancy rates of 39.4% and 30.3%, respectively. Overall, pregnancy rate was greater as well for the 8&6-d heifers compared with the 7&7-d heifers (P = 0.044), with mean overall pregnancy rates of 78.8% and 54.5%, respectively.

PSIII-7 7&7 Synch improved pregnancy rates in beef cows experiencing estrus over 24 h before fixed-time insemination

Abstract Our objectives were to compare estrus response and pregnancy rates between two different estrus synchronization protocols and between cows that experienced estrus more than 24 h before appointment breeding and those that experienced estrus less than 24 h before breeding. Over each of 2 yr, postpartum beef cows (n = 1,108) from two different locations were ranked by days postpartum and randomly selected to receive either; 1) 7-d CO-Synch + CIDR (CO-Synch, n = 547), or; 2) 7 & 7 Synch (7&7, n = 561). For the CO-Synch treatment, cows received a controlled internal drug release insert (CIDR; 1.38g progesterone) and 100 µg gonadotropin-releasing hormone (GnRH) i.m., followed in 7 d by CIDR removal and 25 mg prostaglandin F2alpha (PGF) i.m., followed in 60 to 66 h with AI and 100 µg GnRH i.m. For the 7&7 Synch protocol, cows received a CIDR (1.38g progesterone) and 25 mg PGF i.m., followed in 7 d by 100 µg GnRH i.m., followed in 7 d by CIDR removal and 25 mg PGF i.m., followed in 60 to 66 h with AI and 100 µg GnRH i.m. Heat detection patches were applied at the time of CIDR removal to determine estrus response by the time of AI, with cows having patches ≥ 50% activated defined as being in estrus and cows with patches < 50% activated defined as not experiencing estrus. In yr 2, an additional heat detection was performed 24 h before breeding, at which time cows with ≥ 50% activated heat detection patches were defined as having an early estrus (i.e., estrus more than 24 h before breeding) and cows with activated patches first observed at the time of AI were defined as having an on-time estrus. Pregnancy status was determined via transrectal ultrasonography 35 d after AI. Data were analyzed using the GLIMMIX procedure of SAS. As expected, cows in the 7&7 treatment had greater (P ≤ 0.005) estrus response and pregnancy rates than those in the CO-Synch treatment (83% vs. 62% and 65% vs. 55%, respectively). The proportion of cows experiencing early estrus was similar (P > 0.60) between treatments (20.4 and 22.9 for Co-Synch and 7&7, respectively). Interestingly, pregnancy rate was impacted by a treatment × estrus timing interaction (P = 0.04), where cows in the Co-Synch treatment that experienced an early estrus (52.9%) had reduced pregnancy rates compared with 7&7 cows that experienced early estrus (74.0%), Co-Synch cows that experienced on-time estrus (73.7%) and 7&7 cows that experienced on-time estrus (70.5%). Our results indicate the 7&7 Synch protocol can effectively increase pregnancy rates to fixed-time AI, and that pregnancy rates of cows experiencing early estrus are influenced by synchronization protocol.

Combining Embryo Transfer and Artificial Insemination to Achieve Twinning in Beef Cattle, and Effects of Different Twin Calf-Raising Methods on Neonatal Behavior and Growth

As the beef industry moves towards efficient animal production to improve sustainability in agriculture, new production and management approaches are emerging. Among the many facets of the beef industry, cow–calf operations have the most opportunity for efficiency improvement, including improvements in fertility. This project accounts for measures and methods of (1) twinning reproductive technologies and (2) twin calf perinatal care and pre-weaning rearing. The overall objective was to produce twin calves using two reproductive technologies—embryo transfer and artificial insemination. The subobjectives were to determine accuracy of twin pregnancies embryo/fetal losses using ultrasonography, evaluate parturition and dystocia, and determine the effects of different twin-raising methods on neonatal behavior and growth. A fixed-time artificial insemination (FTAI) protocol was applied to 77 multiparous Angus-cross cows from a commercial beef herd in northcentral South Dakota during the summer of 2019. Cows were assigned to two different treatments groups: only artificially inseminated (AI) or received an embryo transfer following artificial insemination (ET + AI). They were estrous-synchronized, artificially inseminated (AI) with black Angus semen at day 0, and received and embryo transfer (ET) at day 7. Ultrasound examination detected 56% pregnancy risk for both groups, with sensitivity, specificity, and accuracy of 75%, 100%, and 90.5%, respectively, for bilateral twin detection. Calves were born during spring 2020. Twin calves (n = 34) and singleton calves (n = 11) were assigned to one of three raising methods: (1) twin born and twin raised (TT; n = 16), (2) twin born and single raised (TS; n = 18), and (3) single born and single raised (S; n = 11). Neonatal nursing behavior and birth weights were recorded, and adjusted day 200 and day 280 were calculated measures of vitality and growth. Blood samples were collected at age 24 h for colostrum intake measures (total serum protein, IgG1, and IgM). Twin calves were born 20% (p < 0.05) lighter in body weight than singletons; however, weights did not differ at day 280 between TT and S calves. TS calves had the shortest average latency to stand, but immunoglobulin concentrations did not differ among treatments. At weaning, cows that had birthed and raised twins produced more kilograms of live weight per pregnancy than cows birthing and raising singletons. Using ET + AI proved to increase twinning rate, and growth was maintained when raising both twins with their dam.

Blood Plasma Biochemical Changes and Conception Rate in Anoestrus Buffaloes Treated with Various Hormone Protocols

To study certain blood biochemical parameters (total cholesterol, total protein, calcium and inorganic phosphorus) at various periods of treatment. Total 40 anestrous buffalo heifers with history of anoestrus were selected from the semi-arid area of Banaskanth and randomly divided into four equal groups. (1)Group-I:Heatsynch protocol (n=10), (2)Group-II:Ovsynch protocol(n=10), (3)Group-III: Doublesynch protocol (n=10) and (4)Group-IV: Control(n=10) followed by fixed time artificial insemination in treated buffalo heifers and insemination at spontaneous estrus in buffalo heifers of group-IV. The overall conception rate following 3 consecution services was maximum in Group II (50%) followed by Group III (40%), Group I (30%) and Group IV (30%). The blood plasma was collected at the time of initiation of treatment (P1), at the time of inj. PGF2α (P2), at the time of 1st FTAI (P3) and day 21st post insemination (P4) for the estimation of biochemical parameters. The blood biochemical profiles include total protein (range: 4.54± 0.19 to 6.11± 0.52 g/dl) which in all group did not show any significant difference during the different period but significant (P<0.05) difference between the groups was noticed. Similarly calcium and phosphorus found ranging between 5.73± 0.20 to 9.53± 0.95 mg/dl and 5.46± 0.22 to 6.95± 0.30 mg/dl, respectively in all four groups. The cholesterol estimated values (range: 34.12 ± 1.75 to 41.97± 2.43 mg/dl) were found not-significantly varying between the groups as well as among the different periods. Heatsynch, Ovsynch and Doublesynch protocol can be successfully used for the induction of estrus in true anoestrus buffalo heifers with Better conception rate would be achieved following Ovsynch protocol in buffalo heifers.

15 Influence of early progesterone removal on follicular development, estrus expression, and pregnancy rates in presynchronized beef cows

Abstract The objective of this study was to evaluate the impact of early progesterone removal on pregnancy rates to fixed-time artificial insemination (FTAI) in presynchronized beef cows. Postpartum beef cows (n = 922; body condition score = 4.9 ± 0.77; days postpartum = 77.3 ± 20.77 d) from 7 locations were enrolled in a completely randomized design. Within location, cows were stratified by parity and days postpartum before being randomly assigned to 1 of 2 treatments: 1) 7&7 Synch: Cows received a controlled internal drug release insert (CIDR) and a 25-mg injection of prostaglandin F2α (PG1; d 0). Cows received 100 μg of GnRH on d 7 and the CIDR was removed 7 d later, concurrently with a second prostaglandin F2α (PG2) injection (d 14). Cows received a second injection of GnRH and were FTAI 60-66 h after PG2 (d 17); 2) 7&6 Synch: Cows received the same treatment as 7&7 Synch cows; however, CIDR removal and PG2 were performed on d 13, and cows were FTAI 60-66 h after CIDR removal. Ovarian ultrasonography was performed to determine follicle diameter and CL presence at PG2 and FTAI in a subset of cows (n = 40). Estrus detection patches were utilized to evaluate estrus expression between PG2 and FTAI. Pregnancy diagnosis was performed 28-35 d after FTAI. Cows exposed to the 7&7 Synch tended to have larger follicle diameter at PG2 compared with 7&6 Synch cows (P = 0.09); however, there were no differences in follicle diameter at FTAI. Cows that expressed estrus tended to have larger follicle diameter at FTAI compared with cows that failed to express estrus (P = 0.10). Follicular growth between PG2 and FTAI, percentage of cows with a corpus luteum at PG2, and ovulation rate were not impacted by treatment (P ≥ 0.56). No differences were determined between treatments for estrus expression (7&7 Synch: 77.1 ± 5.91; 7&6 Synch: 74.6 ± 6.28; P = 0.50) or pregnancy rates to FTAI (7&7 Synch: 60.1 ± 4.32; 7&6 Synch: 58.2 ± 4.26; P = 0.69). In conclusion, early removal of progesterone did not impact pregnancy rates in presynchronized beef cows. Thus, the 7&6 Synch protocol described herein may be used as an alternative to the conventional 7&7 Synch protocol without compromising fertility.

13 Effects of an acclimation protocol during the handling events of the 7 d CO-synch + CIDR protocol on temperament and reproductive performance of Bos taurus commercial beef heifers

Abstract It has been previously reported that cattle temperament significantly impacts fertility to estrous synchronization (ES) protocols. In addition, enrolling heifers to acclimation protocols following weaning resulted in improved reproductive parameters with an increased proportion of acclimated heifers reaching puberty earlier when compared with non-acclimated cohorts. The objective of the present experiment was to assess the effects of an acclimation protocol during the handling events of an ES on changes in temperament and fertility to the ES protocol in Bos taurus commercial beef heifers. A total of 361 Angus-influenced commercial heifers from 5 different locations were enrolled in the experiment. Before enrolling heifers in the 7-d CO-Synch + CIDR protocol (d -10), individual heifers were evaluated for reproductive tract score (RTS), and chute score (CS) and stratified to receive acclimation (TRT) or to serve as control (CTRL). On d -10, heifers from TRT and CTRL groups received ear tags of different colors and individuals from CTRL and TRT were kept together in their pastures. Before each handling event of the ES (d 0,7,10), TRT heifers were sorted from CTRL and submitted to acclimation, which consisted of running heifers through the handling facilities without catching individuals in the restraining chute. Treatment heifers were then comingled with CTRL heifers, and all heifers were brought to the handling facilities for the ES event. Individual CS and exit velocities (EV) were recorded for all animals at all days (-10,0,7,10). Chute scores were recorded in a 1 to 5 scale, where 1 = calm, no movement; 2 = restless movements; 3 = frequent movement, vocalization; 4 = constant moving, vocalization, shaking of the chute; 5 = violent, continuous struggling. Individual estrus detection patch scores were assessed at the day of fixed-time artificial insemination (FTAI) based on percentage of activation, where 0 = lost; 1 = less than 25% activated, 2 = less than 50% activated, 3 = less than 75% activated, 4 = more than 75% activated. Pregnancy status was determined by rectal ultrasonography approximately 40 d after FTAI. Estrus detection patch scores did not differ between CTRL and TRT heifers (P = 0.1906). In addition, pregnancy rates to FTAI did not differ between TRT and CTRL heifers (56.81% and 52.74%; P = 0.3563). Reproductive tract scores (P = 0.777), EV (P = 0.9607) and CS (P = 0.990) did not differ between CTRL and TRT on d -10. However, CS, but not EV was decreased on d 7 (P = 0.001) and 10 (P = 0.0002) for TRT heifers when compared with CTRL. We conclude that enrolling heifers to an acclimation protocol during the handling events of the ES protocol was effective at decreasing excitability of heifers by the day of FTAI, as evidenced by decreased CS observed for TRT heifers on d 7, and 10 but did not support an increase in fertility to the ES protocol.

28 Investigating the effects of Chromium Propionate supplementation on reproductive performance of beef cows

Abstract The objective of this study was to explore the effects on reproductive performance of beef cows supplemented with Chromium Propionate during the post-partum and breeding periods. A total of 955 multiparous suckled beef cows located at eight locations were enrolled in the study. Within location cows were stratified based on parity, body condition score (BCS), and days postpartum (DPP) and randomly assigned to one of two treatments: 1) Control- supplementation of a mineral product at 113 g animal/d (n = 484 cows; 16 experimental units); or 2) CrP - supplementation of mineral product at 113 g animal/d containing Chromium Propionate (0.4% animal/d, KemTRACE, Kemin Industries; n = 471 cows; 16 experimental units). Mineral supplementation was offered starting at 45 d pre-breeding, during the breeding season, and until weaning for a total of approximately 150 d. Cows were enrolled in a fixed-time artificial insemination (TAI) using the 7-d CO-Synch+CIDR protocol followed by natural service with clean-up bulls 7 d after TAI, for a 70-d total breeding season. Pregnancy diagnosis was performed by ultrasonography 55 d after TAI and again approximately 40 d after the end of the breeding season. Cow body weight (BW) and BCS were recorded at the beginning of the experiment (d -45), at the initiation of the TAI protocol (d -10), at each pregnancy diagnosis (d 55 and 110), and at weaning (d 150). Mineral intake was estimated based on the difference in weight between offered mineral and refusals. Data were analyzed using the GLIMMIX, for binomial data, and MIXED, for continuous data, procedures of SAS. Cow initial BW and DPP were similar (P > 0.10) between treatments (584 ± 198 kg and 78 ± 3 d, respectively). Estimated mineral intake was similar (P = 0.52) between treatments (119.6 ± 10.3 g and 124.5 ± 10.4 g animal/d, for Control and CrP, respectively). Supplementation of CrP did not influence estrus expression (58.3% vs 61.0% ± 2.5 %; P = 0.45), TAI pregnancy rates (63.1% vs 60.1 % ± 2.9 %; P = 0.56), or final breeding season pregnancy rates (94.2% vs 94.7% ± 2.9 %; P = 0.86), for CrP and Control, respectively. Chromium Propionate supplementation did not affect (P > 0.10) final cow BCS and BW (5.2 ± 0.03; 562.9 ± 169.93 kg). In conclusion, Chromium Propionate supplementation delivered via an oral mineral supplement during the pre-breeding and breeding periods did not influence reproductive performance and changes in BW and BCS of beef cows.

130 Effect of an appeasing substance application on growth, stress, handling quality and reproduction of Bos indicus beef heifers synchronized to fixed-time AI

Abstract This experiment evaluated the effects of an analogue of bovine appeasing substance (BAS) on growth, stress, handling quality, and reproduction of Nellore heifers. Twenty-nine heifers [body condition score (BCS) = 4.84 ± 0.13; body weight (BW) = 371 ± 0.77 kg; 23 ± 2 months of age)] were assigned to 1 of 2 treatments: 1) Saline solution (0.9 NaCl; n = 14) or BAS (Secure Cattle, IRSEA Group, Quartier Salignan, France; n = 15). Both solutions were applied topically in the nuchal skin area (5 mL/heifers) at the beginning (d -9) of the fixed-time artificial insemination protocol (FTAI). Heifers were allocated in two different pastures according to the treatment (one pasture per treatment) for 15 d (time of action of BAS) and then combined in a single group (both treatments in the same pasture). Heifers were inseminated on d 0, and BW and BCS were collected on d -9 and 40. The temperament in the squeeze chute (entry and exit scores; scale 1 – 3; chute score; scale 1 – 5; greater values mean more reactive heifers) and blood samples were collected on d -9, 0, 7, 14 and 30. The percentual of aggression, accident, and dirtiness in the perineal region were evaluated on d 0 along with time to inseminate, estrus expression scores (scale 1 – 3; 1 = no expression, 2 = low expression, 3 = greater expression), and dominant follicle size. The corpus luteum size was measured on d 7 and 14, and the pregnancy and embryo size on d 40. The BAS vs. Saline-treated heifers tended to have greater BW change (23.6 vs. 20.8 ± 1.03 kg; P = 0.09) and had greater BCS change (0.05 vs. -0.21 ± 0.05; P < 0.01). The BAS vs. Saline-treated heifers tended to have lesser entry scores (1.42 vs. 1.67 ± 0.09; P = 0.09) and percentual of dirtiness in the perineal region (48.3 vs. 71.1 ± 8.67 %; P = 0.06), and no effects of treatment were detected (P ≥ 0.39) for chute score, exit score, aggression, accident and time to inseminate. The BAS vs. Saline-treated heifers had greater estrus scores (2.00 vs. 1.27 ± 0.21; P = 0.03), and no effects were detected (P ≥ 0.11) for ovarian variables, pregnancy rate, and conceptus size. Furthermore, no treatment effects were detected (P ≥ 0.25) for serum concentration of cortisol and plasma concentration of haptoglobin, ceruloplasmin, and progesterone. Therefore, BAS application at the beginning of the FTAI protocol could decrease the temperament and dirtiness in the perineal region and increase the growth and estrus expression of beef heifers.

82 Effects of a red-clover containing mineral on heifer behavior and fertility

Abstract Heat stress reduces reproductive efficiency in beef cattle. Recent evidence suggests that supplementation of red clover can alleviate the physiological impacts of fescue toxicosis which could decrease heat stress. Our objective was to determine the effect of supplementing heifers grazing endophyte-infected tall fescue (E+ TF) with a conventional mineral containing red-clover leaf. We hypothesized that heifers supplemented with the red-clover leaf mineral would have greater conception rates to AI (CRAI) in times of possible heat stress compared with heifers supplemented with conventional mineral. A grazing study was conducted from May – August in 2021, 2022, and 2023. Yearling beef heifers (n = 32, 36, 40 heifers yr-1, respectively) were blocked by body weight, hair coat score and color into 4 grazing blocks that were assigned to 1 of 2 treatments (2 blocks treatment-1): 1. conventional non-medicated loose mineral control (0%-RC), or 2. conventional loose mineral + 20% wt/wt red clover leaf (20%-RC). Mineral treatments were provided in ground, 3-compartment mineral feeders ad libitum. Each grazing block was assigned to four, 0.8- ha. pastures consisting of predominantly E+ TF and were rotationally managed over the course of the study. Each year estrus was synchronized suitable for fixed time AI in May and all heifers resynchronized in June. In addition, each heifer was fitted with an ear-tag sensor (CowManager SensOor, Agis Automatisering BV, Harmelen, the Netherlands) for continuous behavioral monitoring (including high activity and activity). The CRAI were analyzed with the GLIMMIX procedure of SAS using the binomial distribution link logit function (v.9.4., SAS Inst. Inc., Cary, NC). Total daily activity and high activity reported by CowManger during May and June were analyzed by the MIXED procedure with repeated measures of SAS. The CRAI were similar for 20%-RC (67.30 ± 9.80%) and 0%-RC (60.70 ± 9.80%) heifers following the first service of AI (P = 0.51) during a thermoneutral period in May. In contrast, CRAI was greater (P = 0.02) for 20%-RC heifers (48.67 ± 13.47%) than CM (15.10 ± 13.47%) in June when temperatures increased to potential heat stress levels. Overall, conception rate after two rounds of AI for 20%-RC (78.30 ± 9.30%) heifers tended to be greater (P = 0.08) than CM (62.20 ± 9.30 %). While high activity (associated with estrous behavior) reported by CowManager did not differ between treatments (20%-RC: 203.94; 0%-RC: 213.90; SEM: 8.65 min d-1; P = 0.25), normal daily activity of 20%-RC heifers (150.69 ± 10.40 min d-1) tended to be greater (P = 0.06) than CM (126.33 ± 8.65 min d-1). Results of this study suggest supplementing red-clover containing mineral may mitigate negative effects of consuming E+ TF including decreased conception rates during times of heat stress.

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

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

Human Chorionic Gonadotrophin (hCG) induces changes in IFN-pathway and Interferon-Stimulated Genes (ISGs) on the bovine endometrium at Day 18 of pregnancy.

We hypothesized that the hCG modulates the expression of IFNT-pathway and ISGs in bovine endometrium during early pregnancy. The aim of the current study is to evaluate the effect of hCG on IFNT-pathway signals and ISGs expression in endometrial cells. For this, 29 non-lactating cross-bread cows were used in the study and submitted to a 9-day fixed-time artificial insemination (FTAI) protocol. The day of the AI was considered Day 0 (D0), and five days (D5) after the FTAI, the cows were allocated into two groups: Control and hCG group, when a hCG group received a single dose of 2.500UI of hCG. On day 18 after FTAI (D18) cows were slaughtered and endometrial tissue samples were collected. There was no difference between the embryo recovery rate of the cows in C compared to the hCG. The hCG group increased the accessory corpus luteum formation rate. The hCG resulted in greater serum progesterone concentration in the hCG group compared to the C on Day 14. Only the expression of IFNAR2 and STAT1 were upregulated on pregnant cows of the hCG group compared to the C group. The pathway genes (JAK1, STAT2, and IRF9) were not regulated. The mRNA abundance of ISG15, MX1, MX2, and OAS1 was upregulated in pregnant cows for hCG group, compared to C group. The results show that the administration of hCG, 5 days after AI, in addition to increasing the serum progesterone, modulates the expression of IFNT-pathway and ISGs on bovine endometrium on Day 18 of pregnancy.

Influence of Ovarian Follicle Sizes and Estrous Signs on Pregnancy Following Progesterone-Based Fixed Time Artificial Insemination in Water Buffaloes

The objectives of the present study were to elucidate the importance of follicle sizes and estrous signs during Controlled Internal Drug Release-Synch-human Chorionic Gonadotropin (CIDR-Synch-hCG) protocol for Fixed Time Artificial Insemination (FTAI) and to evaluate their association with pregnancy in water buffaloes. Data from riverine buffaloes (n = 207) under the CIDR-Synch-hCG protocol were analyzed. Buffaloes were administered with Gonadotropin-Releasing Hormone (GnRH) with insertion of CIDR on Day 0. Prostaglandin (PGF2α) was given on Day 7 with the removal of CIDR. hCG was given on Day 9, and AI was performed on Day 10. Follicle measurements by ultrasonography were done on Days 0, 7, and 10, and follicle sizes on those days were categorized into I, II, and III. Estrus signs were taken on the day of AI. The pregnancy diagnosis was done on Day 30-35 post-AI. The average size of follicles in Category III is significantly higher than those of Categories I and II, regardless of the Days of the protocol. Pregnancy is significantly (P<0.001) associated with Pre-Ovulatory Follicle (POF) size and uterine tonicity on the Day of AI but not with follicle sizes on Days 0 and 7, nor with mucus discharge discharge (P>0.05). The overall pregnancy rate is 44.44% while performing AI with POF size ≥12.0 mm increased the probability of pregnancy rate to 56.25%. In conclusion, the present study demonstrated a follicle size-based CIDR-Synch-hCG protocol providing new fertility indicators to improve FTAI efficiency in buffaloes with huge application in other livestock species.

Effects of cortisol levels on reproductive success in cattle of different temperaments during fixed-time artificial insemination (FTAI)

The present study was designed to assess the effect of stress on the reproductive performance of cattle. For this purpose, a total of 137 cows were divided into two groups: 65 belonging to the Azores Lydia breed and 72 crossing Aberdeen-Angus with Limousine, free of IBR, Chlamydia and BVD diseases and with a body condition score ranging from 2.75 to 4.0. Eight days before starting the experimental procedures, animals passed through the containment sleeve every other day for routine and human presence. For the synchronization protocol, a progesterone-impregnated controlled internal drug release (CIDR®) insert was placed intravaginally, and an injection of GnRH was given. After 7 days, PGF2α was administered, and the CIDR® was removed. Sixty hours after removal, cows were inseminated once after another injection of GnRH to induce ovulation, and pregnancy diagnosis was performed by ultrasonography 30 days after artificial insemination. Peripheral blood was collected in 5 ml tubes with a Z Serum Sep Clot Activator, and cortisol was measured using the IMMULITE 2000 Immunoassay System ®. Our results indicated significant differences (p≤0.05) in the cortisol levels among both groups (beef crossbred cows vs Azores Lydia) of 4.3±0.3 ng/dl and 5.8±0.4 ng/dl, respectively. The pregnancy results were also significantly different: 63.7% vs 45.6%, respectively, for beef crossbred and "Azores Lydia" cows. The present study clearly demonstrated a negative correlation between cortisol levels and pregnancy rates after fixed-time artificial insemination. The low levels of cortisol observed in the animals and particularly in the Azores Lydia breed, when compared to other studies carried out on these animals, must be due to the passage of the animals in the sleeve, allowing them to habituate to a routine, as well as to human presence.

178 Optimization of the time of insemination and presynchronization treatment in beef heifers submitted to fixed-time artificial insemination

178 Optimization of the time of insemination and presynchronization treatment in beef heifers submitted to fixed-time artificial insemination

181 Expression of estrus and fertility of different presynchronization strategies for fixed-time artificial insemination in suckled beef cows

181 Expression of estrus and fertility of different presynchronization strategies for fixed-time artificial insemination in suckled beef cows

Comparative evaluation of natural tupping with fixed-time laparoscopic and cervical insemination techniques using chilled semen in estrous synchronized sheep

The present study was conducted in crossbred ewes to compare the efficiency of different artificial insemination techniques with natural tupping following fixed time artificial insemination using fresh semen. Crossbred ewes (n=29) were randomly divided into three groups with 10 animals in natural tupping (NT) and laparoscopic artificial insemination (LAI) groups and nine animals in cervical artificial insemination (CAI) group. Ewes in all the treatment groups were subjected to the same estrus induction protocol: insertion of intravaginal progesterone sponges for 10 days followed by an intramuscular injection of 500 IU eCG at the time of sponge withdrawal. Immediately after removal of sponges, animals of NT group were kept with proven breeding rams up to 72 h. Fixed time cervical or laparoscopic insemination was done at 48 h after sponge removal. The insemination was repeated 12 h later in CAI group. The pregnancy and lambing rates were significantly higher in NT group (90%, 90%), than CAI group (55.5%, 55.5%) and LAI group (20%, 10%). The prolificacy rate was significantly higher in CAI group (180%) than NT group (111.1%) and LAI group (100%). The serum progesterone concentration was higher in pregnant ewes on day 10, day 17 and day 35 than non-pregnant ewes. The fixed time cervical insemination following intravaginal progesterone sponges for 10 day+eCG protocol resulted in better pregnancy rate and prolificacy rate in crossbred ewes during breeding season.

Effects of mineral and vitamin supplementation on pregnancy rates in Nelore cattle submitted to FTAI programs with different reactivity scores

A high-reactivity herd temperament reduces productivity and profitability in cattle farming. In Bos taurus indicus (Nellore) cattle, we hypothesized that a higher reactivity score is associated with a lower pregnancy rate, and that injectable supplementation with minerals (Cu, Zn, Mn, Se) and vitamins (A, E) would increase the pregnancy rate and reduce embryonic mortality in very reactive cows. Injectable supplement or control treatments were administered on days 25 and 10 before fixed-time artificial insemination (FTAI; Day 0 : day of FTAI). We selected 1722 cows 45 ± 7 days postpartum, with different behavioral reactivity levels, and a series of measures related to reproduction were measured. The cows were randomly grouped and received sc 1 mL/50 kg live weight of the supplement (n = 872) or placebo (n = 850), 25 and 10 days before the FTAI protocol (Day–25 and Day–10 respectively). On Day −10, devices containing 1 g progesterone were inserted into the vagina, and 2 mg estradiol benzoate was applied intramuscularly. On Day–2, the devices were removed and 530 µg of sodium d-cloprostenol, 300 IU of equine chorionic gonadotropin, and 1 mg of estradiol cypionate were applied. At 48 h after final injection, the cows were subjected to FTAI (Day 0). On Day - 25 (25 days before IATF), temperament was graded as 3 to 12 using composite reactivity score (RS), which evaluates movement and breathing while the animal is in the cattle chute, and their escape velocity, and was used to classify cows as calm (RS ≤ 4; n = 590), reactive (RS 5 to 8; n = 573), and very reactive (RS ≥ 9; n = 559). On Day 0, calm cows had a higher largest follicle diameter on the FTAI compared to reactive and very reactive (14.4 + 0.2 vs. 13.6 + 0.4 and 13.2 + 0.2 mm, respectively; P = 0.03). Interactions were observed, where supplemented cows compared to those no supplemented had higher pregnancy on Day 30 (P = 0.04; 60.00 vs. 54.10 % calm and 58.00 vs. 52.50 % very reactive) and Day 60 (P = 0.03; 58.70 vs. 47.90 % calm, 52.20 vs. 49.10 % reactive and 48.40 vs. 41.30 % very reactive) and reduced embryonic loss at 30 to 60 d (P = 0.04; 2.00 vs. 6.10 % calm, 4.20 vs. 8.40 % reactive and 9.60 vs. 11.20 % very reactive). Supplementation on Days–25 and–10 improved pregnancy rates and reduced embryonic in calm and very reactive cows.

Effects of a single supplemental dose of gonadotropin-releasing hormone in 2 different fixed-time artificial insemination synchronization protocols among beef cows and heifers

Effects of a single supplemental dose of gonadotropin-releasing hormone in 2 different fixed-time artificial insemination synchronization protocols among beef cows and heifers