Directory UMM :Data Elmu:jurnal:A:Animal Reproduction Science:Vol60-61.Issue1-4.Jul2000:

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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.

Ž . Ž

These include a number of known viability and morphology and unknown factors functional 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 associ-ated with both fertilization rate and embryonic quality. Early insemination results in low

Ž .

fertilization rates low accessory sperm number , but good embryo quality, whereas, late

insemina-Ž .

tion results in high fertilization rates high accessory sperm number , but poor embryo quality. Additional studies will be necessary to substantiate this model; however, if true, future research

)_{Corresponding author.}

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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.}

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

Ž .

problem include repeatability of our laboratory tests Graham, 1978 , our ability to

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accurately measure fertility Oltenacu and Foote, 1976 as well as clearly establish the

Ž .

existence of a variance in fertility among semen samples tested Linford 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

Ž .

Pace et al. 1981 showed viability traits of sperm that could be considered compens-able 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

Ž .

rate. Overstreet et al. 1978 demonstrated that the population of sperm accessing the Ž

site of fertilization under the sustained sperm transport phase potential fertilizing

. Ž

sperm was nearly 100% live. Immotile sperm Mattner, 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. Consider-ing inseminated spermatozoa with misshapen heads, only those with the most subtle

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distortions can cross barriers such as the uterotubal junction Krzanowski, 1974 or

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appear as accessory sperm Saacke 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.

Ž .

ova and embryos Nadir et al., 1993 . These differences have not been explained by conventional viability and morphology semen tests. Undoubtedly important are func-tional 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

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rather than progressively motile sperm are thought to be critical Suarez and Dai, 1992 . Potential fertilizing sperm which colonize the lower oviductal isthmus by binding to the

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mucosa, may also be quantitatively important Lefebvre et.al., 1997 . Similarly, molecu-Ž

lar changes at the sperm surface may involve seminal plasma proteins Killian et al.,

. Ž

1993, 1996 or seminal plasma-related sperm surface binding properties Bellin 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

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regarding cueing of sperm to the ovum see 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 Morton and Glover, 1974; Larsson and .

Larsson, 1986 as well as with the number of sperm at the ovum competing for

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fertilization based upon quantification of accessory sperm Nadir et al., 1993 or

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numbers of ova with accessory sperm Hawk 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 Barth, 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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Ž_{Miller et al., 1982 and after observation of embryo survival in recipients Coleman et}. Ž .

al., 1987 . Bulls were also shown to differ in the development of their embryos Ž

following in vitro fertilization Eyestone 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

Ž .

pronuclear formation was delayed Eid 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 craters, diadem, or nuclear .

pouches, defined by Coulter et al., 1986 on otherwise normally shaped heads are known Ž

to access the ovum following artificial insemination as 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 Miller et al., 1982; DeJarnette .

et al., 1992; Saacke et al., 1994 .

Morphologically, abnormal sperm in semen of bulls has been associated with Ž

subfertility and sterility for many years Williams 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

Ž .

tract or participate in fertilization Saacke et al., 1998a . In addition, the zona pellucida itself appears to provide one of the most formidable barriers against sperm with

Ž .

misshapen heads Howard 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

spermatogene-Ž .

sis affected by the disturbance Vogler 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

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time Gleldhill, 1970 . The chromatin structure assay developed by Evenson et al. Ž_{1980 revealed a strong positive association between heterospermic fertility in bulls}. Ž_{based upon genetic markers at birth and stability of the sperm DNA to acid denatura-}.

Ž . Ž .

tion Ballachey et al., 1988 . Using this same assay, Karabinus et al. 1997 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

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spermiogenesis Sakkas 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

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limitations in disulfide bonding within nuclear proteins Kosower et al., 1992 , a process Ž

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Calvin, 1974 . Such flaws from a testicular perturbation such as elevated testis tempera-ture 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 Vogler et .

al., 1993 . Flaws in sperm DNA have also been incriminated in altered kinetics of Ž nuclear decondensation associated with pronuclear formation following fertilization Qiu

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

Ž .

of pronuclear formation and early cleavage in the bovine Eid 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 for 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. Ovarrrrrembryos 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 for review, Cherr and

. Ž .

Ducibella, 1990 . In ovarembryos presumed morula in embryonic development recov-ered 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 Hunter et .

al., 1998 . In addition, accessory sperm number per ovumrembryo could also be Ž

influenced by rate of penetration of the fertilizing sperm perhaps unique to a given .

male . Nevertheless, accessory sperm do provide, in quality and quantity, insight to the

Ž .

characteristics of potential fertilizing sperm for 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-ovulat-ing 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

Ž .

and fresh semen DeJarnette 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 Ž_{Table 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 most 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

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sperm DeJarnette et al., 1992; Nadir et al., 1993 . If true, this supports the concept

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offered by Howard et al. 1993 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

Ž .

Relationship of accessory sperm per embryorovum to fertilization status and embryo quality ns927

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Embryo quality based upon Lindner and Wright 1983 as modified for degenerate embryos by DeJarnette et

Ž .

al. 1992

Fertilization statusrembryo quality n Mean"SD Median

Excellentrgood 449 24.5"44.1 7

Fairrpoor 213 17.2"32.2 5

Degenerate 80 13.5"38.1 1

DegrUFO 12 2.7"5.7 0.5

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Fig. 2. Histogram showing the numbers of accessory sperm required to maximize embryo quality index for

Ž .

6-day-old embryos morulae derived from artificial insemination of single-ovulating cows. Embryo grading

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was according to Lindner and Wright 1983 as modified by DeJarnette et al. 1992 . 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

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ovum, embryo quality is favored. Soede et al. 1995 found a similar relationship in Ž

swine. Using the same data set as in Fig. 1, but only embryos unfertilized ova and .

degeneraterunfertilized removed , the number of sperm necessary for optimum embryo

Ž .

quality was calculated Fig. 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

Ž .

development day 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 oÕumrembryo

Efforts to raise accessory sperm numbers as well as the determination of factors influencing their numbers have been frustrating. In artificially inseminated cattle, efforts

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. Ž .

al., 1992 , use of fresh vs. frozen semen Nadir et al., 1993 , semen extender

composi-Ž . Ž .

tion milk vs. egg yolk buffer Dalton et al., 1994 and ipsilateral vs. contralateral

Ž .

cornual insemination relative to ovulation Hawk and Tanabe, 1986 . Addition of homologous seminal plasma to ejaculated and cauda epididymal sperm was also without

Ž .

effect Nadir et al., 1996 . In swine, seminal plasma added to frozen inseminates Ž

improved accessory sperm number, presumably via improved sperm transport Weitze et

. Ž .

al., 1990 . Waberski et al. 1995 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

Ž . Ž

number Munkittrick et al., 1992 as did superovulation Hawk and Tanabe, 1986; .

Saacke et al., 1998a . Accessory sperm numbers could be improved most dramatically

Ž 6

by use of specific males or by raising the sperm dosage to very high levels 100=10

. Ž .

cells Nadir et al., 1993 . To a lesser extent, insemination adjacent to the uterotubal junction improved accessory sperm over conventional deposition in the uterine body Ž_{Dalton 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 Dalton et al., .

1998 . Thus, I will give some of the more relevant experimental details. Our evaluation Ž .

of natural service was simply to ask the questions 1 ‘‘what did nature intend’’ the Ž .

accessory sperm number per ovumrembryo to be and 2 how do we compare using contemporary methods and dosages in artificial insemination? For accessory sperm

Ž .

comparison, two studies utilizing 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

Ž .

continuous pressure 2 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

Ž . Ž .

system Walker et al., 1996 . Experimental insemination was at 0 h estrus 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 breeding

w_{. Ž} _.

time post onset of estrus based on HeatWatch System Dalton et al., 1998 a

Treatment n Mean"SD Median Range

Natural service 37 74"100 27 0–340

0 h AI 53 12"30 0 0–162

12 h AI 53 28"53 2 0–216

24 h AI 32 34"49 7 0–209

Ž .

Natural services1 intromission, averaging 2.3 h post first mount 0 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 insemina-tion 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 median number . of sperm per ovumrembryo by natural service for the three bulls was 73, 37 and 2

Ž 6

despite the billions of cells delivered per service. Using artificial insemination 25=10 .

sperm per dose, frozen semen accessory sperm number per ovumrembryo was favored

Ž .

by breeding later, rather than earlier Table 2 .

In the second experiment, only artificial insemination was employed. Fertilization Ž

rate and embryo quality are presented in Fig. 3 for each insemination interval 0, 12 and .

24 h post onset of estrus . From Fig. 3 it is clear that fertilization rate followed

Ž .

accessory sperm number as expected Table 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

Ž . Ž .

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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 24 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

Ž .

in the cow Hunter 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

Ž .

semen. Clearly, more research is necessary to determine if this model Fig. 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

Ž .

excellent–degenerate to constitute a pregnancy according 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 Ž .

into two major categories: 1 those important to sperm transport and function in the female reproductive tract up to and including initiation of the fertilization process and

Ž . Ž .

the block to polyspermy compensable traits and 2 those important to the maintenance

Ž .

of the fertilization event and subsequent embryogenesis, once initiated uncompensable . 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.

Ž .

The 6-day-old bovine embryo morula 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 fertiliza-tion 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 and low accessory .

sperm number , but good embryo quality, whereas, late insemination results in high

Ž .

fertilization rates high 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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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

Ž

embryonic quality. The basis for pregnancy failure by breeding late 24 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

Ž

.

in the cow Hunter 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

Ž

.

semen. Clearly, more research is necessary to determine if this model Fig. 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

Ž .

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4. Conclusions

Seminal traits important to reproductive efficiency in vivo can be considered to fall

Ž .

into two major categories: 1 those important to sperm transport and function in the

female reproductive tract up to and including initiation of the fertilization process and

Ž

.

Ž .

the block to polyspermy compensable traits and 2 those important to the maintenance

Ž

.

of the fertilization event and subsequent embryogenesis, once initiated uncompensable .

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.

Ž

.

The 6-day-old bovine embryo morula 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

fertiliza-tion 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

Ž

compromise. Early insemination results in low fertilization rates and low accessory

.

sperm number , but good embryo quality, whereas, late insemination results in high

Ž

.

fertilization rates high 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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