A. M. Donoghue

Viability assessment of honey bee, Apis mellifera, sperm using dual fluorescent staining

Since the development of instrumental insemination of honey bee (Apis mellifera) queens in the 1930s, there has been interest in the evaluation and in vitro storage of semen. Several fluorescent stains, when used in combination, have been effectively used to assess sperm viability in mammalian and avian species. Our objectives were to test two combinations of living:dead fluorescent stains, SYBR-14 with propidium iodide (PI), or Calcein-AM with PI, and validate the use of these probes with honey bee sperm. SYBR-14 is a nuclear stain producing green fluorescence of the DNA in living sperm, Calcein-AM is a membrane-permeant esterase substrate staining entire sperm green, and PI is a traditional dead cell stain giving a contrasting red color. Both living stains fluoresced bee sperm, but the SYBR-14:PI produced a clearer distinction between the living and dead sperm. A graduated series of known living:dead sperm proportions was used to validate the accuracy of the stains for determining sperm viability in honey bees.

Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species

Approximately 503 of the known species of birds are classified as endangered or critical. Captive propagation programs have proven useful in maintaining genetic diversity and restoring wild populations of certain species, including the Peregrine falcon, California condor and Whooping crane. Artificial insemination (AI) has the potential of solving problems inherent to reproductive management of small, closed populations of endangered birds, including dealing with demographic instability, physical and behavioral disabilities, sexual incompatibility, lack of synchrony, and need to maintain gene diversity. In this review, we address the necessary methods and factors that allow AI to be applied effectively to manage rare bird populations. It is clear that semen availability and quality are the greatest limiting factors to implementing consistently successful AI for birds. Behavioral sensitivity to animal handling and the ability to minimize stress in individual birds also are keys to success. Multiple, deep vaginal inseminations can improve fertility, particularly when semen quality is marginal. Laparoscopic methods of semen transfer also have produced fertile eggs. All of these practices leading to successful AI remain dependent on having adequate basic knowledge on female reproductive status, copulatory behavior, endocrine profiles and duration of fertility, especially as related to oviposition. The overall greatest challenge and highest priority is defining these normative traits, which are highly species-specific.

Prophylactic Supplementation of Caprylic Acid in Feed Reduces Salmonella Enteritidis Colonization in Commercial Broiler Chicks

Salmonella Enteritidis is a major foodborne pathogen for which chickens serve as reservoir hosts. Reducing Salmonella Enteritidis carriage in chickens would reduce contamination of poultry meat and eggs with this pathogen. We investigated the prophylactic efficacy of feed supplemented with caprylic acid (CA), a natural, generally recognized as safe eight-carbon fatty acid, for reducing Salmonella Enteritidis colonization in chicks. One hundred commercial day-old chicks were randomly divided into five groups of 20 birds each: CA control (no Salmonella Enteritidis, CA), positive control (Salmonella Enteritidis, no CA), negative control (no Salmonella Enteritidis, no CA), and 0.7 or 1% CA. Water and feed were provided ad libitum. On day 8, birds were inoculated with 5.0 log CFU of Salmonella Enteritidis by crop gavage. Six birds from each group were euthanized on days 1, 7, and 10 after challenge, and Salmonella Enteritidis populations in the cecum, small intestine, cloaca, crop, liver, and spleen were enumerated. The study was replicated three times. CA supplementation at 0.7 and 1% consistently decreased Salmonella Enteritidis populations recovered from the treated birds. Salmonella Enteritidis counts in the tissue samples of CA-treated chicks were significantly lower (P < 0.05) than those of control birds on days 7 and 10 after challenge. Feed intake and body weight did not differ between the groups. Histological examination revealed no pathological changes in the cecum and liver of CA-supplemented birds. The results suggest that prophylactic CA supplementation through feed can reduce Salmonella Enteritidis colonization in day-old chicks and may be a useful treatment for reducing Salmonella Enteritidis carriage in chickens.

Therapeutic Supplementation of Caprylic Acid in Feed Reduces Campylobacter jejuni Colonization in Broiler Chicks

ABSTRACT Poultry colonized with Campylobacter species are a significant source of human food-borne illness. The therapeutic use of the medium chain fatty acid caprylic acid consistently reduced enteric C. jejuni colonization in chicks by 3 to 4 logs in three separate trials. These results support caprylic acids potential to reduce Campylobacter carriage in poultry.

Assessment of the membrane integrity of fresh and stored turkey spermatozoa using a combination of hypo-osmotic stress fluorescent staining and flow cytometry

Abstract A study was conducted to evaluate the integrity of turkey sperm plasma membrane subjected to various hypo-osmotic conditions, and to develop a test to determine the percentage of viable spermatozoa capable of withstanding hypo-osmotic stress after in vitro storage. Semen from 10 toms was collected and pooled twice weekly for 6 wk, and each trial was repeated 6 times. For Trial I, spermatozoa were subjected to varying osmotic solutions by suspension in 100, 80, 60, 40, 20 or 0% PBS in distilled water (297 to 19 mosm/kg H 2 O) and stained to assess membrane integrity with Calcein-AM (CAL) and propidium iodide (PI). The CAL detected viable spermatozoa (green fluorescence) while the PI stained dead cells (red fluorescence). Spermatozoa were evaluated microscopically and by flow cytometry. The percentage of viable spermatozoa, as determined by flow cytometry, was not different from that in 100% PBS (76.4 ± 3.8) to 20% PBS (74.1 ± 3.5). Fewer viable spermatozoa, however, were detected in 0% PBS (61.1 ± 4.8, P 2 O (Trial II). The percentage of viable spermatozoa was not different for fresh or in vitro-stored spermatozoa in PBS. For spermatozoa stored 24 h in vitro, the percentage of viable cells was lower in H 2 O (48.0 ± 5.1) than in PBS (66.1 ± 5.6, P

Use of plant-derived antimicrobials for improving the safety of poultry products

Salmonella Enteritidis and Campylobacter jejuni are the 2 major foodborne pathogens transmitted through poultry products. Chickens are the reservoir hosts of these pathogens, with their intestinal colonization being the most significant factor causing contamination of meat and eggs. Effective preslaughter strategies for reducing the colonization of birds with these pathogens are critical to improve the microbiological safety of poultry products. An antimicrobial treatment that can be applied through feed represents the most practical and economically viable method for adoption on farms. Additionally, a natural and safe antimicrobial will be better accepted by producers without concerns for toxicity. This symposium talk discussed the potential use of plant-derived, GRAS (generally recognized as safe)-status molecules, caprylic acid, trans-cinnamaldehyde, eugenol, carvacrol, and thymol as feed supplements for reducing cecal populations of Salmonella Enteritidis and C. jejuni in chickens. Additionally, the effect of plant molecules on Salmonella virulence genes critical for cecal colonization in chickens was also discussed.

Caprylic acid reduces Salmonella Enteritidis populations in various segments of digestive tract and internal organs of 3- and 6-week-old broiler chickens, therapeutically

We investigated the efficacy of feed supplemented with caprylic acid (CA), a natural, 8-carbon fatty acid for reducing Salmonella enterica serovar Enteritidis colonization in commercial broiler chickens. In separate 3- and 6-wk trials, 1-d-old straight-run broiler chicks (n = 70 birds/trial) were assigned to a control group (challenged with Salmonella Enteritidis, no CA) and 2 replicates of 0.7 and 1% CA (n = 14 birds/group). Water and feed were provided ad libitum. On d 1, birds were tested for any inherent Salmonella (n = 2 birds/group). For the 3-wk trial, on d 5, birds were challenged with 8 log(10) cfu of Salmonella Enteritidis of a 4-strain mixture by crop gavage, and after 5 d postchallenge, birds (n = 2 birds/group) were euthanized to ensure Salmonella Enteritidis colonization. Caprylic acid was supplemented the last 5 d before tissue collection (n = 10 birds/group). For the 6-wk trial, on d 25, birds were challenged and confirmed for Salmonella Enteritidis colonization. The birds (n = 10 birds/group) were euthanized for tissue samples after CA supplementation for the last 5 d. Caprylic acid at 0.7 or 1% decreased Salmonella Enteritidis populations in cecum, small intestine, cloaca, liver, and spleen in both 3- and 6-wk trials. Body weight of birds did not differ between the groups (P ≥ 0.05). Further, to elucidate a potential antibacterial mechanism of action of CA, we investigated if CA could reduce Salmonella Enteritidis invasion of an avian epithelial cell line and expression of invasion genes hilA and hilD. The cell invasion study revealed that CA reduced invasive abilities of all Salmonella Enteritidis strains by ~80% (P < 0.05). Gene expression studies indicated that CA downregulated (P < 0.001) Salmonella invasion genes hilA and hilD. These results suggest that supplementation of CA through feed could reduce Salmonella Enteritidis colonization in broiler chicken and potentially reduces the pathogens ability to invade intestinal epithelial cells by downregulating key invasion genes, hilA and hilD.

Isolation and Prevalence of Campylobacter in the Reproductive Tracts and Semen of Commercial Turkeys

Abstract Campylobacter is one of the most commonly reported bacterial causes of human foodborne infections in the United States, and epidemiologic evidence indicates that a significant proportion of human infections result from the improper preparation of poultry products. Campylobacter frequently colonizes the avian intestinal tract, but recent research indicates that this organism can also colonize the avian reproductive tract and possibly contaminate eggs and subsequent offspring. The present studies were undertaken to determine the prevalence of Campylobacter in the reproductive systems of commercial turkeys. In the first study, pooled semen samples from seven commercial turkey farms were randomly collected by abdominal massage over a period of 13 wk. The pooled semen samples were serially diluted, and 0.1 ml of each dilution was plated on Campy-Line agar and incubated at 42 C for 48 hr in a microaerophilic environment for enumeration of Campylobacter. Campylobacter was isolated from 57 of the 59 pooled semen samples, and levels ranged from below the limit of detection (<101) to 1.6 × 106 cfu/ml of semen. In the second study, the reproductive tracts of 11 hens and 17 toms were aseptically excised, and the segments (female: vagina, shell gland, isthmus, magnum, and infundibulum; male: ductus deferens and testes) were swabbed with a dry cotton sterile swab. The swabs were incubated for 24 hr in Campylobacter enrichment broth, and 0.1 ml of the enriched sample solution was streaked onto Campy-Line agar plates and incubated at 42 C for 48 hr in a microaerophilic environment. Of the 11 hens sampled, Campylobacter was isolated from the vagina (10/11), the shell gland (7/11), the isthmus (8/11), the magnum (6/11), and the infundibulum (4/11). Of the 17 toms sampled, Campylobacter was isolated from the ductus deferens (8/17) and the testes (2/17). Campylobacter is present in the reproductive tracts and semen of commercial turkeys and may lead to vertical transmission of Campylobacter from the hen to the chick.

Field testing the influence of sperm competition based on sperm mobility in breeder turkey toms

1. Commercial reproduction of turkeys relies on pooling of semen from multiple males for inseminations. Understanding how sperm characteristics influence paternity under commercial breeding conditions is important to improving production efficiency. 2. The objective of this study was to evaluate progeny production of individual toms following commercial practices of pooling semen to determine if sperm mobility influences progeny production in field conditions. 3. A total of 104 toms were evaluated for sperm mobility. A subset of 10 toms were housed together and semen was collected, pooled and used to inseminate hens (n = 28). Hens were inseminated at 30 weeks of age and weekly thereafter. 4. Ejaculates from each tom were evaluated on two separate days for sperm mobility. Semen from each tom was diluted and layered upon 6% (wt/vol) Accudenz® solution. The sperm suspension was incubated at 41°C for 5 min and absorbance was measured with a spectrophotometer. 5. Toms were ranked by absorbance and categorised as high or low if mobility score was ±1 SD from the flock mean (average). 6. For parentage determination, DNA was extracted from tom, hen and poult blood. Poult parentage (n = 276) was determined at one day of age or at 14 weeks by analysis of marker genotypes that were generated by polymerase chain reaction (PCR) amplification of genomic DNA with selected microsatellite markers. 7. Sperm mobility differed across males with absorbance values ranging from 0·147 to 0·366. 8. Findings demonstrate differences in poult production among individual toms when semen from multiple males was pooled and inseminated. Toms classified as high, average and low produced 55, 41 and 4% of the offspring, respectively. 9. It appears that sperm mobility is a trait that influences sperm competition among toms under field conditions where sperm numbers inseminated from individual toms are not controlled or constant and that toms with low sperm mobility produce few offspring.

COMPARATIVE VIABILITY OF BOVINE SPERM FROZEN ON A CRYOMICROSCOPE OR IN STRAWS

The accuracy and repeatability of freezing rates and effects of evaporation were examined using a new cryomicroscope system to establish its usefulness in assessing the development of cryopreservation protocols for bovine semen. Post-thaw sperm plasma membrane integrity, as assessed by using combinations of fluorescent stains and flow cytometry, was used in evaluating protocols for freezing spermatozoa on the cryomicroscope. Semen was diluted in Test-yolk (20%) extender containing 7% glycerol and frozen in 0.5-ml straws, 0.25-ml straws (over liquid nitrogen for 8 min) or in a quartz crucible using a Linkam BCS 196 cryomicroscope. Thawed samples were diluted with Hepes buffered medium containing 0.1% bovine serum albumin (BSA) and stained with either carboxymethylfluorescein diacetate (CMFDA) or SYBR-14 each in combination with propidium iodide (PI). Flow cytometry analysis of the samples revealed 2 major populations: 1) spermatozoa with intense green fluorescence (stained with CMFDA or SYBR-14), which were classified as plasma membrane-intact and 2) spermatozoa with intense red fluorescence, (stained with PI), which were classified as plasma membrane-damaged. Samples frozen using the cryomicroscope contained 29 and 26 % plasma membrane-intact (PMI) sperm cells, as assessed by CMFDA and SYBR-14, respectively. Cryopreservation of spermatozoa in 0.5-ml straws resulted in 22 and 20% plasma membrane- intact sperm cells, while spermatozoa frozen in 0.25-ml straws resulted in 34 and 31% PMI sperm cells for CMFDA and SYBR-14, respectively. No significant difference was observed (P > 0.05) for PMI spermatozoa stained with either CMFDA or SYBR-14. In addition, the ability to recover spermatozoa after freezing on the cryomicroscope establishes the Linkam BCS 196 as a useful tool for the study of sperm cell cryopreservation.