Influence of preovulatory estradiol treatment on the maintenance of pregnancy in beef cattle receiving in vivo produced embryos.

This experiment was designed to determine the role of preovulatory estradiol in pregnancy retention after embryo transfer (ET). Cows were synchronized with the 7-d CO-Synch + CIDR® protocol. On d0 (d-2 =CIDR® removal), cows were grouped by estrual status (estrual [Positive Control] and nonestrual), and nonestrual cows were administered Gonadotropin Releasing Hormone (GnRH) and randomly assigned to either no treatment (Negative Control) or Estradiol (0.1 mg estradiol 17-ß IM). All cows received an embryo on d7. Pregnancy status was retrospectively classified on d56, 30, 24, and 19 by either ultrasonography, plasma pregnancy-associated glycoproteins analysis (PAGs), expression of interferon-stimulated genes, plasma progesterone (P4) concentrations, or a combination of the factors. There was no difference in estradiol concentrations on day 0 h 0 (P > 0.16). At day 0 h 2, Estradiol cows (15.7 ± 0.25 pg/mL) had elevated (P < 0.001) estradiol compared with Positive Controls (3.4 ± 0.26 pg/mL) or Negative Controls (4.3 ± 0.25 pg/mL). On d19, pregnancy rates did not differ (P = 0.14) among treatments. On d24, Positive Controls (47%) had greater (P < 0.01) pregnancy rates than Negative Controls (32%); Estradiol cows were intermediate (40%). There was no difference (P = 0.38) in pregnancy rates between Positive Control (41%) and Estradiol (36%) cows on d30, but Negative Control (27%) cows had (P = 0.01) or tended (P = 0.08) to have decreased pregnancy rates, respectively. Thus, preovulatory estradiol may elicit an effect on early uterine attachment or alter histotroph components, consequently improving pregnancy maintenance through d30.

Effect of supplemental trace minerals on standard and novel measures of bull fertility.

Two studies were conducted to evaluate the effects of trace mineral supplementation on traditional and novel measures of bull fertility. In Experiment 1, 37 mature bulls received one of three dietary supplements daily for 71 d: 1) Supplement without Cu, Zn, and Mn (CON); 2) Supplement with Cu, Zn, and Mn sulfate (SULF); and 3) Supplement with basic Cu chloride, and Zn and Mn hydroxychloride (CHLR). In Experiment 2, 128 Angus or Angus-Hereford bull calves were maintained on a growing diet for 75 d (year 1) or 119 d (year 2) in Calan gate equipped pens without mineral supplementation. Bulls (n = 32 head/treatment) received one of four trace mineral supplements daily for 84 d: 1) Zn with no Cu (ZN), 2) Cu with no Zn (CU), 3) Cu and Zn (ZNCU), or 4) no Cu or Zn (CON). Bull fertility measures included a breeding soundness evaluation (BSE) and novel fertility measures conducted using flow cytometry. In mature bulls, final liver Zn concentration was positively correlated (P = 0.02) with sperm concentration (r = 0.31) and tended (P = 0.06) to be negatively correlated with acrosome damage (r = -0.39). Peripubertal bulls receiving ZNCU had greater ADG than bulls in the CU group (P = 0.05). Each BSE and novel fertility component improved from Day 0-84 in peripubertal bulls and were not affected (P > 0.10) by mineral supplementation. Bulls that received no supplement (CON) had greater (P < 0.01) percentage of sperm with distal midpiece reflex (6.9 vs 4.0% for CON and TM supplement, respectively) and Dag or Dag-like defect (2.6 vs 1.4% for CON and TM supplement, respectively) in their ejaculates. Sperm viability after 30 min of incubation were not affected by trace mineral supplementation, but after 3 h incubation, sperm viability tended to differ (P = 0.06) between treatments and tended to be less for CON bulls (48.5%) compared to ZNCU bulls (55.1%). Among contrast comparisons, trace mineral supplemented bulls had greater (P < 0.05) percentage of viable sperm at 3 h post collection and reactive oxygen resistant sperm than CON bulls. Addition of Zn to trace mineral containing Cu (ZNCU) improved (P < 0.05) percentage of sperm in the ejaculate with high mitochondrial energy potential and viable sperm with intact acrosome membrane. In summary, it appears the homeostasis mechanisms for bull trace mineral maintenance are extremely efficient and mineral supplementation of mature and peripubertal bulls did not have major improvements in any laboratory or chute-side measures of bull fertility, however bulls exposed to breeding or in environments with diet antagonists might respond differently.

Effect of estradiol preceding and progesterone subsequent to ovulation on proportion of postpartum beef cows pregnant.

Two experiments were conducted to examine the effect of plasma concentrations of 17ß-estradiol (E2) preceding and progesterone (P4) subsequent to ovulation on proportions of beef cows pregnant following embryo transfer. Timing of ovulation (d 0) among postpartum cows was synchronized and cows that expressed estrus were removed from each study. In Experiment 1, plasma E2 concentration on d 0 was used to classify cows (n = 353) into Low, Medium, and High E2 groups. Pregnancy rate for cows with Low, Medium, or High E2 concentrations were different (P < 0.05). In Experiment 2, there were multiple administrations of PGF2α to evaluate the independent effects of Low or High E2 before ovulation and Low or Normal (no treatment) P4 after ovulation on proportions of cows pregnant. Treatment groups in Experiment 2, therefore, were: Low E2-Low P4 (LL; n = 71), Low E2-Normal P4 (LN; n = 69), High E2-Low P4 (HL; n = 74), and High E2-Normal P4 (HN; n = 73). Concentrations of P4 on d 7 subsequent to ovulation were less (P < 0.05) in cows of the HL compared to HN, and in LL compared to LN groups. Concentrations of E2 on d -2, 0, and change in E2 (d -2 to d 0) had a positive effect (P < 0.008) on pregnancy rates. In summary, relatively greater E2 concentrations preceding ovulation; and relatively greater P4 concentrations subsequent to ovulation combined with lesser E2 concentrations preceding ovulation had a positive effect on proportions of postpartum cows pregnant.

Loci associated with conception rate in crossbred beef heifers.

The inability of beef cattle to maintain full term pregnancies has become an economic concern for the beef industry. Herd management and nutritional improvements have alleviated environmental impacts on embryonic and fetal loss, yet additional gains can be made through genomic selection. The objectives of this study were to identify loci and gene-sets in crossbred beef heifers associated with the number of services required to become pregnant (TBRD) and heifer conception rate at first service (HCR1). Heifers (n = 709) from a commercial beef operation underwent one round of artificial insemination, before exposure to bulls for natural service for 50 days. Pregnancy and time of conception was determined by ultrasound 35 days after the breeding season. Heifers were genotyped using the GeneSeek (Lincoln, NE) Bovine GGP50K BeadChip prior to genome-wide association analyses (GWAA) conducted using an EIGENSTRAT-like model to identify loci associated (P < 1 × 10-5) with TBRD and HCR1. One locus was associated (P = 8.97 × 10-6) with TBRD on BTA19 and included the positional candidate gene ASIC2, which is differentially expressed in the endometrium of fertility classified heifers, and the positional candidate gene, SPACA3. Gene-set enrichment analyses using SNP (GSEA-SNP) data, was performed and identified one gene-set, oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen as enriched (NES = 3.15) with TBRD and contained nine leading edge genes that contributed to the enrichment of the gene set. The enriched gene-set is involved in catalyzing oxidation-reduction reactions, which have been associated with oxidative stressors impacting pregnancy success. No loci were associated nor gene-sets enriched with HCR1. Identification of loci, positional candidate genes, gene-sets and leading edge genes enriched for fertility facilitate genomic selection that allows producers to select for reproductively superior cattle, reduce costs associated with infertility, and increase percent calf crop.

Pregnancy associated glycoproteins (PAGs) and pregnancy loss in high vs sub fertility heifers.

Reproductive inefficiency and infertility are major financial burdens to domestic livestock. Variables associated with these reproductive losses during early gestation include contributions from the oocyte, uterus, sperm, embryo and placenta. Bovine pregnancy associated glycoproteins (PAG) are produced by the binucleate cells of the ruminant placenta and can be used to diagnose pregnancy. Increased circulating concentrations of PAG early in gestation have been correlated with pregnancy success and decreased concentrations are predictive of impending embryonic mortality in both beef and dairy cattle. The objectives of the current study were to determine whether: 1) heifer fertility status is associated with circulating concentrations of PAG and pregnancy loss; and 2) PAG concentrations within the same animal are repeatable across multiple pregnancies. We hypothesized maternal PAG concentrations would be increased in high fertility compared to subfertile heifers but not repeatable across subsequent pregnancies in the same heifer. Serial embryo transfer (ET; n = 4 rounds) was used to classify predominately Angus heifers (n = 92) as highly fertile (HF = 30; 100% pregnancy success) or subfertile (SF = 62; average = 33%; range = 25-75% pregnancy success) based on day 28 ultrasound diagnosis. Blood samples were collected at both day 28 and 44 for quantification of circulating PAG concentrations by an in house PAG ELISA with antibodies raised against early secreted PAGs. Pregnancy was terminated at day 44 of gestation and heifers were allowed 30 days recovery before synchronization for the next ET. Only heifers that were diagnosed pregnant by ultrasound were used in this study (HF: n = 30, SF: n = 62). Serum concentrations of PAGs were not different between HF (5.90 ±â€¯0.27 ng/mL) and SF (5.56 ±â€¯0.31 ng/mL; P = 0.16) heifers at day 28 of gestation nor was there a difference at day 44 of gestation (P = 0.32). Subfertile heifers had increased pregnancy loss between days 28 and 44 of gestation. Based on odds ratio analysis, SF heifers had a 2.41 times chance to undergo pregnancy loss between day 28-44 compared to HF heifers (P < 0.05). There was no correlation (P > 0.05) in maternal circulating concentrations of PAG between pregnancies on day 28 or 44 of gestation in samples obtained from HF heifers. In summary, circulating concentrations of PAG are not different between HF and SF heifers; however, HF classified heifers have decreased pregnancy loss between days 28 and 44 of gestation.

Role of progesterone concentrations during early follicular development in beef cattle: I. Characteristics of LH secretion and oocyte quality.

Objective was to investigate the effect of different progesterone (P4) concentrations during early follicular development on luteinizing hormone (LH) secretion and oocyte characteristics in beef cows. Primiparous cows (n = 24) were estrous pre-synchronized and follicular ablation was performed (d 0) 6 days following the time of ovulation. At the time of follicular ablation, cows were assigned to either: 1) high P4 treatment - HiP4; a new CIDR was inserted on d 0 to supplement P4 from the existing corpus luteum [CL], or 2) low P4 treatment - LoP4; a previously-used CIDR and two doses of PGF 8 to 12 h apart were given on d 0. Concentrations of P4 were greater (P < 0.01) in the cows of the HiP4 than LoP4 group on d 1.5, 2.5, and 3.5. Peripheral concentrations of E2 were greater (P < 0.05) in the cows of the LoP4 than HiP4 group on d 2.5 and 3.5. Frequency of LH pulses was greater (P <  0.05) in the LoP4 than HiP4 group on d 2.5, but mean LH concentration and pulse amplitude did not differ between treatments. Number of follicles aspirated per cow, total oocytes recovered, recovery rate, percentage of oocytes graded 1 to 3, oocyte diameter, percentage BCB+ oocytes, and relative abundance of oocyte mRNA for FST did not differ (P >  0.10) between treatments. In conclusion, lower P4 concentrations during early follicular development resulted in increased LH pulse frequency and E2 concentrations, but did not affect characteristics of oocyte developmental competence.

Role of progesterone concentrations during early follicular development in beef cattle: II. Ovulatory follicle growth and pregnancy rates.

Two experiments were conducted to investigate the role of relatively lesser and greater progesterone (P4) concentrations during early follicular development on ovulatory follicle growth and pregnancy rate in beef cattle. In Experiment 1, time of ovulation was synchronized with the 5 d CO-Synch + CIDR (Controlled Internal Drug Release) program in multiparous cows (n = 241). Six days after the 2nd GnRH injection of the pre-synchronization program (d 0), ablation of follicles ≥ 5 mm in the ovaries was performed and cows were assigned to receive either a previously used CIDR and 2x-25 mg PGF2α doses 8 h apart (LoP4), or a new CIDR (HiP4). On d 5, CIDR were removed from all cows, 2x-25 mg PGF2α were administered, and estrous detection tail paint was applied. Timed artificial insemination (TAI) was performed on d 8. On d 5, P4 concentrations were greater (P <  0.01) in the HiP4 (4.9 ± 0.13 ng/mL) than LoP4 (1.0 ± 0.06 ng/mL) treatment group. Conversely, d 5 estradiol (E2) concentrations and follicular diameter were greater (P <  0.01) in the LoP4 (5.0 ± 0.23 pg/mL and 8.9 ± 0.20 mm) than HiP4 (1.5 ± 0.12 pg/mL and 7.4 ± 0.15 mm) treatment group. Follicular diameter at TAI (12.0 ± 0.12 mm, Table 1) and TAI pregnancy rate did not differ (P >  0.10) between treatment groups. In Experiment 2, a new follicular wave was induced with estradiol benzoate on d -7, and cows (n = 275) were assigned on d 0 to receive 25 mg PGF2α and either have the CIDR replaced with a new CIDR (HiP4) or the used CIDR was left in place (LoP4).Furthermore, all cows received GnRH on d 0. The CIDRs were removed from all cows on d 5 and two doses of -25 mg PGF2α were administered. Estrous detection combined with AI 12 h later (Estrus-AI) was performed for 60 h after CIDR removal with TAI coupled with GnRH administration at 72 h if estrus was not detected. The concentrations of P4 on d 5 were greater (P <  0.01) in the HiP4 (2.8 ± 0.10 ng/ml) than LoP4 (1.7 ± 0.05 ng/mL) treatment group. For cows that were detected in estrus after PGF2α administration, estrous response (83.5%) and interval to estrus (55.0 ± 0.5 h) did not differ between treatment groups. Pregnancy rate (combined Estrus-AI and TAI) that resulted from breeding at the time of the synchronized time of estrus was similar between treatment groups (HiP4: 77.1%; LoP4: 82.3%). In conclusion, differences in P4 concentrations during early follicular development do not effect pregnancy rate in beef cows when the cows are inseminated at the time of a synchronized estrus if the cows have similar intervals of proestrus.

Glycosylation and immunocytochemistry of binucleate cells in pronghorn (Antilocapra americana, Antilocapridae) show features of both Giraffidae and Bovidae.

Although the pronghorn (Antilocapra americana) resembles an antelope, its nearest relatives are the giraffe and okapi. In this study we have examined the placentae of 6 pronghorns using lectin- and immunocytochemistry to identify giraffid and bovid features. Binucleate cells (BNC) of the placenta exhibited features intermediate between those of the giraffe and bovine; Dolichos biflorus agglutinin binding - strong in the bovine BNC and absent in the giraffe - was evident in only a subpopulation of BNC while binding to blood vessels, as in the giraffe. Binding of Phytolacca americana agglutinin resembled that of the giraffe and okapi whereas many other glycans were found in all four clades. PAG antigens were similar to bovine and okapi but not giraffe. In summary, although the pronghorn outwardly resembles an antelope, placental BNC show giraffid features. Although each clade has its own individual characteristics, there are far more similarities than differences between them, emphasizing the common ancestry of all four clades.

Species differences in hepatic biotransformation of the anthelmintic drug flubendazole.

Flubendazole (FLBZ) is a broad-spectrum benzimidazole anthelmintic used in pigs, poultry, and humans. It has been proposed as a candidate for development for use in elimination programmes for lymphatic filariasis and onchocerciasis in humans. Moreover, FLBZ has shown promise in cancer chemotherapy, particularly for neuroblastoma. This work investigated the hepatic carbonyl-reducing pathway of FLBZ in different species, including humans. Microsomal and cytosolic fractions were obtained from sheep, cattle, pig, hen, rat, and human liver. Both subcellular fractions of each species converted FLBZ into a reduced metabolite (red-FLBZ). The rate of microsomal red-FLBZ production was highest in sheep (1.92 ± 0.13 nmol/min.mg) and lowest in pigs (0.04 ± 0.02 nmol/min.mg); cytosolic red-FLBZ production ranged from 0.02 ± 0.01 (pig) to 1.86 ± 0.61 nmol/min.mg (sheep). Only subcellular fractions from sheep liver oxidized red-FLBZ to FLBZ in a NADP+ -dependent oxidative reaction. Liver microsomes from both pigs and humans transformed FLBZ to red-FLBZ and a hydrolyzed metabolite. Very significant differences in the pattern of FLBZ metabolism were observed among the tested species and humans. These results reinforce the need for caution in extrapolating data on metabolism, efficacy, and safety of drugs derived from studies performed in different species.

Effects of reduced in utero and post-weaning nutrition on milk yield and composition in primiparous beef cows.

Development and long-term retention of replacement beef females in a semi-arid environment are of a major concern for extensive livestock producers. Furthermore, the demand of not only producing a thriving, healthy calf, but having sufficient milk to support that first calf is essential. To address this issue, we conducted a 3-year study measuring milk production and milk constituent yields in primiparous beef heifers (n=48; 16/year reared under two different feeding regimens) raising steer calves. Cows received 1.8 or 1.2 kg/day winter supplementation for ~80 day before parturition and their heifer calves were then randomly assigned to heifer development treatments that provided ad libitum (AL) or 80% (less than ad libitum (LAL)) of ad libitum feed post weaning. Heifers developed on the AL treatment also received 1.8 kg/day winter supplementation for life, whereas heifers developed on the LAL treatment received 1.2 kg/day winter supplementation for life. Milk production of primiparous cows was measured with a portable milking machine every other week from days 27 to 125 postpartum. Milk yield for the 125-day lactation period was calculated from area under the lactation curve approximated by trapezoidal summation. The ANOVA model included in utero winter nutrition, post-weaning heifer development treatment, year and their interaction. Heifers subjected to the AL treatment reached peak milk yield ~12.3 day later (P=0.02) than heifers receiving LAL treatment. In addition, an in utero nutrition×post-weaning heifer treatment×year interaction existed (P⩽0.04) for milk peak yield, average daily milk yield (kg/day) and nutrient composition (protein, lactose, fat, solids non-fat, g/day). These interactions manifest as changes in magnitude and rank across the 3 years of the study. Livestock production in extensive environments is subject to variations in seasonal precipitation patterns and quality and quantity of grazeable forage and these fluctuations have a large impact on milk yield. In summary, the gestational nutritional environment of a heifer's mother may interact with the heifer's nutrient consumption during post-weaning growth and the current year to trigger variation in year-to-year milk production.

Genomic prediction of continuous and binary fertility traits of females in a composite beef cattle breed.

Reproduction efficiency is a major factor in the profitability of the beef cattle industry. Genomic selection (GS) is a promising tool that may improve the predictive accuracy and genetic gain of fertility traits. There is a wide range of traits used to measure fertility in dairy and beef cattle including continuous (days open), discrete (pregnancy status), and count (number of inseminations) responses. In this study, a joint analysis of age of puberty (AOP), age at first calving (AOC), and the heifer pregnancy status (HPS) was performed. Data used in this study consisted of records from 1,365 Composite Gene Combination (CGC; 50% Red Angus, 25% Charolais, 25% Tarentaise) first parity females born between 2002 and 2011. The pedigree file included 5,374 animals. A total of 3,902 animals were genotyped with different density SNP chips (3K to 50K SNP). Animals genotyped with low-density arrays were imputed to higher density (BovineSNP50 BeadChip) using FImpute. Data were analyzed using univariate and multivariate classical quantitative models (pedigree based) and univariate genomic approaches. For the latter, 3 different Bayesian methods (BayesA, BayesB, and BayesCπ) were implemented and compared. Estimates of heritabilities using univariate and multivariate analyses based on pedigree relationships ranged between 0.03 (for AOC) to 0.2 (AOP). Heritability of pregnancy status was 0.15 and 0.09 using the univariate and multivariate analyses, respectively. Genetic correlation between pregnancy status and the other 2 traits was low being 0.08 with age at puberty and -0.10 with age at first calving. Heritability estimates were slightly higher using genomic rather than average additive relationships. The accuracy of genomic prediction was similar across the 3 Bayesian methods with higher accuracies for age of puberty than the age at first calving likely due to the higher heritability of the former. The prediction of the binary pregnancy status measured using the area under the curve increased by 27% to 29% compared to a random classifier. Due to the small size of the data, all estimates have large posterior standard deviations and results should be interpreted with caution.

Developmental and reproductive characteristics of beef heifers classified by pubertal status at time of first breeding.

Data collected for 10 or more years at the West Central Research and Extension Center, North Platte, NE ( = 1,104); the Gudmundsen Sandhills Laboratory, Whitman, NE ( = 1,333); and the USDA, ARS, Fort Keogh Livestock and Range Research Laboratory, Miles City, MT ( = 1,176) were retrospectively analyzed to evaluate growth and reproductive performance of beef heifers classified by pubertal status before first breeding. Concentrations of progesterone in serum from 2 blood samples collected 9 to 11 d apart before the breeding season classified heifers as pubertal (progesterone ≥ 1.0 ng/mL in 1 or both samples) or nonpubertal (progesterone < 1.0 ng/mL in both samples). Average date of birth was earlier ( < 0.06) and proportion born in the first 21 d of the calving season was 10 to 20 percentage points greater for heifers that were pubertal at the start of breeding compared with heifers not pubertal by the start of breeding. Heifers that were pubertal by the start of breeding were 7 to 10 kg heavier ( < 0.01) and 1 cm taller ( < 0.01) at weaning than heifers not pubertal by the start of breeding. Differences in BW persisted through the start of breeding to pregnancy diagnosis. Heifers that achieved puberty by the start of breeding had greater ( < 0.05) feed intake and G:F during postweaning development and had greater ( < 0.01) LM area and fat thickness over the LM at approximately 1 yr of age compared with heifers not pubertal by the start of breeding. Heifers that achieved puberty before the start of breeding had greater ( < 0.01) ADG from birth to weaning but slower ( < 0.10) rates of gain from the start of breeding through pregnancy diagnosis. Pregnancy rate was greater ( < 0.01) for heifers that were pubertal at the start of breeding. In heifers that became pregnant, those that were pubertal before the start of breeding calved earlier ( < 0.01), with a greater ( < 0.01) percentage calving in the first 21 d of calving than heifers not pubertal at the start of breeding. Calves from heifers that achieved puberty before the start of breeding were heavier at weaning ( < 0.01) than calves from heifers that had not achieved puberty by the start of breeding. In summary, heifers that failed to achieve puberty by the start of breeding were less desirable for several traits evaluated. Based on these results, implementing feeding strategies to increase the proportion of heifers that achieve puberty before first breeding could result in propagation of undesirable characteristics.

Primary Heligmosomoides polygyrus bakeri infection induces myeloid-derived suppressor cells that suppress CD4+ Th2 responses and promote chronic infection.

Primary infection with the gastrointestinal nematode Heligmosomoides polygyrus bakeri is chronic in C57BL/6 (B6) mice whereas challenge infection is rapidly eliminated. F4/80-CD11b+Gr+ cells, presumed to be neutrophils, were reported to accumulate around encysting larvae in intestinal tissue during primary infection, but their exact identity and role remain unclear. We observed significant increases in F4/80-CD11bhiGr1hi cells in mesenteric lymph nodes (MLNs) and spleen after primary but not challenge infection; a high proportion of these cells expressed Ly6G and Ly6C. These cells, which phenotypically resemble myeloid-derived suppressor cells (MDSC), increased in lamina propria (LP) early during primary infection. Increased MDSC were associated with low numbers of alternatively activated macrophages (AAMØ) in LP and CD4+GATA3+ T cells and AAMØ in MLN and spleen. Purified CD11c-CD11b+Gr1+ cells from H. polygyrus bakeri-infected mice suppressed OVA-specific CD4+ T-cell proliferation via a nitric oxide-dependent mechanism and parasite-specific IL-4 secretion in vitro. Adoptive transfer of CD11c-CD11b+Gr1+ cells from mice with primary infection resulted in significantly higher adult worm burdens and increased egg production in naïve B6 recipients infected with H. polygyrus bakeri. Altogether, these findings indicate that primary H. polygyrus bakeri infection induces a novel subset of MDSC that suppress CD4+ Th2 responses and promote chronic infection.

Haemonchus contortus: Applications in Drug Discovery.

Haemonchus contortus is an important pathogen of small ruminants and is therefore a crucially important target for anthelmintic chemotherapy. Its large size and fecundity have been exploited for the development of in vitro screens for anthelmintic discovery that employ larval and adult stages in several formats. The ability of the parasite to develop to the young adult stage in Mongolian jirds (Meriones unguiculatus) provides a useful small animal model that can be used to screen compounds prior to their evaluation in infected sheep. This chapter summarizes the use of H. contortus for anthelmintic discovery, offers a perspective on current strategies in this area and suggests research challenges that could lead to improvements in the anthelmintic discovery process.

Effect of supplemental trace mineral level and form on peripubertal bulls.

Objectives were to determine if supplemental trace mineral levels and/or forms (sulfate and metal amino acid complexes) influence age at puberty, semen quality, endocrine status, and scrotal circumference in peripubertal bulls. Fifty peripubertal bulls were blocked by age and scrotal circumference and assigned to one of five treatments: (1) 1x sulfate form (1S); (2) 1x complexed form (1C); (3) 1S+1C (2SC); (4) 1S + 2 × 1 C (3SCC); and (5) 3 × 1S (3S). Each 1x supplementation level contained 360 mg Zn, 125 mg Cu, 200mg Mn and 12.5mg Co. Liver biopsies were collected on d -21 and 100, and scrotal circumference, semen, and blood samples were collected on d -14, 14, 42, 70, and 98. All bulls were deficient in Cu yet adequate in Zn on d -21. Following 100 d on treatment, liver Zn concentrations decreased (P<0.01) and liver Cu concentrations increased (P<0.01) in bulls regardless of treatment. Day 100 liver Zn concentrations were similar (P=0.50) across treatments, but liver Cu concentrations were greater (P=0.07) in 3SCC and 3S bulls compared to 1C and 1S bulls, whereas 2SC bulls were intermediate. Bulls fed complexed minerals tended to reach puberty after fewer (P=0.11) days on treatment (43.9 ± 5.7 d) than bulls fed only sulfate minerals (58.5 ± 6.7 d). Supplementing complexed Cu and Zn to prepubertal bulls may lower the age at puberty, however, no differences (P ≥ 0.40) in semen characteristics or scrotal measurements (P ≥ 0.11) were observed.

Predicting Embryo Presence and Viability.

Pregnancy establishment, followed by birth of live offspring, is essential to all mammals. The biological processes leading up to pregnancy establishment, maintenance, and birth are complex and dependent on the coordinated timing of a series of events at the molecular, cellular, and physiological level. The ability to ovulate a competent oocyte, which is capable of undergoing fertilization, is only the initial step in achieving a successful pregnancy. Once fertilization has occurred and early embryonic development is initiated, early pregnancy detection is critical to provide proper prenatal care (humans) or appropriate management (domestic livestock). However, the simple presence of an embryo, early in gestation, does not guarantee the birth of a live offspring. Pregnancy loss (embryonic mortality, spontaneous abortions, etc.) has been well documented in all mammals, especially in humans and domestic livestock species, and is a major cause of reproductive loss. It has been estimated that only about 25-30% of all fertilized oocytes in humans result in birth of a live offspring; however, identifying the embryos that will not survive to parturition has not been an easy task. Therefore, investigators have focused the identification of products in maternal circulation that permit the detection of an embryo and assessment of its well-being. This review will focus on the advances in predicting embryonic presence and viability, in vivo.

Pharmacokinetic comparison of different flubendazole formulations in pigs: A further contribution to its development as a macrofilaricide molecule.

Despite the well established ivermectin activity against microfilaria, the success of human filariasis control programmes requires the use of a macrofilaricide compound. Different in vivo trials suggest that flubendazole (FLBZ), an anthelmintic benzimidazole compound, is a highly efficacious and potent macrofilaricide. However, since serious injection site reactions were reported in humans after the subcutaneous FLBZ administration, the search for alternative pharmaceutical strategies to improve the systemic availability of FLBZ has acquired special relevance both in human and veterinary medicine. The goal of the current experimental work was to compare the pharmacokinetic plasma behavior of FLBZ, and its metabolites, formulated as either an aqueous hydroxypropyl- ß -cyclodextrin-solution (HPBCD), an aqueous carboxymethyl cellulose-suspension (CMC) or a Tween 80-based formulation, in pigs. Animals were allocated into three groups and treated (2 mg/kg) with FLBZ formulated as either a HPBCD-solution (oral), CMC-suspension (oral) or Tween 80-based formulation (subcutaneous). Only trace amounts of FLBZ parent drug and its reduced metabolite were measured after administration of the different FLBZ formulations in pigs. The hydrolyzed FLBZ (H-FLBZ) metabolite was the main analyte recovered in the bloodstream in pigs treated with the three experimental FLBZ formulations. The oral administration of the HPBCD-solution accounted for significantly higher (P < 0.05) Cmax and AUC (23.1 ± 4.4 µg h/mL) values for the main metabolite (H-FLBZ), compared with those observed for the oral CMC-suspension (AUC = 3.5 ± 1.0 µg h/mL) and injectable Tween 80-based formulation (AUC: 7.5 ± 1.7 µg h/mL). The oral administration of the HPBCD-solution significantly improved the poor absorption pattern (indirectly assessed as the H-FLBZ plasma concentrations) observed after the oral administration of the FLBZ-CMC suspension or the subcutaneous injection of the Tween 80 FLBZ formulation to pigs. Overall, the work reported here indicates that FLBZ pharmacokinetic behavior can be markedly changed by the pharmaceutical formulation.

The effect of follicle age on pregnancy rate in beef cows.

The effect of the age of the ovulatory follicle on fertility in beef cows was investigated. Multiparous (n = 171) and primiparous (n = 129) postpartum beef cows in 2 groups (G1 and G2) received estradiol benzoate (EB; 1 mg/500 kg BW, intramuscular [i.m.]) 5.5 d (G1; n = 162) and 6.5 d (G2; n = 138) after the final GnRH of a synchronization program (5d CO-Synch + CIDR) to induce emergence of a new follicular wave (NFW), followed by prostaglandin F2α (PGF(2α); 25 mg, i.m.) administration either 5.5 d ("young" follicle, YF; n = 155) or 9.5 d ("mature" follicle, MF; n = 145) after EB. Estrous detection coupled with AI 12 h later (estrus-AI) was performed for 60 h (MF) and 84 h (YF) after PGF(2α); cows not detected in estrus within this period received timed AI (TAI) coupled with GnRH at 72 and 96 h, respectively. Within the first 72 h after PGF(2α), more (P < 0.01) cows in the MF (76.3%) than YF treatment (47.7%) exhibited estrus, but through 96 h, the proportion detected in estrus (P < 0.05) and interval from PGF(2α) to estrus (P < 0.01) were greater in the YF than MF treatment (88.6% vs. 76.3%, 78.9 ± 0.8 vs. 57.5 ± 1.6 h, respectively). Age of the ovulatory follicle at AI was greater (P < 0.01) in the MF (9.32 ± 0.04 d) than YF (6.26 ± 0.02 d) treatment, but follicle diameter at AI and pregnancy rates did not differ between MF (13.1 ± 0.2 mm; 72.0%) and YF (12.9 ± 0.1 mm; 67.1%) treatments. Regardless of treatment, the diameter of the ovulatory follicle at AI and pregnancy rate were greater (P < 0.01) with estrus-AI (13.1 ± 0.1 mm; 75.0%) than TAI (12.6 ± 0.2 mm; 55.4%). Cows in the MF treatment that initiated a second NFW after EB but before PGF(2α) (MF2; n = 47) were induced to ovulate with GnRH and TAI at 72h, when ovulatory follicles were 4 d old and 10.2 ± 0.2 mm in diameter. Pregnancy rate for TAI (51.1%) in MF2 did not differ from TAI pregnancy rate (55.4%) across the MF and YF treatments. In summary, the age of the ovulatory follicle affected interval to estrus and AI but did not influence pregnancy rate in suckled beef cows.

Effect of follicle age on conception rate in beef heifers.

The objective of this study was to determine the effect of age of the ovulatory follicle on fertility in beef heifers. Ovulation was synchronized with the 5 d CO-Synch + controlled intravaginal drug release (CIDR) program in heifers in Montana (MT; n = 162, Hereford and Angus Crossbred) and Ohio (OH; n = 170, Angus Crossbred). All heifers received estradiol benzoate (EB; 1 mg/500 kg BW, [i.m.]) 6 d after the final GnRH of the synchronization program to induce follicular atresia and emergence of a new follicular wave (NFW) followed by prostaglandin F2α (PGF(2α); 25 mg, i.m.) administration either 5 d ("young" follicle [YF]; n = 158) or 9 d ("mature" follicle [MF]; n = 174) after EB. Estrous detection was performed for 5 d after PGF(2α) with AI approximately 12 h after onset of estrus. Ovarian ultrasonography (MT location only) was performed in YF and MF at EB, 5 d after EB, PGF(2α), and AI. Heifers in MT (n = 20) and OH (n = 18) that were not presynchronized or did not initiate a NFW were excluded from further analyses, resulting in 142 and 152 heifers in MT and OH, respectively. Heifers from the MF treatment in MT that initiated a second NFW after EB but before PGF(2α) (MF2; n = 14) were excluded from the primary analysis. In the secondary analysis, the MF2 group was compared to MF and YF treatments in MT. Estrous response was similar (90%; 252/280) between treatments and locations. Proestrus interval (from PGF(2α) to estrus) and age of the ovulatory follicle at AI were similar for MF heifers between locations (54.6 ± 1.7 h and 8.3 ± 0.07 h) but were greater (P < 0.01) for YF heifers in OH (78.5 ± 1.4 h and 5.3 ± 0.06 h) than MT (67.4 ± 1.6 h and 4.8 ± 0.06 h; treatment × location, P < 0.01). However, conception rate did not differ for MF (63.8%; 74/116) and YF (67.0%; 91/136) treatments. In the MT heifers, follicle size and follicle age at AI in the YF treatment (10.4 ± 0.15 mm and 4.8 ± 0.06 d, respectively) was less (P < 0.01) than in the MF treatment (11.0 ± 0.18 mm and 8.3 ± 0.11 d, respectively), but conception rate to AI did not differ between treatments in MT. In the MF2 group proestrus interval was greater (P < 0.01); hence, diameter of the ovulatory follicle and age were similar to that for the YF treatment. Conception rate to AI did not differ between MF2, MF, and YF (61.5, 63.3, and 64.7%, respectively) in MT. In conclusion, manipulation of age of the nonpersistent ovulatory follicle at spontaneous ovulation did not influence conception rate.

Identification of Dirofilaria immitis proteins recognized by antibodies from infected dogs.

The identification of excreted-secreted (ES) proteins of filarial nematodes as potential diagnostic reagents is an important requirement for the development of methods to determine level of infection in the host, especially for human filariae. Dirofilaria immitis, the canine heartworm, is a widespread and important veterinary pathogen and is a useful model for filarial parasites of humans. An analysis of proteins released from adult D. immitis (the secretome) in culture is available. We sought to identify D. immitis ES proteins found in vivo to validate the in vitro secretome and to investigate them as potential diagnostic reagents. Cultures of D. immitis adults obtained from infected dogs were maintained for 72 hr with daily changes of media. Proteins were concentrated from spent media by standard methods and were passed through Protein-A columns containing purified IgG antibodies from heartworm-infected dogs. Following extensive washing, heartworm proteins recognized by the antibodies were eluted from these columns and submitted for analysis by tandem mass spectrometry (MS/MS). As a comparison, somatic proteins from adult D. immitis female parasites and microfilaria were also processed and analyzed by the same protocol. Six, 9, and 12 proteins were identified by MS/MS in the ES, adult female, and microfilaria samples, respectively. The identification of the most abundant parasite proteins present in the serum of infected hosts offers a rational approach to the development of new diagnostic assays that may be applicable across the Filarioidea.