Relationships among frozen-thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program.

Frozen semen specimens from 22 Holstein bulls representing a wide range of field fertility levels or nonreturn rates (NRR) were used in this study. Semen specimens were thawed at 37 degrees C for a minimum of 30 sec, followed by assessment via a routine semen analysis (RSA) and other sperm functional tests. The RSA was performed by assessing sperm count, motility and morphological characteristics. Other sperm functional tests were performed by assessing the acrosomal membrane integrity, sperm penetration into the cervical mucus and the sperm membrane functional integrity. Following assessment of sperm characteristics, the fertility data of the various bulls were compared to the RSA and the functional tests results. Bulls of high and low fertility were similar in terms of sperm count and progressive motility (P > 0.05). Other characteristics measured by the RSA and functional tests were significantly higher in high fertility bulls (P < 0.05). Correlation coefficients among the various sperm characteristics and fertility of bulls were highly significant (P < 0.01). The highest correlation coefficients between sperm characteristics and fertility were obtained for motility (r = 0.53; P < 0.01), normal morphology (r = 0.59; P < 0.01) and swollen spermatozoa (r = 0.57; P < 0.01). Analysis of specific sperm swelling patterns showed that those patterns considered to reflect maximal sperm swelling were indicative of high fertility.

Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses.

Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained the green fluorescein fluorophore mainly in the acrosome. A third population, presumed to be degenerate spermatozoa, possessed only red fluorescent nuclei. These populations were quantified using dual parameter flow cytometry in 14 samples of cryopreserved bovine spermatozoa for which fertility and seminal quality data were available. Flow cytometric analyses were highly correlated with other seminal quality measurements. Sequential flow cytometric analyses provided the ability to rapidly quantitate changes in specific fluorescently stained populations. The ability to make rapid quantitative measurements should allow development of new and presumably more reliable information on the functional aspects of spermatozoa.

Evaluation of two seminal collection regimens for mature Holstein bulls.

Twenty mature Holstein bulls (3 to 10 yr old) were used to test the effect of two semen collection regimens on spermatozoal output, post-thaw percentage spermatozoal motility, and time needed to make the collections/week. For both regimens, six ejaculates/wk were collected using either three ejaculates/d, 2 d/wk, or two ejaculates/d, 3 d/wk. A three-period switchback experimental design was used. Each collection period for which measurements were taken was 3 wk and was preceded by a 2 wk period of acclimation. The total number of spermatozoa harvested per week was not significantly different (P greater than .05): 33.2 X 10(9) when the bulls were collected two ejaculates 3 d/wk, compared with 33.9 X 10(9) three ejaculates 2 d/wk. Post-thaw progressive spermatozoal motility was 50.3 and 52.1% (P greater than .05), respectively. The average time per week to collect each bull was 73.6 and 83.7 min (P less than .05), respectively.

Fine structural abnormalities of spermatozoa from a subfertile bull: a case report.

Spermatozoa from a nine-year-old subfertile Holstein bull were evaluated by light and electron microscopy. Morphological abnormalities observed included cytoplasmic droplet-like structures, constriction of the midpiece, missing axial filaments and abnormal spermatozoal head shapes.

Current status of semen preservation in the ram, boar and stallion.

From the studies cited it was concluded that short and long term preservation of stallion semen has encountered major obstacles. Fertilizing capacity of extended or extended and cooled spermatozoa has been impaired. With the hydrogen ion extenders, the fertility was depressed either with or without glycerol when the semen was inseminated immediately after extension. With the cream-gel extender, fertility was not impaired when inseminated immediately after extension, but was impaired after storage at 5 C for 24 hr or in the presence of glycerol. The fertilizing capacity of extended frozen spermatozoa particularly from some stallions has been more adversely affected than that of others. These studies show that the pregnancy rate range was from 50 to 80% for raw semen from the same stallion used in the frozen studies. Pregnancy rate with this magnitude of difference must be carefully weighed in applying the results from a few stallions to the population. Sufficient information has been generated to suggest that the preservation of stallion spermatozoa is possible but the fertilizing capacity is impaired. Causes of this impairment must be further investigated. When this is accomplished, the number of motile spermatozoa needed per insemination and the frequency of insemination required for optimal fertilization reported in this review must then be reevaluated.

Effect of timing of insemination, numbers of spermatozoa and extender components on the pregnancy rate in mares inseminated with frozen stallion semen.

Fertilization rate was highest in mares inseminated with frozen semen within 12 hr of ovulation. Foaling rate was improved (P less than 0-05) by increasing the number of motile spermatozoa inseminated from 40 X 10(6) to 80 X 10(6) but was not further improved by increasing the number to 160 X 10(6) or by increasing the frequency of insemination from once to twice daily. The fertilizing capacity of spermatozoa frozen in one of the hydrogen ion extenders studied was dependent upon relative osmotic pressure and method of freezing (ampoules or pellets). Adjusting glycerol concentration from 7% to 2%, addition of glycerol 15 min before freezing and freezing 2 hr after extension all enhanced the fertilizing capacity of spermatozoa. Pregnancy rate per mare service period was higher (P less than 0-01) for mares inseminated with unextended semen (75%) than for extended semen containing no glycerol (50%) or extended semen containing 7% glycerol (35%). Although wide variability in the fertilizing capacity of spermatozoa from individual stallions was observed when semen was extended or extended and frozen, pregnancy rate was depressed in all stallions. It was concluded, therefore, that hydrogen ion extenders depress fertilizing capacity of stallion spermatozoa immediately after extension and show little promise as semen extenders for short- or long-term storage of stallion semen.

Effect of centrifugation and seminal plasma on motility and fertility of stallion and bull spermatozoa.

The effect of centrifugation of diluted and undiluted semen on equine and bovine spermatozoan motility and fertility was examined, as was the effect of seminal plasma and dilution on stallion spermatozoa during incubation before and after freezing. Centrifugation at 370 g or 829 g was not detrimental (P greater than 0.05) to prefreeze or postfreeze motility if a final concentration of 10% seminal plasma was present. A reduction of seminal plasma from 10% to 2% significantly (P smaller than 0.05) reduced motility. A centrifugal force of 956 g significantly reduced prefreeze but not postfreeze motility of spermatozoa in undiluted semen, regardless of seminal plasma concentration. With a dried skim milk extender, prefreeze and postfreeze motility was greater in samples containing 20% seminal plasma. Motility was depressed by high and low concentrations of seminal plasma. The fertility of frozen or unfrozen stallion spermatozoa was not depressed (P greater than 0.05) by centrifugation at 310 g for 3.5 minutes. In contrast, the fertility of bull semen was significantly (P smaller than 0.05) lowered by centrifugation at 270 g for three minutes. Further, the fertility of centrifuged, diluted bovine semen was lower (P smaller than 0.05) than centrifuged, undiluted semen.