Effect of subclinical endometritis and flunixin meglumine administration on pregnancy in embryo recipient beef cows.

Fertility of recipient beef cows with subclinical endometritis (SCE) that did or did not receive flunixin meglumine (FM) treatment were compared following transfer of d 7 embryo. The study population comprised of 600 Angus cross cows that expressed estrus following Select-Synch + CIDR (Controlled Internal Drug Release) estrus synchronization protocol. At the time of embryo transfer, approximately 3 wk after sampling for subclinical endometritis, cows were randomly allocated either to receive FM treatment (500 mg of Banamine®; n = 300) or not (Control; n = 300). The effect of subclinical endometritis (at ≥ 1% PMN on endometrial cytology by cytobrush method) and FM treatment on pregnancy/embryo transfer (P/ET, %) were evaluated by mixed model. Of the 600 cows, 323 (53.8%) became pregnant; 55.0% (165/300) cows that received FM treatment vs. 52.7% (158/300) control cows (P > 0.1), and 55.9% (266/476) normal vs. 46.0% (57/124) subclinical endometritis cows (P < 0.05). There was a trend for treatment by subclinical endometritis for P/ET (P = 0.09). Pregnancy was recorded in 55.3% (134/242) of normal and 53.4% (31/58) of subclinical endometritis cows that received FM treatment, and in 56.4% (132/234) of normal and 39.4% (26/66) of subclinical endometritis cows that did not receive FM treatment (P = 0.09). In conclusion, subclinical endometritis in recipient beef cows resulted in lower P/ET. Though not significant in cows with subclinical endometritis, FM treatment resulted in 14.0% points more pregnancy compared with control.

Timed artificial insemination strategies with or without short-term natural service and pregnancy success in beef heifers.

Objective was to compare estrous response, and first service and breeding season pregnancy rates in Angus cross beef heifers that received four progesterone (CIDR, vaginal insert) -based estrous-synchronization treatment regimens for timed artificial insemination (TAI) with or without short-term natural service (NS). Heifers (n = 1744; 4 locations) were assigned a reproductive tract score (RTS: 1 = immature, acyclic to 5 = mature, cyclic), body condition score (BCS: 1 = emaciated to 9 = obese) and temperament score (0 = calm to 1 = excitable). Within location, heifers were assigned to either of four treatment regimens. Heifers in Select-Synch + CIDR (C) + short-term NS (SSC + NS) treatment (n = 438) received 100 µg GnRH im + CIDR on Day -7 and CIDR removal + 25 mg PGF2α (im) + estrus-detection patches in the morning of Day 0 and were subjected to NS (bull to heifer ratio, 1:25) from the morning of Day 2 (48 h from CIDR removal) to evening of Day 8. Heifers in CO-Synch + C (COSC) +TAI + NS treatment (n = 433) received 100 µg GnRH + CIDR on Day -7 and CIDR removal + 25 mg PGF2α im + estrus-detection patches in the morning of Day 0 + 100 µg GnRH im + TAI on Day 2, 60 h from CIDR removal, and were subjected to natural service (bull: heifer, 1:25) from the evening of Day 2 immediately after AI through Day 8. Heifers in COSC + TAI treatment (n = 443) received 100 µg GnRH + CIDR on Day -7 and CIDR removal + 25 mg PGF2α im + estrus-detection patches in the morning of Day 0 + 100 µg GnRH im + TAI on Day 2, 60 h from CIDR removal. Heifers in SSC + split time AI (STAI) treatment (n = 430) received 100 µg GnRH + CIDR on Day -7 and CIDR removal + 25 mg PGF2α im + estrus-detection patches in the morning of Day 0. Heifers determined to be in estrus were inseminated at 64 h after PGF2α (25 mg, im) administration and non-estrous heifers determined to be in estrus 20 h later (at 84 h) were inseminated at that time, whereas remaining non-estrous heifers at 84 h received GnRH (100 µg) and were inseminated concomitantly. In SSC + NS and COSC + TAI + NS treatments, natural service bulls were removed and reintroduced (@ bull: heifer, 1:40) on Day 22 and kept until Day 63. In COSC + TAI and SSC + STAI bulls (bull: heifer, 1:40) were introduced on Day 14 and retained until Day 63. All heifers, irrespective of treatment regimens, were observed for estrus three times a day from Day 2 through Day 8. Pregnancy diagnosis was performed on Day 93, 30 days from end of breeding season. Accounting for temperament (P < 0.0001), BCS (P < 0.0001) and RTS (P < 0.0001), the breeding program influenced the estrous response (P < 0.0001). Heifers in COSC + TAI + NS (86.8%), SSC + STAI (84.9%), and SSC + NS (86.5%) treatments had greater estrous response compared with heifers in COSC + TAI (75.8%) treatment. Accounting for temperament (P < 0.01), BCS (P < 0.05) and RTS (P < 0.01), the treatment regimens influenced first service pregnancy rate (P < 0.05). Heifers in COSC + TAI + NS treatment had greater first service pregnancy rate (60.3%) compared with COSC + TAI (54.2%) (P < 0.05). First service pregnancy rate for heifers in SSC + STAI (59.3%) and SSC + NS (57.3%) treatments did not differ from COSC + TAI + NS and COSC + TAI treatments. Accounting for temperament (P < 0.001), BCS (P < 0.0001) and RTS (P < 0.0001), the treatment regimens influenced breeding season pregnancy rate (P < 0.05). Breeding season pregnancy rate for heifers in COSC + TAI + NS (95.4%) and COSC + STAI (94.5%) treatments were greater than that of heifers in COSC + TAI (90.8%) treatment (P < 0.05), and heifers in SSC + NS (94.1%%) treatment did not differ from heifers in other treatments. In conclusion, progesterone-based CO-Synch timed artificial insemination with short-term natural service treatment regimen resulted in proportionately more pregnancies than without short-term natural service treatment regimen. In addition, 64/84 h split-time AI or natural service following Select-Synch treatment regimen could be implemented as an alternative as these treatment regimens resulted in similar pregnancy rate as progesterone based CO-Synch timed artificial insemination with short-term natural service treatment regimen.

IFNT, ISGs, PPARs, RXRs and MUC1 in day 16 embryo and endometrium of repeat-breeder cows, with or without subclinical endometritis.

Interferon-τ (IFNT), IFN stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), Peroxisome proliferator activated receptors (PPARA, D, and G), Retinoic acid receptors (RXRA, B, and G), and Mucin-1 (MUC1) play decisive roles in embryo elongation. The objective was to elucidate expressions of these genes in day 16 embryo [tubular (n = 4) vs. filamentous (n = 4)] and corresponding endometrium [without (n = 4) vs. with subclinical endometritis (SCE; n = 4)] of repeat breeder Holstein cows (2 × 2 factorial design). Results showed that the mRNA abundances (except PPARA and RXRB) were greater (P < 0.05) in filamentous embryo and endometrium without SCE compared with tubular embryo and endometrium with SCE, respectively. Overall, the mRNA abundances (except RSAD2, PPARA and RXRA) in filamentous embryo and corresponding endometrium of cows without SCE were greater (P < 0.05) than tubular embryo and corresponding endometrium of cows with SCE. Proteins IFNT, ISGs, PPARs and RXRs (except RXRB) were greater (P < 0.05) and protein MUC1 was lower (P < 0.01) in filamentous embryo and corresponding endometrium of cows without SCE compared to tubular embryo and corresponding endometrium of cows with SCE. On pairwise comparison, mRNA and protein abundances of MUC1 significantly differed between tubular embryo in uterus with or without SCE, and corresponding endometrium with or without SCE (P < 0.05). In conclusion, the mRNA and protein abundances of IFNT, ISG15, CXCL10, PPARG and MUC1 differed among filamentous and tubular conceptuses, and endometrium with or without SCE of repeat breeder cows on Day 16, indicating that these genes and their downstream signaling cascades play important roles in embryo elongation. Perhaps, interruptions in cross-talk between endometrium and conceptus impaired conceptus elongation in repeat breeder cows with SCE. In addition to disrupted signaling, the tubular conceptus (compared to filamentous conceptus) was unable to downregulate MUC1 (anti-adhesive glycocalyx) in repeat breeder cows with or without SCE, resulting in early embryonic demise.

Metabolic biomarkers, body condition, uterine inflammation and response to superovulation in lactating Holstein cows.

The objective was to determine associations between response to superovulation and body condition, subclinical endometritis and circulating metabolic biomarkers [adiponectin, leptin, insulin, IGF1, tumor necrosis factor (TNF) α, interleukin (IL) 1ß, IL6, and urea] in lactating dairy cows. Ten multiparous lactating Holstein cows in each body condition score (1-5; 1 emaciated; 5 obese) category (BCSC) 2.00 to < 2.50 (BCSC1), 2.50 to < 3.00 (BCSC2), 3.00 to <3.50 (BCSC3), 3.50 to <4.00 (BCSC4) and 4.00 to 5.00 (BCSC5) groups (total n = 50) were randomly selected and superovulated, timed artificially inseminated with frozen-thawed semen from three sires and embryos collected (n = 50 collections). At embryo collection, blood samples and embryo recovery fluid were collected for determination of metabolic markers and presence of subclinical endometritis (lavage technique; > 6% PMN). In total, 379 embryos were collected (average of 7.6 embryos per superovulation). Mean numbers of total ova and embryos was greater for cows in BCSC2, BCSC3 and BCSC4 groups compared with cows in BCSC1 and BCSC5 groups (P < 0.01). Total number of transferrable embryos were greater for cows in BCSC 2 and BCSC3 groups compared with cows in BCSC1, BCSC4 and BCSC5 groups (P < 0.01). Mean number of total ova and embryos and of transferrable embryos was higher for cows with 0 or 1-6% PMN compared to cows with >6% PMN (P < 0.01). In addition, there was a quadratic association between blood urea nitrogen concentrations and % transferrable embryos (r2 = 0.85; P < 0.05) and between BCS and % transferrable embryos (r2 = 0.73; P < 0.05). Circulating adiponectin, leptin, insulin, IGF1 and TNFα were greater in cows with moderate to good body condition compared to thin or obese cows (P < 0.05). Circulating adiponectin, leptin, IGF1 and insulin were greater in normal cows (≤6% PMNs), whereas, TNFα and IL1ß and IL6 were greater in cows with subclinical endometritis (P < 0.05). In conclusion, BCS and subclinical endometrial inflammation were associated with superovulatory response and embryo quality. Further, circulating metabolic biomarkers were associated with superovulatory response and embryo quality, likely due to donor's metabolic status and uterine environment. Optimizing superovulatory responses and embryo quality in lactating dairy cows requires management of nutrition and uterine health.

Cyclicity, estrus expression and pregnancy rates in beef heifers with different reproductive tract scores following progesterone supplementation.

Two experiments were conducted to determine effects of progesterone (P4) on cyclicity, estrus expression rate (EER) and artificial insemination pregnancy rate (AIPR) in beef heifers with various reproductive tract scores (RTS; 1 to 5; 1, immature, acyclic; 5, mature, cyclic). In Experiment 1, Angus-cross heifers (n = 100, 20 per RTS category; mean (±SEM) age, 15 ± 0.8 mo) were randomly assigned to receive a CIDR (Days -17 to -10) or no CIDR (untreated control), with weekly blood samples and ultrasonography (Days 0-85). Among heifers with RTS 2 to 4, median interval to cyclicity were shorter (P < 0.05) for heifers in CIDR versus control. In Experiment 2, Angus-cross heifers (n = 11,098) were assigned RTS, body condition score (BCS; 1 to 9; 1, emaciated; 9, obese) and temperament score (calm versus excitable). Heifers with RTS 2-5 (n = 10,569) were allocated to CO-Synch (n = 5099) or CO-Synch + CIDR (n = 5470). Estrus was detected until AI (72 h after PGF2α), with pregnancy diagnosis ∼70 d later. Controlling for RTS (P < 0.0001), BCS (P < 0.0001), temperament (P < 0.0001), age (P < 0.0001), treatment by RTS (P < 0.01), treatment by BCS (P < 0.01), and treatment by age, EER differed between CO-Synch and CO-Synch + CIDR (71.0 vs 75.9%, respectively, P < 0.0001). Accounting for RTS (P < 0.0001), BCS (P < 0.0001), temperament (P < 0.0001), age (P < 0.0001), heifers detected in estrus (P < 0.0001), RTS by treatment (P < 0.01), BCS by treatment (P < 0.01), and age by treatment, AIPR differed between CO-Synch versus CO-Synch + CIDR (55.3 vs 61.0%, P < 0.0001). In conclusion, exogenous P4 hastened cyclicity in pre- and peri-pubertal beef heifers. Further, it increased EER and AIPR. However, RTS, BCS and age influenced EER and AIPR. Among RTS 4 and 5, EER was greater for CO-Synch + CIDR vs CO-Synch. Among RTS 3 to 5, AIPR was greater for CO-Synch + CIDR versus CO-Synch. Progesterone status or supplementation at onset of synchronization protocols was critical to pregnancy outcomes, emphasizing heifer development for early puberty or progesterone supplementation.

Endometrial expression of various genes (ISGs, PPARs, RXRs and MUC1) on day 16 post-ovulation in repeat breeder cows, with or without subclinical endometritis.

Our objective was to elucidate differences in endometrial mRNA expressions of interferon-stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), peroxisome proliferator activated receptors (PPARA, PPARD, and PPARG), retinoic acid receptors (RXRA, RXRB, and RXRG), and mucin 1 (MUC1) in repeat breeder cows, with or without subclinical endometritis (RB + SE and RB, respectively) and normal cows on day 16 post-ovulation (n = 4 cows per group). The CXCL10 and SLC27A6 mRNA abundances were greater for normal cows compared to RB and RB + SE cows (P < 0.05 and P < 0.01 respectively) whereas ISG15 and SLC2A1 mRNA abundances were greater for normal cows compared to RB + SE (P < 0.05). The SLC27A6 mRNA abundances were greater for RB versus RB + SE (P < 0.01). Similarly, PPARD, PPARG, RXRA and RXRG mRNA abundances were greater for normal cows compared to RB and RB + SE (P < 0.01 and P < 0.05, respectively). Abundances of PPARD, PPARG, RXRA and RXRG mRNA were greater for RB versus RB + SE (P < 0.05) and MUC1 was lower in abundance in normal cows compared to RB or RB + SE (P < 0.05). Key predicted molecular functions were binding, signal transducer and transporter; key biological processes were cellular, localization and metabolic; key cellular components were cell part, membrane and organelle components; and key protein classes were nucleic acid binding, receptor, and transcription factors. Gene networking analysis highlighted interactions and pathways involving PAPRs, RXRs, and MUC1, notably among PPARD, PPARG, and MUC1. In conclusion, endometrial mRNA expressions of ISGs (CXCL10 and SLC27A6), PPAR isomers (PPARD and PPARG), and RXRs (RXRA and RXRG) were in lower abundances, whereas MUC1 expression was more abundant in RB or RB + SE compared to normal cows on day 16. In addition, ISG15 and SLC2A1 genes were less abundant in RB + SE versus RB or normal cows. Altered expression of these uterine genes and associated potential impairment in embryo elongation and implantation may promote embryonic loss in repeat breeder cows. Furthermore, interactions among PPARD, PPARG and MUC1 may be therapeutically exploitable.

Injectable or transdermal flunixin meglumine improves pregnancy rates in embryo transfer recipient beef cows without altering returns to estrus.

OBJECTIVES: were to determine effects of 1) injectable or transdermal flunixin meglumine (FM) at embryo transfer (ET) compared to an untreated control group on pregnancy per ET (P/ET; ∼35 d after ET); 2) embryo and recipient factors on P/ET; 3) FM on hormone concentrations; and 4) FM on returns to estrus. Angus-cross beef cows (n = 1145) at five locations were scored for body condition (BCS; 1-9) and temperament (0 or 1) and given Select-Synch + CIDR. Recipient cows with a corpus luteum (CL) ≥1.5 cm received a frozen-thawed embryo 7 d after estrus and were concurrently given 1.1 mg/kg injectable FM im (INJFM; n = 384), 3.3 mg/kg transdermal FM pour on (TDFM; n = 388), or nothing (CON group; n = 373). Blood samples were collected at ET and 7 d later (60 cows). Accounting for temperament (P < 0.05), ET difficulty score (1-3, easy to difficult; P < 0.01), treatment by temperament (P < 0.001) and treatment by embryo quality (P < 0.05), FM treatments affected P/ET (P < 0.05). The P/ET for cows given INJFM [62.8% (241/384)] or TDFM [58.7% (228/388)] were not different (P = 0.26), but they were greater (P = 0.01 and P = 0.04, respectively) than P/ET for controls [51.2% (191/373)]. The P/ET was greater for calm versus excitable cows, 60.2 (463/769) and 52.4% (197/376), respectively (P < 0.01) and was lower for difficulty score 3 [49.2% (156/317)] compared to score 1 [62.7% (254/405; P < 0.001) or score 2 [59.1% (250/423; P < 0.01)]. There was no effect (P > 0.1) of cow age, BCS, or stage of embryo development on P/ET. Pregnancy rates for embryo quality grade 1 (excellent/good) and grade 2 (fair) were 60.4% (314/520) and 55.4% (346/625), respectively (P > 0.05). Percentages of non-pregnant recipient cows in estrus from Days 18-26 did not differ among treatment groups (P > 0.1). Control cows had lower progesterone concentrations and greater substance-P, PGFM and 8-isoprostane PGF2α concentrations at 7 d after ET compared to FM-treated cows (P < 0.05). In conclusion, injectable or transdermal FM improved pregnancy rates in ET recipients, without affecting nonpregnant cows return to estrus.

Sperm and seminal plasma proteomics of high- versus low-fertility Holstein bulls.

Sperm are highly specialized compartmentalized cells, with unique compositional, morphological and functional properties, including a plasma membrane that undergoes dynamic protein remodeling and surface modifications. Seminal plasma is a highly complex biological fluid containing proteins, amino acids, enzymes, fructose and other carbohydrates, lipids, major minerals and trace elements. Seminal plasma proteins are involved in regulation of osmotic pressure and pH of seminal plasma, transport of ions, lipid and hormones. The objective was to compare sperm and seminal plasma proteomes of bulls with differing fertility and to relate differences to biological processes. Semen was collected from bulls with high or low fertility (4 bulls in each category). Sperm and seminal plasma proteins were isolated, purified, subjected to 2-D gel electrophoresis, protein identification and ontology. In sperm and seminal plasma, binder of sperm proteins (BSP)-1, -3 and -5, and spermadhesin-1, ALB, TIMP, AKI and PEBP1 were higher for high-versus low-fertility bulls (P < 0.05), whereas proteins CLU, CCT5 and 8, ELSPbP1, and PSMA6 were more abundant in sperm and seminal plasma of low- versus high-fertility bulls (P < 0.05). Further, HSP90, ZFP34, IFNRF4, BCL62, NADHD, TUBB3 and Histone H1 were in greater abundance in sperm of high- compared with low-fertility bulls. The two key biological processes of proteins differentially expressed in high- and low-fertility bulls were metabolic processes and biological regulation. The most prominent molecular functions for proteins that differed are binding, catalytic and receptor activities. The main cellular components for proteins that differed are cellular, extracellular, and plasma membrane. Since protein content differed in high- versus low-fertility bulls, we inferred that the efficiency of associated sperm functions that are necessary for fertility may also differ between high- and low-fertility semen. In conclusion, differences between high- and low-fertility bulls regarding abundance of sperm and seminal plasma proteins likely contributed to differences in fertility.

Predictors of beef calf temperament at weaning and its impact on temperament at breeding and reproductive performance.

Two experiments were conducted to determine (i) factors influencing calf temperament at weaning, (ii) association between heifer-calf temperament at weaning and temperament at breeding and (iii) effect of heifer-calf temperament on pregnancy rate per artificial insemination (P/AI). In experiment 1, beef cows and their calves (n = 285) from three farms were used. Sire docility estimated progeny difference (EPD) score, birth type (normal or assisted), calf gender, calf behaviour (during 1st 4 weeks) and calf health status (until weaning) were recorded. Cows and calves were assigned a temperament score (0-calm; 1-excitable), and all cows were given a body condition score (BCS, 1-9; 1-emaciated; 9-obese) at weaning. Calf's illness (p < .05), low sire docility EPD score (p < .05), altered gait (p < .05), altered resting behaviour (p < .01), reduced/no play behaviour (p < .05) and cow excitable temperament (p < .001) increased calf excitable temperament at weaning. In experiment 2, replacement heifer-calves (n = 758) from 12 farms were assigned a temperament score at weaning and later at breeding. Blood from 40 calves at weaning and 31 heifers at initiation of synchronization (same animals) was collected by coccygeal venipuncture for determination of circulating cortisol and substance P concentrations. Heifers were assigned a BCS and reproductive tract score (RTS, 1-5; 1-immature, acyclic; 5-mature, cyclic), synchronized for fixed time AI, observed for oestrus and were artificially inseminated. Cortisol concentrations were increased in excitable heifer-calves compared to calm heifer-calves at weaning (p < .05), and substance P was increased in excitable compared to calm females both at weaning and breeding (p < .05). Low sire EPD docility score (p < .01), heifer-calf excitable temperament at weaning increased excitable temperament at breeding (p < .01). Controlling for BCS categories (p < .01), oestrous expression (p < .0001) and temperament at breeding by oestrous expression (p < .05), the calf's excitable temperament at weaning (p < .001) reduced P/AI (Calm, 62.7 (244/389) vs. Excitable, 53.4% (197/369); p < .01). In conclusion, selection of docile cows and sires with greater docility EPD score should be given consideration to reduce calf excitement. Temperament in beef female can be detected earlier in their life and could be used as a tool in the selection process and to improve their performances.

Flunixin meglumine improves pregnancy rate in embryo recipient beef cows with an excitable temperament.

Objectives were to determine effects of: 1) handling temperament and administration of flunixin meglumine, an inhibitor of prostaglandin F2a (PGF2a) synthesis, given at the time of embryo transfer, on pregnancy rates in beef cattle embryo transfer recipients; 2) handling temperament and flunixin meglumine on peripheral concentrations of progesterone, cortisol, substance-P, prostaglandin F metabolites (PGFM, (13,14-dihydro-15-keto-PGF2a) and isoprostane 8-epi PGF2a; and 3) flunixin meglumine treatment on proportion of non-pregnant recipient cows returning to estrus within an expected interval. Angus cross beef cows (n = 710) at 7 locations were assigned a body condition score (BCS: 1, emaciated; 9, obese) and a temperament score [0, calm, slow chute exit; walk (n = 352), 1, excited, fast chute exit; jump, trot or run (n = 358)] and were synchronized with Select-Synch with a controlled internal drug release (CIDR) protocol. Estrus detection aids were applied at CIDR removal and cows were observed thrice daily for estrus until 72 h. Recipient cows that expressed estrus and had a corpus luteum received a frozen-thawed embryo on Day 7 after estrus. At the time of transfer, recipient cows were randomly allocated to receive 10 mL of flunixin meglumine im, immediately after transfer (n = 365) or served as an untreated control (n = 345). In a subset of cows (n = 80), ovarian ultrasonography was performed on the day of embryo transfer to determine corpus luteum volume and blood samples were collected twice, at the time of embryo transfer and 7 d later. All cows received estrus detection aids again on Day 14 (7 d after embryo transfer) and were observed for estrus twice daily until Day 24. Accounting for treatment (P > 0.1), embryo transfer difficulty score (P < 0.1), temperament by treatment interaction (P < 0.05), recipient cows with calm temperament had a higher pregnancy rate compared to those with an excited temperament [59.4 (209/352) vs 51.7% (185/358)]. The pregnancy rate for excitable cows without flunixin meglumine was lower (46.3% 81/175) compared to excitable cows that did received flunixin meglumine [56.8% (104/183)], and calm cows that did [59.3% (108/182)] or did not [59.4% (104/170)] receive flunixin meglumine. Proportions of non-pregnant recipient cows returning to estrus on Days 18-24 were not different between flunixin meglumine and control groups, 87.6% (134/153) and 84.0% (137/163), respectively (P > 0.1). At the time of embryo transfer and 7 d later, there were moderate to strong correlations among circulating concentrations of progesterone, cortisol, substance-P, PGFM and isoprostane 8-epi PGF2a. Among excitable cows, progesterone concentrations were lower and cortisol, substance-P, PGFM and isoprostane 8-epi PGF2a concentrations were greater for cows in the control group compared to cows that received flunixin meglumine. In conclusion, administration of flunixin meglumine improved pregnancy rates in excitable recipient cows following embryo transfer without affecting the proportion of non-pregnant cows returning to estrus.

Impact of delayed insemination on pregnancy rates to gender selected semen in a fixed-time AI system.

The objectives of the current experiment were to determine if delaying insemination by 8 h in a FTAI protocol would alter estrus expression and pregnancy rates in cows inseminated with sex-sorted semen, characterize bull variation in pregnancy rates to sex-sorted semen and examine the impact of repeated years of FTAI to sex-sorted semen on calving distribution. Over three breeding seasons, postpartum cows (n = 839) were estrous synchronized using the 5-day CO-Synch + CIDR system. Cows were given GnRH (100 µg i.m., Factrel) at time of insertion of a controlled internal drug releasing device (CIDR; Eazi-Breed CIDR). Five d later CIDR was removed and PGF2α (25 mg i.m., Lutalyse) was given at removal and 8 h later. Estrus detection aids were applied at CIDR removal. Cows were inseminated with X-sorted or Y-sorted sex-sorted semen at 72 h (NORM) or 80 h (DELAY) after CIDR removal, and GnRH was administered at AI. At insemination, estrus status was categorized as positive (YES), partial (QUES), unknown (NR) or negative (NO). Bulls were introduced to cows at 14 d and removed at 60 d after FTAI. Pregnancy diagnosis was performed by ultrasound at d 60 after FTAI and via palpation at 60 d after bull removal. There was no difference (P > 0.05) in pregnancy rates to sex-sorted semen or final pregnancy rates between NORM and DELAY cows. Pregnancy to sex-sorted semen averaged 35.2% whereas final pregnancy rates were 90.6%. More cows (P < 0.05) in the DELAY group expressed estrus before FTAI, but this increase did not alter pregnancy rates to sex-sorted semen. Expression of estrus before FTAI increased (P < 0.02) pregnancy rates to sex-sorted semen across treatments with differences being YES > QUES or NR > NO. There was considerable variation in pregnancy rate by bull (P < 0.05) with pregnancy rates ranging from 55.6% to 19.3%. Whole herd calving distribution was altered (P < 0.05) after 3 y of use of sex-sorted semen compared to the previous 3 y when conventional semen was used. We conclude that delaying insemination by 8 h in an FTAI protocol did not improve pregnancy rates to sex-sorted semen despite more cows exhibiting estrus before FTAI. In addition, a high bull to bull variation in pregnancy rates to sex-sorted semen is a limitation in FTAI systems. Further research into FTAI strategies for use with sex-sorted semen is warranted.

Relationship among circulating anti-Müllerian hormone, insulin like growth factor 1, cadmium and superovulatory response in dairy cows.

The objectives of this study were 1. to determine the associations among circulating anti-Mullerian hormone (AMH), insulin like growth factor 1 (IGF1) and cadmium (Cd) concentrations of lactating Holstein cows at the time of superovulation and 2. to determine the effect of circulating AMH, IGF1 and Cd concentrations on the superovulatory response in Holstein dairy cows. Holstein cows (n = 30) were milked thrice daily and housed and fed in free stall barn as a separate group. All animals were synchronized for superovulation and flushed. Three blood samples for AMH, IGF1 and Cd analysis were collected prior to superovulation, at estrus and at the time of embryo collection. The concentrations of blood makers prior to superovulation were highly correlated to superovulatory response. Circulating concentrations of AMH, IGF1 prior to superovulation were negatively correlated to Cd concentrations (P < 0.05). There was no correlation between circulating concentrations of AMH and IGF1. The number of corpus luteum (r = 0.71), total embryo (r = 0.67), total transferable embryo (r = 0.51) and total grade 1 embryo (r = 0.5) were positively correlated to AMH concentrations (P < 0.05). There was a trend for negative correlation found between circulating cadmium concentrations and total grade 1 embryo yield (P < 0.1). When cows were classified into quartiles (Q) of circulating AMH concentration, number of corpus luteum, and total embryos, total transferable embryos and total grade 1 embryos yield was significantly different for AMH quartiles. The superovulatory response parameters evaluated were increased with increased AMH concentrations; particularly we observed a >2-fold difference between first and fourth AMH quartiles in total transferable embryo yield and total grade 1 embryo yield. In conclusion, circulating AMH concentration was strongly associated with superovulatory response. Measuring AMH before enrolling cows in superovulation programs will likely allow practitioners to improve numbers of embryos produced and, thereby, reduce costs per embryo produced.

Associations of hypoosmotic swelling test, relative sperm volume shift, aquaporin7 mRNA abundance and bull fertility estimates.

Mammalian sperm are exposed to a natural hypoosmotic environment during male-to-female reproductive tract transition; although this activates sperm motility in vivo, excessive swelling can harm sperm structure and function. Aquaporins (AQPs) is a family of membrane-channel proteins implicated in sperm osmoregulation. The objective was to determine associations among relative sperm volume shift, hypoosmotic swelling test (HOST), sperm aquaporin (AQP) 7 mRNA abundances, and sire conception rate (SCR; fertility estimate) in Holstein bulls at a commercial artificial insemination center. Three or four sires for each full point SCR score from -4 to +4 were included. Each SCR estimate for study bulls (N = 30) was based on > 500 services (mean ± SEM) of 725 ± 13 services/sire). Sperm from a single collection day (two ejaculates) from these commercial Holstein bulls were used. Relative mRNA expression of AQP7 in sperm was determined by polymerase chain reaction. Mean relative sperm volume shift and percentage of sperm reacted in a HOST (% HOST) were determined (400 sperm per bull) after incubating in isoosmotic (300 mOsm/kg) and hypoosmotic (100 mOsm/kg) solutions for 30 min. There was no correlation between %HOST and SCR (r = 0.28 P > 0.1). However, there was a positive correlation between relative sperm volume shift and SCR (r = 0.65, P < 0.05). Furthermore, AQP7 mRNA abundance was positively correlated to both relative volume shift (r = 0.73; P < 0.05) and to SCR (r = 0.67; P < 0.05). The mRNA expressions of AQP7 and relative sperm volume shift differed (P < 0.05) among low- (<2 SCR), average- (-2 to +2) and high- (>2) fertility sire groups. In conclusion, bulls with higher SCR had significantly greater AQP7 mRNA abundance in frozen-thawed sperm. This plausibly contributed to greater regulation of sperm volume shift, which apparently conferred protection from detrimental swelling and impaired functions.

Two split-time artificial insemination programs in suckled beef cows.

Our objective was to determine which of 2 split-time AI programs applied to suckled beef cows would result in greater pregnancy risk. Suckled beef cows (n = 1,062) at 12 locations in 4 states (CO, KS, MY, and WA) were enrolled. Cows were treated on d -7 with a progesterone insert concurrent with 100 µg GnRH and on d 0 with 25 mg PGF plus removal of the insert. Estrus-detection patches were affixed to cows at insert removal. The study was designed as a completely randomized experiment of 2 treatment combinations. Within location and balanced for parity (primiparous vs. multiparous), cows were assigned randomly to 2 treatment times (55 vs. 65 h after CIDR insert removal) at which time estrus-detection patches were assessed. Estrus was defined to have occurred when an estrus-detection patch was > 50% colored (activated). Cows determined to be in estrus were inseminated at either 55 or 65 h, whereas the residual nonestrous cows in both treatment times received GnRH at 55 or 65 h but were inseminated 20 h later at 75 or 85 h, respectively. Pregnancy outcomes were determined at 36 d after AI and at the end of the breeding season. Thus, pregnancy outcomes of interest were compared between the 55 + 75-h treatment combination and that of the 65+85-h combination. Expression of estrus was greater ( = 0.001) by 65 h after PGF than by 55 h (62.0% vs. 41.9%), respectively, and this proportion was influenced by parity (time x parity interaction; = 0.006). As a result, proportionally more ( < 0.001) cows received the timed AI at 75 than 85 h (59.4% vs. 40.6%). Similar proportions of cows not in estrus by 55 or 65 h were detected in estrus by 75 or 85 h (40.1% vs. 39.3%), respectively. The cumulative proportion of cows in estrus by 75 h was less ( < 0.001) than that by 85 h (66.7% vs. 76.7%), respectively. Pregnancy risks at 36 d differed among treatments, with cows detected in estrus and inseminated at 55 or 65 h having greater pregnancy risks than their time-inseminated herd mates at 75 or 85 h (62.3% vs.49.7%), respectively. Overall pregnancy risk for cows in the 65+85-h treatment combination was greater at 36 d than for cows in the 55 + 75-h treatment combination (61.0% vs. 51.4%), respectively. We conclude that the 65 + 85-h treatment combination produced more pregnancies than the 55 + 75-h combination, but its implementation may be somewhat less convenient in terms of cow handling times.

Fertility of Angus cross beef heifers after GnRH treatment on day 23 and timing of insemination in 14-day CIDR protocol.

This study compared artificial insemination pregnancy rate (AI-PR) between 14-day CIDR-GnRH-PGF2α-GnRH and CIDR-PGF2α-GnRH synchronization protocol with two fixed AI times (56 or 72 hr after PGF2α). On day 0, heifers (n = 1311) from nine locations assigned body condition score (BCS: 1, emaciated; 9, obese), reproductive tract score (RTS: 1, immature, acyclic; 5, mature, cyclic) and temperament score (0, calm; and 1, excited) and fitted with a controlled internal drug release (CIDR, 1.38 g of progesterone) insert for 14 days. Within herd, heifers were randomly assigned either to no-GnRH group (n = 635) or to GnRH group (n = 676), and heifers in GnRH group received 100 µg of GnRH (gonadorelin hydrochloride, IM) on day 23. All heifers received 25 mg of PGF2α (dinoprost, IM) on day 30 and oestrous detection aids at the same time. Heifers were observed for oestrus thrice daily until AI. Within GnRH groups, heifers were randomly assigned to either AI-56 or AI-72 groups. Heifers in AI-56 group (n = 667) were inseminated at 56 hr (day 32 PM), and heifers in AI-72 group (n = 644) were inseminated at 72 hr (day 33 AM) after PGF2α administration. All heifers were given 100 µg of GnRH concurrently at the time AI. Controlling for BCS (p < .05), RTS (p < .05), oestrous expression (p < .001), temperament (p < .001) and GnRH treatment by time of insemination (p < .001), the AI-PR differed between GnRH treatment [GnRH (Yes - 60.9% (412/676) vs. No - 55.1% (350/635); p < .05)] and insemination time [AI-56 - 54.6% (364/667) vs. AI-72 - 61.8% (398/644); (p < .01)] groups. The GnRH treatment by AI time interaction influenced AI-PR (GnRH56 - 61.0% (208/341); GnRH72 - 60.9% (204/335); No-GnRH56 - 47.9% (156/326); No-GnRH72 - 62.8% (194/309); p < .001). In conclusion, 14-day CIDR synchronization protocol for FTAI required inclusion of GnRH on day 23 if inseminations were to be performed at 56 hr after PGF2α in order to achieve greater AI-PR.

Detection of genes encoding multidrug resistance and biofilm virulence factor in uterine pathogenic bacteria in postpartum dairy cows.

Reckless use of antibiotics and/or development of biofilm are the rationale for the development of multidrug resistance (MDR) of pathogenic bacteria. The objective of the present study was to detect MDR genes in Trueperella pyogenes and to detect biofilm virulence factor (VF) genes in Escherichia coli isolated from the uterus of postpartum dairy cows. Uterine secretions from different parity postpartum Holstein cows (n = 40) were collected using cytobrush technique after a sterile procedure from cows with varying degree of uterine inflammatory conditions. The cytobrush was stored in a specimen collector, placed in a cooler with ice, and transported to the laboratory within 2 hours. The pathogens were isolated and were identified initially by their colony morphology and biochemical characteristics. To further identify and classify the single species, and to determine the presence of MDR and VF genes, the genes fragments were amplified using the respective primers by either singleplex or multiplex polymerase chain reaction protocol, and amplicons were detected by electrophoresis method. T pyogenes was isolated in 17 of 40 (42.5%) cows in the study population as recognized by the 16S rRNA gene. Of the positive T pyogenes samples, 8 of 17 (42.1%) were positive for integron type 1 (intI I), and none were positive for integron type 2 (intI II). Of those 8 positive for intI I, six of eight (66.7%) were positive for amplicons aadA5 and aadA24-ORF1 at 1048 and 1608 bp, respectively, associated with specific drug resistance. Presence of addA5 indicated resistance to sulfadiazine, bacitracin, florfenicol, and ceftiofur. Presence of addA24-ORF1 indicated resistant to sulfadiazine, bacitracin, penicillin, clindamycin, and erythromycin. E coli was isolated in 18 of 40 (45.0%) cows in the study population. The genes for VF, Agn43a, and Agn43 b, associated with biofilm production, were found in 6 of 18 (33.3%) of the positive isolates. Both T pyogenes MDR gene and E coli biofilm VF existed in more severe form of uterine diseases than subclinical endometritis. In conclusion, 35% of T pyogenes isolates found were positive for a gene cassette associated with antibiotic resistance, and 33% of the E coli isolates contained genes for the VF associated with biofilm production.

Fertility after implementation of long- and short-term progesterone-based ovulation synchronization protocols for fixed-time artificial insemination in beef heifers.

Two experiments were conducted to evaluate the effect of long-term (LT; a 14-day controlled internal drug release insert [CIDR]-PGF2α [PGF]-GnRH) and short-term (ST; 5-day CO-Synch + CIDR) progesterone-based protocols on pregnancy rate to fixed-time artificial insemination (FTAI) in beef heifers. In experiment 1, Angus cross beef heifers (N = 1887) at nine locations received a body condition score and a reproductive tract score (RTS). Within the herd, heifers were randomly assigned to LT-72 and ST-56 protocol groups. Heifers in the LT-72 group received a CIDR from Days 0 to 14, followed by 25 mg of PGF 16 days later (Day 30). Heifers in the ST-56 group received a CIDR and 100 µg of gonadorelin hydrochloride (GnRH) on Day 25 followed by 25 mg of PGF at CIDR removal on Day 30 and a second dose of PGF 6 hours later (Day 30). Artificial insemination was performed at 56 hours (Day 32) after CIDR removal for the ST-56 group and at 72 hours (Day 33) after CIDR removal for the LT-72 group, and all heifers were given GnRH (100 µg, intramuscular) at the time of AI. In experiment 2, Angus cross beef heifers (N = 718) at four locations received a body condition score and an RTS. Within the herd, heifers were randomly assigned to LT-72 and ST-72 protocol groups. The protocol was similar to experiment 1 except that AI was performed at 72 hours after CIDR removal for both LT-72 and ST-72 groups. In experiment 1, no difference in AI pregnancy rates between the LT-72 and ST-56 groups was observed (54.5% [489 of 897] and 55.5% [549 of 990], respectively; P = 0.92) after accounting for the RTS. The AI pregnancy rates for heifers with RTS 3 or less, 4, and 5 were 52.6%, 53.6%, and 59.9%, respectively (P < 0.05). In experiment 2, controlling for the RTS, no difference in AI pregnancy rates was observed between the LT-72 and ST-72 groups, 56.9% (198 of 347) and 57.8% (214 of 371), respectively (P = 0.87). The AI pregnancy rates for heifers with RTS 3 or less, 4, and 5 were 49.3%, 58.4%, and 62.1%, respectively (P < 0.05). In conclusion, heifers synchronized for fixed-time AI with LT and ST protocols resulted in a similar AI pregnancy rate. Approximately, 55% of the herd was pregnant to one insemination in 33 days with the LT protocol compared with just 8 days with the ST protocol.

Calm temperament improves reproductive performance of beef cows.

Profitability of a beef operation is determined by the proportion of cows attaining pregnancy early in the breeding season and those that are pregnant at the end of breeding season. Many factors, including temperament, contribute to those reproductive parameters. The objective of this study was to evaluate effects of temperament on reproductive performance of beef cows. In Experiment 1, Angus and Angus-cross beef cows (n = 1546) from eight locations were assigned a body condition score (BCS; 1 = emaciated; 9 = obese) and chute exit and gait score (1 = slow exit, walk; calm temperament; 2 = jump, trot or run; excitable temperament). Cows were grouped with bulls (1 : 25 to 1 : 30; with satisfactory breeding potential and free of venereal disease) for an 85-day breeding season. Pregnancy status and stage of gestation were determined (transrectal palpation) 35 days after the end of the breeding season. Controlling for BCS (p < 0.01) and handling facility (p < 0.0001) and handling facility by temperament score interaction (p < 0.001), breeding season pregnancy rate was lower in excited versus calm cows [88.6% (798/901) vs 94.1% (607/645); p < 0.001]. Cows with an excitable temperament took 24 more days to become pregnant compared to calm cows (median days to pregnancy, 35 vs 59 days; p < 0.0001). In Experiment 2, Angus and Angus-cross beef cows (n = 1407) from 8 locations were assigned scores for body condition and chute exit and gait (as described in Experiment 1) and assigned to bulls (breeding sound and free of venereal disease; 1 : 25 to 1 : 30) for 85 days. Pregnancy status was determined by transrectal palpation at 2 and 6 months after the onset of the breeding season. Controlling for BCS (p < 0.05), pregnancy loss was higher in excited versus calm cows [5.5% (36/651) vs 3.2% (20/623), p < 0.0001]. In conclusion, beef cows with an excitable temperament had significantly lower reproductive performance than calmer cows. The modified two-point chute exit-gait scoring method was repeatable and identified cattle with an excitable temperament.

Influence of temperament score and handling facility on stress, reproductive hormone concentrations, and fixed time AI pregnancy rates in beef heifers.

The objectives were (i) to evaluate the effect of temperament, determined by modified 2-point chute exit and gait score, on artificial insemination (AI) pregnancy rates in beef heifers following fixed time AI and (ii) to determine the effect of temperament on cortisol, substance-P, prolactin and progesterone at initiation of synchronization and at the time of AI. Angus beef heifers (n = 967) at eight locations were included in this study. At the initiation of synchronization (Day 0 = initiation of synchronization), all heifers received a body condition score (BCS), and temperament score (0 = calm; slow exit and walk or 1 = excitable; fast exit or jump or trot or run). Blood samples were collected from a sub-population of heifers (n = 86) at both synchronization initiation and the time of AI to determine the differences in serum progesterone, cortisol, prolactin and substance-P concentrations between temperament groups. Heifers were synchronized with 5-day CO-Synch+ controlled internal drug release (CIDR) protocol and were inseminated at 56 h after CIDR removal. Heifers were examined for pregnancy by ultrasound 70 days after AI to determine AI pregnancy. Controlling for synchronization treatment (p = 0.03), facility design (p = 0.05), and cattle handling facility design by temperament score interaction (p = 0.02), the AI pregnancy differed between heifers with excitable and calm temperament (51.9% vs 60.3%; p = 0.01). The alley-way with acute bends and turns, and long straight alley-way had lower AI pregnancy rate than did the semicircular alley-way (53.5%, 56.3% and 67.0% respectively; p = 0.05). The serum hormone concentrations differed significantly between different types of cattle handling facility (p < 0.05). The cattle handling facility design by temperament group interactions significantly influenced progesterone (p = 0.01), cortisol (p = 0.01), prolactin (p = 0.02) and substance-P (p = 0.04) both at the initiation of synchronization and at the time of AI. Inter- and intra-rater agreement for temperament scoring were moderate and good (Kappa = 0.596 ± 0.07 and 0.797 ± 0.11) respectively. The predictive value for calm and pregnant to AI was 0.87, and excited and non-pregnant to AI was 0.76. In conclusion, the modified 2-point temperament scoring method can be used to identify heifers with excitable temperament. Heifers with excitable temperament had lower AI pregnancy. Further, cattle handling facility design influenced the temperament and AI pregnancy.

Mucin 1 and cytokines mRNA in endometrium of dairy cows with postpartum uterine disease or repeat breeding.

Mucin (MUC) 1 is an inducible innate immune effector, an important component of defense against bacterial invasion, and is linked with infertility in humans. The objectives were to evaluate messenger RNA (mRNA) expression of MUC1 and cytokine genes in the endometrium of cows with various postpartum uterine inflammatory conditions or with a history of repeat breeding. Endometrial samples were collected from lactating dairy cows diagnosed with metritis (n = 4), endometritis (n = 4), subclinical endometritis (n = 4), or no uterine pathology (normal; n = 4). In addition, endometrial samples were collected from repeat breeder cows with (n = 4) or without (n = 4) subclinical endometritis, and unaffected cows (n = 4). Quantitative polymerase chain reaction was used to determine mRNA abundances of MUC1, Toll-like receptor (TLR) 4, interleukin (IL) 1ß, IL6, IL8, tumor necrosis factor (TNF) α, insulin-like growth factor (IGF) 1, and IGF-binding protein (BP) 2. The mRNA expressions were significantly greater for cows with metritis and clinical endometritis compared with cows with no uterine inflammation, except for IL6. However, mRNA expressions for these target genes were not different for cows with subclinical endometritis, compared with cows without uterine inflammation, except for IL1ß and TNFα mRNA (P < 0.01). All mRNA expressions were greater (P < 0.001) for repeat breeder cows with subclinical endometritis compared with normal cows. However, in repeat breeder cows without subclinical endometritis, only expressions of MUC1, IGF1, and IGF BP2 were greater compared with normal cows (P < 0.01). Based on functional protein networks, there were significant associations between these transcripts. In conclusion, endometrial expressions of MUC1 and cytokine genes differed among normal, fertile versus diseased, and subfertile dairy cows. Perhaps, these altered gene expressions contribute to endometrial insufficiency and consequently pregnancy wastage.