Measurement of intracellular pH in mammalian sperm cells under physiological conditions.

The goal of this study was to develop a technique for the measurement of intracellular pH (pHi) in living mammalian (bovine) sperm cells under physiological conditions. Like many other biochemical measurements, pHi measurements have typically been made under non-physiological conditions on cells whose immediate functional status is not readily assessed and may even be non-viable. Additionally, many pHi measurement techniques may themselves alter the pHi of the cells being measured. Such measurements could yield misinformation. The sperm cell is unique in that its functional status can be easily and continuously monitored by means of its motility, which is directly affected by pHi. In this respect, the sperm cell provides an ideal model system for evaluating pHi measurement techniques. In this article we summarize the validation of a ratiometric absorbance technique for the measurement of pHi of mammalian (bovine) sperm cells under physiological conditions which does not affect their functional status. The pHi of ejaculated bovine sperm cells was calculated to be 6.9 +/- 0.05 (11 replicates). This approach may also be suitable for pHi measurements in other cell types.

Paternal influence on S-phase in the first cell cycle of the bovine embryo.

The objective of this study was to determine whether the initiation and length of the zygotic S-phase differs for embryos sired by bulls demonstrating high versus low fertility in vivo. Bovine oocytes were matured in vitro for 24-27 h and fertilized with frozen-thawed bovine semen. Six bulls that differed in fertility level were used in the study. The bulls were classified into two groups: those demonstrating high fertility in vivo (in vivo high-fertility bulls; n = 3), with a group mean +/- SEM lifetime nonreturn rate of 78 +/- 2%, and those demonstrating low fertility in vivo (low-fertility bulls; n = 3), with a group mean +/- SEM nonreturn rate of 69 +/- 1%. The S-phase in zygotes was identified by means of an immunocytochemical technique after pronuclear-stage zygotes were labeled with 5'bromo-2'deoxyuridine (BrdU). To visualize all pronuclei, presumptive zygotes were also stained with propidium iodide. In the first experiment, zygotes were labeled with BrdU at 2-h intervals from 8 to 20 h after sperm addition. There were no differences between bull fertility groups in the time course of pronuclear formation (p > 0.05). The beginning of S-phase was earlier in zygotes sired by high- compared to low-fertility bulls (p < 0.05). The end of S-phase was not affected by sire fertility group (p > 0.05). In the second experiment, zygotes were labeled with BrdU continuously from 8 to 20 h after sperm addition.(ABSTRACT TRUNCATED AT 250 WORDS)

A new antibiotic combination for frozen bovine semen. 2. Evaluation of seminal quality.

Amikacin, clindamycin, gentamicin, Linco-Spectin, minocin and tylosin were added individually and in combinations at various concentrations to bovine neat semen and to egg-yolk citrate, egg yolk-tris or heated whole milk extenders (nonglycerol fractions) prior to final processing for freezing to -196 degrees C. After thawing samples, seminal quality was measured by progressive motility and acrosomal integrity evaluations. Studies were performed in parallel with microbiological efficacy studies of Shin et al. (7). The antibiotic combination including gentamicin, tylosin and Linco-Spectin at 500 ug/ml, 100 ug/ml and 300/600 ug/ml, respectively, was not detrimental to seminal quality and, in the parallel studies, was most efficacious in controlling microorganisms potentially present in bovine semen.

A new antibiotic combination for frozen bovine semen, 3. Evaluation of fertility.

Field fertility (nonreturn rate) studies were performed independently by three artificial insemination organizations to evaluate bovine semen processed for freezing using the antibiotics gentamicin, tylosin and Linco-Spectin at concentrations of 500 ug, 100 ug, and 300/600 ug, respectively, per milliliter of neat semen and per milliliter of nonglycerol portion of the extender. The antibiotic combination including penicillin, dihydrostreptomycin, polymyxin B sulfate, with/without Linco-Spectin (500 units/ml, 2000 ug/ml, 1000 units/ml and 300/600 ug/ml, respectively) was used as the control treatment. Results indicated no significant effect on seminal quality as measured by field fertility under the conditions of these experiments using heated whole milk or egg yolk-sodium citrate seminal extenders. Use of the new antibiotic combination has been adopted by Certified Semen Services.

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.

Influence of bovine cervical mucus samples and storage conditions on sperm migration in vitro.

In vitro sperm migration assays were performed using bovine spermatozoa and cervical mucus. Experiments were designed to test the effects of storage temperature, method of storage, duration of storage, and source of cervical mucus. Significant variation in migration of spermatozoa was due both to differences in mucus samples and to short-term mucus storage at temperatures ranging from ambient to -196 degrees C. The parallel-orienting effect of cervical mucus on migrating sperm was shown to be a major factor in quantitative assays based upon migration distance. Thus, comparisons of migration among different specimens of semen likely will be biased unless the tests are run simultaneously. Implications of these results are discussed relative to the performance of quantitative sperm migration assays in the clinical or research laboratory.

Polyacrylamide as a substitute for cervical mucus in sperm migration tests.

A synthetic migration medium for capillary sperm migration studies was developed. Parallel sperm migration in 1.8% polyacrylamide cross-linked with 0.042% N,N'-methylene bis acrylamide was similar to sperm migration in bovine cervical mucus. Bull spermatozoa varying widely in migration distances in bovine cervical mucus maintained similar relative migration distances in this synthetic medium. The advantages of the synthetic medium are its availability in large quantities, its uniformity, and its stability. There was no change in parallel sperm migration distances in the synthetic medium stored at 4 degrees C for up to 4 months. Use of this synthetic medium for human or bovine sperm migration studies would appear to overcome problems associated with the variability of cervical mucus.

Radiolabeling of mammalian spermatozoa and their use to monitor sperm transport in females.

Radiolabeling of mammalian spermatozoa with 131I, 67Ga, 111In, and 99mTc was investigated. Spermatozoa were labeled with 99mTc for in vivo studies because of a high labeling yield (70 to 90%) combined with the lack of impairment of sperm motility. Ovariectomized sheep were brought into estrus by sequential administration of progesterone and estradiol cyprionate. Sheep were necropsied up to 6 hours after insemination with 99mTc-labeled ram sperm and their reproductive tracts were resected and examined with a rectilinear scanner. Radioactivity was clearly observed in the fallopian tubes, with larger amounts in the vaginas, cervices, and uteri. In contrast, when 99mTc-spermatozoa were replaced with 99mTcO4-, much less radioactivity remained in the reproductive tract at resection and this was evenly distributed. Some label left the 99mTc-spermatozoa in vivo, but radioactivity remained on the cells long enough to consider attempting to monitor sperm transport in vivo in a suitable species.

Inhibition of sperm migration through cervical mucus in vitro.

The effectiveness of inhibiting bovine sperm migration through cervical mucus in vitro by prior treatment of semen with 45 to 150 micrograms of soybean trypsin inhibitor, univalent (papain-digested, nonagglutinating) and bivalent (undigested) rabbit anti-bovine sperm immunoglobulin, and heat-treated heifer serum was studied. Sperm head-to-head agglutination resulted from treatment of semen with bivalent immune antibody and heat-treated heifer serum. Migration through cervical mucus was inhibited only by treatment resulting in spermagglutination. It is postulated that in vivo inhibition of sperm migration may be influenced by secretory immunoglobulins from the cervix.