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Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen using three commercial semen extenders. Sperm motility, membrane integrity, acrosome integrity and apoptosis were evaluated before and after freezing for each aliquot. In addition, the percentage of ASA-bound sperm was evaluated post-thaw. In Experiment 2, semen from 22 stallions was frozen immediately after collection a single formulation of semen extender. Sperm motility and ASA binding were evaluated post-thaw. The results of both experiments showed similar findings. The frequency of ASA-positive samples was higher among stallions with poor sperm cryosurvival (Exp. 1 and 2 = 6/11, 54.5%) than for good sperm cryosurvival (Exp. 1 = 0/16, 0%; Exp. 2 = 1/11, 9.1%). The percentage of IgG- and IgA-bound sperm was also higher in stallions with poor sperm cryosurvival in both experiments (P

History A 2-year-old sexually intact male Vietnamese potbellied pig that weighed 113 kg (249 lb) was admitted and examined to determine the cause of inappetance, irritability, and an enlargement in the inguinal region. The clinical signs had become more evident and the pig’s condition had gradually worsened during the preceding week. No treatment had been initiated prior to our examination. The owners reported that the boar would attempt to mate with a piece of polyvinyl chloride pipe. Physical examination revealed massive distention (40 X 25 cm [15.7 X 9.8 inches]) in the inguinal region and caudoventral portion of the abdomen. Palpation of the enlargement revealed it was firm and excessively warm but could be compressed, which suggested it was filled with fluid. The prepuce was edematous. There was evi dence of irritation of the skin over the right inguinal region. Question

The use of 4‐day CoSynch + Controlled internal drug release (CIDR) + timed artificial insemination (TAI) in dairy heifers has resulted in adequate pregnancy rates compared with the 5‐day CoSynch + CIDR + TAI protocol. The objective of this study was to compare follicular growth, timing of ovulation and serum progesterone (P4), estradiol (E2) and luteinizing hormone (LH) concentrations in dairy heifers treated with modified 4‐ or 5‐day CoSynch + CIDR protocols (CIDR for 4 or 5 days, PGF 2 α at CIDR removal and GnRH + TAI 72 h later). Twelve cycling Holstein heifers were randomly assigned to either the 4‐ or 5‐day Co‐Synch+CIDR (n = 6/treatment) to receive an intravaginal insert CIDR ® containing 1.38 g of P4 for 4 or 5 days, respectively. At CIDR removal, 25 mg of PGF 2 α was injected IM; 72 h after CIDR removal, heifers received 100 μg of GnRH IM and timed artificial insemination (TAI). Follicular growth and timing of ovulation were assessed using transrectal ultrasonography. Blood samples were collected at the time of CIDR insertion and at frequent time points after CIDR removal for determination of P4 (at TAI), E2 (every 12 h) and LH (every 6 h during the first and second day and every 2 h on the third day). Heifers in the 4‐day group had smaller follicles from CIDR insert removal to ovulation compared with heifers in the 5‐day treatment. Five of six heifers (83.3%) in the 4‐day treatment ovulated at 90–96 h post CIDR insert removal, whereas most heifers in the 5‐day treatment (4/6; 66.6%) ovulated at 84–90 h post CIDR insert withdrawal. Heifers in the 5‐day treatment reached greater peak LH concentration between 48 and 72 h after CIDR insert removal and lesser E2 concentration at TAI than heifers in the 4‐day treatment. In conclusion, heifers in the 4‐day treatment had smaller follicular diameter at 0, 30, 36, 42 and 48 h after CIDR insert removal, longer interval from CIDR insert removal to ovulation, greater E2 concentrations at TAI, and lesser peak LH concentration than heifers in the 5‐day treatment. These results represent a baseline for further studies to determine if prolonging the interval to TAI by 6 h in the 4‐day CoSynch+CIDR would improve pregnancy risk.

The objective was to compare pregnancy per AI and follicular dynamic in suckled Bos taurus beef cows treated with either a 7-day progesterone + estradiol-based protocol or a 5-day progesterone CoSynch protocol for timed artificial insemination (TAI) during four breeding seasons. We hypothesized that estrous cycle status, days postpartum (DPP), fat depth and plasma progesterone concentration differentially modify the effect of treatments. Every year, 9 days before initiation of each breeding season, cows were randomly assigned to one of two groups. Cows in the 7-d P + E group (n = 428) received a progesterone intravaginal device (DIB) and estradiol benzoate on Day −9. On Day −2 the device was removed, and cows received cloprostenol and estradiol cypionate. Forty-eight hours later (Day 0) cows received TAI. Cows in the 5-d P + CoS group (n = 428) received a DIB, and GnRH on Day −8. On Day −3, the device was removed, and cows received cloprostenol. A second dose of cloprostenol was given on Day −2. Cows received GnRH and TAI 72 h after device removal (Day 0). On Day −9, estrous cycle status was determined. In a subset of cows (n = 79) the size of the dominant follicle was determined between Days −2 and 0. In another subset of cows (n = 340), DPP, fat depth (mm) and plasma progesterone concentration (ng/mL) were evaluated on Day −9. Pregnancy per AI was determined 30 d after TAI. Pregnancy per AI was greater for cows in the 5-d P + CoS group than for cows in the 7-d P + E group (50.9% vs. 41.3%, P = 0.01) and was also greater in cyclic than in anestrus cows (54.3% vs. 33.2%, P < 0.0001). There was also a significant effect of breeding season (P = 0.0002) and sire (P = 0.03), and an interaction between treatment group and breeding season (P = 0.03). The dominant follicle was larger (P < 0.0001) in cows in the 5-d P + CoS group than the 7-d P + E group (10.7 ± 0.29 mm vs. 9.0 ± 0.28 mm). Pregnancy per AI was greater in cows with ≥55 DPP (47.0% vs. 29.6%, P = 0.001), fat depth ≥0.50 mm (44.7% vs. 29.7%), and with plasma progesterone concentration ≥1 ng/mL (47.2% vs. 28.7%, P = 0.01). In cows with plasma progesterone ≥1 ng/mL on Day −9, pregnancy per AI was greater in the 5-d P + CoS group (60.5%) than in the 7-d P + E group (34.9%), but there was no difference between treatment groups in cows with plasma progesterone < 1 ng/mL (P = 0.07). In conclusion, the 5-d P + CoS protocol resulted in greater size of the dominant follicle and pregnancy per AI in suckled Bos taurus beef cows subjected to TAI. Fil: Bilbao, María Guillermina. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Confluencia; Argentina Fil: Zapata, Luis Oscar. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; Argentina Fil: Romero Harry, H.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional La Pampa-San Luis. Estación Experimental Agropecuaria Anguil; Argentina Fil: Perez Wallace, S.. Zoetis SRL; Argentina Fil: Farcey, Maria Florencia. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; Argentina Fil: Gelid, Lucas Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro Regional La Pampa-San Luis. Estación Experimental Agropecuaria Anguil; Argentina Fil: Palomares, R. A.. University of Georgia. College of Veterinary Medicine; Estados Unidos Fil: Ferrer, M. S.. University of Georgia. College of Veterinary Medicine; Estados Unidos Fil: Bartolome, Julian. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; Argentina

This study was performed to elucidate whether the route of booster vaccination affects the immune response against respiratory vaccine viruses in pre-weaning beef calves that receive primary intranasal (IN) vaccination during the first month of life. The objective was to compare the serum neutralizing antibody (SNA) titers to BHV1, BRSV, and BPI3V, cytokine mRNA expression and mucosal BHV1- and BRSV-specific IgA in nasal secretions following administration of IN or subcutaneous (SC) modified-live virus (MLV) booster vaccines 60 days after primary IN vaccination in young beef calves. Twenty-one beef calves were administered 2 mL of an IN MLV vaccine containing BHV1, BRSV, and BPI3V (Inforce3®) between one and five weeks of age. Sixty days after primary vaccination, calves were randomly assigned to one of two groups: IN-MLV (n = 11): Calves received 2 mL of the same IN MLV vaccine used for primary vaccination and 2 mL of a SC MLV vaccine containing BVDV1 & 2 (Bovi- Shield GOLD® BVD). SC-MLV (n = 10): Calves were administered 2 mL of a MLV vaccine containing, BHV1, BRSV, BPI3V, and BVDV1 & 2 (Bovi-Shield GOLD® 5). Blood and nasal secretion samples were collected on days -61 (primary vaccination), -28, -14, 0 (booster vaccination), 14, 21, 28, 42 and 60 for determination of SNA titers, cytokine gene expression analysis and nasal virus-specific IgA concentrations. Statistical analysis was performed using a repeated measures analysis through PROC GLIMMIX of SAS®. Booster vaccination by neither IN nor SC routes induced a significant increase in SNA titers against BHV1, BRSV, and BPI3V. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA titers (on day 42) and IgA concentration in nasal secretions (on days 21 and 42) compared to calves receiving IN booster vaccination. Both IN and SC booster vaccination were able to stimulate the production of BHV1-specific IgA in nasal secretions. In summary, booster vaccination of young beef calves using either SC or IN route two months after IN MLV primary vaccination resulted in comparable SNA titers, cytokine gene expression profile and virus-specific IgA concentration in nasal secretions. Only a few differences in the systemic and mucosal immune response against BHV1 and BRSV were observed. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA and secretory IgA titers compared to IN booster vaccination.

Bovine antisperm antibodies (ASAs) have been associated with teratospermia and asthenospermia. It was hypothesized here that scrotal insulation induces the formation of ASAs and deterioration of sperm function. Scrotal insulation bags were placed in 10 bulls for 8 days. Semen was collected on days −29, −22 and −2, twice weekly from days 5 to 54, and thereafter weekly until day 96 (day 0 = first day of scrotal insulation). On each collection day, scrotal circumference, sperm motility, morphology, membrane integrity, acrosome integrity, apoptosis, lipid peroxidation, mitochondrial membrane potential, ASA binding and DNA integrity were evaluated. The percentage of IgG- and IgA-bound sperm increased between days 12 and 96 (P < 0.0001), in association with poor motility (days 19–30, P < 0.005) and morphology (days 8–40, P < 0.0001). Mean scrotal circumference decreased between days 15 and 75 (P < 0.0001). There was also a deterioration in sperm membrane integrity (days 19–40, P < 0.0001), acrosome integrity (days 26–89, P < 0.0001), lipid peroxidation (days 5–12, P < 0.0001), and mitochondrial membrane potential (days 12–96, P = 0.001). In contrast, a decrease in apoptotic cells (days 37–83, P = 0.0002) and lipid peroxidation (days 19–96, P < 0.0001) was noticed. Most bulls recovered normospermia by day 96. However, the persistence of ASAs, acrosomal damage and dysfunctional mitochondria suggest a long term effect of scrotal insulation on sperm function and the homeostasis of the reproductive immune system.

Freezing cooled-transported semen allows veterinarians and breeders to collect and process the semen of stallions on farm, and then ship the semen to a semen freezing center. There, however, is a lack of standardization of shipping and freezing protocols. The objectives were to optimize and simplify protocols to freeze cooled-shipped semen. In Experiment 1, cooled-transported semen was centrifuged at room temperature or 5 °C before freezing. Sperm variables (motility, membrane integrity, acrosome integrity, membrane fluidity) were evaluated before and after freezing. Centrifugation temperature had no effect on post-thaw semen quality. In Experiment 2, cooled-transported semen was centrifuged at room temperature and cryopreserved in three semen freezing extenders. With use of the improved modified French formula, there was less post-thaw total and progressive motility compared with use of Botucrio or the improved lactose-EDTA formula (P0.0001). Semen cryopreserved in the improved modified French formula also had a lesser percentage of sperm with intact membranes compared with lactose-EDTA, and a greater percentage of sperm with capacitation-like changes compared with Botucrio (P0.0001). In Experiment 3, semen diluted in each extender was frozen conventionally or placed directly in a -80 °C ultra-freezer. Freezing in the ultra-freezer resulted in a lesser post-thaw sperm motility, but not membrane and acrosome integrity and capacitation-like changes. In conclusion, centrifugation and addition of freezing extender to cooled transported semen can be performed at room temperature or 5 °C. The Botucrio and lactose-EDTA formula are recommended for conventional cryopreservation of cooled-transported stallion semen as compared with the modified French formula.

Antisperm antibodies (ASAs) have been associated with infertility in stallions. The objectives of this study were to investigate the frequency of ASA-positive semen samples in satisfactory and non-satisfactory breeder stallions, the association between ASA binding and semen quality, and factors that may affect the diagnosis. Breeding soundness examinations were performed in 21 stallions and the percentage of IgG- and IgA-bound spermatozoa was evaluated using flow cytometry. Median IgG and IgA binding did not differ between the first and second ejaculates. The percentage of IgA-bound spermatozoa was higher in non-satisfactory (n = 10) than satisfactory breeder stallions (n = 11). However, IgG binding or frequency of IgG-positive ejaculates did not differ with stallion classification. The IgG-positive stallions had significantly lower total sperm motility, concentration and total numbers than IgG-negative stallions in the first ejaculate, and lower sperm concentration in the second ejaculate. The IgA-positive stallions had lower total sperm motility, normal spermatozoa and total numbers than IgA-negative stallions in the first ejaculate, and lower total sperm motility, normal spermatozoa and total numbers in the second ejaculate. While IgG binding did not differ with season, IgA binding was higher in the non-breeding season (n = 6 stallions) than the breeding season (n = 15 stallions) in the first ejaculate. Stallion age did not differ with ASA classification. In conclusion, IgG binding was highly prevalent in both groups of stallions, while IgA binding was higher and more prevalent in non-satisfactory breeders. Both isotypes were associated with poor semen quality. Season and sexual rest had an effect on IgA but not IgG binding.

While changes in semen quality after heat stress are well characterized in the bull, changes in endocrine function have not been critically evaluated. It was hypothesized here that scrotal insulation results in alterations in Sertoli cell and Leydig cell function, as measured by changes in serum testosterone and anti-Müllerian hormone (AMH) concentration. Scrotal insulation bags were placed in 10 bulls for 8 d. Blood was collected on days -22 and -2, and weekly from days 5 to 96 (day 0 = first day of scrotal insulation) for measurement of serum concentration of AMH and testosterone using ELISA. The concentration of AMH decreased on day 5, followed by an increase on day 54 (P = 0.014). When AMH concentration was normalized to pre-insulation values, the percent increase in serum concentration of AMH was significant between days 26 and 54, with another peak at 75 d (P = 0.031). The serum concentration of testosterone (P = 0.0001) and the percentage of change in testosterone concentration (P0.0001) increased on day 5, followed by a decrease from days 33 to 96. Scrotal insulation was associated with Sertoli and Leydig cell dysfunction, as measured by serum testosterone and AMH concentration. The persistently low concentration of testosterone at the end of the study suggests a long term effect of scrotal insulation on Leydig cell function.

The objective of this study was to evaluate the effect of paternal high energy diets on blastocyst development during in vitro embryo production (IVP). Eight sires were stratified by body weight (initial BW = 946 ± 85 kg) and randomly assigned to the same diet (NEm = 2.10, NEg = 1.44, CP = 14.1%, NDF = 16.6%, DM basis) fed at two different inclusion rates while having ad libitum access to bermudagrass hay (NEm = 1.02, NEg = 0.45, CP = 10.2%, NDF = 71.6). After a 10-d adaptation period, sires were individually fed to receive 0.5% (MAINT) or 1.25% [High gain (HG)] of their BW daily for 67 days. At the end of the feeding period, semen was collected through electroejaculation and frozen. Antral follicles were aspirated from ovaries obtained from a slaughterhouse and utilized for IVP in 4 independent replicates (n = 2,227 total oocytes). Cleavage rates were evaluated 48 h after fertilization and blastocyst development rates were evaluated after 7 days of embryo culture. The proposed treatments successfully induced differences in BW gain (P < 0.01; 2.28 vs -0.04 kg/d) and carcass composition (Rump fat: 1.63 vs. 0.41 cm, P = 0.08; Rib fat: 1.06 vs. 0.41 cm, P = 0.02; intramuscular fat: 3.5 vs. 3.0%, P = 0.36; for HG vs. MAINT sires, respectively). There was a significant decrease in cleavage rates (69.9 ± 2.5 vs. 65.0 ± 2.7; P < 0.04), blastocyst rate as a percentage of oocytes (16.7 ± 2.9 vs. 11.5 ± 2.1; P < 0.01), and blastocyst rates as a percentage of cleaved structures (24.1 ± 3.8 vs. 11.5 ± 2.1; P < 0.01) for HG compared with MAINT sires. In conclusion, sires fed diets that induce highly anabolic conditions had impaired blastocyst development compared to sires fed a maintenance diet.

SummaryBackground The potential for life-threatening complications of metritis requires prompt initiation of antimicrobial treatment, often before microbiological test results are reported. However, published studies to guide first-line antibiotic selection are scarce. Objectives To report the most frequent bacterial species, antimicrobial susceptibility and prevalence of multidrug resistance (MDR) in cases of equine metritis. Study design Retrospective analysis of uterine bacterial culture and antimicrobial susceptibility test (AST) results from mares with metritis. Methods Data from uterine culture and AST from 45 mares and 88 bacterial isolates were analysed. The frequency of single and mixed infections, bacterial species and susceptibility to antimicrobials was reported. Results Mixed growth (62.2% mares) was more frequent than pure growth. The most commonly isolated bacterial species was Escherichia coli (30.7%) and mares with mixed growth most commonly grew a combination of Gram negative and Gram positive bacteria (65.5%). Gentamicin with penicillin was an appropriate choice for 65.1% of the mares. Trimethoprim/sulfonamide was effective in only 48.8% of the mares. Effective antimicrobial therapy was provided by the combination of penicillin with amikacin (90.7%) or with enrofloxacin (81.4%). Some organisms were also sensitive to tetracyclines, cephalosporins and chloramphenicol. MDR was more frequent in Gram negative (85.4%) than Gram positive bacteria (23.5%). Main limitations Low number of bacteria, limited number of veterinary specific interpretive criteria for equine metritis. Conclusions Mixed infections were common in equine metritis. Gram negative bacteria and Enterococci were often associated with resistance to the most frequently used antimicrobials. Potentiated sulfonamides are not an appropriate first choice for mares with metritis. While the combination of penicillin and gentamicin may be an appropriate first-line treatment for some mares, use of amikacin or enrofloxacin instead of gentamicin may be preferential. This article is protected by copyright. All rights reserved.

The cytobrush is considered the method of choice to obtain endometrial samples. Rigid brush fibers, however, may induce endometrial irritation and bleeding, or cell fragmentation, decreasing quality and diagnostic value of the samples. It was hypothesized that samples collected using a novel cytotape would provide sample smears of greater quality and less blood contamination than the cytobrush. Endometrial samples were collected with a cytotape and a cytobrush from ten mares without endometritis. Endometritis was then induced with artificial insemination, and samples were again collected 6 h after insemination. A cytology smear and bacterial culture were prepared from each sample. The collection methods and times were compared in terms of number and integrity of endometrial cells; number, integrity, and percentage of neutrophils; number of red blood cells, and number of colony-forming units. Frequency of positive cytology and culture was compared when there was use of each technique. The sensitivity, specificity, positive predictive value, and negative predictive value of cytology and culture for each technique was calculated using endometrial biopsy as the gold standard. While all samples had adequate and comparable cellularity and cell integrity, cytotape samples had less red blood cell contamination compared to cytobrush samples (P < 0.05). The number and percentage of PMNs, frequency of positive cytology diagnosis, number of colony-forming units and frequency of positive cultures did not differ between collection methods. In conclusion, the cytotape is a rapid, easy, and practical technique that can provide endometrial samples with similar diagnostic value to the cytobrush, but with less blood contamination.

This study aimed to describe and compare semen parameters (pre-freeze and post-freezing) and antisperm antibodies (ASA) of donkeys with epididymal sperm granuloma and healthy controls. Feral donkeys (n = 10) castrated in a concurrent study were enrolled in the present experiment. Three donkeys had unilateral granulomas, two donkeys had bilateral granulomas, whereas the remaining five had grossly normal epididymides. The granulomas were either single or multiple, firm, well-circumscribed, tan to red, and 1–5 mm in size. Upon incision, abundant, thick, tan to white-yellow fluid was recovered. Histopathology revealed epithelioid macrophages, multinucleated giant cells, and abundant sperm cell fragments with mineralized cellular debris. Each epididymis was dissected, and semen harvested for cryopreservation. Semen was assessed for sperm motility parameters, plasma membrane integrity, and mitochondrial membrane potential. All donkeys had semen cryopreserved in a standard manner. In addition, post-thaw semen from all donkeys was assessed for ASA (IgG and IgA), acrosome integrity and morphology. Post-thaw, the progressive sperm motility and the percentage of sperm with an intact plasma membrane were reduced in donkeys with sperm granuloma (P = 0.04). There was no difference in total sperm motility, morphology, or damaged acrosome across groups (P > 0.05). Three donkeys with sperm granuloma (60%) displayed increased IgG and IgA ASA. In conclusion, sperm granulomas only marginally affected sperm quality and resulted in IgG ASA binding to sperm with damaged plasma membrane. It remains to be determined if sperm granuloma and ASA affect fertility in donkeys.

Criollo Limonero is a tropical Bos taurus breed for sustainable dual purpose (milk and beef) production in the South-American tropics, which is currently threatened with extinction. The objective was to perform a clinical evaluation and histopathological assessment of uterine biopsy samples of repeat breeder (RB) Criollo Limonero cattle to determine the occurrence of pathological conditions as potential causes of subfertility. Twenty-four Criollo Limonero cattle [18 cows (5-13 years old) and 6 heifers (6-7.5 years old)] that had failed to conceive after four or more services were considered for this study. Additionally, five cows with history of adequate reproductive performance were used as a control group. Animals were submitted to physical exam, vaginoscopy, and ultrasonographical evaluation of the reproductive tract. Uterine biopsy samples were collected for histopathological evaluation. Vaginoscopy revealed that 41.7% of the RB cattle had abnormal vaginal secretions, while abnormal secretions were not observed in any control cow. Ultrasonographical examination of the uterus revealed the presence of free uterine fluid in 20.8% of the RB animals, while none of the control cows had fluid in the uterine lumen. In addition, ovarian cysts were observed in 25.0% of the RB animals. Histopathological evaluation of the endometrial biopsies revealed that mononuclear leukocyte infiltration, dilated uterine glands, and periglandular fibrosis were the most prevalent lesions in the sub-fertile animals. Chronic endometritis characterized by inflammatory (mononuclear leukocyte infiltration) and degenerative (dilated glands and periglandular fibrosis) endometrial lesions, and ovarian cysts were the most frequent reproductive pathologies observed in the studied subfertile Criollo Limonero cattle, suggesting a strong association with their reduced fertility.

The objective of this study was to compare the pregnancy rate after timed artificial insemination (P/TAI) in dairy heifers treated with 4- versus 5-day Co-Synch + controlled internal drug release (CIDR) protocols. A total of 120 Holstein heifers were randomly assigned to one of two groups. The heifers received an intravaginal CIDR insert containing 1.38 g of progesterone for 4 days (Monday-Friday 4-day Co-Synch + CIDR; n = 60) or 5 days (5-day Co-Synch + CIDR; n = 60). At the time of CIDR removal, 25 mg of PGF2α was injected intramuscularly, and 72 hours after CIDR removal, the heifers received 100 μg of GnRH intramuscularly and were artificially inseminated. Artificial insemination was performed by an experienced technician, using commercial frozen-thawed semen from a single sire. Pregnancy diagnosis was performed by ultrasonography per rectum 32 days after TAI. Categorical data were analyzed using proc logistic and the chi-square test, whereas continuous variables were analyzed using the t-test of Statistical Analysis Systems. Heifers in the 4-day Co-Synch + CIDR group had an acceptable P/TAI32 (55.0%, 33 of 60), which was not different (P = 0.35) from that observed in the 5-day Co-Synch + CIDR group (63.3%, 38 of 60). Progesterone concentration at CIDR insertion or estradiol concentration at TAI did not influence the pregnancy outcomes. Interestingly, estradiol concentration at TAI was greater in the 4-day Co-Synch + CIDR group compared to the 5-day Co-Synch + CIDR group (P < 0.01). In conclusion, the Monday to Friday 4-day Co-Synch + CIDR protocol resulted in adequate P/TAI in dairy heifers, which was similar to that of the 5-day Co-Synch + CIDR protocol. This novel protocol might represent a promising hormonal treatment for TAI in dairy heifers, facilitating their reproductive management routine, while maintaining an adequate fertility.

The objectives of this study were to determine reference intervals (RIs) for sperm-bound immunoglobulins G and A (IgG and IgA), prevalence of antisperm antibodies (ASAs) in satisfactory and nonsatisfactory breeders, and association between ASAs and semen quality in beef bulls. It was hypothesized that ASA binding differed with breeding soundness classification and semen quality. The percentage of IgG- (IgGperc) and IgA-bound (IgAperc) spermatozoa was evaluated in satisfactory (n = 134) and nonsatisfactory (n = 71) breeder beef bulls using flow cytometry. The RI for IgGperc was 0% to 13.5%. The RIs for IgAperc were 0% to 25.8% in yearling Aberdeen Angus bulls and 0% to 12% in all other bulls. The prevalence of IgA-positive samples was higher in nonsatisfactory (14.1%) than that in satisfactory (1.5%) breeders (P = 0.0003). However, the prevalence of IgG-positive samples did not differ. Similarly, IgA binding was higher in nonsatisfactory (median; interquartile range; 2.18; 0.77%-8.57%) than that in satisfactory breeders (median; interquartile range; 1.11; 0.32%-3.16%; P = 0.0035), but IgG binding did not differ. Among ASA-positive bulls, median IgA and IgG binding was 39.7% (range, 18.8%-96.2%) and 24.8% (range, 14.2%-33.1%), respectively. Immunoglobulin A binding correlated with the percentage of total (P < 0.0001; r(2) = -0.345) and progressively motile spermatozoa (P < 0.0001; r(2) = -0.329), morphologically normal spermatozoa (P = 0.0004; r(2) = -0.256), sperm head abnormalities (P = 0.0416; r(2) = 0.149), proximal droplets (P = 0.0227; r(2) = 0.167), and coiled tails (P = 0.0338; r(2) = 0.156). Immunoglobulin G binding correlated with the percentage of total (P < 0.0001; r(2) = -0.373) and progressively motile spermatozoa (P < 0.0001; r(2) = -0.455) and sperm concentration (P = 0.0332; r(2) = -0.195). Reference intervals were established for determination of cutoffs for clinically significant sperm-bound IgA and IgG with flow cytometry. Immunoglobulin A binding was both higher and more prevalent in nonsatisfactory breeder bulls. Although IgG binding did not differ with breeding soundness classification, detection of surface-bound IgG and IgA was associated with changes in semen quality.

The use of sexed semen (SS) for AI of cattle has resulted in lower pregnancy per timed AI (P/TAI) compared with conventional semen. This has been attributed to lower sperm dose and cell damage during the cell sorting-freezing process. Horn AI is a common practice in equines and canines to improve pregnancy rate when using low sperm doses. The objective was to compare P/TAI and pregnancy loss (PL) in dairy heifers inseminated with SS deposited in the uterine horn (UH) ipsilateral to the ovary containing the preovulatory follicle (POF) versus TAI in the uterine body (UB). This study was performed on two dairy farms in Georgia (A and B). On farm A, 74 Holstein heifers (12 months old) received a 5-day Cosynch + controlled internal drug release (CIDR) protocol including an intravaginal insert (Eazi-Breed CIDR, Zoetis Animal Health) containing 1.38 g of progesterone for 5 days and a dose of gonadotrophin-releasing hormone (GnRH) intramuscularly (100 µg, 2 mL of Factrel, Zoetis Animal Health). At CIDR removal, heifers received a dose of prostaglandin F2α (PGF2α) intramuscularly (25 mg, 5 mL of Lutalyse, Zoetis Animal Health) and again 24 h later; 72 h after CIDR removal, heifers received 100 µg of GnRH intramuscularly and TAI with frozen-thawed SS. On farm B, 237 Holstein × Jersey heifers (12 months old) received a modified 5-day Cosynch + CIDR. This protocol was similar to that on farm A but did not include GnRH at the time of CIDR insertion or a second dose of PGF2α 24 h after CIDR removal. Immediately before TAI, heifers were examined using transrectal ultrasonography (TRUS) to determine on which ovary the POF was present. Each heifer was randomly assigned to TAI using SS in either the UH ipsilateral to the ovary containing the POF (n = 150; farm A, n = 32; farm B, n = 118) or the UB regardless of where the POF was identified (n = 161; farm A, n = 42; farm B, n = 119). Pregnancy was diagnosed 32 days after TAI using TRUS to determine P/TAI. Heifers diagnosed pregnant were re-examined using TRUS at 60 days of gestation to assess PL. Data were analysed using PROC LOGISTIC and the chi-square test of SAS (SAS Institute Inc.). Heifers that received SS in the UH had adequate P/TAI (50.6% (76/150); farm A: 50.0% (16/32); farm B: 50.8% (60/118)), which tended to be numerically greater (P = 0.12) than that observed in heifers receiving SS in the UB (43.4% (70/161); farm A: 40.5% (17/42); farm B: 44.5% (53/119)). In addition, PL was not different between groups and was within normal ranges for dairy heifers (10.0 and 9.4% for UH and UB, respectively). In conclusion, TAI of dairy heifers with SS deposited in the UH ipsilateral to the ovary containing the POF resulted in adequate P/TAI, which was 7.2% greater than TAI in the UB. Horn AI with SS might become a valuable tool for optimising reproductive efficiency and genetic selection of dairy herds.

The objectives of this study were to identify prognostic indicators of calf survival in SCNT-derived beef calves. Ultrasonographic parameters of fetal well-being and development, maternal clinical parameters, and neonatal parameters were evaluated as predictors of calf survival in cows carrying SCNT-derived beef fetuses (n = 38). Calf survival was 61.5% (88.2% female and 40.9% male calves; P = 0.0026). Cow respiratory rate and cow temperature were significantly greater in the nonsurviving (NS) group 1 week prepartum. In surviving (S) calves, fetal heart rate (FHR) decreased during the last 2 weeks of gestation (P < 0.01). However, this final deceleration was not observed in NS calves, resulting in higher FHRs in this group (P < 0.0001). Fetal movement and fluid scores did not differ with calf classification. Mean amniotic fluid depth was smaller in S (5.5 ± 0.7 cm) than NS (8.7 ± 1.4 cm) calves (P = 0.0398). However, mean allantoic fluid depth did not differ (P = 0.6120). There was a significant association between the body weight of calf and the diameter of the fetal aorta (P = 0.0115; r(2) = 0.3762). Surviving calves were lighter at birth (P = 0.0028) and were born later (P = 0.007) than NS calves. Calves born vaginally had a smaller fetal aorta (2.1 ± 0.1 cm vaginal and 2.4 ± 0.1 cm Cesarean) (P = 0.0487) and a lighter birth weight (41.4 ± 4.2 kg vaginal and 60.4 ± 2.1 kg Cesarean) (P = 0.0001) than calves born by Cesarean. Also, calves that underwent spontaneous labor (52.2% S and 0% NS; P = 0.0029) had a lighter birth weight (44.9 ± 3.8 kg) than calves that did not initiate labor (61.6 ± 2.2 kg) (P = 0.0004). Frequent ultrasonographic fetal monitoring allowed identification of differences between S and NS calves. Calves without a final decrease in FHR or with a large aortic diameter were more likely to require a Cesarean because of failure to initiate labor or fetomaternal disproportion. Parameters of fetal well-being and development during the last 3 weeks of gestation were first described in SCNT-derived beef calves.

Olives were introduced in Argentina from Europe over 400 years ago. Severalcultivars are grown in Mendoza province and problems exist on misclassification ofplants in orchards and nurseries. This work was aimed to develop a cost effectiveprotocol for fingerprinting olive germplasm using microsatellites (SSRs) andgenerate a database in order to help in the identification of olives trees in Mendoza.Four highly informative polymorphic SSRs (DCA3, DCA9, DCA11 and DCA16)were selected to fingerprint olive cultivars. A protocol using a single dye-labeledforward common primer with the M13 (-21) sequence was used in combination withthe reverse specific SSR primers. PCR amplifications were multiplexed and resolvedin automated capillary genetic analyzer equipment. Profiles of the cultivars and alist of alleles with the sizes obtained by capillary electrophoresis are provided.Several genetic parameters were calculated to characterize the microsatellite loci. Adatabase of SSR markers for eleven of the more distributed olive cultivars inMendoza was developed. Fil: Torres, M. R.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Cuyo Mendoza - San Juan; Argentina Fil: Cornejo, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina Fil: Bertoldi, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina Fil: Ferrer, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina Fil: Masuelli, Ricardo Williams. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Cuyo Mendoza - San Juan; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina

It was hypothesized here that sperm-bound antisperm antibodies (ASAs) impair the ability of bovine spermatozoa to undergo capacitation, bind to the zona pellucida, and complete the acrosome reaction. The effect of ASA binding on these functions was evaluated in frozen/thawed spermatozoa from four bulls before and after induction of ASAs. Ejaculates were divided into ASA negative ( R 2 = −0.48) but not IgG binding. Sperm binding to the zona pellucida was lower in IgA-positive (six spermatozoa/oocyte; 3–10 spermatozoa/oocyte) than IgA-negative ejaculates (seven spermatozoa/oocyte; 4–13 spermatozoa/oocyte) (P = 0.019). Zona binding was negatively correlated with the percentage of IgA-bound spermatozoa (P = 0.04; R 2 = −0.24) but not IgG-bound spermatozoa. The percentage of acrosome-reacted spermatozoa was higher in calcium ionophore A23187-treated than control aliquots in both ASA-negative and ASA-positive ejaculates (P

Conventional centrifugation protocols result in important sperm losses during removal of the supernatant. In this study, the effect of centrifugation force (400 or 900 × g), duration (5 or 10 min), and column height (20 or 40 mL; Experiment 1); sperm concentration (25, 50, and 100 × 10(6)/mL; Experiment 2), and centrifugation medium (EZ-Mixin CST [Animal Reproduction Systems, Chino, CA, USA], INRA96 [IMV Technologies, Maple Grove, MN, USA], or VMDZ [Partnar Animal Health, Port Huron, MI, USA]; Experiment 3) on sperm recovery and survival after centrifugation and cooling and storage were evaluated. Overall, sperm survival was not affected by the combination of centrifugation protocol and cooling. Total sperm yield was highest after centrifugation for 10 min at 400 × g in 20-mL columns (95.6 ± 5%, mean ± SD) or 900 × g in 20-mL (99.2 ± 0.8%) or 40-mL (91.4 ± 4.5%) columns, and at 900 × g for 5 min in 20-mL columns (93.8 ± 8.9%; P0.0001). Total (TMY) and progressively motile sperm yield followed a similar pattern (P0.0001). Sperm yields were not significantly different among samples centrifuged at various sperm concentrations. However, centrifugation at 100 × 10(6)/mL resulted in significantly lower total sperm yield (83.8 ± 10.7%) and TMY (81.7 ± 6.8%) compared with noncentrifuged semen. Centrifugation in VMDZ resulted in significantly lower TMY (69.3 ± 22.6%), progressively motile sperm yield (63.5 ± 18.2%), viable yield (60.9 ± 36.5%), and survival of progressively motile sperm after cooling (21 ± 10.8%) compared with noncentrifuged semen. In conclusion, centrifuging volumes of ≤ 20 mL minimized sperm losses with conventional protocols. With 40-mL columns, it may be recommended to increase the centrifugal force to 900 × g for 10 min and dilute the semen to a sperm concentration of 25 to 50 × 10(6)/mL in a milk- or fractionated milk-based medium. The semen extender VMDZ did not seem well suited for centrifugation of equine semen.

The objectives were to develop a transplantation assay for equine testicular cells using busulfan-treated prepubertal immunocompetent rats as recipients, and to determine if putative equine spermatogonial stem cells (SSCs) could be enriched by flow cytometric cell sorting (based on light scattering properties), thereby improving engraftment efficiency. Four weeks after transplantation of frozen/thawed PKH26-labeled equine testicular cells, 0.029 ± 0.045% (mean ± SD) of viable donor cells transplanted had engrafted. Donor cells were present in seminiferous tubules of all recipient rats forming chains, pairs, mesh structures, or clusters (with two to >30 cells/structure). Cells were localized to the basal compartment by the basement membrane. Although equine cells proliferated within rat seminiferous tubules, no donor-derived spermatogenesis was evident. Furthermore, there was no histologic evidence of acute cellular rejection. No fluorescent cells were present in control testes. When equine testicular cells were sorted based on light scattering properties, the percentage of transplanted donor cells that engrafted was higher after injection of cells from the small, low complexity fraction (II; 0.169 ± 0.099%) than from either the large, high complexity fraction (I; 0.046 ± 0.051%) or unsorted cells (0.009 ± 0.007%; P < 0.05). Seminiferous tubules of busulfan-treated prepubertal immunocompetent rats provided a suitable niche for engraftment and proliferation, but not differentiation, of equine testicular cells. Sorting equine testicular cells based on light scattering properties resulted in a 19-fold improvement in colonization efficiency by cells with high forward scatter and low side scatter, which may represent putative equine SSCs.

The typical dose for insemination into the uterine body of the mare is > 300 x 10(6) progressively motile spermatozoa (PMS) and an insemination dose of > 200 x 10(6) PMS is recommended for frozen-thawed semen. Low-dose insemination techniques allow for a drastic reduction in the numbers of spermatozoa required to achieve pregnancy. Acceptable pregnancy rates can be achieved with doses ranging from 1 to 25 x 10(6) PMS in volumes ranging from 20 to 1000 microL. Two techniques have been described: hysteroscopic insemination and transrectally guided deep horn insemination using a pipette. Similar pregnancy rates can be attained by either method when 5 x 10(6) PMS are used. Hysteroscopic insemination may provide an advantage when the dose is 1-3 x 10(6) PMS. These techniques have the potential to make more efficient use of frozen-thawed or sex-sorted semen from certain stallions. The use of low-dose insemination to improve fertility of infertile stallions warrants further investigation.

In this study, a database of fetal and maternal parameters was developed in pregnant alpacas from 120 days of gestation to term for evaluation of fetal well-being and development. Ten pregnancies were examined every 2 weeks from 120 to 300 days of gestation, then weekly until parturition. Five more pregnancies were evaluated weekly during the last 4 weeks prepartum. Maternal weight and heart rate were recorded. Indicators of fetal well-being (mean, minimum, and maximum fetal heart rate, fetal movement, and tone), development (size of the aorta, thorax, abdomen, stomach, kidney, head, eye orbit, radius, and umbilical vessels), and uterine environment (combined thickness of the uterus and placenta, depth and character of allantoic and amnionic fluids) were assessed using transabdominal ultrasound. Reference values and fetal growth curves were created. Weights of the cria, fetal membranes, and dam at parturition were associated with parameters of fetal growth. Mean (R(2) = 0.65), minimum (R(2) = 0.57), and maximum (R(2) = 0.64) fetal heart rate decreased from midgestation to term (P0.0001). Fetal activity also decreased as pregnancy advanced (P0.0001). Fetal presentation was anterior in all fetuses by 10 months. Increases in depth of allantoic (R(2) = 0.11) and amniotic (R(2) = 0.02) fluid, combined thickness of the uterus and placenta (R(2) = 0.31), and maternal weight (R(2) = 0.09) were significantly but poorly associated with gestation length. All parameters of fetal development were positively associated with gestational age (P0.0001). Size of the aorta (R(2) = 0.88), abdomen (R(2) = 0.87), kidney (R(2) = 0.83), and thorax (R(2) = 0.82) provided the best estimates of gestational age. Birth weight was associated with the size of the aorta (R(2) = 0.71), kidney (R(2) = 0.61), and radius (R(2) = 0.73) (P0.05). This information might be of value in the assessment of fetal well-being and development in alpacas.

The objective of the present study was to characterize ovarian activity in non-mated vicunas, relating ovarian structures (evaluated by transrectal ultrasonography, daily for 30 days) to changes in plasma concentrations of estradiol-17beta and progesterone. Ovarian follicular activity occurred in waves, characterized by the follicle emergence, growth and regression. The mean duration of follicular waves was 7.2+/-0.5 days (mean+/-S.E.M.), with a range of 4-11 days. The follicular growth phase averaged 3.0+/-0.2 days, the static phase 1.4+/-0.1, the regression phase 2.9+/-0.3 days, and the inter-wave interval was 4.2+/-0.3 days. The mean growth rate during the growing phase was 1.8+/-0.1mm/day, while the duration of the interval from 6mm to maximum diameter was 1.4+/-0.1 days. The mean maximum diameter of the dominant follicle was 8.4+/-0.3mm (range: 6.2-11.2) and mean diameter of the largest subordinate follicle was 5.4+/-0.1mm. There was an inverse relationship between the size of the largest follicle and the total number of follicles (r=-0.21, P=0.002). Follicle activity alternated between ovaries in 77% of the waves, with 40% of dominant follicles present in the left ovary and 60% in the right ovary. Plasma estradiol-17beta concentrations also had a wave-like pattern, varying between 12.0 and 62.8 pmol/l. Plasma progesterone concentrations remained below 5.0 nmol/l and there was no ultrasonographic evidence of ovulation during the study.

Table 1. ASA-bound spermatozoa and ASA-positive samples in twenty Information on anti-sperm antibodies (ASAs) in stallion populations is scarce. The objectives were to investigate presence of sperm-bound ASAs in a group of stallions, the association of ASA with semen quality and other factors that may affect the diagnosis. It was hypothesized that ASA binding differed with breeding soundness classification and semen quality. Stallion age, breed, sexual rest and season were proposed as determinants. After a breeding soundness examination, twenty stallions (2 to 26 years old) were classified as satisfactory or non-satisfactory breeders. One ejaculate per stallion was analyzed for ASA binding. Spermatozoa were labeled with fluorescein isothiocyanate (FITC)-labeled anti-equine IgG or IgA, and propidium iodide. Isotype controls were included. Percentages of IgGand IgA-bound live spermatozoa were analyzed with flow cytometry. Dead spermatozoa were excluded from the analysis since dead cells uptake labeling antibodies nonspecifically. Regional specificity of antibody binding had previously been confirmed with confocal laser microscopy but was not evaluated in this study. The percentage of ASAbound spermatozoa was compared between satisfactory (n1⁄411) and non-satisfactory breeders (n1⁄49), semen collected during the breeding (n1⁄414) or non-breeding (n1⁄46) season, sexually (n1⁄417) or non-sexually rested stallions (n1⁄43), or stallions of Quarterhorse (n1⁄414) and Warmblood (n1⁄46) breeds using a Wilcoxon rank test. ASA binding was compared among age groups with a Friedman test. Reference intervals (RIs) for ASA binding were established using 95% of the distributionwithin the 0.975 fractile of satisfactory breeders. Semen samples with a percentage of ASA-bound spermatozoa above the upper limit of the RIs