R.H. Foote

Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: A consensus report☆

Reproductive toxicity studies are increasingly including assessments of sperm parameters including motility, morphology, and counts. While these assessments can provide valuable information for the determination of potential reproductive toxicity, the methods for conducting the assessments have not been well developed in all laboratories and are continually evolving. The use of different methods in different laboratories makes comparison of data among laboratories difficult. To address the differences in methods, a working group was convened to discuss methods currently in use, share data, and try to reach consensus about optimal methods for assessing sperm parameters in rats, rabbits, and dogs. This article presents the consensus report, as well as future research needs, with the hope that optimized common methods will aid in the detection of reproductive effects and enhance interlaboratory comparisons.

QUANTIFICATION OF BULL SPERM CHARACTERISTICS MEASURED BY COMPUTER-ASSISTED SPERM ANALYSIS (CASA) AND THE RELATIONSHIP TO FERTILITY

Two experiments were conducted to evaluate semen quality of bulls housed under controlled conditions at a large AI facility and relate results to fertility. In Experiment 1 semen was collected from six 6-yr-old bulls twice daily at 3- to 4-d intervals for 3 d. In Experiment 2 eleven 6- to 11-yr-old bulls were used. Extensive breeding information was available and semen was collected as in Experiment 1 but replicated 4 times. Standard semen analysis and computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, model 10 unit, were performed on 36 first and second ejaculates in Experiment 1 and on 44 first ejaculates in Experiment 2. Sixteen fields (2 chambers with 8 fields per chamber) were examined per sample. In Experiment 1 the correlation between estimated sperm concentration by spectrophotometry and CASA was 0.91 (P < 0.01). Among bulls the range in the percentage of motile spermatozoa was 52 to 82 for CASA versus 62 to 69 for subjective measurements made by highly experienced technicians. Thus, CASA, with high repeatability, provided a more discriminating estimate of the percentage of motile sperm cells than did the subjective procedure. Bull effect was much greater than any other variable in the experiments. Chamber differences were small and so the results for the 2 chambers with 8 fields each were combined. One to five CASA values were correlated with bull fertility, defined as 59-day nonreturn rates corrected for cow and herd effects. The percentage of motile spermatozoa accounted for a small fraction of the total variation in fertility (r2 = 0.34). However higher r2 values (0.68 to 0.98) were obtained for 2 to 5 variables used in the multiple regression equations. The results are promising, and further testing will determine more precisely which of these CASA variables are most useful in estimating bull fertility potential.

Effect of donor-embryo-recipient interactions on pregnancy rate in a large-scale bovine embryo transfer program

The effects on pregnancy rate of 23 factors relating to time, embryos, donors, and recipients in a commercial bovine embryo transfer program were analyzed retrospectively. Over 6 12 years, embryos were recovered on 1625 occasions from 825 different cows and heifers. Unfrozen embryos were transferred surgically to virgin Holstein heifers of a relatively uniform size and age, maintained under a single management system in the state of Pennsylvania in the USA. Transfer of 7652 embryos resulted in an average pregnancy rate of 71.3% with small, but significant differences among years and months. There were no differences among years, however, in the mean quality, stage, or age of the embryos transferred, or in the mean age of donors, number of embryos recovered, or the mean number of times that a recipient was used. The pregnancy rate was not affected by breed, fertility or lactational status of the donor, or day of the estrous cycle on which superovluatory treatment began; however, embryos from cows over 15 years of age resulted in lower pregnancy rates. While there was no influence of total number of ova recovered or percent fertilization, the number of fertilized ova had a small effect. Moreover, there were differences due to the morphological quality and stage of development of the embryos, and due to the interval from the donors estrus to recovery. Pregnancy rates were lower with embryos recovered 9 or more days after estrus. Although duration of culture had no effect, Hams F-10 was superior to modified PBS. Although the interval from treatment to estrus as well as the number of previous estrus synchronization treatments had no influence, recipients induced with prostaglandin to be in estrous-cycle synchrony with the donor had a distinctly higher pregnancy rate than those in natural synchrony. Estrous synchrony between the donor and recipient and the interval from the recipients estrus to transfer affected the pregnancy rate, but the side and quality of the corpus luteum of the recipient and the number of times that a heifer had been used as a recipient did not. Interactions between the folowing factors influenced the pregnancy rate: 1) day of flush and embryo quality, 2) day of flush and stage of development of the embryo, 3) embryo quality and estrous synchrony, 4) stage of embryonic development and interval from the recipients estrus to transfer, and 5) stage of embryonic development and estrous synchrony.

Motility and fertility of bull sperm in whole milk extender containing antioxidants

Bull sperm are exposed to aerobic conditions during processing before freezing, and they have little endogenous antioxidant to protect them against reactive oxygen species that may be present. Seventeen laboratory studies and two field trials were conducted with 174 semen collections from bulls in an artificial breeding cooperative. More than 250 combinations and concentrations of reduced glutathione (GSH), superoxide dismutase (SOD), ascorbic acid, hypotaurine (HPT), 2,2,6,6-tetramethylpeperidine-1-oxyl (Tempo) and 4-hydroxy-2, 2, 6, 6-tetramethylpeperidine (Tempol) were tested by adding these compounds to fresh semen, and to a whole milk (WM) glycerol extender. Semen packaged in straws in the WM extender was frozen with liquid nitrogen. The motility of frozen-thawed sperm during storage at 25 or 5 degrees C after freezing was compared with semen stored without freezing. Antioxidants generally were not beneficial, except the percentage of motile sperm was improved by 6-11% units (P<0.05) when sperm were stored unfrozen or after freezing when 0.5mM of GSH with or without SOD was added. In two field trials, non-return rates were 71.9, 69.5 and 70.9% (P>0.05) with WM containing 0.0, 0.5 and 1.0mM of GSH, respectively, and 74.0 and 73.9% with WM and WM plus 0.5mM of GSH and 100U/ml of SOD (P>0.05). WM contains an abundant supply of casein which is an antioxidant, and additional antioxidants were ineffective in improving motility of sperm immediately after freezing and thawing or in affecting fertility. However, sperm responses were different in egg yolk-Tris extender. Sperm in this egg yolk extender tolerated substantial concentrations of Tempo and Tempol compared with toxic effects in WM (P<0.05). Therefore, optimal combinations of antioxidants tested here may have more useful applications in organizations using an egg yolk-based semen extender.

Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing

Diluents containing sonicated liposomes of purified phosphatidylserine (PS), phosphatidylcholine (PC) with varying fatty acyl chain lengths and double bonds and cholesterol (CH) alone or in combination, or egg yolk lecithin were evaluated for protection of bull sperm during cold shock produced by rapid cooling from 25 to 0 degrees C and during freezing and thawing. Bull semen was washed twice and diluted to 50 X 10(6) sperm/ml in diluents containing no lipid, 0.5 or 5 mM sonicated lipid or 20% egg yolk and plunged into ice water to cold shock the sperm. Sperm so treated were frozen using conventional methods. The percentage of progressively motile sperm (MS) was estimated prior to cooling, after cold shock, and after freezing and thawing. Lipids with fatty acyl chains of less than 12 carbons were toxic to sperm cells. Phosphatidylserine alone or in combination with PC or CH, but not PC or CH alone, protected sperm from cold shock as well as did egg yolk lecithin liposomes or egg yolk. Liposomes of PS/PC or PS/CH were not better than PS in protecting sperm from cold shock. Lipid concentrations of 0.5 mM were more effective than liposomes at 5 mM in protecting sperm during freezing and thawing. During freezing, PS alone or in combination with PC partially protected sperm, but only PS/CH was as effective as egg yolk in protecting sperm from freeze-thaw damage. It is concluded that defined diluents, particularly those containing PS, may be useful in studies of cryobiology of spermatozoa.

Superovulatory responses of Holstein cows

Abstract Approximately 1000 registered cows and heifers were superovulated one to 10 times. Nonsurgical embryo recoveries were performed on all donors which exhibited estrus. Healthy donors produced more total ova and cleaving embryos and had a higher ovum recovery rate, fertilization rate and pregnancy rate from embryos transferred than did cows classified as infertile. While ovum number was not affected during 10 repeated superovulations, fertilization rate and embryo number decreased. The number of ova recovered from healthy cows was affected by season, and from infertile cows by the day of the estrous cycle on which FSH was started and by the number of days since calving. More ova were recovered from infertile cows synchronized with prostaglandins prior to superovulation than following a natural estrous cycle. The number of embryos recovered from infertile cows was affected by age and from healthy cows by daily milk production. Fertilization rates in both healthy and infertile cows were affected by age, time since calving, daily milk production, day of cycle FSH was injected and season. There was no effect of the day of recovery on the number of ova or embryos recovered from healthy or infertile cows.

Fertility estimation: a review of past experience and future prospects

Fertility has many components and stages which require that males and females be functionally capable of carrying out all critical stages if each generational reproductive cycle is to be completed. To accomplish this, the male must produce and ejaculate normal fertile sperm. The female must produce, store and ovulate normal fertilizable oocytes. Furthermore, the female must provide a reproductive system compatible with sperm transport, capacitation, and fertilization of the oocytes, embryo and fetal development, and finally birth of healthy young. Reproductive success or failure at several of these points can be estimated quantitatively on a population basis, and in a few situations on an individual basis. It is important that fertility estimates be determined accurately and with precision to be most useful to researchers and managers of animal enterprises. Many studies have underestimated the biological relationship of fertility to other traits because the estimates lacked precision. Many in vitro manipulations of sperm in artificial insemination, of gametes in various assisted reproductive technologies, and of embryos in embryo transfer are utilized in animal breeding programs. Accurate estimation of reproductive efficiency of these in vitro procedures also is important. Conditions surrounding different sets of fertility estimates almost certainly will be different. These conditions should be described as precisely as possible, and appropriate controls included in all experiments. When possible, experiments should be replicated over time and place to determine the repeatability of the various criteria used to estimate fertility and reproductive efficiency. Advances in genomic information and molecular biology should facilitate characterizing more fully inherent potential fertility of animals at birth. In vitro tests will improve, and automated techniques will facilitate making multiple determinations possible on a large scale. Reliability of fertility estimates will increase, with the potential for enhanced animal reproductive performance through more accurate selection, genetic engineering, and enlightened animal care. Simultaneously, it is important to recognize that prediction of future fertility is more hazardous than estimating fertility, as a completely new set of circumstances may occur which are not predictable. Because fertility estimation may be applied under a myriad of conditions, principles and factors affecting fertility will be emphasized in this review as being more useful than a compilation of numerical examples.

The rabbit as a model for reproductive and developmental toxicity studies

The rabbit has many advantages as a nonrodent and second model for assessing the effects of toxic agents on semen quality, fertility, developmental toxicity, and teratology. The male and female reproductive systems of the rabbit are described, and data on growth, sexual development and reproduction are compared with mice, rats, and humans. Techniques for semen collection and evaluation in the male, and artificial insemination, superovulation, embryo culture, and embryo transfer in the female are included as useful procedures in toxicity testing. Examples of the use of rabbits and experimental replication for toxicity testing are given. Special features of the visceral yolk sac and development of the chorioallantoic placenta of the rabbit are compared with rodents. The rabbit extraembryonic membranes more closely resemble the human than do the rodents, in some respects. The use of the rabbit in developmental toxicity and teratology studies is discussed.

Milk Progesterone as a Diagnostic Aid

SUMMARY Methods of obtaining, storing and assaying milk samples have been studied. Under field conditions last milk has been preferable, with either whole milk or butter-fat used in the RIA. Progesterone is very stable in storage. Herds using artificial insemination, and free from known diseases, have tended to have lower fertility as herd size and level of milk production increased. Milk progesterone analysis revealed that most cows were cycling normally by 50 days post partum, but frequently were not seen in oestrus or oestrus was inaccurately detected. Cows on a high, medium or low energy ration had their first post-partum milk progesterone cycle start on average 24, 24 and 31 days after calving respectively. Observed oestrus occurred later and was more variable. With the selected use of heat mount detectors, chalk, electronic probes and computerized reproduction management guides, the problem of missed oestrus can largely be overcome. Milk progesterone can serve as a non-pregnancy test with 98% accuracy about three weeks after insemination. This focuses attention on cows requiring reinsemination and frequently results in observed oestrus and insemination without missing a cycle. Milk progesterone also was useful in distinguishing between cows with follicular cysts, luteinized follicles and persistent corpora lutea, and in evaluating experimental and clinical trials using GnRH. Palpation of the reproductive organs per rectum at about six weeks is recommended as a final check of pregnancy. Embryo mortality between 28 and 75 days was estimated by progesterone analyses to be 7.2%.

Synergistic effect of ethanol and cycloheximide on activation of freshly matured bovine oocytes

Bovine follicular oocytes were collected from ovarian antral follicles (2 to 7 mm in diameter) from slaughtered cattle. They were matured in vitro (IVM) for 23 to 24 h and then activated. In Experiment 1, 4 concentrations of ethanol were compared. The activation rates of oocytes were 4, 12, 36 and 27%, respectively, following exposure for 7 min to 0, 5, 7 and 10% ethanol. In Experiment 2, 7% ethanol was tested with exposure times of 0, 5, 7.5 and 10 min, and 6, 32, 27 and 33% of the oocytes were activated, respectively. In Experiment 3 the synergistic effect of ethanol and electric pulse was compared within 4 treatments: A) 7% ethanol alone, B) electric pulse alone, C) ethanol first and then electric pulse treatment, and D) electric pulse first followed by ethanol exposure. Of the oocytes activated, 37, 31, 28 and 51%, respectively, were from Treatments A through D. In Experiments 4 and 5 the possible synergistic effect of ethanol and a protein synthesis inhibitor, cycloheximide, was studied within 4 treatments: A) parthenogenetic control with no activation treatment, B) ethanol alone, C) cycloheximide alone, and D) ethanol treatment followed by cycloheximide. The oocyte activation rates in Experiment 4 in Treatments A through D, respectively, were 9, 44, 43 and 84%. Corresponding values for development of oocytes to the 2 to 8-cell stage after culture for 3 d (Experiment 5) were 9, 20, 14 and 45%, respectively (P<0.05). In conclusion, exposure to 7% ethanol for 5 min followed by incubation with cycloheximide was the best activation treatment for bovine IVM oocytes.