. 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
Ž 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
Ž 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. Ova r r
r r
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
Ž 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.
Ž 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
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
Ž 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
Ž .
sperm DeJarnette et al., 1992; Nadir et al., 1993 . If true, this supports the concept Ž
. 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
Ž .
Embryo quality based upon Lindner and Wright 1983 as modified for degenerate embryos by DeJarnette et Ž
. al. 1992
Fertilization statusrembryo quality n
MeanSD Median
Excellentrgood 449
24.544.1 7
Fairrpoor 213
17.232.2 5
Degenerate 80
13.538.1 1
DegrUFO 12
2.75.7 0.5
Unfertilized 173
1.616.5
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
Ž .
Ž .
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
Ž .
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 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
Ž without effect include: blockage of retrograde sperm loss at insemination DeJarnette et
. 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 Ž
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 HeatWatch
w
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 Ž
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
MeanSD Median
Range Natural service
37 74100
27 0–340
0 h AI 53
1230 0–162
12 h AI 53
2853 2
0–216 24 h AI
32 3449
7 0–209
a
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 2.00.9, 12.10,6 and 24.20.7 h, respectively.
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
Ž
. DeJarnette 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 Ž
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
. insemination ns117 , adapted from Dalton et al. 1998 .
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
. 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.
4. Conclusions
