Single-step genomic predictions for heat tolerance of production yields in US Holsteins and Jerseys.

The physiological stress caused by excessive heat affects dairy cattle health and production. This study sought to investigate the effect of heat stress on test-day yields in US Holstein and Jersey cows and develop single-step genomic predictions to identify heat tolerant animals. Data included 12.8 million and 2.1 million test-day records, respectively, for 923,026 Holstein and 153,710 Jersey cows in 27 US states. From 2015 through 2021, test-day records from the first 5 lactations included milk, fat, and protein yields (kg). Cow records were included if they had at least 5 test-day records per lactation. Heat stress was quantified by analyzing the effect of a 5-d hourly average temperature-humidity index (THI5d¯) on observed test-day yields. Using a multiple trait repeatability model, a heat threshold (THI threshold) was determined fowr each breed based on the point that the average adjusted yields started to decrease, which was 69 for Holsteins and 72 for Jerseys. An additive genetic component of general production and heat tolerance production were estimated using a multiple trait reaction norm model and single-step genomic BLUP methodology. Random effects were regressed on a function of 5-d hourly average (THI5d¯) and THI threshold. The proportion of test-day records that occurred on or above the respective heat thresholds was 15% for Holstein and 10% for Jersey. Heritability of milk, fat, and protein yields under heat stress for Holsteins increased, with a small standard error, indicating that the additive genetic component for heat tolerance of these traits was observed. This was not as evident in Jersey traits. For Jersey, the permanent environment explained the same or more of the variation in fat and protein yield under heat stress indicating that nongenetic factors may determine heat tolerance for these Jersey traits. Correlations between the general genetic merit of production (in the absence of heat stress) and heat tolerance genetic merit of production traits were moderate in strength and negative. This indicated that selecting for general genetic merit without consideration of heat tolerance genetic merit of production may result in less favorable performance in hot and humid climates. A general genomic estimated breeding value for genetic merit and a heat tolerance genomic estimated breeding value were calculated for each animal. This study contributes to the investigation of the impact of heat stress on US dairy cattle production yields and offers a basis for the implementation of genomic selection. The results indicate that genomic selection for heat tolerance of production yields is possible for US Holsteins and Jerseys, but a study to validate the genomic predictions should be explored.

Review: Integrating a semen quality control program and sire fertility at a large artificial insemination organization.

The technology available to assess sperm population characteristics has advanced greatly in recent years. Large artificial insemination (AI) organizations that sell bovine semen utilize many of these technologies not only for novel research purposes, but also to make decisions regarding whether to sell or discard the product. Within an AI organization, the acquisition, interpretation and utilization of semen quality data is often performed by a quality control department. In general, quality control decisions regarding semen sales are often founded on the linkages established between semen quality and field fertility. Although no one individual sperm bioassay has been successful in predicting sire fertility, many correlations to various in vivo fertility measures have been reported. The most powerful techniques currently available to evaluate semen are high-throughput and include computer-assisted sperm analysis and various flow cytometric analyses that quantify attributes of fluorescently stained cells. However, all techniques measuring biological parameters are subject to the principles of precision, accuracy and repeatability. Understanding the limitations of repeatability in laboratory analyses is important in a quality control and quality assurance program. Hence, AI organizations that acquire sizeable data sets pertaining to sperm quality and sire fertility are well-positioned to examine and comment on data collection and interpretation. This is especially true for sire fertility, where the population of AI sires has been highly selected for fertility. In the December 2017 sire conception rate report by the Council on Dairy Cattle Breeding, 93% of all Holstein sires (n=2062) possessed fertility deviations within 3% of the breed average. Regardless of the reporting system, estimates of sire fertility should be based on an appropriate number of services per sire. Many users impose unrealistic expectations of the predictive value of these assessments due to a lack of understanding for the inherent lack of precision in binomial data gathered from field sources. Basic statistical principles warn us of the importance of experimental design, balanced treatments, sampling bias, appropriate models and appropriate interpretation of results with consideration for sample size and statistical power. Overall, this review seeks to describe and connect the use of sperm in vitro bioassays, the reporting of AI sire fertility, and the management decisions surrounding the implementation of a semen quality control program.

Impact of a timed-release follicle-stimulating hormone treatment from one to three months of age on endocrine and testicular development of prepubertal bulls.

In prepubertal bulls, FSH facilitates testis maturation and a transient proliferation of Sertoli cells. Two experiments examined the effects of exogenous FSH on hormone secretion and testis development in Angus bulls. Exogenous FSH treatment consisted of an intramuscular injection (i.m.) of 30 mg FSH (Folltropin-V) in a 2% hyaluronic acid solution (FSH-HA). In Exp. 1, bulls (50 ± 6.5 d of age) received either FSH-HA ( = 5) or saline (control; = 5) on d 50 and 53.5. Blood samples were collected via jugular venipuncture to assess FSH concentrations every 6 h for 24 h after treatment and every 12 h until 84 h. After each treatment, peripheral FSH concentrations were greater ( < 0.05) in the FSH-HA-treated bulls than in the control bulls 6 h after treatment and tended to be greater ( ≤ 0.08) 12 h after treatment. The FSH concentration from 18 to 84 h after treatment did not differ between treatments. In Exp. 2, bulls were treated with FSH-HA ( = 11) or saline (control; = 11) every 3.5 d from 35 to 91 ± 2 d of age. Blood samples were collected before each treatment to quantify FSH, testosterone, and activin A concentrations. Scrotal circumference (SC) and BW were measured weekly. Bulls were castrated at 93 ± 2 d of age. Seminiferous tubule diameter, testis composition, and the number of Sertoli cells per tubule cross section (GATA-4 positive staining) were determined from fixed and stained histological sections. Follicle-stimulating hormone concentrations within the FSH-HA-treated bulls increased ( < 0.05) on d 70 from prior sampling and remained elevated. The FSH concentration did not differ between treatments from 35 to 66.5 d of age but were greater ( < 0.05) in the FSH-HA-treated bulls than in the control bulls from 70 to 91 d of age. Serum concentration of activin A on d 35, 70, and 91 did not differ between treatments. The FSH-HA and control bulls did not differ ( > 0.1) in BW, SC, testis weight, testis volume, percent of parenchyma composed of tubules, tubule diameter, and concentration of testosterone. The number of Sertoli cells per tubule cross section was greater in the FSH-HA-treated bulls than in the control bulls (33.35 ± 0.9 vs. 28.27 ± 0.9 cells; ˂ 0.05). In summary, the FSH-HA treatment from 35 to 91 d of age resulted in increased endogenous FSH from 70 to 91 d and increased numbers of Sertoli cells at 93 d of age. Exogenous FSH altered endocrine mechanisms regulating endogenous FSH secretion and augmented Sertoli cell proliferation in young bulls, but this effect was apparently not caused by increased activin A concentration in the FSH-HA-treated bulls.

Design and production of antibodies for the detection of Streptococcus uberis.

Streptococcus uberis (S. uberis) is an important environmental pathogen causing mastitis in dairy cattle. Mastitis or mammary gland infection is the most common disease in milking cows and cause significant economic burden to farmers due to the reduction of the amount of milk and its quality. The development of rapid analytical methods for the determination of mastitis-causing pathogens in milk is of utmost importance for the early identification of the causes of mastitis and the beginning of timely treatment of cows. Combining in silico bioinformatic analysis and solid phase peptide synthesis using Fmoc chemistry, we have made two different peptides to mimic the adhesion protein of S. uberis, which is promoting the attachment of bacteria to epithelial cells. After purification with RP-HPLC, the peptides were conjugated with a larger carrier protein (KLH) and used for immunization of rabbits to produce specific antibodies. The separation of anti-S. uberis antibodies from rabbit blood antisera was carried out with affinity chromatography, using the synthetic peptides as affinity ligands. The purified antibodies showed high affinity and specificity towards S. uberis and were used for rapid bio-recognition and identification of S. uberis with an immunobiosensing system.

Effects of dietary energy on sexual maturation and sperm production in Holstein bulls.

In prepubertal bulls and heifers of dairy and beef breeds, puberty can be induced to occur earlier than typical with targeted high-energy diets due to precocious activation of the endocrine mechanisms that regulate puberty. Precocious activation of puberty in bulls intended for use in the AI industry has the potential to hasten and perhaps increase sperm production. It was hypothesized that feeding bulls a high-energy diet beginning at 8 wk of age would advance the prepubertal rise in LH and lead to advanced testicular maturation and age at puberty. From 58 to 230 ± 0.3 d of age, Holstein bulls received either a high-energy diet (HE;n = 9; targeted ADG 1.5 kg/d) or a control diet (CONT;n = 10; targeted ADG 0.75 kg/d). Thereafter, all bulls were fed a similar diet. The HE treatment increased LH secretion at 125 d of age, testosterone concentrations from 181 to 210 d, and scrotal circumference (SC) from 146 to 360 d of age relative to the CONT treatment. Beginning at 241 ± 5 d of age, semen collection (artificial vagina) was attempted every 14 d in bulls from the HE (n = 8) and CONT (n = 7) treatment until each bull attained puberty (ejaculate containing 50 × 10 spermatozoa with 10% motility). To assess semen production as mature bulls, semen was collected thrice weekly beginning at 541 ± 5 d of age until slaughter at 569 ± 5 d of age. After slaughter, epididymal and testicular measurements were collected and testicular tissue was fixed to determine seminiferous tubule diameter. Age at puberty did not differ between treatments (310 ± 35 d). Although testis and epididymal weight and testis volume were greater (P < 0.05) in the HE than the CONT treatment, sperm production of mature bulls did not differ between treatments. Diameter of seminiferous tubules also did not differ between treatments. We conclude that the HE advanced aspects of sexual maturation and increased testes size, but this was not reflected in hastened puberty or sperm production in the present experiment.

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.

Triennial Reproduction Symposium: the obstacle course to successful establishment of pregnancy in domestic livestock species.

The 2012 Triennial Reproduction Symposium (TRS), "Impediments to Fertility in Domestic Animals," was held immediately before the Joint Annual Meeting of the American Society of Animal Science, American Dairy Science Association, Canadian Society of Animal Science, Western Section of the American Society of Animal Science, and the Asociación Mexicana de Producción Animal in Phoenix, AZ, in July, 2012. The theme of the symposium highlighted key impediments or opportunities in the process of creating a pregnancy, beginning with male and female gametes and ending with a viable fetus. The 2012 TRS was designed to focus on areas of current and exciting investigation across a variety of species and to include 8 presentations from a mix of established and early-career scientists. The TRS was also the venue for presentation of the 2012 L. E. Casida Award for Graduate Education; the recipient was R. D. Randel (Texas A&M University). The symposium provided an excellent opportunity for reproductive biologists to consider the broad spectrum of factors that limit fertility in domestic species and contemplate the current status of knowledge relative to several of the significant obstacles to pregnancy.

Use of Saccharomyces cerevisiae fermentation product on growth performance and microbiota of weaned pigs during Salmonella infection.

Anaerobically fermented yeast products are a rich source of nutritional metabolites, mannanoligosaccharides, and ß-glucans that may optimize gut health and immunity, which can translate into better growth performance and a reduced risk of foodborne pathogens. The objective of this study was to quantify the effects of Saccharomyces cerevisiae fermentation product (Diamond V Original XPC) inclusion in nursery diets on pig performance and gastrointestinal microbial ecology before, during, and after an oral challenge with Salmonella. Pigs (n = 40) were weaned at 21 d of age, blocked by BW, and assigned in a 2 × 2 factorial arrangement consisting of diet (control or 0.2% XPC) and inoculation (sterile broth or Salmonella). Pigs were fed a 3-phase nursery diet (0 to 7 d, 7 to 21 d, and 21 to 35 d) with ad libitum access to water and feed. On d 14, pigs were orally inoculated with 10(9) cfu of Salmonella enterica serovar Typhimurium DT104 or sterile broth. During d 17 to 20, all pigs were treated with a 5 mg/kg of BW intramuscular injection of ceftiofur-HCl. Growth performance and alterations in the gastrointestinal microbial ecology were measured during preinoculation (PRE; 0 to 14 d), sick (SCK; 14 to 21 d), and postinoculation (POST; 21 to 35 d). Body weight and ADG were measured weekly. Rectal temperature (RT) was measured weekly during PRE and POST, and every 12 h during SCK. Diet had no effect on BW, ADG, or RT during any period (P = 0.12 to 0.95). Inclusion of XPC tended (P < 0.10) to increase Salmonella shedding in feces during SCK. Consumption of XPC altered the composition of the gastrointestinal microbial community, resulting in increased (P < 0.05) populations of Bacteroidetes and Lactobacillus after Salmonella infection. Pigs inoculated with Salmonella had decreased ADG and BW, and increased RT during SCK (P < 0.001). Furthermore, fecal Salmonella cfu (log(10)) was modestly correlated (P = 0.002) with BW (r = -0.22), ADFI (r = -0.27), ADG (r = -0.36), G:F (r = -0.18), and RT (r = 0.52) during SCK. After antibiotic administration, all Salmonella-infected pigs stopped shedding. During POST, an interaction between diet and inoculation (P = 0.009) on ADG indicated that pigs infected with Salmonella grew better when eating XPC than the control diet. The addition of XPC to the diets of weanling pigs resulted in greater compensatory BW gains after infection with Salmonella than in pigs fed conventional nursery diets. This increase in BW gain is likely associated with an increase in beneficial bacteria within the gastrointestinal tract.

The evaluation of corpus luteum blood flow using color-flow Doppler ultrasound for early pregnancy diagnosis in bovine embryo recipients.

The objective of this experiment was to evaluate corpus luteum blood flow (CLBF) as an early indicator of pregnancy status in bovine embryo recipients. Fifty crossbred beef cows were submitted to embryo transfer on Day 7 after estrus. On Days 7, 11, 13, 15, 17, 19, 21, 26, 33, and 40, a blood sample was taken, the CL examined using a color-flow Doppler ultrasound scanner, and video was recorded of each scanning session. Ultrasound data were grouped by the first day progesterone concentrations were <1 ng/mL (indicating early embryo loss, EEL) through Day 21 (EEL-17, n=3; EEL-19, n=9; EEL-21, n=3), absence of an embryo on Days 26, 33, or 40 (late embryo loss; LEL; n=12), or remained pregnant (P; n=23). The first decrease in CLBF of EEL-17, EEL-19, and EEL-21 cows compared to P cows occurred on Days 17, 19, and 21, respectively (P<0.05). There was no difference in CLBF between LEL and P cows on Days 17, 19, and 21. Six evaluators diagnosed pregnancy from randomized video clips on Days 17, 19, and 21. Evaluators made more (P<0.004) correct diagnoses on Day 19 than Day 17. Sensitivity (82.9+/-10.1%) was not affected by day. From Days 17 to 19, diagnostic specificity increased (P=0.046) from 43.2+/-3.0 to 54.3+/-3.0% but remained unchanged thereafter. Due to low specificity and sensitivity, evaluation of CLBF alone was insufficient for early pregnancy diagnosis.

Negative effect of estradiol on luteinizing hormone throughout the ovulatory luteinizing hormone surge in mares.

The negative effect of estradiol-17beta (E2) on LH, based on exogenous E2 treatments, and the reciprocal effect of LH on endogenous E2, based on hCG treatments, were studied throughout the ovulatory follicular wave during a total of 103 equine estrous cycles in seven experiments. An initial study developed E2 treatment protocols that approximated physiologic E2 concentrations during the estrous cycle. On Day 13 (ovulation = Day 0), when basal concentrations of E2 and LH precede the ovulatory surges, exogenous E2 significantly depressed LH concentrations to below basal levels. Ablation of all follicles > or = 10 mm when the largest was > or =20 mm resulted in an increase in percentage change in LH concentration within 8 h that was greater (P < 0.03) than for controls or E2-treated/follicle-ablated mares. Significant decreases in LH occurred when E2 was given when the largest follicle was either > or =25 mm, > or =28 mm, > or =35 mm, or near ovulation. Treatment with 200 or 2000 IU of hCG did not affect E2 concentrations during the initial portion of the LH surge (largest follicle, > or =25 mm), but 2000 IU significantly depressed E2 concentrations before ovulation (largest follicle, > or =35 mm). Results indicated a continuous negative effect of E2 on LH throughout the ovulatory follicular wave and may be related to the long LH surge and the long follicular phase in mares. Results also indicated that a reciprocal negative effect of LH on E2 does not develop until the E2 surge reaches a peak.

Acute effects of prostaglandin F(2alpha) on systemic oxytocin and progesterone concentrations during the mid- or late-luteal phase in mares.

The acute effects of prostaglandin F(2alpha) (PGF) on circulating oxytocin and progesterone concentrations were characterized in mares during the mid- or late-luteal phase. Pony mares were randomly assigned to the following experimental groups based on treatment with PGF (2.5mg) or saline on Day 8 or Day 13 (Day 0=ovulation): PGF-8, PGF-13, saline-8, or saline-13 (n=7/group). Mares were fitted with indwelling, jugular vein catheters and two blood samples (-5 and 0 min) were collected prior to treatment. Treatments were administered into the jugular vein (0 min) and blood collection continued thereafter at 1 min intervals until 5 min and then at 5 min intervals until 60 min. Based on the combined data of -5 and 0 min samples, mares on Day 8 had greater (P<0.05) oxytocin concentrations than mares on Day 13. On Day 8, PGF treatment resulted in a biphasic pattern of oxytocin release. Oxytocin concentrations increased (P<0.05) 1 min after PGF treatment, decreased (P<0.05) from 1 to 10 min, and increased (P<0.05) from 10 to 30 min. Oxytocin concentrations were greater (P<0.05) from 1 to 3 min in PGF-treated than saline-treated mares and at most sample times from 15 to 60 min. On Day 13, oxytocin concentrations were greater (P<0.05) in PGF-treated than in saline-treated mares for most sample times. Mares treated with PGF on Day 8 had greater (P<0.05) oxytocin concentrations at 25, 30, and 40 min than mares on Day 13. Progesterone concentrations on Day 8 also increased by 1 min after PGF, decreased toward basal concentrations by 2-3 min, and then increased to a maximum 10 min after treatment. Subsequently, circulating progesterone decreased (P<0.05) below pretreatment concentrations by 40-50 min after PGF. In conclusion, treatment with PGF resulted in an immediate and biphasic increase in progesterone concentrations prior to the expected decrease. Treatment of mares with PGF on Day 8 resulted in an overall greater increase in systemic oxytocin concentrations compared to treatment on Day 13, and the increase on Day 8 was biphasic.

Luteal blood flow and progesterone production in mares.

The temporal relationships between blood flow in the corpus luteum (CL) and circulating progesterone concentrations were studied in 20 mares. Retrospective inspection of plasma progesterone concentrations indicated that a precipitous decrease occurred during Days 15-17 (Day 0 = ovulation) and was defined as the luteolytic period. Mean percentage of CL with color-Doppler signals for blood flow was maximum on Day 10 (77.3%), and Days 10-14 (49.8%) were defined as the preluteolytic period. The cross-sectional area of the CL decreased progressively from Day 4 (9.0 cm2) to Day 19 (1.5 cm2). Progesterone reached maximum concentration on Day 8 (12.8 ng/ml) and thereafter CL area and plasma progesterone decreased in parallel until the onset of luteolysis. During the luteolytic period, the decrease in plasma progesterone was about sixfold greater than during the preluteolytic period, whereas the decrease in CL area and in percentage of CL with blood-flow area were about twofold greater. There was no indication that an acute increase or decrease in luteal blood flow occurred prior to the precipitous decrease in plasma progesterone.

Follicle deviation and diurnal variation in circulating hormone concentrations in mares.

The temporal relationships between follicle deviation and systemic hormone concentrations were studied in mares. Blood samples were obtained at 01:00, 07:00, 13:00, and 19:00 h from nine mares throughout an interovulatory interval. Diurnal variation in progesterone occurred on Days 4-12 and in LH on Days 4 and 5; the lowest concentration for both hormones was at 13:00 h. Ultrasonically observed deviation in the ovulatory follicular wave began on Day 15.7+/-0.5 (ovulation=Day 0). An increase (P<0.002) in LH began on Day 14 before the beginning of deviation, and an increase (P<0.05) in estradiol began at the beginning of deviation. Testosterone concentrations began to increase (P<0.05) 2 days after the beginning of deviation and reached maximum 1 day before the next ovulation. The beginning of deviation was encompassed by a decline (P<0.003) in cortisol concentrations, and the concentrations remained low during the preovulatory period.

Controlling interrelationships of progesterone/LH and estradiol/LH in mares.

The interrelationships of progesterone, estradiol, and LH were studied in mares (n=9), beginning at the first ovulation (Day 0) of an interovulatory interval. An increase in mean progesterone concentrations began on Day 0 and reached maximum on Day 6, with luteolysis beginning on Day 14. A common progesterone threshold concentration of about 2 ng/ml for a negative effect on LH occurred at the beginning and end of the luteal phase. Progesterone and LH concentrations decreased at a similar rate from Day 6 until the onset of luteolysis on Day 14, consistent with a decreasing positive effect of LH on progesterone. Concentrations of LH during the increase in the ovulatory surge consisted of two linear regression segments involving a rate of 0.4 ng/ml/day for Days 14-22 and 1.8 ng/ml/day for Day 22 to 1 day after the second ovulation. The end of the first segment and beginning of the second segment was 2 days before ovulation and was the day the ovulatory estradiol surge was at a peak.

Effects of varying the holding temperature and interval from collection to freezing on post-thaw development of bovine embryos in vitro.

The objective of this study was to evaluate the in vitro development of frozen-thawed bovine embryos held at room temperature or refrigerated for 2, 6 or 12 h prior to freezing. After recovery, embryos were randomly assigned to be placed in holding media for 2 h (n=131), 6 h (n=136) or 12h (n=133) prior to freezing. Approximately one-half of the embryos were refrigerated (5 degrees C; n=203) while the remaining half were held at room temperature (22 degrees C; n = 197) until freezing. Embryos were frozen in 10% ethylene glycol and stored in liquid nitrogen. After thawing, embryos were cultured for 72 h in Ham's F-10 media supplemented with 4% fetal bovine serum. Embryos were evaluated for quality and stage of development prior to freezing and after culture. At the end of culture, it was determined if each embryo had developed beyond the stage recorded prior to freezing and if the embryo had hatched from the zona pellucida. The percentage of embryos that developed during culture was greater (P < 0.001) for Grade 1 (81%) than for either Grade 2 (65%) or Grade 3 (48%) embryos. Likewise, a greater proportion (P < 0.001) of Grade 1 embryos developed to hatched blastocysts (60%) than either Grade 2 (40%) or Grade 3 (24%) embryos. The holding temperature from collection to freezing did not influence embryo development, regardless of the interval from embryo collection to freezing. The proportion of embryos that developed to expanded blastocysts and hatched was greater (P < 0.005) for embryos held 2 h prior to freezing (64%) than for embryos held for 12 h (33%). Hatching rate of embryos held 6 h prior to freezing (54%) tended (P < 0.08) to be lower than the hatching percentage for embryos held for 2 h. Thus, post-thaw embryonic development was impaired the longer embryos were held prior to freezing and temperature during the interval from collection to freezing did not affect post-thaw development.

The effects of varying the interval from follicular wave emergence to progestin withdrawal on follicular dynamics and the synchrony of estrus in beef cattle.

The objective of this experiment was to examine the effects of varying the interval from follicular wave emergence to progestin (controlled internal drug-releasing insert, CIDR) withdrawal on follicular dynamics and the synchrony of estrus. A secondary objective was to assess the effects of causing the dominant follicle (DF) to develop in the presence or absence of a corpus luteum (CL) on follicular dynamics and the synchrony of estrus and ovulation. The experiment was designed as a 2 x 2 x 2 factorial arrangement of treatments with injection of GnRH or estradiol-17 beta and progesterone (E2 + P4) at treatment initiation, duration of CIDR treatment, and injection of PG (prostaglandin F2 alpha) or saline at the time of CIDR insertion as main effects. Estrous cycles (n = 49) in Angus cows were synchronized, and treatments commenced on d 6 to 8 of the estrous cycle. Cows were randomly assigned to receive a CIDR containing 1.9 g of P4 for 7 or 9 d. Approximately half the cows from each CIDR group received either GnRH (100 micrograms) or E2 + P4 (1 mg of E2 + 100 mg of P4) at CIDR insertion. Cows in GnRH or E2 + P4 groups were divided into those that received PG (37.5 mg) or saline at CIDR insertion. All cows received PG (25 mg) 1 d before CIDR removal. Daily ovarian events were monitored via ultrasound. The intervals from GnRH or E2 + P4 treatment to follicular wave emergence were 1.4 and 3.3 d, respectively (P < 0.05). The interval from follicular wave emergence to CIDR removal was longer (P < 0.05) for cows treated with GnRH (6.6 d) than those treated with E2 + P4 (4.7 d) and longer (P < 0.05) for those fitted with a CIDR for 9 d (6.5 d) than those with a CIDR in place for 7 d (4.8 d). Cows treated with PG or GnRH at CIDR insertion had a larger (P < 0.05) DF at CIDR removal than those treated with saline or E2 + P4. Treatment with a CIDR for 9 d also resulted in a larger (P < 0.07) DF at CIDR removal compared with cows fitted with a CIDR for 7 d. The interval from CIDR removal to estrus was shorter (P < 0.05) in cows treated with PG than those treated with saline. The synchrony of estrus and ovulation was not affected by any of the treatments (P > 0.05). Altering the interval from follicular wave emergence to progestin removal or creating different luteal environments in which the DF developed caused differences in the size of the DF at CIDR removal and the timing of the onset of estrus, but it did not affect the synchrony of estrus or ovulation.

Separation and surveys of proteins of Helicobacter pylori.

The analysis of Helicobacter pylori proteins is a demanding task for the elucidation of virulence factors, antigens and vaccines, all important for diagnosis, therapy and protection. In the "pre-genomic era" the purification of proteins was mostly performed by using various techniques such as detergent treatment of the bacterial cells, ultra-centrifugation, various chromatographic methods, antibody detection, N-terminal sequence determination and finally cloning and identification of the corresponding gene. In this review, the most representative methods used for purification, separation and identification of H. pylori proteins will be presented as well as some important developments in the "post-genomic era" that have improved the performance of these characterisation techniques.

Identification of novel immunogenic proteins of Helicobacter pylori by proteome technology.

Cell surface proteins of the human gastric pathogen Helicobacter pylori, reference strain CCUG 17874, were extracted with acid glycine and fractionated by heparin affinity chromatography. The extracts were subsequently analysed using high-resolution two-dimensional gel electrophoresis (2-DE) and immunoblotting. Four proteins of low molecular masses (25-30 kDa) stained by Coomassie R-350, were identified by peptide ESI-MS/MS sequencing after in-gel tryptic digestion. The identified proteins were recognised by sera from H. pylori-infected patients. Two of them are now described for the first time as immunogenic proteins of which one protein was determined to be distinct from all H. pylori proteins previously described. In addition, the specificity of the identified peptides was evaluated using both 1-D and 2-D immunoblotting against a panel of sera from patients with various bacterial infections. The present identification of highly specific antigens of H. pylori will encourage the improvement of serological diagnostic tests to diagnose and monitor H. pylori infection.

Helicobacter pylori vacuolating cytotoxin binding to a putative cell surface receptor, heparan sulfate, studied by surface plasmon resonance.

The Helicobacter pylori vacuolating cytotoxin or VacA toxin is a major virulence factor in H. pylori infection and type B gastritis. We predicted heparin/heparan sulfate (H/HS) binding properties of the 58-kDa subunit of VacA cytotoxin using bioinformatics tools and showed this by surface plasmon resonance (SPR)-based biosensor studies. Putative H/HS binding peptides were synthesized and binding to HS was shown by SPR in the absence or presence of trifluoroethanol. We found that a recombinant cytotoxin VacA polypeptide binds to surface-immobilized HS and propose that HS might be a receptor/co-receptor for H. pylori VacA cytotoxin.

Seropositivity to Helicobacter pylori heat shock protein 60 is strongly associated with intensity of chronic inflammation, particularly in antrum mucosa: an extension of an 18-year follow-up study of chronic gastritis in Saaremaa, Estonia.

Helicobacter pylori is a cause of chronic gastritis and leads to development of atrophy in some cases. There is evidence that the heat shock protein 60 (HSP60) of H. pylori is involved in induction of chronic inflammation. Seroprevalence of IgG antibodies to H. pylori HSP60 in an adult cohort from Saaremaa, Estonia (68 persons, median age 57 years), with a high prevalence of antibodies to cell surface proteins of H. pylori (92%) and a well characterized dynamics of chronic gastritis in an 18-year follow-up study, was tested using purified H. pylori HSP60 at a concentration of 1 microg ml(-1) with ELISA. The state of the gastric mucosa and the presence of H. pylori in histological sections in the samples of 1979 and 1997 were assessed in accordance with the Sydney system. Seropositivity for H. pylori HSP60 was 65%. Immunological response to H. pylori HSP60 is associated with the morphological presence of H. pylori in the antrum and corpus (P=0.01) and is strongly correlated with the grade of chronic inflammation, particularly in the antrum mucosa (r=0.34; P=0.003; OR=5.97 (95% CI 1.21-29.3)), but is not associated with development of atrophy during 18 years of follow-up, or with the activity of gastritis. This finding supports the evidence that immunological response to H. pylori HSP60 may play a role in triggering of the inflammatory process in the gastric mucosa.