Artificial Insemination Protocol Research Articles

Association of genomic prediction for twin pregnancies with the incidence of double ovulation in primiparous dairy cows.

Twin pregnancies compromise the health and well-being of dairy cattle. A recent genomic prediction model for twin pregnancies has been developed based on twin calving or abortion. However, the incidence of double ovulation is significantly higher than that of twin births. This study aimed to evaluate whether genomic prediction values for twin pregnancies are associated with the incidence of double ovulation in primiparous dairy cows. Factors influencing the double ovulation rate were analysed using binary logistic regression on 676 cows: 475 (70.3%) inseminated at spontaneous estrus and 201 following one of two different estrus synchronization protocols for fixed-time artificial insemination (FTAI). The odds ratio for double ovulations was 0.92 (p = .002) per unit increase in prediction value and 2 (p = .01) for cows subjected to an FTAI protocol. Our findings suggest that genomic prediction values for twin pregnancies can effectively identify the risk of double ovulation at the herd level.

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.

Metabolic imprinting in beef calves supplemented with creep feeding on performance, reproductive efficiency and metabolome profile

This experiment evaluated the influence of creep feeding supplementation on productive and reproductive performance and on serum metabolome profile in Nelore (Bos indicus) heifers. Female calves were assigned to treatments: Creep (n = 190), with ad libitum access to a nutritional supplement from 70 to 220 days after birth, or Control (n = 140), without supplementation. After weaning (Day 220), both groups followed the same pasture and nutritional management. Body weight (BW) and backfat thickness (BFAT) were measured over time. Blood samples were collected at 220 and 360 days for LC–MS/MS targeted metabolomics. On day 408, during the synchronization timed artificial insemination (TAI) protocol, reproductive status (RS: diameter of uterine horn and largest follicle, and presence of CL) was assessed. Creep feeding increased BW and BFAT at weaning, but no differences in BW, BFAT, or RS after weaning were observed. Nonetheless, the pregnancy per AI (P/AI) for 1st service was 28.9% higher in the Creep group. On day 220, 11 significant metabolites influenced five metabolic pathways: Glucose-alanine cycle, alanine, glutathione, phenylalanine and tyrosine metabolism, and urea cycle. On day 360, 14 significant metabolites influenced eight metabolic pathways: Malate-aspartate shuttle, arginine and proline metabolism, urea cycle, aspartate, beta-alanine, glutamate metabolism, ammonia recycling and citric acid cycle. In conclusion, creep feeding supplementation improved calf performance and induced metabolic changes at weaning and 360 days of age. Although heifers had similar productive performance and reproductive status, when submitted to TAI, those supplemented with creep feeding had greater P/AI.

Biochemical profile and oocyte quality of primiparous Bos indicus cows submitted to a timed artificial insemination protocol

The objective of this study was to verify the causes of the lower response of primiparous Bos indicus cows to the ovulation synchronization protocol. Two experiments were performed to evaluate the biochemical profile, oocyte and follicular cell quality (Experiment 1) and pregnancy (Experiment 2). In Experiment 1, suckled primiparous (n = 24) and multiparous cows (n = 24) were submitted to ovum pick up (OPU). On Day 0 (D0), all cows received 2 mg of estradiol benzoate (EB) and an intravaginal progesterone insert (P4). Five days (D5) after the first hormonal administration (EB + P4), all follicles larger than 3 mm were counted on each ovary, and ovum pick-up (OPU) was performed. On day 12 (D12), the intravaginal progesterone insert was removed, and measurement and aspiration of the largest follicle were performed. Blood samples were collected on D5 and D12 to evaluate the concentrations of glucose, cholesterol, NEFA, IGF-1 and insulin. In Experiment 2, suckled primiparous (n = 50) and multiparous (n = 50) cows were subjected to an ovulation synchronization protocol based on E2/P4 (D0: 2 mg EB plus P4 intravaginal insert; D8: 500 μg of cloprostenol, 1 mg cypionate estradiol and 300UI of eCG; D10: artificial insemination). In addition, blood samples were collected on D10 for evaluation of the same hormones and metabolites described in Experiment 1. In all studies, calves remained with the cows during the experimental period. In experiment 1, the number of oocytes grade 1 (P = 0.83), grade 2 (P = 0.23) and grade 3 (P = 0.51), total number of retrieved oocytes (P = 0.14), oocyte quality index (P = 0.93) and total viable oocytes (P = 0.38) did not differ between primiparous and multiparous cows. The number of follicles at the time of follicular aspiration (20.7 ± 1.5 vs. 18.0 ± 1.9; P = 0.05) and the diameter of the largest follicle on D12 (13.5 ± 0.6 vs. 11.4 ± 0.6; P = 0.04) were greater in multiparous cows, and the number of degenerated oocytes was greater in primiparous cows (1.9 ± 0.7 vs. 1.2 ± 0.3; P = 0.05). On D5, the concentrations of IGF-1 (P = 0.47), insulin (P = 0.08), total cholesterol (P = 0.47), NEFA (P = 0.77) and glucose (P = 0.55) in the blood and IGF-1 (P = 0.97) and insulin (P = 0.11) in the follicular fluid did not differ between parity groups. On D12, there was no difference in the concentrations of IGF-1 (P = 0.26), total cholesterol (P = 0.32), NEFAs (P = 0.31) and glucose (P = 0.93) in the blood between primiparous and multiparous cows, however, the serum insulin concentration (P = 0.04) was higher in primiparous cows. There was no correlation between serum and follicular fluid insulin concentrations (r = 0.17; P = 0.31), however, there was a low correlation between serum and follicular fluid IGF-1 concentrations (r = 0.47; P = 0.002). Quantification of transcripts did not differ between parity groups. In experiment 2, concentrations of NEFA (P = 0.12) and insulin (P = 0.16) in the blood and P/AI (P = 0.93) did not differ between parity [60 % (30/50) primiparous vs. 60 % (30/50) multiparous]. In contrast, blood concentrations of IGF-1 (P = 0.0001), total cholesterol (P = 0.005) and glucose (P = 0.01) were greater in primiparous cows. It was concluded that the oocyte quality and expression of the genes evaluated in the granulosa cells were not different between primiparous and multiparous cows. Unexpectedly, the pregnancy rate did not differ between parity. Nevertheless, the blood concentrations of IGF-1, total cholesterol and glucose were greater in primiparous cows.

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.

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.

Insemination timing influences reproductive performance rather than eCG (Equine Chorionic Gonadotropin) or synchronisation protocol in Murciano-Granadina does

Counteracting goat reproduction seasonality and ensuring acceptable fertility rates are critical points of the success of selection of synchronisation protocols. The present study aims to investigate the factors that affect the performance of specific protocols to increase the probability of pregnancy results in the Murciano-Granadina breed within the official breeding program and way in which such factors interrelate. This study evaluated 11,008 artificial inseminations carried out between 2010 and 2013, using three different synchronisation protocols and eCG combinations. A categorical regression model (CATREG) was used to determine the impact of farm, doe age, birth number, kidding type, kid number, number of alive and dead born kids, synchronisation protocol, kidding month, type of semen, kidding season, eCG concentration, and their inter-actions. Results indicated that all variables, except for the number of alive and dead kids and semen type, significantly (p < 0.05) contributed to explaining the variability in pregnancy rates. Conclusively, rather than the role of eCG levels and artificial insemination (AI) protocol separately, Farm x eCG x AI Protocol interaction and the multifactorial influences on pregnancy rates should be considered when selecting does for insemination to maximise the outcomes of reproductive performance.

Comparative effectiveness of different methods of using the analogue of gonadotropin-releasing hormone for ovulation stimulation in rabbit does

Modern reproductive technologies require artificial insemination (AI) protocols that are safe for the health of animals and do not violate their welfare. Stress factors arising in the reproduction process of industrial rabbit breeding negatively affect the sexual function and reproductive performance of rabbits and their offspring. In recent years, researchers have focused on alternative methods of ovulation stimulation, including intravaginal introduction of hormonal means of its induction. Therefore, the aim of our study was a comparative evaluation of the effectiveness of the intramuscular method of administration of the gonadotropin-releasing control (GnRH) analogue and its addition to the sperm dose for AI on Hyplus rabbit (n = 30), which were randomly divided into three groups. For this, buserelin acetate was administered intramuscularly at a dose of 1 μg per rabbit immediately after artificial insemination (control group). Experimental animals were inseminated with sperm doses with the addition of a GnRH analogue in the amount of 25 μg (experimental group 1) and 15 μg (experimental group 2). The influence of the method of administration of GnRH analogue on reproductive function was evaluated by indicators of the effectiveness of AI, fertility, and the dynamics of the progesterone level during pregnancy. A decrease in fecundity was observed in females; for example, in experimental group 1, the effectiveness of AI in terms of the number of rabbits was 64.3 %; in animals of experimental group 2 – 41.2 %, and the number of inseminations in this group of rabbits was 41.7% higher compared to control data. The fertility of rabbits of all groups was high, reaching the maximum value in the control group (10.42 ± 0.52 heads/female). Fertility according to live newborns in experimental group 1 was the highest among all groups of rabbits and was 9.67 ± 0.49 heads/female, which showed an increasing trend compared to the control. Also, in the animals of experimental group 1, an increase in the level of progesterone in blood serum was observed (on the 7th day of pregnancy by 13.2 %, on the 14th day by 14.3 %, on the 21st day by 16.4 %). In contrast, in experimental group 2, a significant decrease in the progesterone level was noted. Thus, 25 μg of GnRH analogue added to the extender is optimal. It is worth noting that there were no advantages of intravaginal administration over intramuscular administration in terms of reproductive performance. However, the number of live births and progesterone levels were improved.

Economic viability of using OvSynch and fixed timed artificial insemination protocol in breeding improvement of pastoral herds in the rangelands.

Though using Assisted Reproductive Technologies (ARTs) can improve oestrus detection, conception and pregnancy success, thus benefit breeding program implementation, empirical evidence of their economic viability is lacking to inform investment decisions in pastoral herds. This study assessed economic viability of using OvSynch and fixed Timed Artificial Insemination (TAI) protocol in Sahiwal upgrading breeding program under two hypothetical cases of best and worst in activity-based money allocations when pastoral herds deploy either optimal or low input husbandry practices. From herd owners' assessment of milk production, best-case scenarios attain on average 10 L/cow/day with optimal husbandry and 5 L/cow/day with low input husbandry. The worst-case scenarios attain 5 L/cow/day with optimal husbandry and 1 L/cow/day with low input husbandry. Benefit- Cost Analysis (BCA) estimated Net Present Value (NPV), Benefit-Cost ratio (BCR) and Internal Rate of Return (IRR) to establish economic viability of using OvSynch and TAI Protocol in pastoral breeding programs. Both best-case scenarios retuned positive NPVs (82,028 and 6,912), BCR values (1.68 and 1.08) and IRR (27.46% and 8.08%) while worst-case scenarios returned negative NPVs (-135,855 and -141,025), BCR values of below 1 (0.87 and 0.66) and IRR values below the minimum rate of returns. These economic parameters were sensitive to price changes in inputs and outputs, under both optimal and low input husbandry practices. Results indicate that using OvSynch and TAI Protocol is a profitable and economically viable investment under optimal husbandry practices but not under low input husbandry practices. By implications, use of OvSynch and TAI Protocol in Sahiwal upgrading breeding programs need be accompanied with improved husbandry practices and de-risking pastoral herd owners from price changes in input and output markets.

Rumen-protected methionine modulates body temperature and reduces the incidence of heat stress temperatures during the hottest hours of the day of grazing heat-stressed Bos indicus beef cows.

This study evaluated the effects of supplementation of rumen-protected methionine (RPM) on body thermoregulation and conception rate of Nelore cows exposed to high temperature-humidity index (THI). On -31 days before the artificial insemination protocol, 562 lactating, multiparous cows were assigned to receive (MG) or not (CG) RPM supplementation (3 g/cow mixed into 100 g of mineral supplement). Both groups remained in tropical pastures and received supplementation for 77 days. A subset of cows (n= 142) remained with an intravaginal thermometer collecting intravaginal temperature (IT). The respective minimum, average, and maximum environmental THI were 72.8, 78.0, and 83.3. Effects of treatment × hour of the day were detected (P< 0.0001) for IT. From 1330 to 1730 h and 1830 to 1900 h, IT was higher (P< 0.05) for CG versus MG cows when exposed to moderate and high THI. The supplementation with RPM did not affect conception rate (CG = 64.4% vs. MG = 58.2%; P> 0.05). In conclusion, 3g of RPM supplementation lowered internal body temperature and possibly altered critical THI threshold in Nelore cows with no impact on reproduction.

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.

176 Luteolytic efficiency of a single or double dose of cloprostenol sodium in a 5-day artificial insemination protocol in dairy cows

176 Luteolytic efficiency of a single or double dose of cloprostenol sodium in a 5-day artificial insemination protocol in dairy cows

Characterizing pregnancy losses in Bos indicus beef females receiving a fixed-timed artificial insemination protocol

This experiment evaluated pregnancy losses from day 30 of gestation to calving in Bos indicus females, and evaluated if serum haptoglobin concentrations during early gestation impacts subsequent pregnancy losses. A total of 4926 Nelore females were used, being 1802 nulliparous heifers inseminated as yearlings (precocious heifers), 1356 nulliparous heifers inseminated at 24 months of age (conventional heifers), 887 suckling primiparous cows, and 881 suckling multiparous cows. Cows were assigned to an ovulation synchronization + fixed-time artificial insemination (FTAI) protocol from day −11 to 0 of the experiment. Pregnancy status was verified using transrectal ultrasonography on days 30 and 60 after FTAI, via transrectal palpation on day 150 after FTAI, and according to calf birth. Blood samples were collected from all animals diagnosed pregnant on day 30 after FTAI, and analyzed for serum concentrations of haptoglobin and pregnancy associated glycoproteins (PAG). Pregnancy loss was greater (P < 0.01) from day 60–150 of gestation (10.2 %) compared with day 30–60 of gestation (6.0 %) and with day 150 of gestation to calving (7.4 %), and differed (P = 0.04) between these latter two periods. Pregnancy loss from day 30–60 of gestation did not differ (P = 0.26) among parities, whereas total pregnancy losses (day 30 to calving) were greater (P < 0.01) in precocious (28.4 %) and conventional (27.1 %) heifers compared with primiparous (16.4 %) and multiparous (13.0 %) cows. Serum PAG concentrations on day 30 after FTAI were less (P ≤ 0.03) in cows that lost the pregnancy (5.63 ng/mL) from day 30–60 of gestation, as well as those that lost the pregnancy from day 30 to calving (8.59 ng/mL) compared with cohorts that maintained the pregnancy (9.39 and 9.32 ng/mL, respectively). No differences in serum PAG concentrations on day 30 after FTAI were noted (P ≥ 0.23) according to pregnancy losses from day 60 to calving. Serum haptoglobin concentration on day 30 after FTAI also did not differ (P ≥ 0.48) between cows that maintained or lost the pregnancy. This experiment provides novel information about pregnancy losses after day 30 of gestation in B. indicus cattle, with most losses occurring as fetal mortality and not affected by systemic inflammation during early gestation. Pregnancy losses were nearly doubled in precocious and conventional heifers compared with parous cows, demonstrating the relevance of this reproductive failure to B. indicus replacement heifers.

Uterine microbial profiles in healthy postpartum dairy cows do not vary with sampling techniques or phases of estrous cycle

In this study, we aimed to compare uterine microbial profiles in postpartum dairy cows, determined by bacteriological culture and next-generation sequencing, using three uterine sampling techniques (swab, cytobrush, and lavage) and induced phases of the estrous cycle (estrus and diestrus). Fifteen healthy postpartum dairy cows at 53 ± 5 days postpartum were enrolled in the study. Uterine samples were collected during a fixed-time artificial insemination protocol. Viable bacteria were aerobically cultured from part of each sample, and bacterial isolates were identified through Sanger sequencing of the 16S rRNA gene. Total genomic DNA was extracted from the remainder of undiluted samples to quantify bacterial load using 16S rRNA qPCR and characterize the microbiome by metagenomic sequencing of the V1–V3 region of the 16S rRNA gene. Microbial profiles and composition were analyzed using the Shannon-Weaver diversity index and principal component analysis, respectively. Out of 87 samples, 88 % (77/87) were culture positive. The proportion of culture-positive uterine samples did not differ between sampling techniques (P = 0.39) or estrous cycle phases (P = 0.99). However, swab, cytobrush, and lavage techniques yielded 1.5, 9 and 9 times greater bacterial loads (P < 0.01), respectively, during diestrus than estrus phase. Moreover, during diestrus phase, the cytobrush method yielded 3 and 6 times more bacteria (P < 0.01) than both the lavage and swab methods. The most abundant bacterial genera identified from both bacteriological culture and metagenomic sequencing were Bacillus and Enterococcus, regardless of sampling technique or phases of the estrous cycle. Bacterial genera in moderate to low abundance through metagenomic sequencing included Streptococcus, Oscillospiraceae, and Lachnospiraceae. Notably, the uterine microbial profiles and composition, determined by metagenomic sequencing, did not differ by sampling techniques (P = 0.55 and P = 0.60, respectively) or estrous cycle phases (P = 0.34 and P = 0.17, respectively). In conclusion, our results suggest that any of the sampling techniques can be reliably used to study the uterine microbiome of healthy cows at random phases of the estrous cycle. However, it is important to consider potential differences in bacterial yield as a confounding factor.

171 Supplementing Ca Salts of Soybean Oil Via Low-Moisture Molasses-Based Blocks to Improve Reproductive Performance and Overall Productivity of Beef Cows

Abstract This experiment evaluated reproductive and productive responses of beef cows receiving self-fed low-moisture blocks (LMB) enriched or not with Ca salts of soybean oil (CSSO) throughout the breeding season. Non-pregnant, suckling multiparous Angus-influenced cows (n = 554) were assigned to a fixed-time artificial insemination (AI) protocol (day -10 to 0) followed by natural service (day 15 to 70). Cows were managed in 12 groups (46 ± 4 cows/group) maintained in individual pastures, and groups received LMB enriched with 25% (as-fed basis) of CSSO or ground corn (CON) from day -10 to 100. Both treatments were designed to yield a daily LMB intake of 0.454 kg/cow (as-fed basis). Groups were enrolled in an estrus-synchronization + fixed-time AI protocol from day -10 to 0 (AI occurred on day 0) of the experiment and beginning 15 days after AI cows were exposed to mature Angus bulls for 55 d. Pregnancy status was verified on day 55 and 100 of the experiment, and cows that gave birth during the initial two weeks were considered pregnant to fixed-time AI. Calf birthdate and weight was recorded during the subsequent breeding season. All calves were weaned at the same time, and a calf BW and age were recorded. Blood samples were also collected from a subsample of cows on days -10, 0 (fixed-time AI), and 55 (pregnancy diagnosis). All data were analyzed using group as the experimental unit and Satterthwaite approximation to determine the denominator df for the tests of fixed effects. Quantitative data were analyzed with the MIXED procedure of SAS, and binary data analyzed with the GLIMMIX procedure of SAS with a binomial distribution and logit link function. Data were analyzed with group (treatment × location), cow(group), and location as random variables. Significance was set at P ≤ 0.05 and tendencies were determined if P &amp;gt; 0.05 and ≤ 0.10. Cows receiving CSSO had greater (P &amp;lt; 0.01) mean concentrations of ω-6 fatty acids in plasma samples collected on days 0 and 55. Cows receiving CSSO had greater (P = 0.05) pregnancy rate to fixed-time AI (67.2 vs. 59.3%), whereas final pregnancy rate did not differ (P = 0.92) between treatments. Pregnancy loss was less (P = 0.03) in CSSO cows (4.50 vs. 9.04%), which also calved earlier during the calving season (treatment × week; P = 0.04). Weaning rate tended to be greater (P = 0.09) in CSSO (84.8 vs. 79.4%), although calf weaning age and weight did not differ (P ≥ 0.72) between treatments. Kilos of calf weaned/cow exposed was greater (P = 0.04) in CSSO cows (234 vs. 215 kg). Therefore, supplementing CSSO to beef cows via LMB during the breeding season improved their reproductive and overall productivity during a cow-calf cycle.

PSIX-20 Differential Gene Expression in Peripheral White Blood Cells of Fertile and Sub-Fertile Beef Heifers

Abstract Infertility in beef heifers remains a major economic loss to the cow-calf production industry. Identifying beef heifers with superior genetic potential for improved fertility would increase profitability. Therefore, the study aimed to identify differences in transcriptome profile from peripheral white blood cells (pWBCs) of beef heifers with varying reproductive potential. To accomplish this, Angus-Simmental crossbred heifers were subjected to an estrus synchronization and fixed-time artificial insemination (AI) protocol (7-Day CO-Synch + CIDR). Fourteen days after AI, all heifers were exposed to fertile bulls for a 60-day natural breeding season. Depending on the presence or absence of conceptus 120 days post-AI, heifers were classified as fertile (pregnant heifers) or sub-fertile (non-pregnant by AI or bull-breeding). Pregnancies were terminated, and all animals in both groups were cycling when pWBCs were isolated. Total RNA was extracted from the PWBCs and subjected to the library preparation and sequencing on the Nova-Seq platform. The read counts were obtained after data filtering and quality control using FastQC v0.11.9 and MultiQC v1.12 and alignment to the Ensemble’s ARS UCD1.2 Bos taurus genome reference using STAR aligner v2.7.5 for fertile (n = 8) and sub-fertile (n = 5) beef heifers. The filtered data were subjected to differential expression analysis using DESeq2. We identified 300 significantly differentially expressed genes (DEGs) with P-value ≤ 0.05 and absolute (log2 fold change) ≥ 0.5. Among them, 34 genes including FKBP5, ADCY3, REPS1, LAMP1, CTSH, ADGRE5 were identified with padj &amp;lt; 0.05. DEGs were over-represented for pathways such as response to thyroid stimulus, immune response, response to lipid, neutrophil degranulation, and calcium ion binding. Some of these genes were reported as fertility-related, while the other genes are novel and have the potential to predict heifers with varying reproductive potential. A detailed understanding of the underlying biological mechanisms of the top genes and a follow-up study with a higher sample size at different time points could validate the potential candidates identified in this study.

180 Differences in Gene Expression in Granulosa Cells Harvested from Fertile and Sub-Fertile Beef Heifers

Abstract Infertility in beef replacement heifers is one of the leading contributors to economic losses in the cow-calf sector of the beef production industry. Successful reproduction is complex, involving multiple tissues and biological systems. This complexity makes identifying the leading causes of subfertility difficult. One potential source of fertility issues is poor oocyte health. As developing oocytes are dependent on granulosa cells (GCs) and physically coupled with GCs, we hypothesized that the gene expression profile would be different between the GCs from fertile heifers and those from sub-fertile heifers. The objective of this study was to identify differences in gene expression in GCs collected from beef heifers of differing fertility status, either fertile or sub-fertile. To gather this information, Angus-Simmental crossbred heifers underwent an estrus synchronization and fixed-time artificial insemination (AI) protocol (7-Day CO-synch + CIDR). At 41 days post-AI, heifers were pregnancy checked through ultrasound and palpation. Heifers pregnant to AI were classified as fertile (n = 8). Any heifers that remained non-pregnant following AI were exposed to bulls of proven fertility for 61 days. Bull-exposed heifers were evaluated for pregnancy 34 days after the bulls were removed. The non-pregnant heifers following both AI and natural service were classified as sub-fertile (n = 5). Pregnancies were terminated in fertile heifers, and heifers from both groups were allowed to resume normal cyclicity before GCs being harvested. RNA was extracted from GCs and quality was checked for each sample. Total RNA from GCs of 13 heifers was subjected to the library preparation and sequencing on the Nova-Seq platform. Through the application of DESeq2, 79 genes were identified as significantly differentially expressed genes (DEGs) with P-value &amp;lt; 0.05 and absolute (log2 fold change) &amp;gt; 0.5 including DNER, HOXD10, CD52, CXCL12, and HOXD11. DEGs, along with co-expressed genes, were over-represented in pathways associated with the regulation of MAP kinase activity, regulation of serine/threonine kinase activity, MHC protein binding, and plasma membrane signaling receptor complex. Through the pursuit of knowledge regarding the function of top DEGs, increased sample size, and more diverse sample collection time points, we may improve our understanding of the causation of heifer fertility issues. Improving our knowledge of heifer infertility has the potential to develop therapeutic options, and in turn, increase the efficiency and sustainability of cow-calf production systems.

PSVI-21 Effects of Calcium Salts of Soybean Oil Supplementation to Nellore Heifers During Second and Third Trimesters of Gestation and Offspring Sex on Milk Production and Performance of the Offspring

Abstract The study aimed to evaluate the effects of supplementing calcium salts of soybean oil to Nellore heifers during the second and third trimesters of gestation and offspring sex on cow productivity and their male and female offspring growth. Forty-eight pregnant Nellore heifers [body weight (BW) = 387 ± 38.4 kg] were assigned to this experiment at day 123 ± 13 of gestation. All heifers were conceived during the same fixed-time artificial insemination protocol at 14 and 24 months of age. Heifers were randomly used in a completely randomized block design with a 2 × 2 factorial treatment arrangement, consisting of supplementing or not (CON) calcium salts of soybean oil (CSSO; 3 g/kg of BW; Table 1) and the sex of the offspring (male or female), totaling four treatments and twelve reps per treatment. Throughout the experimental period, heifers were maintained in a pasture composed of Brachiaria brizantha cv.Marandu. After birth, all animals remained under the same pasture conditions and received the same supplementation until weaning. Data were analyzed using the MIXED procedure of SAS and heifer as the experimental unit. Treatment × offspring sex interaction was not detected (P &amp;gt; 0.36) for any of the variables evaluated. Calves born from heifers supplemented with CCSO were 2 kg heavier (P = 0.03) at birth and 5 kg heavier (P = 0.03) at 120 days of age compared with calves born from CON heifers. At weaning (210 days), no dietary treatment effects were observed (P = 0.17) in offspring BW. Male offspring were 3.5 kg, 9 kg, and 12 kg heavier (P ≤ 0.01) at birth, day 120, and at weaning, respectively, compared with female offspring (Table 2). Throughout the experiment, male offspring gained 0.040 kg more daily (P = 0.01) than female offspring. The female offspring had 0.1 thicker subcutaneous fat per 100 kg of BW (P = 0.02) than the male offspring. Heifer milk production was not impacted (P &amp;gt; 0.10) by the dietary treatments or sex of the offspring in any of the periods evaluated (days 30 and 210; Table 3). Hence, supplementing CSSO to Nellore heifers during the second and third trimester of gestation altered offspring BW up to 120 days after birth, suggesting programming effects on postnatal offspring growth resultant from the CSSO treatment. Male offspring were heavier from birth to weaning compared with female offspring. These outcomes indicate that supplementing CSSO to Nellore heifers during pregnancy might be a feasible alternative to optimize offspring productivity, and carcass merit in grazing systems.

PSIII-7 Effect of Time of Estrus on Conception Rate for a Sexed Semen Artificial Insemination Protocol in Multiparous Beef Cows

Abstract Sexed semen artificial insemination is a breeding technique that allows producers to shorten breeding seasons, optimize genetics through use of superior semen, create calves based on sex preference, and increase profitability. Sexed semen is a technology that sorts semen by differences in DNA content between X (female) and Y (male) bearing sperm. However, sexed semen practices have yet to be widely used in beef cows due to reduced sperm numbers, low conception rates, and increased costs. The objective of this study was to determine the effect of time of estrus upon fixed-time artificial insemination (FTAI) of sex-sorted semen on conception rate using two different estrous synchronization protocols. Multiparous commercial beef cows (n = 73) were randomized by age, body condition score (BCS), and days post-partum at the University of Arkansas Livestock and Forestry Research Station in Batesville. Both synchronization treatments (TRT) used a controlled internal drug releasing device (Eazi-breed CIDR; 1.38g progesterone) inserted intravaginally on d -14 of the protocol simultaneous to administration of prostaglandin F2α (PGF; 500µg cloprostenol sodium; Estrumate). In the first treatment (TRT 1; 7&amp;7 Synch), gonadotropin-releasing hormone (GnRH; 86µg gonadorelin; Fertagyl) was administered on d -7. In the second treatment (TRT 2; 8&amp;6 Synch), GnRH was administered on d -6. Both treatments were then administered PGF simultaneous to CIDR removal on d 0, 66 ± 2h before FTAI and administration of GnRH. Standing heat (estrus) was visually detected every 3 h for 30 min intervals between CIDR removal and FTAI. Heat detection patches were used in conjunction with visual detection of estrus. Time of estrus was signified as time of standing immobile when mounted in combination with presenting a patch score of 4 (&amp;gt;75% color removed). Time of estrus (TD) was deviated from time of FTAI and categorized into three equal time groups (Grp 1 = 0 to 11.0 h; Grp 2 = 11.01 to 22.0 h; and Grp 3 = 22.01 to 33.0 h). Conception rate (CR) was determined via ultrasound 37 ± 2 d after FTAI. Data were analyzed using the GLIMMIX procedure of SAS 9.4. CR did not differ by age of dam (P = 0.58), BCS (P = 0.48), or TRT (P = 0.83). However, TD had an effect on CR (P = 0.002); and resulted in CR of 45% (Grp 1), 87% (Grp 2), and 42% (Grp 3). In conclusion, these results indicate artificially inseminating cows with sexed semen between 11.01 and 22.0 h after time of standing estrus will increase pregnancy rates for multiparous commercial beef cattle.