D R Holsberger’s research while affiliated with Maryland Department Of Agriculture and other places

Since all domestic turkeys are produced through artificial insemination, a measurable sperm characteristic that would be predictive of fertility would allow for the culling of poor males, resulting in improved reproductive efficiency. The sperm mobility test (SMT), which quantifies sperm penetration into an Accudenz solution, has been shown to correlate highly with fertilization potential of individual turkeys. Since this sire-selection test is based on the differences in sperm mobility between whole ejaculates from individual males, the objective of this study was to determine whether specific sperm velocity parameters would correlate with the SMT and to determine whether these characteristics could account for phenotypic differences in sperm mobility observed between males. The SMT was used to rank males within a flock (n = 110) in triplicate and to classify them into high, average, and low sperm mobility phenotypes on the basis of the sperm mobility index. Several sperm velocity parameters were evaluated for each male by a computer-aided sperm analysis (CASA) system, the Hobson Sperm Tracker. The types of measurements taken of 200 sperm tracks/ejaculate included the following: curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), linearity (LIN), beat-cross frequency (BCF), and mean angular displacement (MAD). Significant positive correlations were found between VSL, LIN, BCF, and sperm mobility, and a significant negative correlation was seen between MAD and sperm mobility. Subpopulations of sperm that had penetrated the Accudenz solution were isolated from each mobility phenotype and were analyzed by CASA, and significant correlations were again observed between VSL, LIN, BCF, and sperm mobility. We conclude that sperm velocity and linearity contribute to overall sperm mobility phenotype and are important characteristics of turkey sperm function. Key words: Motility, computer, spermatozoa.

We evaluated the utility of the commercial version of a new sperm-egg binding assay for detection of differences in sperm quality in samples of turkey semen from individual toms. Each sample had a swirl of 2 or more on a scale of 0 to 4. For assays conducted with fresh semen at 4 x 10(6) sperm per well, values ranged from 0.11 to 12% sperm bound to an extract of perivitelline membrane. Within-male variation averaged 0.17 percentage units, based on three ejaculates per male evaluated. Two experiments compared fertility and hatch for hens after weekly insemination with pooled semen from subpopulations of toms classified as having sperm with LOW or HIGH binding. Average fertility and hatch were lower (P < 0.05) for eggs laid by hens inseminated with semen from LOW toms in one experiment. In another experiment, hen fertility was not different between treatments after insemination during Weeks 32 to 39; however, a sharp decline in hatch was observed only for hens inseminated with semen from LOW toms after 40 wk of age. With semen from HIGH toms, hatch remained at > or = 80%. For these experiments, approximately 7% more poults were obtained from hens inseminated with semen from HIGH toms. We demonstrated that the sperm-egg binding assay detects differences in sperm quality between individuals, and these differences influence fertility.

Given a pool of genetically superior male turkeys, the subsequent selection of toms as semen donors for artificial insemination should be based upon criteria that are predictive of the tom's fertility and fecundity over the course of a production cycle. Recently, sperm mobility phenotype has been shown to correlate highly with fertility. Therefore, the objectives of this study were to determine: 1) whether sperm mobility phenotypes of individual toms were independent of time, and 2) the extent to which traditional semen quality variables changed as a function of time during the study interval. Sperm mobility phenotype was determined by objectively measuring the ability of sperm to penetrate 2% Accudenz solution at body temperature. During the initial assessments of a flock (n = 94), sperm mobility indexes (SMI) were significantly higher for the High Mobility Phenotype toms (56.61 +/-1.03% SMI) compared to the Low Mobility Phenotype (30.46+/-1.27% SMI, P < or = 0.0001) toms. Over the 5 mo of this study, the High Mobility Phenotype toms consistently had higher (P < or = 0.05) SMI values than the Low Mobility Phenotype toms, with SMI values similar to those of the initial screen. Ejaculate volume, concentration, and plasma membrane integrity showed no significant differences between phenotypes (P > or = 0.05). Sperm viability remained significantly lower (P < or = 0.05) over the course of this study for the High Mobility Phenotype toms than for the Low Mobility Phenotype toms. Because sperm mobility phenotype remained consistent through time, the Sperm Mobility Test provides a potentially important tool for selecting semen donors in turkeys.

Commercial turkey production relies on the artificial insemination (AI) of pooled semen. However, semen quality ultimately depends on the quality of individual ejaculates. The purpose of this study was to evaluate semen from individual toms by means of an objective sperm-mobility assay. Semen was then pooled by mobility phenotype and inseminated into hens, and the percentages of fertile and hatched eggs were determined after egg incubation. To indirectly evaluate hens' sperm storage, we determined the number of sperm holes in the perivitelline layer (PL) of freshly laid eggs. Semen from individual ejaculates (two trials, total of 169 toms) was evaluated by use of the sperm-mobility test (SMT). Semen was diluted to 1 x 10(9) sperm/ml, in prewarmed N-tris-[hydroxymethyl] methyl-2-amino-ethanesulfonic acid (TES)-buffered saline, and was placed over 3 ml of a 2% (w/v) Accudenz solution at 41degrees C. After a 5-minute incubation period, the cuvette was placed in a densimeter, and percentage transmission was recorded after 1 minute. Semen samples from toms ranked, according to sperm mobility, in the highest 10% and the lowest 10%, after three evaluations, were pooled by group and were used to inseminate hens weekly (trial 1: n = 20 hens/group, for 10 weeks, AI dose 150 x 10(6) spermatozoa inseminated fresh and after 24-hour in vitro storage at 5 degrees C; trial 2: n = 60 hens/group, for 16 weeks, AI dose = 75 x 10(6) spermatozoa inseminated fresh). Each week, eggs from day 6 post-AI were evaluated for holes in the PL, vestiges of acrosomal induced hydrolysis. Spermatozoa from toms of different mobility phenotypes were also evaluated individually, for sperm chromatin structure and motility variables, by use of the Hobson Sperm Tracker. Toms characterized by high and low in vitro sperm-mobility phenotype were categorized as "high mobility" and "low mobility," respectively. The percentage of fertile eggs from hens inseminated with semen from the high-mobility toms was higher than the percentage of fertile eggs from the low-mobility group, in each trial (95.8+/-1.3% vs. 90.4+/-2.2%, and 88.7+/-4.0% vs. 82.4+/-0.4%, trials 1 and 2, respectively; P < 0.05). More sperm holes were observed in the PL of eggs fertilized by the high-mobility toms than in the PL of eggs fertilized by the low-mobility toms (P < 0.05). No differences in susceptibility of sperm nuclear DNA to denature in situ, as measured by the flow-cytometric sperm chromatin-structure assay, were detected between toms of differing mobility phenotypes. Sperm-motility variables, straight-line velocity, and average-path velocity were significantly greater for high-mobility toms compared to low-mobility toms (P < 0.05). Sperm-mobility differences between toms (detected by means of the SMT) influenced sperm storage, as indicated by the number of sperm in the PL and by the percentage of fertile eggs produced.

A progressive decline in fertility over the course of egg production may be observed when turkey hens are inseminated weekly with semen stored for 24 h. In vitro storage of spermatozoa before insemination results in lower fertilization, possibly because fewer spermatozoa survive selection and storage in the hen's sperm storage tubules in vivo; alternatively, stored spermatozoa may be as capable of reaching the egg as are fresh spermatozoa, but unable to penetrate and fertilize the egg normally. The objective of this study was to determine whether this decline in fertility is a result of fewer spermatozoa reaching the egg after insemination with spermatozoa stored in vitro. Hens were inseminated weekly over the first 12 weeks of egg production with either fresh semen (n = 30 hens) or semen stored for 24 h (n = 30 hens). A total of 301 eggs was evaluated by determining the density distribution of spermatozoa embedded in the outer perivitelline layer. For the 12 weeks of egg production, the fertility of hens inseminated with fresh semen remained greater than 94%. Conversely, the percentage fertility of eggs from hens inseminated with stored semen in weeks 1-3 was greater than 94% but thereafter fertility averaged 86%. There was no difference in hatchability of fertile eggs between the two treatments over all weeks combined, and weekly throughout the study (P > 0.05). The mean number of spermatozoa in the perivitelline layer was higher (P < 0.001) when hens were inseminated with fresh (12.1 +/- 1.3 spermatozoa per 5.5 mm2 membrane) versus stored semen (2.5 +/- 0.3 spermatozoa per 5.5 mm2 membrane) over all weeks combined, and weekly throughout the study (P < 0.05). As a result of storage for 24 h, fewer spermatozoa are stored in the sperm storage tubules and, consequently, fewer spermatozoa are present at the site of fertilization, thus contributing to the depressed fertility.