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Relationship of seminal traits and insemination time to fertilization rate and embryo quality
Animal Reproduction Science 60–61 Ž2000. 663–677 www.elsevier.comrlocateranireprosci
Relationship of seminal traits and insemination time to fertilization rate and embryo quality R.G. Saacke ) , J.C. Dalton, S. Nadir, R.L. Nebel, J.H. Bame Department of Dairy Science, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061-0315, USA
Abstract The nature of subfertility due to the male or inseminate is as complex as that of the female. Fertilization failure or failure in embryogenesis are both documented to be of seminal origin. Males also differ in the numbers of sperm required to reach their maximum fertilization rate. Males requiring more sperm would be considered to have compensable seminal deficiencies. These include a number of known Žviability and morphology. and unknown factors Žfunctional or molecular traits. precluding sperm access to the ovum or ability to engage the ovum sufficiently to initiate fertilization and the block to polyspermy. Differences in fertility among males or inseminates independent of sperm dosage are considered uncompensable. These deficiencies would be associated with fertilizing sperm that are incompetent to maintain the fertilization process or subsequent embryogenesis once initiated, with most failures occurring prior to maternal recognition of pregnancy. Such sperm would preempt fertilization by competent sperm. Chromatin aberrations in morphologically normal or near normal spermatozoa from abnormal semen samples appear to be the best candidates for the uncompensable deficiency. However, recognition of uncompensable or incompetent fertilizing sperm has not been achieved. Six-day-old non-surgically recovered bovine ovarembryos have been used to evaluate compensable and uncompensable seminal deficiencies as well as to test reproductive strategies. These ovarembryos provide information on fertilization status and embryo quality as well as quantitative and qualitative data regarding associated accessory sperm. Thus, they permit the separation of reproductive failure by fertilization from that by embryonic development. Accessory sperm number is positively associated with both fertilization rate and embryonic quality. Early insemination results in low fertilization rates Žlow accessory sperm number., but good embryo quality, whereas, late insemination results in high fertilization rates Žhigh accessory sperm number., but poor embryo quality. Additional studies will be necessary to substantiate this model; however, if true, future research
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0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 1 3 7 - 8
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designed to improve results to artificial insemination should be tested by breeding early in estrus where sperm viability is most limiting and embryo quality is best. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Semen quality; Fertility; Embryo quality; Accessory sperm
1. Introduction There is a wealth of published research showing positive relationships of seminal traits, as measured in the laboratory, to fertility. It is clear, however, that the modest correlations obtained leave little doubt that we still lack sufficient knowledge to predict the outcome of matings based upon seminal quality as currently judged. Sources of the problem include repeatability of our laboratory tests ŽGraham, 1978., our ability to accurately measure fertility ŽOltenacu and Foote, 1976. as well as clearly establish the existence of a variance in fertility among semen samples tested ŽLinford et al., 1976.. Another aspect of the problem is our tendency to oversimplify the biological nature of subfertility due to the male or inseminate, often assuming that a single test or pair of tests will embrace the majority of factors important to fertility. Therefore, I would like to orient this paper toward our growing understanding of the nature of reproductive failure due to the male or inseminate. If we understand the principles involved, we then should be able to place laboratory evaluation of semen traits in proper perspective and improve our ability to reach the stage of predicting pregnancy rate.
2. Compensable and uncompensable seminal deficiencies It is now recognized that seminal deficiencies reflected in reduced pregnancy rates can be categorized as compensable or uncompensable. Seminal differences among males in numbers of sperm required to reach their respective maximum pregnancy rates are considered compensable. Once the maximum pregnancy rate of a male is met and further sperm inseminated are without effect, differences in pregnancy rates due to the male are then considered uncompensable. 2.1. Compensable semen traits Pace et al. Ž1981. showed viability traits of sperm that could be considered compensable included percent progressively motile sperm and percent of spermatozoa with intact cell or acrosomal membranes. Up to a threshold, they demonstrated that it was the number, and not percent of such cells in the inseminate that was important to pregnancy rate. Overstreet et al. Ž1978. demonstrated that the population of sperm accessing the site of fertilization under the sustained sperm transport phase Žpotential fertilizing sperm. was nearly 100% live. Immotile sperm ŽMattner, 1963; Overstreet et al., 1978; Mullins and Saacke, 1989. or sperm with tail defects or protoplasmic droplets ŽKoeford-Johnsen, 1972; Mitchell et al., 1985. are markedly impaired at barriers in the
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female tract precluding their access to the site of fertilization in the oviducts. Considering inseminated spermatozoa with misshapen heads, only those with the most subtle distortions can cross barriers such as the uterotubal junction ŽKrzanowski, 1974. or appear as accessory sperm ŽSaacke et al., 1998a.. Morphologically, spermatozoa appear to be excluded from contact with the ovum in vivo, more by severity rather than type of head distortion. Clearly, there are compensable seminal traits that cannot be explained by the conventional assessments mentioned above. In the bovine, males can differ 10-fold or more in ability to access the ovum in vivo based upon fertility at low insemination dose Žden Daas et al., 1998., or based on accessory sperm numbers in 6-day-old recovered ova and embryos ŽNadir et al., 1993.. These differences have not been explained by conventional viability and morphology semen tests. Undoubtedly important are functional changes of sperm in the oviducts involving motility patterns or molecular events on the surface of sperm associated with capacitation, spermregg recognition or the acrosome reaction. For penetration of the viscoelastic zona pellucida, hyperactivated rather than progressively motile sperm are thought to be critical ŽSuarez and Dai, 1992.. Potential fertilizing sperm which colonize the lower oviductal isthmus by binding to the mucosa, may also be quantitatively important ŽLefebvre et.al., 1997.. Similarly, molecular changes at the sperm surface may involve seminal plasma proteins ŽKillian et al., 1993, 1996. or seminal plasma-related sperm surface binding properties ŽBellin et al., 1994; Ax et al., 1996. that have been positively related to fertility in bulls. Numbers of sperm reaching the site of fertilization are small in relation to the number inseminated ŽHunter and Wilmut, 1984.. Governing this number are many factors involving the physiology of the Fallopian tube and the adjacent ovary which appear to work in concert regarding cueing of sperm to the ovum Žsee Hunter, 1998 for concepts and review.. However, there is also a positive relationship between the number of sperm inseminated and the number of sperm reaching the oviduct ŽMorton and Glover, 1974; Larsson and Larsson, 1986. as well as with the number of sperm at the ovum competing for fertilization based upon quantification of accessory sperm ŽNadir et al., 1993. or numbers of ova with accessory sperm ŽHawk et al., 1988.. The importance of compensable traits to artificial insemination is the fact that such traits govern the minimal numbers of sperm required for an inseminate to reach maximum fertility. 2.2. Uncompensable semen traits Observations in a variety of species indicate that factors associated with lowered sperm quality as measured by viability and morphology, result in low embryo quality or very early embryonic failure prior to maternal recognition of pregnancy ŽBarth, 1992; Courot and Colas, 1986; DeJarnette et al., 1992; Orgebin-Crist and Jahad, 1977; Setchell et al., 1988, see Setchell, 1998 for review.. Clearly, semen or sperm traits leading to this reproductive wastage suggests spermatozoal incompetence after fertilization. Such a deficiency could not be eliminated by sperm dosage alone, thus, the suggestion that the deficiency is uncompensable. Differences among bulls in embryonic development of their conceptuses have been reported at the time of routine recovery for embryo transfer
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ŽMiller et al., 1982. and after observation of embryo survival in recipients ŽColeman et al., 1987.. Bulls were also shown to differ in the development of their embryos following in vitro fertilization ŽEyestone and First, 1989; Hillery et al., 1990; Shi et al., 1990; Eid et al., 1994.. In low fertility bulls, early cleavage rates were reduced and pronuclear formation was delayed ŽEid et al., 1994.. This represents strong evidence for the uncompensable component in the low fertility bulls. Incompetent fertilizing sperm that can be labeled as uncompensable have not been identified using contemporary laboratory semen tests. However, sperm with subtly misshapen heads as well as those with nuclear vacuoles Žcraters, diadem, or nuclear pouches, defined by Coulter et al., 1986. on otherwise normally shaped heads are known to access the ovum following artificial insemination Žas determined from accessory sperm, Saacke et al., 1998a.. In addition, semen with sperm having these traits has been shown to yield higher frequencies of low quality embryos and lower fertilization rates than controls where such traits are missing or minimized ŽMiller et al., 1982; DeJarnette et al., 1992; Saacke et al., 1994.. Morphologically, abnormal sperm in semen of bulls has been associated with subfertility and sterility for many years ŽWilliams and Savage, 1925, 1927; Lagerlof, 1934.. We now recognize that sperm with classically misshapened heads, described by these early workers using simple microscopes, do not traverse the female reproductive tract or participate in fertilization ŽSaacke et al., 1998a.. In addition, the zona pellucida itself appears to provide one of the most formidable barriers against sperm with misshapen heads ŽHoward et al., 1993.. On this basis, a majority of abnormal sperm would be considered a compensable deficiency. However, males having disturbances in spermatogenesis resulting in ejaculation of abnormal sperm usually provide a broad spectrum in severity of morphological forms dependent upon the stage of spermatogenesis affected by the disturbance ŽVogler et al., 1991, 1993.. Thus, we believe that recognition of classically abnormal sperm in semen may represent the ‘‘tip of the iceberg’’ where disturbances in spermatogenesis signified by these sperm extend to otherwise normal or near-normal appearing sperm in the same ejaculates. These normal and near-normal appearing sperm that can access the ovum in vivo are the most likely candidates for the ‘‘uncompensable’’ traits causing pregnancy wastage through very early embryonic death. Finally, it should be recognized that sperm with microscopically normal morphology but with defective chromatin have been implicated in cases of male subfertility for some time ŽGleldhill, 1970.. The chromatin structure assay developed by Evenson et al. Ž1980. revealed a strong positive association between heterospermic fertility in bulls Žbased upon genetic markers at birth. and stability of the sperm DNA to acid denaturation ŽBallachey et al., 1988.. Using this same assay, Karabinus et al. Ž1997. have shown that sperm ejaculated before a mild thermal insult of the testis by scrotal insulation have more stable DNA than those ejaculated after scrotal insulation where abnormal sperm are also evident. Flaws in packaging and condensation of sperm chromatin during spermiogenesis ŽSakkas et al., 1995 and 1996. has been speculated to be involved in the instability of DNA of subfertile semen. The instability of the DNA may be due to limitations in disulfide bonding within nuclear proteins ŽKosower et al., 1992., a process that continues throughout spermiogenesis and epididymal maturation ŽBedford and
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Calvin, 1974.. Such flaws from a testicular perturbation such as elevated testis temperature early in spermiogenesis would be expected to influence sperm head shape; however, the same perturbation late in spermiogenesis or during epididymal maturation could impact DNA condensation without effect on head shape, which is already set ŽVogler et al., 1993.. Flaws in sperm DNA have also been incriminated in altered kinetics of nuclear decondensation associated with pronuclear formation following fertilization ŽQiu et al., 1995.. Regarding the embryological impact of an incompetent sperm, this timing agrees quite well with the uncompensable component in bulls affecting kinetics of rate of pronuclear formation and early cleavage in the bovine ŽEid et al., 1994. discussed earlier. Potentially underlying normal condensation and decondensation of DNA in sperm formation, maturation, and subsequent fertilization are optimum disulfide–thiol relationships within the nuclear protein. These relationships, in turn, are dependent upon the redox status of the sperm environment at each step Žfor reviews of this concept, see Sakkas et al., 1999; Levine, 1996.. Clearly, chromatin status in the sperm head presents a strong focal point for research designed to reveal the ‘‘uncompensable’’ component in semen causing early pregnancy wastage.
3. Ova r embryos and associated accessory sperm as biomonitors of seminal traits and reproductive strategy In the bovine, accessory sperm are those sperm trapped in the zona pellucida of the ovum or embryo. Once the fertilizing sperm enters the vitellus, the ovum reacts precluding further penetration and binding by additional sperm Žfor review, Cherr and Ducibella, 1990.. In ovarembryos Žpresumed morula in embryonic development. recovered on day 6 post insemination, accessory sperm are thought to represent the number of sperm competing for fertilization during the time the ovum was receptive. This implies that accessory sperm fulfill the structural and physiological requirements necessary for traversing the female tract as well as ovum recognition, binding and partial penetration. Clearly, it is not that simple. Quantitatively, the window of time the ovum is receptive could also vary among females due to permissiveness of the zona pellucida to sperm penetration as well as the speed of the block to polyspermy following sperm penetration. However, once established, the block to polyspermy in the cow is quite stable ŽHunter et al., 1998.. In addition, accessory sperm number per ovumrembryo could also be influenced by rate of penetration of the fertilizing sperm Žperhaps unique to a given male.. Nevertheless, accessory sperm do provide, in quality and quantity, insight to the characteristics of potential fertilizing sperm Žfor review, Saacke et al., 1998b.. 3.1. Interaction of compensable and uncompensable seminal deficiencies Through several years of experimentation in our laboratory, we recovered nearly 1000 single-ovulated ovarembryos 6 days post artificial insemination. Twenty-five bulls, all of which provided acceptable semen, are represented in this data. Fig. 1 shows the distribution of accessory sperm found in the zonae of embryos and ova from these cows as being very skewed, having a mean, median and mode of 12.0, 2.4, and 0 sperm
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Fig. 1. Frequency distribution of accessory sperm per embryo or ovum in artificially inseminated single-ovulating cows. Quality and quantity of semen used varied, but was within acceptable standards for commercial artificial insemination. Similar distributions have been reported for individual experiments utilizing both frozen and fresh semen ŽDeJarnette et al., 1992; Nadir et al., 1993..
per ovumrembryo, respectively. The association of accessory sperm number per ovumrembryo to fertilization status and embryo quality is of reproductive importance ŽTable 1.. Clearly, unfertilized ova are ‘‘sperm hungry’’ having a median accessory sperm number of 0. These data also show that embryo quality is positively related to accessory sperm number Žmost evident by comparing the median accessory sperm number per embryo with embryo quality.. This has been interpreted to indicate that the larger accessory sperm numbers per embryo are most likely associated with higher embryo quality because they represent increasing competition among potential fertilizing sperm ŽDeJarnette et al., 1992; Nadir et al., 1993.. If true, this supports the concept offered by Howard et al. Ž1993. regarding the role of the zona pellucida in selection against misshapen sperm. The concept also further incriminates sperm with subtly misshapen heads as ‘‘uncompensable’’ or incompetent in sustaining normal embryogen-
Table 1 Relationship of accessory sperm per embryorovum to fertilization status and embryo quality Ž ns927. Embryo quality based upon Lindner and Wright Ž1983. as modified for degenerate embryos by DeJarnette et al. Ž1992. Fertilization statusrembryo quality
n
Mean"SD
Median
Excellentrgood Fairrpoor Degenerate DegrUFO Unfertilized
449 213 80 12 173
24.5"44.1 17.2"32.2 13.5"38.1 2.7"5.7 1.6"16.5
7 5 1 0.5 0
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Fig. 2. Histogram showing the numbers of accessory sperm required to maximize embryo quality index for 6-day-old embryos Žmorulae. derived from artificial insemination of single-ovulating cows. Embryo grading was according to Lindner and Wright Ž1983. as modified by DeJarnette et al. Ž1992.. Embryo quality index was the average embryo quality based upon the numerical score listed above. A minimum of 10–20 accessory sperm per embryo was required to maximize embryo quality index. The number within each bar is the number of embryos recovered in that accessory sperm category.
esis. Regardless of the mechanism, there appears to be a potential interaction between compensable and uncompensable traits in semen. By increasing sperm numbers at the ovum, embryo quality is favored. Soede et al. Ž1995. found a similar relationship in swine. Using the same data set as in Fig. 1, but only embryos Žunfertilized ova and degeneraterunfertilized removed., the number of sperm necessary for optimum embryo quality was calculated ŽFig. 2.. In this model, between 10 and 20 accessory sperm per embryo are required for embryo quality to be optimized. Since the median value from all inseminations in our studies was 2.4 sperm per ovumrembryo and the median for all embryos was approximately 5.9 sperm per embryo, this would indicate that successful strategies to increase accessory sperm would prove beneficial to pregnancy rate. Fig. 2 also shows that once embryo quality is optimized, further increases in accessory sperm number are without effect. This indirectly suggests that, up to the morula stage of development Žday 6., polyspermy at fertilization in vivo does not seem to be a factor affecting embryonic development in the bovine. 3.2. Factors affecting accessory sperm numbers and efforts to raise accessory sperm numbers per oÕum r embryo Efforts to raise accessory sperm numbers as well as the determination of factors influencing their numbers have been frustrating. In artificially inseminated cattle, efforts without effect include: blockage of retrograde sperm loss at insemination ŽDeJarnette et
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al., 1992., use of fresh vs. frozen semen ŽNadir et al., 1993., semen extender composition Žmilk vs. egg yolk buffer. ŽDalton et al., 1994. and ipsilateral vs. contralateral cornual insemination relative to ovulation ŽHawk and Tanabe, 1986.. Addition of homologous seminal plasma to ejaculated and cauda epididymal sperm was also without effect ŽNadir et al., 1996.. In swine, seminal plasma added to frozen inseminates improved accessory sperm number, presumably via improved sperm transport ŽWeitze et al., 1990.. Waberski et al. Ž1995. have shown that seminal plasma administered at estrus onset advanced time of ovulation in this species. Microencapsulation of bovine sperm, designed to prolong availability of sperm in the inseminate, reduced accessory sperm number ŽMunkittrick et al., 1992. as did superovulation ŽHawk and Tanabe, 1986; Saacke et al., 1998a.. Accessory sperm numbers could be improved most dramatically by use of specific males or by raising the sperm dosage to very high levels Ž100 = 10 6 cells. ŽNadir et al., 1993.. To a lesser extent, insemination adjacent to the uterotubal junction improved accessory sperm over conventional deposition in the uterine body ŽDalton et al., 1999.. Our most recent research examines the effect of natural service and time of artificial insemination on accessory sperm numbers and fertilization statusrembryo quality. At the time of this writing, the work has only been presented in abstract form ŽDalton et al., 1998.. Thus, I will give some of the more relevant experimental details. Our evaluation of natural service was simply to ask the questions Ž1. ‘‘what did nature intend’’ the accessory sperm number per ovumrembryo to be and Ž2. how do we compare using contemporary methods and dosages in artificial insemination? For accessory sperm comparison, two studies Žutilizing the same 3 males. were conducted and the data were combined for presentation in Table 2. In these experiments, all cows were continuously monitored for behavioral estrus by HeatWatchw which utilizes radio frequency data communications. Transmitters were contained in a nylon pouch, glued to the hair of the sacral region, and were activated by continuous pressure Ž2 s. from a mounting herdmate. Data transmitted to the computer included transmitter number, date, time, and duration of standing events. On this basis, exact time of first mount could be recorded and was used to mark estrus onset. In lactating Holsteins, ovulation occurs 27.6 " 5.4 h following the first mount using this system ŽWalker et al., 1996.. Experimental insemination was at 0 h Žestrus onset., 12 h, or 24 h following the first mount. However, due to logistics associated with monitoring
Table 2 Effect of artificial insemination time and natural service on accessory sperm per embryo or ovum Žbreeding time post onset of estrus based on HeatWatch System w . ŽDalton et al., 1998. Treatment a
n
Mean"SD
Median
Range
Natural service 0 h AI 12 h AI 24 h AI
37 53 53 32
74"100 12"30 28"53 34"49
27 0 2 7
0–340 0–162 0–216 0–209
a
Natural services1 intromission, averaging 2.3 h post first mount Ž0 h. or AI at 0 h or 12 h or 24 h following first mount; actual time of inseminations at 2.0"0.9, 12.1"0,6 and 24.2"0.7 h, respectively.
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the computer every 3 h followed by retrieving the cow for insemination, actual insemination times were: 2.0 "0.9 h, 12.1 " 0.6 and 24.2 " 0.7 h following first mount, respectively. Natural service was used in the first experiment only at 0 h. Actual time of service was 2.3 " 0.7 h following first mount. Six days following insemination, the embryo was recovered non-surgically and examined for fertilization statusrembryo quality and numbers of accessory sperm according to previously published methods ŽDeJarnette et al., 1992.. Artificial insemination was to one of three bulls used at random and balanced by number of resulting embryosrova recovered for each insemination interval. From the data in Table 2, it is clear that natural service delivered more sperm to the ovum than did artificial insemination at any interval. However, only three bulls were used and bull variation in accessory sperm number is large Žmedian number of sperm per ovumrembryo by natural service for the three bulls was 73, 37 and 2. despite the billions of cells delivered per service. Using artificial insemination Ž25 = 10 6 sperm per dose, frozen semen. accessory sperm number per ovumrembryo was favored by breeding later, rather than earlier ŽTable 2.. In the second experiment, only artificial insemination was employed. Fertilization rate and embryo quality are presented in Fig. 3 for each insemination interval Ž0, 12 and 24 h post onset of estrus.. From Fig. 3 it is clear that fertilization rate followed accessory sperm number as expected ŽTable 2., being highest at the 24 h insemination. However, examination of embryo quality in relation to time of insemination shows a shift from high quality embryos achieved by inseminations at onset of estrus to low
Fig. 3. Effect of time of insemination on fertilization status and embryo quality judged 6 days following insemination Ž ns117., adapted from Dalton et al. Ž1998..
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quality embryos resulting from insemination at 24 h following estrus onset. On the basis of these data, it appears that we are obtaining a reproductive compromise in pregnancy rate to our current techniques and recommendations for artificial insemination. This compromise is illustrated in Fig. 4 where success in breeding early appears to be limited by sperm life leading to fertilization failure and breeding late is limited by declining embryonic quality. The basis for pregnancy failure by breeding late Ž24 h post heat onset. could reside in the fact that we would often have an aging ovum awaiting sperm arrival. In this scenario we assume ovulation did occur 27.6 " 5.4 h post heat onset and that sustained sperm transport to the site of fertilization requires a minimum of 4 to 8 h in the cow ŽHunter and Wilmut, 1984.. On the other hand, the high embryo quality associated with early insemination suggests that duration of sperm residence in the female tract may result in exertion of additional selection pressure favoring fertilization by a more competent sperm, particularly when there are uncompensable sperm in the semen. Clearly, more research is necessary to determine if this model ŽFig. 4. deserves further consideration. If this model of response to insemination time is correct in the optimization of fertilization rate vs. optimization of embryonic quality, future research efforts toward improving pregnancy rates to artificial insemination need revising. For one example, future efforts in refining semen cryopreservation methods or evaluating semen extender additives would be best served by insemination early in estrus where accessory sperm numbers and fertilization rates clearly are the limiting factor to optimum pregnancy rate and where embryonic quality is highest. Historically, new technology has been tested by inseminating at mid to late estrus where this model would indicate pregnancy rate is limited by the aging ovum or insufficient time for sperm selection, not by low fertilization rate or accessory sperm number. This work also underlines the importance of distinguishing between fertilization failure and embryonic failure in addressing problems relative to pregnancy rate.
Fig. 4. Calculated pregnancy rate from data presented in Fig. 3 based upon the ability of embryos classified excellent–degenerate to constitute a pregnancy Žaccording to Lindner and Wright, 1983.. AI as a compromise is based upon early inseminations being inadequate due to high levels of unfertilized ova, and late inseminations characterized by poor embryo quality. However, high embryo quality appears to be associated with early insemination and high fertilization rates are associated with late inseminations.
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4. Conclusions Seminal traits important to reproductive efficiency in vivo can be considered to fall into two major categories: Ž1. those important to sperm transport and function in the female reproductive tract up to and including initiation of the fertilization process and the block to polyspermy Žcompensable traits. and Ž2. those important to the maintenance of the fertilization event and subsequent embryogenesis, once initiated Žuncompensable.. Both sperm viability and morphology are important to the compensable traits because aberrations in either result in complete or partial exclusion at several barriers in the female tract of which the zona pellucida may be the most formidable. Differences among males or semen samples exist with respect to accessibility of sperm to the ovum that cannot be explained by conventional sperm viability or morphological assessment. Spermatozoal traits at the functional or molecular level important to colonization of the oviduct and to binding and traversing the ovum vestments remain to be identified before we have a full appreciation of the compensable factors. Uncompensable traits affecting embryo quality have been associated with errors in spermatozoal chromatin. The errors appear to be most important in morphologically normal or near-normal spermatozoa that have been shown to access the ovum in vivo. Although use of semen with morphologically misshapen or vacuolated sperm heads results in higher frequency of low quality embryos, recognition of deficiencies in the incompetent fertilizing sperm has not been achieved. However, the positive association of accessory sperm number and embryo quality indicates that competition at the zona pellucida favors fertilization by a more competent sperm and that selection appears to be morphologically based. The 6-day-old bovine embryo Žmorula. can serve as a biomonitor for compensable and uncompensable seminal traits as well as testing reproductive strategies. This non-surgically recovered ovumrembryo provides an approach which separates fertilization rate from embryogenesis, both of which impact pregnancy rate. It also permits quantitative evaluation of accessory sperm, which positively reflects both fertilization rate and embryo quality, thus providing a basis to judge reproductive strategies. Using this approach, the optimum time of artificial insemination in cattle appears to be a compromise. Early insemination results in low fertilization rates Žand low accessory sperm number., but good embryo quality, whereas, late insemination results in high fertilization rates Žhigh accessory sperm number., but poor embryo quality. Additional research is necessary to substantiate this model. Nevertheless, our current data suggests that future research designed to improve results to artificial insemination should be tested by breeding early where sperm viability is most limiting and embryo quality is best.
Acknowledgements We very much appreciate the research support of the following organizations for our work on accessory sperm and male fertility reported in this paper: Select Sires, Plain
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City, OH; the National Association of Animal Breeders, Columbia, MO and the Virginia Agriculture Council, Richmond, VA.
References Ax, R.L., Bellin, M.E., Hawkins, H.E., 1996. Utility of measuring seminal heparin binding protein to predict fertility of cattle. In: Proc. 16th Tech. Conf. on Artif. Insem. and Reprod. Nat’l Assoc. Animal Breeders, Columbia, MO. pp. 61–64. Ballachey, B.E., Evenson, D.P., Saacke, R.G., 1988. The sperm chromatin structure assay: relationship with alternate tests of semen quality and heterospermic performance of bulls. J. Androl. 9, 109–115. Barth, A.D., 1992. The relationship between sperm abnormalities and fertility. In: Proc. 14th Tech. Conf. on Artif. Insemin. and Reprod., Nat’l, Assoc. Animal Breeders, Columbia, MO. pp. 47–63. Bedford, J.M., Calvin, H.I., 1974. The occurrence and possible functional significance of s-s crosslinks in sperm heads with particular reference to eutherian mammals. J. Exp. Zool. 188, 137–156. Bellin, M.E., Hawkins, H.E., Ax, R.L., 1994. Fertility of range beef bulls grouped according to presence or absence of heparin-binding proteins in sperm membranes and seminal fluid. J. Anim. Sci. 72, 2441–2448. Cherr, G.N., Ducibella, T., 1990. Activation of the mammalian egg; Cortical granule distribution, exocytosis, and the block to polyspermy. In: Bavister, B.D., Cummins, J., Roldan, E.R.S. ŽEds.., Fertilization in Mammals. Serono Symp. Norwell, MA, pp. 309–330. Coleman, D.A., Dailey, R.E., Leffel, R.E., Baker, R.D., 1987. Estrous synchronization and establishment of pregnancy in bovine embryo transfer recipients. J. Dairy Sci. 70, 858–866. Coulter, G.H., Oko, R.J., Costerton, J.W., 1986. Ultrastructure of ‘‘crater’’ defect of bovine spermatozoa. Theriogenology 9, 165–168. Courot, M., Colas, G., 1986. The role of the male in embryonic mortality Žcattle and sheep.. In: Greenan, J.M., Diskin, M.G. ŽEds.., Embryonic Mortality in Farm Animals. Martinus Nijhoff, Dordrecht, pp. 95–203. Dalton, J.C., Nadir, S., Degelos, S., Bame, J.H., Saacke, R.G., 1994. Effect of cream added to the inseminate on accessory sperm number in cattle. J. Anim. Sci. 72, 172, ŽSuppl. 1.. Dalton, J.C., Nadir, S., Bame, J.H., Noftsinger, M., Nebel, R.L., Saacke, R.G., 1998. Effect of artificial insemination time and natural service on accessory sperm number, fertilization status and embryo quality in cattle. J. Anim. Sci. 76, 239, ŽSuppl. 1.. Dalton, J.C., Nadir, S., Bame, J.H., Saacke, R.G., 1999. Effect of deep uterine insemination on spermatozoal accessibility to the ovum in cattle: a competitive insemination study. Theriogenology 51, 883–890. DeJarnette, J.M., Saacke, R.G., Bame, J.H., Vogler, C.J., 1992. Accessory sperm: their importance to fertility and embryo quality, and attempts to alter their numbers in artificially inseminated cattle. J. Anim. Sci. 70, 484–491. den Daas, J.H.G., DeJong, G., Lansbergen, L., Van Wagtendonk-De Leeuw, A.M., 1998. The relationship between the number of spermatozoa inseminated and the reproductive efficiency of individual bulls. J. Dairy Sci. 81, 1714–1723. Eid, L.N., Lorton, S.P., Parrish, J.J., 1994. Paternal influence on S-phase in the first cell cycle of the bovine embryo. Biol. Reprod. 51, 1232–1237. Evenson, D.P., Darznikiewicz, Z., Melamed, M.R., 1980. Relation of mammalian sperm chromatin heterogeneity of fertility. Science 240, 1131–1134. Eyestone, W.H., First, N.L., 1989. Variation in bovine embryo development in vitro due to bulls. Theriogenology 31, 191–196. Gleldhill, B.L., 1970. Enigma of spermatozoal DNA and male infertility: a review. Am. J. Vet. Res. 31, 539–549. Graham, E.F., 1978. Fundamentals of the preservation of spermatozoa. In: The Integrity of Frozen Spermatozoa. Nat’l Acad. Sci., Washington, DC, pp. 4–44. Hawk, H.W., Tanabe, T.Y., 1986. Effect of unilateral cornual insemination upon fertilization rate in superovulating and single-ovulating cattle. J. Anim. Sci. 63, 551–560.
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Hawk, H.W., Conley, H.H., Wall, R.J., Whitaker, R.O., 1988. Fertilization rates in superovulating cows after deposition of semen on the infundibulum, near the uterotubal junction or after insemination with high numbers of sperm. Theriogenology 29, 1131–1138. Hillery, F.L., Parrish, J.J., First, N.L., 1990. Bull specific effect on fertilization and embryo development in vitro. Theriogenology 33, 249. Howard, J.G., Donoghue, A.M., Johnston, L.A., Wildt, D.E., 1993. Zona pellucida filtration of structurally abnormal spermatozoa and reduced fertilization in teratospermic cats. Biol. Reprod. 49, 131–139. Hunter, R.H.F., 1998. Sperm–epithelial interactions in the isthmus and ampulla of the Fallopian tubes and their ovarian control. In: Lauria, A., Gandolfi, F., Enne, G., Gianaroli, L. ŽEds.., Gametes: Development and Function. Serono Symp., Milano, pp. 355–367. Hunter, R.H.F., Wilmut, I., 1984. Sperm transport in the cow: periovulatory redistribution of viable cells within the oviduct. Reprod. Nutr. Dev. 24, 597–603. Hunter, R.H.F., Vajta, G., Hyttel, P., 1998. Long-term stability of the bovine block to polyspermy. J. Exp. Zool. 280, 182–188. Karabinus, D., Vogler, C.J., Saacke, R.G., Evenson, D.P., 1997. Chromatin structural changes in bovine sperm after scrotal insulation of Holstein bulls. J. Androl. 18, 549–555. Killian, G.J., Chapman, D.A., Rogowski, L.A., 1993. Fertility-associated proteins in Holstein bulls. Biol. Reprod. 49, 1202–1208. Killian, G.J., Chapman, D.A., Cash, E., Griel, L., Allen, C.H., Gilbert, G., Kaproth, M., Lenz, R., Marshall, C.E., 1996. Conditions which affect the abundance of fertility associated proteins in seminal plasma. In: Proc. 16th Tech. Conf. Artif. Insem. and Reprod., the Nat’l Assoc. Animal Breeders, Columbia, MO. pp. 57–64. Koeford-Johnsen, H.H., 1972. Cervical secretions as a selective filter for abnormal types of spermatozoa. Arsberetnig Inst. For Sterilitetsforskning, Konelige Veterinaer-og Landbohojskole 15, 171–176. Kosower, N.S., Katayose, H., Yanagamachi, R., 1992. Thiol–disulfide status and acridine orange fluorescence of mammalian sperm nuclei. J. Androl. 13, 342–348. Krzanowski, H., 1974. The passage of abnormal spermatozoa through the uterotubal junction of the mouse. J. Reprod. Fertil. 38, 81–90. Lagerlof, N., 1934. Morphological studies on the changes in the sperm structure and in the testes of bulls with decreased or abolished fertility. Acta Path. Microbiol. Scand. 19, 254–267. Larsson, B., Larsson, K., 1986. Sperm localization in the oviducts of artificially inseminated dairy cattle. Acta Vet. Scand. 27, 303–312. Lefebvre, R., Lo, M.C., Suarez, S.S., 1997. Bovine sperm binding to oviductal epithelium involves fucose recognition. Biol. Reprod. 56, 1198–1204. Lindner, G.M., Wright, R.O. Jr., 1983. Bovine embryo morphology and evaluation. Theriogenology 20, 407–411. Linford, E., Glover, F.A., Bishop, C., Stewart, D.L., 1976. The relationship between semen evaluation methods and fertility in the bull. J. Reprod. Fertil. 47, 283–291. Mattner, P.E., 1963. Spermatozoa in the genital tract of the ewe: II. Distribution after coitus. Aust. J. Biol. Sci. 16, 688–693. Miller, D., Hrudka, M., Cates, W.F., Mapletoft, R., 1982. Infertility in a bull with a nuclear sperm defect: a case report. Theriogenology 17, 611–621. Mitchell, J.R., Senger, P.L., Rosenberger, J.L., 1985. Distribution and retention of spermatozoa with acrosomal and nuclear abnormalities in the cow genital tract. J. Anim. Sci. 61, 956–963. Morton, D.B., Glover, J.D., 1974. Sperm transport in the female rabbit: the effect of inseminate volume and sperm density. J. Reprod. Fertil. 38, 139–142. Mullins, J., Saacke, R.G., 1989. Study of the functional anatomy of the bovine cervical mucosa with special reference to mucus secretion and sperm transport. Anat. Rec. 225, 106–117. Munkittrick, T.W., Nebel, R.L., Saacke, R.G., 1992. Effect of microencapsulation on accessory sperm in the zona pellucida. J. Dairy Sci. 75, 725–731. Nadir, S., Dalton, J.C., Bame, J.H., Pandolfi, S., Saacke, R.G., 1996. Seminal plasma: how does it aid sperm function in cattle? In: Proc. 16th Tech. Conf. Artif. Insem. and Reprod. Nat’l Assoc. Animal Breeders, Columbia, MO. pp. 65–70.
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Nadir, S., Saacke, R.G., Bame, J.H., Mullins, J., Degelos, S., 1993. Effect of freezing semen and dosage of sperm on number of acessory sperm, fertility and embryo quality in artificially inseminated cattle. J. Anim. Sci. 71, 199–204. Oltenacu, E.A.B., Foote, R.H., 1976. Monitoring fertility of AI programs: can non-return rate do the job? In: Proc. 6th Tech. Conf. Artif. Insem. and Reprod. Nat’l. Assoc. Animal Breeders, Columbia, MO. pp. 25–33. Orgebin-Crist, M., Jahad, C., 1977. Delayed cleavage of rabbit ova after fertilization by young epididymal spermatozoa. Biol. Reprod. 16, 358–363. Overstreet, J.W., Cooper, W., Katz, D.F., 1978. Sperm transport in the reproductive tract of the female rabbit: II. The sustained phase of transport. Biol. Reprod. 19, 115–132. Pace, M.M., Sullivan, J.J., Elliott, F.I., Graham, E.F., Coulter, G.H., 1981. Effects of thawing temperature, number of spermatozoa and spermatozoal quality on fertility of bovine spermatozoa packaged in 0.5-ml French straws. J. Anim. Sci. 53, 693–701. Qiu, J., Hales, B.F., Robaire, B., 1995. Effects of chronic low-dose cyclophosphamide exposure on the nuclei of rat spermatozoa. Biol. Reprod. 52, 33–40. Saacke, R.G., DeJarnette, J.M., Bame, J.H., Karabinus, D.S., Whitman, S., 1998a. Can spermatozoa with abnormal heads gain access to the ovum in artificially inseminated super- and single-ovulating cattle? Theriogenology 51, 117–128. Saacke, R.G., Dalton, J.C., Nadir, S., Bame, J., Nebel, R.L., 1998b. Spermatozoal characteristics important to sperm transport, fertilization and early embryonic development. In: Lauria, A., Gandolfi, F., Enne, G., Gianaroli, L. ŽEds.., Gametes: Development and Function. Serono Symp., Milano, pp. 320–335. Saacke, R.G., Nadir, S., Dalton, J.C., Bame, J.H., DeJarnette, J.M., Degelos, S., Nebel, R.L., 1994. Accessory sperm evaluation and bull fertility: an update. Proc. 15th Tech. Conf. Artif. Insem. and Reprod. Nat’l. Assoc. Animal Breeders, Columbia, MO, 57–67. Sakkas, D., Manicardi, G., Bianchi, P.G., Bizzaro, D., Bianchi, U., 1995. Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa. Biol. Reprod. 52, 1140–1155. Sakkas, D., Urner, F., Bianchi, P.G., Bizzaro, D., Wagner, I., Jaquenoud, N., Manicardi, G., Campana, A., 1996. Sperm Chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Human Reprod. 11, 837–843. Sakkas, D., Mariethoz, E., Manicardi, G., Bizzaro, D., Bianchi, P.G., Bianchi, U., 1999. Origin of DNA damage in ejaculated human spermatozoa. Rev. Reprod. 4, 31–37. Setchell, B.P., 1998. Heat and the testis. J. Reprod. Fertil. 114, 179–194. Setchell, B.P., Occhio, M.J., Hall, M.S., Lourie, M.S., Tucker, M.J., Zupp, J.L., 1988. Is embryonic mortality increased in normal female rats mated to subfertile males? J. Reprod. Fertil. 83, 567–574. Shi, K.S., Lu, K.H., Gordon, I., 1990. Effect of bulls on fertilization of bovine oocytes and their subsequent development in vitro. Theriogenology 33, 324. Soede, N.M., Wetzels, C.C.H., Zondag, W., Hazeleger, W., Kemp, B., 1995. Effects of a second insemination after ovulation on fertilization rate and accessory sperm count in sows. J. Reprod. Fertil. 105, 135–140. Suarez, S.S., Dai, X., 1992. Hyperactivation enhances mouse sperm capacity for penetrating viscoelastic media. Biol. Reprod. 46, 686–691. Vogler, C.J., Saacke, R.G., Bame, J.H., DeJarnette, J.M., McGilliard, M.L., 1991. Effects of scrotal insulation on viability characteristics of cryopreserved bovine semen. J. Dairy Sci. 74, 3827–3835. Vogler, C.J., Bame, J.H., DeJarnette, J.M., McGilliard, M.L., Saacke, R.G., 1993. Effects of elevated testicular temperature on morphology characteristics of ejaculated spermatozoa in the bovine. Theriogenology 40, 1207–1219. Waberski, D., Sudhoff, H., Hahn, T., Jungblut, P.W., Kallweit, E., Calvete, J.J., Ensslin, M., Happen, H.O., Wintergalen, N., Weitze, K.F., Topfer-Petersen, E., 1995. Advanced ovulation in gilts by the intrauterine application of a low molecular mass pronase-sensitive fraction of boar seminal plama. J. Reprod. Fertil. 105, 247–254. Walker, W.L., Nebel, R.L., McGilliard, M.L., 1996. Time of ovulation relative to mounting activity in dairy cattle. J. Dairy Sci. 79, 1555–1561. Weitze, K.F., Rabeler, J., Willmen, T., Waberski, D., 1990. Interaction between inseminate, uterine and
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ovarian function in the sow. I. Influence of seminal plasma and estrogens in the inseminate on intragenital transport, time of ovulation and fertility in gilts. Reprod. Domest. Anim. 25, 191–196. Williams, W.W., Savage, A., 1925. Observations on the seminal micropathology of bulls. Cornell Vet. 15, 353–375. Williams, W.W., Savage, A., 1927. Methods of determining the reproductive health and fertility of bulls. A review with additional notes. Cornell Vet. 17, 374–376.
Relationship of seminal traits and insemination time to fertilization rate and embryo quality R.G. Saacke ) , J.C. Dalton, S. Nadir, R.L. Nebel, J.H. Bame Department of Dairy Science, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061-0315, USA
Abstract The nature of subfertility due to the male or inseminate is as complex as that of the female. Fertilization failure or failure in embryogenesis are both documented to be of seminal origin. Males also differ in the numbers of sperm required to reach their maximum fertilization rate. Males requiring more sperm would be considered to have compensable seminal deficiencies. These include a number of known Žviability and morphology. and unknown factors Žfunctional or molecular traits. precluding sperm access to the ovum or ability to engage the ovum sufficiently to initiate fertilization and the block to polyspermy. Differences in fertility among males or inseminates independent of sperm dosage are considered uncompensable. These deficiencies would be associated with fertilizing sperm that are incompetent to maintain the fertilization process or subsequent embryogenesis once initiated, with most failures occurring prior to maternal recognition of pregnancy. Such sperm would preempt fertilization by competent sperm. Chromatin aberrations in morphologically normal or near normal spermatozoa from abnormal semen samples appear to be the best candidates for the uncompensable deficiency. However, recognition of uncompensable or incompetent fertilizing sperm has not been achieved. Six-day-old non-surgically recovered bovine ovarembryos have been used to evaluate compensable and uncompensable seminal deficiencies as well as to test reproductive strategies. These ovarembryos provide information on fertilization status and embryo quality as well as quantitative and qualitative data regarding associated accessory sperm. Thus, they permit the separation of reproductive failure by fertilization from that by embryonic development. Accessory sperm number is positively associated with both fertilization rate and embryonic quality. Early insemination results in low fertilization rates Žlow accessory sperm number., but good embryo quality, whereas, late insemination results in high fertilization rates Žhigh accessory sperm number., but poor embryo quality. Additional studies will be necessary to substantiate this model; however, if true, future research
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Corresponding author.
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 1 3 7 - 8
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designed to improve results to artificial insemination should be tested by breeding early in estrus where sperm viability is most limiting and embryo quality is best. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Semen quality; Fertility; Embryo quality; Accessory sperm
1. Introduction There is a wealth of published research showing positive relationships of seminal traits, as measured in the laboratory, to fertility. It is clear, however, that the modest correlations obtained leave little doubt that we still lack sufficient knowledge to predict the outcome of matings based upon seminal quality as currently judged. Sources of the problem include repeatability of our laboratory tests ŽGraham, 1978., our ability to accurately measure fertility ŽOltenacu and Foote, 1976. as well as clearly establish the existence of a variance in fertility among semen samples tested ŽLinford et al., 1976.. Another aspect of the problem is our tendency to oversimplify the biological nature of subfertility due to the male or inseminate, often assuming that a single test or pair of tests will embrace the majority of factors important to fertility. Therefore, I would like to orient this paper toward our growing understanding of the nature of reproductive failure due to the male or inseminate. If we understand the principles involved, we then should be able to place laboratory evaluation of semen traits in proper perspective and improve our ability to reach the stage of predicting pregnancy rate.
2. Compensable and uncompensable seminal deficiencies It is now recognized that seminal deficiencies reflected in reduced pregnancy rates can be categorized as compensable or uncompensable. Seminal differences among males in numbers of sperm required to reach their respective maximum pregnancy rates are considered compensable. Once the maximum pregnancy rate of a male is met and further sperm inseminated are without effect, differences in pregnancy rates due to the male are then considered uncompensable. 2.1. Compensable semen traits Pace et al. Ž1981. showed viability traits of sperm that could be considered compensable included percent progressively motile sperm and percent of spermatozoa with intact cell or acrosomal membranes. Up to a threshold, they demonstrated that it was the number, and not percent of such cells in the inseminate that was important to pregnancy rate. Overstreet et al. Ž1978. demonstrated that the population of sperm accessing the site of fertilization under the sustained sperm transport phase Žpotential fertilizing sperm. was nearly 100% live. Immotile sperm ŽMattner, 1963; Overstreet et al., 1978; Mullins and Saacke, 1989. or sperm with tail defects or protoplasmic droplets ŽKoeford-Johnsen, 1972; Mitchell et al., 1985. are markedly impaired at barriers in the
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female tract precluding their access to the site of fertilization in the oviducts. Considering inseminated spermatozoa with misshapen heads, only those with the most subtle distortions can cross barriers such as the uterotubal junction ŽKrzanowski, 1974. or appear as accessory sperm ŽSaacke et al., 1998a.. Morphologically, spermatozoa appear to be excluded from contact with the ovum in vivo, more by severity rather than type of head distortion. Clearly, there are compensable seminal traits that cannot be explained by the conventional assessments mentioned above. In the bovine, males can differ 10-fold or more in ability to access the ovum in vivo based upon fertility at low insemination dose Žden Daas et al., 1998., or based on accessory sperm numbers in 6-day-old recovered ova and embryos ŽNadir et al., 1993.. These differences have not been explained by conventional viability and morphology semen tests. Undoubtedly important are functional changes of sperm in the oviducts involving motility patterns or molecular events on the surface of sperm associated with capacitation, spermregg recognition or the acrosome reaction. For penetration of the viscoelastic zona pellucida, hyperactivated rather than progressively motile sperm are thought to be critical ŽSuarez and Dai, 1992.. Potential fertilizing sperm which colonize the lower oviductal isthmus by binding to the mucosa, may also be quantitatively important ŽLefebvre et.al., 1997.. Similarly, molecular changes at the sperm surface may involve seminal plasma proteins ŽKillian et al., 1993, 1996. or seminal plasma-related sperm surface binding properties ŽBellin et al., 1994; Ax et al., 1996. that have been positively related to fertility in bulls. Numbers of sperm reaching the site of fertilization are small in relation to the number inseminated ŽHunter and Wilmut, 1984.. Governing this number are many factors involving the physiology of the Fallopian tube and the adjacent ovary which appear to work in concert regarding cueing of sperm to the ovum Žsee Hunter, 1998 for concepts and review.. However, there is also a positive relationship between the number of sperm inseminated and the number of sperm reaching the oviduct ŽMorton and Glover, 1974; Larsson and Larsson, 1986. as well as with the number of sperm at the ovum competing for fertilization based upon quantification of accessory sperm ŽNadir et al., 1993. or numbers of ova with accessory sperm ŽHawk et al., 1988.. The importance of compensable traits to artificial insemination is the fact that such traits govern the minimal numbers of sperm required for an inseminate to reach maximum fertility. 2.2. Uncompensable semen traits Observations in a variety of species indicate that factors associated with lowered sperm quality as measured by viability and morphology, result in low embryo quality or very early embryonic failure prior to maternal recognition of pregnancy ŽBarth, 1992; Courot and Colas, 1986; DeJarnette et al., 1992; Orgebin-Crist and Jahad, 1977; Setchell et al., 1988, see Setchell, 1998 for review.. Clearly, semen or sperm traits leading to this reproductive wastage suggests spermatozoal incompetence after fertilization. Such a deficiency could not be eliminated by sperm dosage alone, thus, the suggestion that the deficiency is uncompensable. Differences among bulls in embryonic development of their conceptuses have been reported at the time of routine recovery for embryo transfer
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ŽMiller et al., 1982. and after observation of embryo survival in recipients ŽColeman et al., 1987.. Bulls were also shown to differ in the development of their embryos following in vitro fertilization ŽEyestone and First, 1989; Hillery et al., 1990; Shi et al., 1990; Eid et al., 1994.. In low fertility bulls, early cleavage rates were reduced and pronuclear formation was delayed ŽEid et al., 1994.. This represents strong evidence for the uncompensable component in the low fertility bulls. Incompetent fertilizing sperm that can be labeled as uncompensable have not been identified using contemporary laboratory semen tests. However, sperm with subtly misshapen heads as well as those with nuclear vacuoles Žcraters, diadem, or nuclear pouches, defined by Coulter et al., 1986. on otherwise normally shaped heads are known to access the ovum following artificial insemination Žas determined from accessory sperm, Saacke et al., 1998a.. In addition, semen with sperm having these traits has been shown to yield higher frequencies of low quality embryos and lower fertilization rates than controls where such traits are missing or minimized ŽMiller et al., 1982; DeJarnette et al., 1992; Saacke et al., 1994.. Morphologically, abnormal sperm in semen of bulls has been associated with subfertility and sterility for many years ŽWilliams and Savage, 1925, 1927; Lagerlof, 1934.. We now recognize that sperm with classically misshapened heads, described by these early workers using simple microscopes, do not traverse the female reproductive tract or participate in fertilization ŽSaacke et al., 1998a.. In addition, the zona pellucida itself appears to provide one of the most formidable barriers against sperm with misshapen heads ŽHoward et al., 1993.. On this basis, a majority of abnormal sperm would be considered a compensable deficiency. However, males having disturbances in spermatogenesis resulting in ejaculation of abnormal sperm usually provide a broad spectrum in severity of morphological forms dependent upon the stage of spermatogenesis affected by the disturbance ŽVogler et al., 1991, 1993.. Thus, we believe that recognition of classically abnormal sperm in semen may represent the ‘‘tip of the iceberg’’ where disturbances in spermatogenesis signified by these sperm extend to otherwise normal or near-normal appearing sperm in the same ejaculates. These normal and near-normal appearing sperm that can access the ovum in vivo are the most likely candidates for the ‘‘uncompensable’’ traits causing pregnancy wastage through very early embryonic death. Finally, it should be recognized that sperm with microscopically normal morphology but with defective chromatin have been implicated in cases of male subfertility for some time ŽGleldhill, 1970.. The chromatin structure assay developed by Evenson et al. Ž1980. revealed a strong positive association between heterospermic fertility in bulls Žbased upon genetic markers at birth. and stability of the sperm DNA to acid denaturation ŽBallachey et al., 1988.. Using this same assay, Karabinus et al. Ž1997. have shown that sperm ejaculated before a mild thermal insult of the testis by scrotal insulation have more stable DNA than those ejaculated after scrotal insulation where abnormal sperm are also evident. Flaws in packaging and condensation of sperm chromatin during spermiogenesis ŽSakkas et al., 1995 and 1996. has been speculated to be involved in the instability of DNA of subfertile semen. The instability of the DNA may be due to limitations in disulfide bonding within nuclear proteins ŽKosower et al., 1992., a process that continues throughout spermiogenesis and epididymal maturation ŽBedford and
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Calvin, 1974.. Such flaws from a testicular perturbation such as elevated testis temperature early in spermiogenesis would be expected to influence sperm head shape; however, the same perturbation late in spermiogenesis or during epididymal maturation could impact DNA condensation without effect on head shape, which is already set ŽVogler et al., 1993.. Flaws in sperm DNA have also been incriminated in altered kinetics of nuclear decondensation associated with pronuclear formation following fertilization ŽQiu et al., 1995.. Regarding the embryological impact of an incompetent sperm, this timing agrees quite well with the uncompensable component in bulls affecting kinetics of rate of pronuclear formation and early cleavage in the bovine ŽEid et al., 1994. discussed earlier. Potentially underlying normal condensation and decondensation of DNA in sperm formation, maturation, and subsequent fertilization are optimum disulfide–thiol relationships within the nuclear protein. These relationships, in turn, are dependent upon the redox status of the sperm environment at each step Žfor reviews of this concept, see Sakkas et al., 1999; Levine, 1996.. Clearly, chromatin status in the sperm head presents a strong focal point for research designed to reveal the ‘‘uncompensable’’ component in semen causing early pregnancy wastage.
3. Ova r embryos and associated accessory sperm as biomonitors of seminal traits and reproductive strategy In the bovine, accessory sperm are those sperm trapped in the zona pellucida of the ovum or embryo. Once the fertilizing sperm enters the vitellus, the ovum reacts precluding further penetration and binding by additional sperm Žfor review, Cherr and Ducibella, 1990.. In ovarembryos Žpresumed morula in embryonic development. recovered on day 6 post insemination, accessory sperm are thought to represent the number of sperm competing for fertilization during the time the ovum was receptive. This implies that accessory sperm fulfill the structural and physiological requirements necessary for traversing the female tract as well as ovum recognition, binding and partial penetration. Clearly, it is not that simple. Quantitatively, the window of time the ovum is receptive could also vary among females due to permissiveness of the zona pellucida to sperm penetration as well as the speed of the block to polyspermy following sperm penetration. However, once established, the block to polyspermy in the cow is quite stable ŽHunter et al., 1998.. In addition, accessory sperm number per ovumrembryo could also be influenced by rate of penetration of the fertilizing sperm Žperhaps unique to a given male.. Nevertheless, accessory sperm do provide, in quality and quantity, insight to the characteristics of potential fertilizing sperm Žfor review, Saacke et al., 1998b.. 3.1. Interaction of compensable and uncompensable seminal deficiencies Through several years of experimentation in our laboratory, we recovered nearly 1000 single-ovulated ovarembryos 6 days post artificial insemination. Twenty-five bulls, all of which provided acceptable semen, are represented in this data. Fig. 1 shows the distribution of accessory sperm found in the zonae of embryos and ova from these cows as being very skewed, having a mean, median and mode of 12.0, 2.4, and 0 sperm
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Fig. 1. Frequency distribution of accessory sperm per embryo or ovum in artificially inseminated single-ovulating cows. Quality and quantity of semen used varied, but was within acceptable standards for commercial artificial insemination. Similar distributions have been reported for individual experiments utilizing both frozen and fresh semen ŽDeJarnette et al., 1992; Nadir et al., 1993..
per ovumrembryo, respectively. The association of accessory sperm number per ovumrembryo to fertilization status and embryo quality is of reproductive importance ŽTable 1.. Clearly, unfertilized ova are ‘‘sperm hungry’’ having a median accessory sperm number of 0. These data also show that embryo quality is positively related to accessory sperm number Žmost evident by comparing the median accessory sperm number per embryo with embryo quality.. This has been interpreted to indicate that the larger accessory sperm numbers per embryo are most likely associated with higher embryo quality because they represent increasing competition among potential fertilizing sperm ŽDeJarnette et al., 1992; Nadir et al., 1993.. If true, this supports the concept offered by Howard et al. Ž1993. regarding the role of the zona pellucida in selection against misshapen sperm. The concept also further incriminates sperm with subtly misshapen heads as ‘‘uncompensable’’ or incompetent in sustaining normal embryogen-
Table 1 Relationship of accessory sperm per embryorovum to fertilization status and embryo quality Ž ns927. Embryo quality based upon Lindner and Wright Ž1983. as modified for degenerate embryos by DeJarnette et al. Ž1992. Fertilization statusrembryo quality
n
Mean"SD
Median
Excellentrgood Fairrpoor Degenerate DegrUFO Unfertilized
449 213 80 12 173
24.5"44.1 17.2"32.2 13.5"38.1 2.7"5.7 1.6"16.5
7 5 1 0.5 0
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Fig. 2. Histogram showing the numbers of accessory sperm required to maximize embryo quality index for 6-day-old embryos Žmorulae. derived from artificial insemination of single-ovulating cows. Embryo grading was according to Lindner and Wright Ž1983. as modified by DeJarnette et al. Ž1992.. Embryo quality index was the average embryo quality based upon the numerical score listed above. A minimum of 10–20 accessory sperm per embryo was required to maximize embryo quality index. The number within each bar is the number of embryos recovered in that accessory sperm category.
esis. Regardless of the mechanism, there appears to be a potential interaction between compensable and uncompensable traits in semen. By increasing sperm numbers at the ovum, embryo quality is favored. Soede et al. Ž1995. found a similar relationship in swine. Using the same data set as in Fig. 1, but only embryos Žunfertilized ova and degeneraterunfertilized removed., the number of sperm necessary for optimum embryo quality was calculated ŽFig. 2.. In this model, between 10 and 20 accessory sperm per embryo are required for embryo quality to be optimized. Since the median value from all inseminations in our studies was 2.4 sperm per ovumrembryo and the median for all embryos was approximately 5.9 sperm per embryo, this would indicate that successful strategies to increase accessory sperm would prove beneficial to pregnancy rate. Fig. 2 also shows that once embryo quality is optimized, further increases in accessory sperm number are without effect. This indirectly suggests that, up to the morula stage of development Žday 6., polyspermy at fertilization in vivo does not seem to be a factor affecting embryonic development in the bovine. 3.2. Factors affecting accessory sperm numbers and efforts to raise accessory sperm numbers per oÕum r embryo Efforts to raise accessory sperm numbers as well as the determination of factors influencing their numbers have been frustrating. In artificially inseminated cattle, efforts without effect include: blockage of retrograde sperm loss at insemination ŽDeJarnette et
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al., 1992., use of fresh vs. frozen semen ŽNadir et al., 1993., semen extender composition Žmilk vs. egg yolk buffer. ŽDalton et al., 1994. and ipsilateral vs. contralateral cornual insemination relative to ovulation ŽHawk and Tanabe, 1986.. Addition of homologous seminal plasma to ejaculated and cauda epididymal sperm was also without effect ŽNadir et al., 1996.. In swine, seminal plasma added to frozen inseminates improved accessory sperm number, presumably via improved sperm transport ŽWeitze et al., 1990.. Waberski et al. Ž1995. have shown that seminal plasma administered at estrus onset advanced time of ovulation in this species. Microencapsulation of bovine sperm, designed to prolong availability of sperm in the inseminate, reduced accessory sperm number ŽMunkittrick et al., 1992. as did superovulation ŽHawk and Tanabe, 1986; Saacke et al., 1998a.. Accessory sperm numbers could be improved most dramatically by use of specific males or by raising the sperm dosage to very high levels Ž100 = 10 6 cells. ŽNadir et al., 1993.. To a lesser extent, insemination adjacent to the uterotubal junction improved accessory sperm over conventional deposition in the uterine body ŽDalton et al., 1999.. Our most recent research examines the effect of natural service and time of artificial insemination on accessory sperm numbers and fertilization statusrembryo quality. At the time of this writing, the work has only been presented in abstract form ŽDalton et al., 1998.. Thus, I will give some of the more relevant experimental details. Our evaluation of natural service was simply to ask the questions Ž1. ‘‘what did nature intend’’ the accessory sperm number per ovumrembryo to be and Ž2. how do we compare using contemporary methods and dosages in artificial insemination? For accessory sperm comparison, two studies Žutilizing the same 3 males. were conducted and the data were combined for presentation in Table 2. In these experiments, all cows were continuously monitored for behavioral estrus by HeatWatchw which utilizes radio frequency data communications. Transmitters were contained in a nylon pouch, glued to the hair of the sacral region, and were activated by continuous pressure Ž2 s. from a mounting herdmate. Data transmitted to the computer included transmitter number, date, time, and duration of standing events. On this basis, exact time of first mount could be recorded and was used to mark estrus onset. In lactating Holsteins, ovulation occurs 27.6 " 5.4 h following the first mount using this system ŽWalker et al., 1996.. Experimental insemination was at 0 h Žestrus onset., 12 h, or 24 h following the first mount. However, due to logistics associated with monitoring
Table 2 Effect of artificial insemination time and natural service on accessory sperm per embryo or ovum Žbreeding time post onset of estrus based on HeatWatch System w . ŽDalton et al., 1998. Treatment a
n
Mean"SD
Median
Range
Natural service 0 h AI 12 h AI 24 h AI
37 53 53 32
74"100 12"30 28"53 34"49
27 0 2 7
0–340 0–162 0–216 0–209
a
Natural services1 intromission, averaging 2.3 h post first mount Ž0 h. or AI at 0 h or 12 h or 24 h following first mount; actual time of inseminations at 2.0"0.9, 12.1"0,6 and 24.2"0.7 h, respectively.
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the computer every 3 h followed by retrieving the cow for insemination, actual insemination times were: 2.0 "0.9 h, 12.1 " 0.6 and 24.2 " 0.7 h following first mount, respectively. Natural service was used in the first experiment only at 0 h. Actual time of service was 2.3 " 0.7 h following first mount. Six days following insemination, the embryo was recovered non-surgically and examined for fertilization statusrembryo quality and numbers of accessory sperm according to previously published methods ŽDeJarnette et al., 1992.. Artificial insemination was to one of three bulls used at random and balanced by number of resulting embryosrova recovered for each insemination interval. From the data in Table 2, it is clear that natural service delivered more sperm to the ovum than did artificial insemination at any interval. However, only three bulls were used and bull variation in accessory sperm number is large Žmedian number of sperm per ovumrembryo by natural service for the three bulls was 73, 37 and 2. despite the billions of cells delivered per service. Using artificial insemination Ž25 = 10 6 sperm per dose, frozen semen. accessory sperm number per ovumrembryo was favored by breeding later, rather than earlier ŽTable 2.. In the second experiment, only artificial insemination was employed. Fertilization rate and embryo quality are presented in Fig. 3 for each insemination interval Ž0, 12 and 24 h post onset of estrus.. From Fig. 3 it is clear that fertilization rate followed accessory sperm number as expected ŽTable 2., being highest at the 24 h insemination. However, examination of embryo quality in relation to time of insemination shows a shift from high quality embryos achieved by inseminations at onset of estrus to low
Fig. 3. Effect of time of insemination on fertilization status and embryo quality judged 6 days following insemination Ž ns117., adapted from Dalton et al. Ž1998..
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quality embryos resulting from insemination at 24 h following estrus onset. On the basis of these data, it appears that we are obtaining a reproductive compromise in pregnancy rate to our current techniques and recommendations for artificial insemination. This compromise is illustrated in Fig. 4 where success in breeding early appears to be limited by sperm life leading to fertilization failure and breeding late is limited by declining embryonic quality. The basis for pregnancy failure by breeding late Ž24 h post heat onset. could reside in the fact that we would often have an aging ovum awaiting sperm arrival. In this scenario we assume ovulation did occur 27.6 " 5.4 h post heat onset and that sustained sperm transport to the site of fertilization requires a minimum of 4 to 8 h in the cow ŽHunter and Wilmut, 1984.. On the other hand, the high embryo quality associated with early insemination suggests that duration of sperm residence in the female tract may result in exertion of additional selection pressure favoring fertilization by a more competent sperm, particularly when there are uncompensable sperm in the semen. Clearly, more research is necessary to determine if this model ŽFig. 4. deserves further consideration. If this model of response to insemination time is correct in the optimization of fertilization rate vs. optimization of embryonic quality, future research efforts toward improving pregnancy rates to artificial insemination need revising. For one example, future efforts in refining semen cryopreservation methods or evaluating semen extender additives would be best served by insemination early in estrus where accessory sperm numbers and fertilization rates clearly are the limiting factor to optimum pregnancy rate and where embryonic quality is highest. Historically, new technology has been tested by inseminating at mid to late estrus where this model would indicate pregnancy rate is limited by the aging ovum or insufficient time for sperm selection, not by low fertilization rate or accessory sperm number. This work also underlines the importance of distinguishing between fertilization failure and embryonic failure in addressing problems relative to pregnancy rate.
Fig. 4. Calculated pregnancy rate from data presented in Fig. 3 based upon the ability of embryos classified excellent–degenerate to constitute a pregnancy Žaccording to Lindner and Wright, 1983.. AI as a compromise is based upon early inseminations being inadequate due to high levels of unfertilized ova, and late inseminations characterized by poor embryo quality. However, high embryo quality appears to be associated with early insemination and high fertilization rates are associated with late inseminations.
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4. Conclusions Seminal traits important to reproductive efficiency in vivo can be considered to fall into two major categories: Ž1. those important to sperm transport and function in the female reproductive tract up to and including initiation of the fertilization process and the block to polyspermy Žcompensable traits. and Ž2. those important to the maintenance of the fertilization event and subsequent embryogenesis, once initiated Žuncompensable.. Both sperm viability and morphology are important to the compensable traits because aberrations in either result in complete or partial exclusion at several barriers in the female tract of which the zona pellucida may be the most formidable. Differences among males or semen samples exist with respect to accessibility of sperm to the ovum that cannot be explained by conventional sperm viability or morphological assessment. Spermatozoal traits at the functional or molecular level important to colonization of the oviduct and to binding and traversing the ovum vestments remain to be identified before we have a full appreciation of the compensable factors. Uncompensable traits affecting embryo quality have been associated with errors in spermatozoal chromatin. The errors appear to be most important in morphologically normal or near-normal spermatozoa that have been shown to access the ovum in vivo. Although use of semen with morphologically misshapen or vacuolated sperm heads results in higher frequency of low quality embryos, recognition of deficiencies in the incompetent fertilizing sperm has not been achieved. However, the positive association of accessory sperm number and embryo quality indicates that competition at the zona pellucida favors fertilization by a more competent sperm and that selection appears to be morphologically based. The 6-day-old bovine embryo Žmorula. can serve as a biomonitor for compensable and uncompensable seminal traits as well as testing reproductive strategies. This non-surgically recovered ovumrembryo provides an approach which separates fertilization rate from embryogenesis, both of which impact pregnancy rate. It also permits quantitative evaluation of accessory sperm, which positively reflects both fertilization rate and embryo quality, thus providing a basis to judge reproductive strategies. Using this approach, the optimum time of artificial insemination in cattle appears to be a compromise. Early insemination results in low fertilization rates Žand low accessory sperm number., but good embryo quality, whereas, late insemination results in high fertilization rates Žhigh accessory sperm number., but poor embryo quality. Additional research is necessary to substantiate this model. Nevertheless, our current data suggests that future research designed to improve results to artificial insemination should be tested by breeding early where sperm viability is most limiting and embryo quality is best.
Acknowledgements We very much appreciate the research support of the following organizations for our work on accessory sperm and male fertility reported in this paper: Select Sires, Plain
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City, OH; the National Association of Animal Breeders, Columbia, MO and the Virginia Agriculture Council, Richmond, VA.
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