Effect of serum sources and colostral whey on bovine semen quality and spermatozoa immunoglobulin G immunofluorescence

Heifer, bull, fetal calf sera, and colostral whey were used to evaluate the influence of protein concentrations on percent progressive motility, head-to-head agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence of bovine spermatozoa using ejaculates from 10 bulls. In the first experiment, 10% (vol/vol) addition of undiluted colostral whey resulted in the highest head-to-head agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence. Ten percent (vol/vol) addition of whey diluted to a protein concentration equivalent to fetal calf serum produced significantly lower agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence. Fetal calf serum was unable to produce agglutination and immunoglobulin G immunofluorescence of bovine spermatozoa. Heifer and bull sera produced similar responses for all seminal measurements. In Experiment 2, unheated whey and heifer serum resulted in higher response for all variables than heat inactivated whey and heifer serum. Whey treatment produced greater spermatozoal motility, agglutination, acrosomal integrity; and immunoglobulin G immunofluorescence than treatment with heifer serum. Spermatozoal immunofluorescence indicated antibodies in normal whey, bull, and heifer serum bound to spermatozoal membranes at the acrosomal region. Colostral whey was an effective source of agglutinin factor. Normal unheated whey and heifer serum did not cause sperm damage or immobilization.     

Survival and fertility of antibiotic-treated bovine spermatozoa.

Motility of spermatozoa stored at 5 C with up to 1000 units or mug of chloramphenicol, polymyxin, kanamycin, tylosin, ampicillin, lincomycin, spectinomycin, erythromycin, novabiocin, or terramycin per ml of extender was compared to that with penicillin plus dihydrostreptomycin. Novabiocin and terramycin were toxic, but other antibiotic treatments had no effect. However, erythromycin and tylosin, as well as colymycin, depressed motility of frozen thawed spermatozoa. Spermatozoal motility was equivalent, following freezing in ampules or straws. All of the antibiotics which were non-toxic when added singly to frozen semen were also not harmful to frozen spermatozoa when as much as 2000 units or mug were added per ml of extender containing penicillin and dihydrostreptomycin. The addition of 1000 units or mug of ampicillin, chloramphenicol, kanamycin, lincomycin, polymyxin, or spectinomycin per ml of extender containing 750 units penicillin and 750 mug dihydrostreptomycin per ml did not influence the fertility of frozen spermatozoa in a field test involving 19,663 first inseminations.     

Induction of bovine sperm capacitation by TEST-yolk semen extender

Ejaculated bull semen was diluted 1:10 in the TEST-yolk buffer, cooled slowly to 4 degrees C, and stored for up to 48 h. Aliquots were taken at 0, 4, 8, 16, 24, and 48 h and washed once or three times in bovine serum albumin-saline and the sperm pellets resuspended in this saline. Fertilization of zona-free hamster oocytes was used to assess sperm capacitation. Motility differed between samples washed once or three times (53.7 vs. 21.7%). Motility was highest at 4 h storage but did not differ between 16, 24, or 48 h of storage. More sperm without intact acrosomes were found at 4 h than at 0 h, but the percentage did not change further until after 24 h. Penetration of oocytes was not different between sperm washed once or three times (28.5 vs. 26.9%). No penetration occurred at 0 h, and highest penetration rates occurred at 4 and 8 h of storage (32.1 and 33.4%). Penetration rates at 16, 24, and 48 h were not different (25.3, 25.2, 22.5%). In conclusion, storage of bull sperm in TEST-yolk buffer for 4 to 48 h resulted in capacitation. Even though capacitation was induced by 4 h, at least 71% of the sperm population had not undergone an acrosome reaction by 48 h of storage. This may explain why penetrability was maintained over this period.     

Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae

The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the sperm through Percoll did not increase sperm binding to the ZP but did remove any positive effect of EZM on sperm-ZP binding. In Experiment IV, exposure of either sperm or ZP to EZM before co-incubation did not increase sperm binding to ZP. In Experiment V, sperm diluted in TALP containing skim milk, EZM, or INRA96 bound more efficiently to the ZP than sperm diluted in TALP without milk proteins. In Experiment VI, sodium caseinate, native phosphocaseinate, and caseinoglycopeptide increased sperm binding to the ZP. In conclusion, diluents containing milk or milk proteins markedly enhanced the number of sperm bound to both equine and bovine ZP.     

Antibiotics for elimination of mycoplasmas and ureaplasma from bovine semen.

Minocin at 500 micrograms/ml of semen extender eliminated ureaplasma from naturally or artificially infected bovine semen. Minocin with lincospectin eliminated Acholeplasma laidlawii, Mycoplasma bovigenitalium, bovis, canadense, and arginini from artificially infected semen. Stabilization times of 15 min at 35 C and 3 h at 4 C are important considerations to maximize antibiotic activity.     

Effects of scrotal insulation on viability characteristics of cryopreserved bovine semen.

The effect of a 48-h scrotal insulation on spermatozoal viability (motility and acrosomal integrity), before and after semen cryopreservation, was studied in six young Holstein bulls whose semen was collected twice in succession at 3-d intervals. Motility and acrosomal integrity were measured before and after incubation of semen at 37 degrees C for 3 h. For assessment of results, collection days were grouped: period 1 (control) = d -6, -3, and 0, where d 0 = initiation of scrotal insulation after semen collection; period 2 = d 3, 6, and 9 (sperm presumed in the epididymis or rete testis during scrotal insulation); period 3 = d 12, 15, ... 39 (sperm presumed in spermatogenesis during scrotal insulation). Semen was cryopreserved each collection day until morphologically abnormal cells exceeded 50% of the ejaculate (d 12 to 21). Semen viability before and after freezing was lower in period 3 than in period 1 (P less than .05). These differences coincided with the appearance in period 3 of abnormal sperm morphology and depressed undiluted semen motility, which began on d 12 (P less than .01). Semen collected during period 2 that was extended but unfrozen did not differ from that collected during period 1 in morphology or viability. However, for frozen semen, period 2 was significantly poorer than period 1 for both viability measurements, but only after incubation for 3 h at 37 degrees C postthaw (P less than .05). We conclude that epididymal sperm are adversely affected by elevated testicular temperatures, as noted by their decreased ability to maintain motility and acrosomal integrity following cryopreservation.     

Transport and fate of spermatozoa after insemination of cattle

Sperm capable of fertilizing ova reach the isthmus of cows about 8 h after mating and remain in the caudal 2 cm of the isthmus until ovulation. Then small numbers of sperm move to the site of fertilization at the junction of the isthmus and ampulla. Within a few hours after deposition of semen in the uterine body, most sperm have drained to the exterior in cervical mucus. By 12 to 24 h after insemination, only a few percent of the sperm remain in the reproductive tract, and most of these are in the vagina. Contractions of the reproductive tract appear to be the primary mechanism of sperm transport. Flagellation of sperm is probably required for sperm to enter the folds of the cervix, and flagellation may be helpful or essential for sperm to pass through the uterotubal junction, move from the isthmus to the ampulla, and penetrate ova. High proportions of sperm undergo the acrosome reaction only in the ampulla on the side of ovulation and only after ovulation. The fertilization rate in cattle can be improved by use of semen from high fertility bulls and perhaps by timing insemination with semen from lower fertility bulls after the end of estrus.     

Alteration of seminal proteins during freeze-drying of bovine semen

Bovine semen in TEST-yolk extender was frozen, freeze-dried to 50, 25, 12, 6, and less than 2% residual moisture, and stored at -196 degrees C. The freeze-dried semen was rehydrated, sampled for protein analysis, and used to inseminate cattle. Agar gel electrophoresis revealed no moisture was reduced to less than 6%. At that point, the percent of cationic proteins and neural proteins decreased with a concurrent increase in anionic migrating proteins. Immunodiffusion data with antisera against spermatozoa and seminal plasma revealed no difference in formation of precipitin lines if residual moisture was at least 6%. However, semen freeze-dried to 2% residual moisture was modified antigenically as certain precipitin lines were lost, new lines appeared, and concentration of other seminal antigens decreased. The absence of fertility with semen freeze-dried to 2% residual moisture is hypothesized to 2% residual moisture is hypothesized to be from alteration of the tertiary structure of certain essential seminal proteins.