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From the Departments of * Dairy Science and
Large Animal Clinical Sciences, Virginia
Polytechnic Institute and State University, Blacksburg, Virginia.
| Correspondence to: Dr F. C. Gwazdauskas, Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0315 (e-mail: guaz{at}vt.edu). |
| Received for publication August 11, 2003; accepted for publication February 4, 2004. |
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Abstract |
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Key words: Percoll, swim-up, abnormal spermatozoa, thermal insult, in vitro fertilization
Kot and Handel (1987) suggested that the interaction between abnormal murine spermatozoa and the oocyte is different from the interaction between normal spermatozoa and the oocyte, resulting in decreased penetration or fertilization and, subsequently, increased embryonic death. Further in vivo bovine studies by Saacke et al (1998) demonstrated that severe and moderately misshapen sperm heads were excluded from participating in fertilization, but subtly misshapen sperm heads appear as accessory sperm on fertilized ova, regardless of their proportion in the inseminate. However, since spermatozoa with the pyriform defect apparently have normal acrosomes and normal motility, they could be expected to penetrate the zona pellucida and enter the ooplasm under IVF conditions. Moreover, Thundathil et al (1999) reported that with bovine IVF, the percentage of pyriform spermatozoa that penetrate the zona pellucida and gain access to the ooplasm was lower than that of normal-shaped sperm. They found that most pyriform-shaped spermatozoa that bound to the zona pellucida also managed to penetrate it, suggesting that pyriform-shaped spermatozoa are primarily discriminated against during zona binding.
Interference with the normal scrotal thermoregulation of bulls resulted in decreased percentages of sperm with normal head and tail morphology and reduced pregnancy rates when they were used for natural mating (Lunstra and Coulter, 1997). Furthermore, Saacke et al (1994, 2000) defined compensable seminal traits as those deficiencies in semen that preclude the availability of sperm for fertilization in vivo but stated that the effect can be minimized by increasing the sperm dosage. This is opposed to the uncompensable seminal deficiencies that cannot be overcome by increased sperm dosages and that most likely impair embryonic development.
For IVF purposes, the removal of extender components, including cryoprotectant, from cryopreserved bovine semen is necessary to obtain a high percentage of normal, viable spermatozoa. Methodological factors such as sperm preparation could easily influence IVF results within the same bull. Several methods for sperm selection have been described, but the most widely used are swim-up and Percoll gradient separation techniques (Rodriguez-Martinez et al, 1997; Palomo et al, 1999). Sapienza et al (1993) and Dode et al (2002) reported no differences between the quality of the swim-up–separated and Percoll-separated spermatozoa. However, other researchers have reported a lower recovery but a better quality of spermatozoa using swim-up, whereas, according to these researchers, Percoll separation resulted in a higher recovery rate and percentage of acrosome-intact spermatozoa (Parrish et al, 1995; Somfai et al, 2002). Moreover, IVF results were not affected when high concentrations of spermatozoa were used, which is contrary to when low concentrations were used (Ward et al, 2003). Furthermore, spermatozoa must undergo capacitation in order to bind to the zona and undergo the acrosome reaction. The exact cellular mechanism by which spermatozoa undergo capacitation is still largely unknown, but several studies have shown that the addition of heparin to the IVF medium is an effective method for inducing capacitation in vitro of bovine sperm and that heparin concentrations can have an effect on IVF results (Parrish et al, 1988, 1995; Saeki et al, 1995).
The effects of sperm preparation procedures and heparin concentrations on bovine IVF are not well established when abnormal sperm are used. Our hypothesis was that a short-term thermal insult caused by scrotal insulation would induce abnormal spermatozoa production that would, in turn, provide semen to test the effects of semen preparation methods and heparin addition when using IVF on early bovine embryonic development. Therefore, the objectives of this study were to 1) evaluate the effect of sperm separation methods on semen samples collected from bulls that were subjected to scrotal insulation (to induce abnormal sperm morphology) on embryonic development after IVF, and 2) determine whether morphologically abnormal and normal bovine spermatozoa would be differentially affected by variation in heparin concentrations.
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Methods |
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Semen Cryopreservation
On each semen collection day, the volume of each ejaculate was determined.
The 2 ejaculates from each bull were pooled and diluted with egg
yolk–citrate extender. Ejaculates were prepared for cryopreservation at
a concentration of 50 x 106 sperm per milliliter with egg
yolk–citrate-glycerol (Robbins et
al, 1976).
Sperm Morphology
The collected semen samples were evaluated for morphological abnormalities
according to the methods of Barth and Oko
(1989). Abnormalities were
classified by defects in the shape of the sperm head (pyriform, tapered, and
asymmetrical) and completely distorted head shapes, plus nuclear vacuolization
(apical vacuoles, diadems, and random vacuoles), which were all classified as
primary abnormalities that, if produced, appeared in a specific chronological
order (Vogler et al, 1993).
Thus, the scrotal insulation provided a means of varying the quality of the
sperm population within bulls as well as across bulls
(Vogler et al, 1993). The
bulls were then ranked according to the severity of their response to scrotal
insulation on the basis of the morphological assessment of the semen samples
collected. Semen samples from 3 of these bulls were used in these studies.
Semen Separation Procedures
The standard protocols for the Percoll gradient (90%/45%) separation
(Pertoft et al, 1978) and the
swim-up method (Parrish et al,
1986) were used to separate fractions for IVF. Both the pellet
(Ap) and the interphase layer between the 45%/90% gradient
(Bp) were isolated from the Percoll gradient; from the swim-up
method, both the supernatant (As) and the interphase
(Bs) were isolated and used for IVF. In short, a Percoll gradient
was prepared in a 15-mL conical tube with 2 mL of 90% Percoll (1:9 [vol/vol]
mixture of Percoll and 10x synthetic oviductal fluid buffered with
HEPES; SOF HEPES; Sigma Chemical Co, St Louis, Mo) added to the tube, which
was layered with 2 mL of 45% Percoll (1:1 [vol/vol] mixture of 90% Percoll and
1x SOF HE-PES). Frozen-thawed semen was layered on the Percoll gradient
and centrifuged at 700 x g for 30 minutes. Both the
Ap and Bp fractions were then washed in SOF HEPES by
centrifugation at 500 x g for 10 minutes at room temperature.
For the swim-up method, each of 4 tubes was filled with 1.0 mL of SOF HEPES
medium, and a 1.0-mL volume of frozen-thawed semen was layered under the
medium. After a 1-hour incubation at 38.7°C, the upper 0.8 mL of the
medium was collected as the As fraction, and the lower portion (0.8
mL) was collected as the Bs fraction. Both fractions were washed
twice by centrifugation at room temperature (700 x g for 10
minutes).
IVF and Embryonic Culture
For each replicate, slaughterhouse-collected oocytes (n = 400) were matured
in tissue culture medium 199 (TCM 199; Gibco, Grand Island, NY) supplemented
with 10% bovine calf serum (Gibco), 0.01 U/mL luteinizing hormone (Sigma), and
0.01 U/mL follicle-stimulating hormone (Sigma) for a 24-hour period
(Thompson et al, 1998).
IVF was performed with frozen-thawed semen samples from the different bulls according to their responses to scrotal insulation, which were based on the morphological assessment of each sample after collection. Matured oocytes in a quantity sufficient to perform 3 replicates per day were then washed in SOF HEPES medium and placed in a 47-µL SOF-IVF drop (10 oocytes per drop) supplemented with a heparin concentration of 10 µg/mL, and a 3-µL sample of each separation fraction was added to the appropriate drop for a final concentration of 1 x 106 spermatozoa per milliliter (Parrish et al, 1995). Each treatment was replicated on each of 3 days.
After 18 hours of sperm–oocyte incubation at 38.7°C in an atmosphere of 5% CO2, the presumptive embryos were denuded by vortexing for 5 minutes in 1.0 mL of SOF HEPES medium. Embryos were washed and placed in a 30-µL SOF–in vitro culture (SOF-IVC) drop and cultured at 38.7°C in an atmosphere of 5% CO2, 10% O2, and 85% N2 (Thompson et al, 1998). Subsequent development was recorded on day 8 (IVF = day 0), and a developmental score (0 = degenerate, 1 = 2-cell embryo through 5 = blastocyst stage) was assigned to each embryo.
Sperm Morphology Evaluation
In addition to the morphological evaluation conducted at the time of
collection and freezing, a morphological assessment was conducted after the
IVF procedures in 2 of the replicates for each sample, with care being taken
to keep the individual fractions (Ap, Bp, As,
and Bs) separated. Wet smears were prepared for analysis by placing
a drop from each fraction onto a microscope slide and immobilizing the
spermatozoa with a drop of 40 mM sodium fluoride and then covering the drop
with a coverslip (Mitchell et al,
1985). Morphology was evaluated on 200 cells using differential
interference contrast microscopy at 1000x magnification under oil. The
percentage of morphologically normal spermatozoa and the percentage of
specific primary abnormalities that characterize a disturbance in
spermatogenesis due to scrotal insulation were recorded for each sample.
Spermatozoa with multiple abnormalities were counted only once, but each
abnormality of a particular spermatozoa was classified separately
(Barth and Oko, 1989) and
included pyriform, tapered, vacuolated, decapitated, and severely misshapen
spermatozoa (Table 1). Data for
asymmetrical shape and diadem defects made up less than 10% of the total and
are not listed in the table.
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Experiment I: Separation Protocols
Semen Samples—
Three types of frozen-thawed semen
samples were used for IVF. The first treatment was semen collected from a
bull, predetermined to be effective for IVF in our laboratory (control). A
second sample was obtained from one of the more extreme responders to scrotal
insulation (D -5 prior to the 48-hour scrotal insulation). A third semen
sample was taken from the same bull on day 27 after scrotal insulation (D
+27). The D +27 sample was selected because it had the smallest overall
percentage of normal spermatozoa, being reduced from about 70% to about 20%.
This sampling protocol permitted an internal and external control for IVF.
Experiment II: Heparin Effectiveness
Semen from the bulls that displayed the greatest and least responses to
scrotal insulation was chosen to evaluate differences in sperm morphology. For
each of these bulls, 3 semen samples were used for IVF: a preinsult sample
collected 5 days before insulation (D -5) and 2 postinsult samples, one
collected 23 days later (D +23), when the percentages of pyriform, tapered,
vacuolated, and severely abnormal spermatozoa were observed to be the greatest
for bull I (the bull that displayed the greatest response), and the other
collected 34 days after insult (D +34) at the end of the collection period
(when sperm morphology had returned to near-normal levels). The bull that
displayed the least response (bull II) to scrotal insulation showed no
differences in sperm morphology during the same time period. Within each
treatment group, semen samples separated using Percoll procedures
(Ap) (Pertoft et al,
1978) were exposed to 3 different heparin concentrations (0.1,
1.0, and 10 µg/mL) that had been added to the SOF-IVF medium.
Slaughterhouse-collected oocytes were randomly assigned to each heparin
concentration within each treatment group (n = 30) after a 24-hour maturation
period, and this was replicated on 3 different days. After an 18-hour
sperm–oocyte incubation, the presumptive zygotes were cultured as in
experiment I; on day 8 (IVF = day 0), a developmental score (0 = degenerate, 1
= 2-cell embryo through 5 = blastocyst stage) was assigned to each embryo.
Statistical Analyses
Data from both experiments were analyzed by 1-way analysis of variance
using the PROC GLM and/or PROC MIXED procedure of Statistical Analysis Systems
(1999) and chi-square
analysis. Cleavage and developmental rates were determined for both
experiments. The effects included in the model for experiment I were semen
sample collection day, separation method, separation fractions, repetitions,
and interactions among semen sample, separation fraction, and separation
method. Replicate effects were not significant (P > .05) and were
deleted from the final models. Pearson simple correlations were conducted
between the development of sperm abnormalities and the percentages of
abnormalities in each sample using the PROC CORR procedure of SAS
(Statistical Analysis Systems,
1999). In experiment II, the model included the main effects of
bull, semen sample collection day, heparin concentration, and interactions
among bull and heparin concentration, bull and semen sample collection day,
and semen sample collection day and heparin concentration. Replicate effects
were not significant and were deleted from the final models.
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Results |
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Cleavage rates were significantly (P < .01) different among semen samples and were significantly affected (P < .01) by the method of separation. For the control semen, the cleavage rate was 64 ± 2.1%, which was higher (P < .01) than the 51 ± 2.3% that was observed for the D -5 semen sample and the 31 ± 2.2% cleavage that was observed for the D +27 semen sample, which, in turn, was the lowest and different (P < .01) from the D -5 sample. The cleavage rates for semen from the control bull were highest (75 ± 3%) for the swim-up method and 53 ± 3% for the Percoll separation (P < .01) compared to 59 ± 3 and 45 ± 3% for the D -5 semen collections (Figure 1). Cleavage rates were lowest and different (P < .01) for Percoll (26 ± 3%) and swim-up separation (35 ± 3%) procedures for D +27. Cleavage rates were affected (P < .01) by separation fractions (A and B). The control bull semen fraction A (Ap and As) was higher (P < .01) than the D -5 fraction A (79 ± 3% vs 55.1 ± 3.2%), which was not different from the control fraction B (49.1 ± 2.9%) and the D -5 fraction B (48.6 ± 3.2%). These fractions had greater cleavage rates (P < .01) than the D +27 fractions A (33.5 ± 3.0%) and B (28.3 ± 3.1%), which were not different from each other.
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The cleavage rates (Table 2) were significantly lower (P < .01) between the Ap and the Bp fractions for control bull semen and semen from the D -5 sample, but not for semen from the D +27 sample, while the there was no difference in the cleavage rates between the As and Bs fractions. However, within each fraction, As and Bs, the cleavage rates were significantly different (P < .01) only among all 3 semen samples for As fractions and decreased from 81% for the control As fractions to 38% for the D +27 samples, while for the Bs fractions, cleavage rates decreased from 67% (control and D -5) to 34% at D +27.
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The overall embryonic developmental scores were significantly different (P < .01) among the 3 semen samples, with the control having the highest development at 1.8 ± 0.06, and it was different from D -5 samples (1.5 ± 0.06), which was greater than D +27 samples (0.6 ± 0.06). Additionally, there was a significant difference (P < .01; Table 3) in the developmental score between preparation methods, Percoll vs swim-up. Notably, the swim-up separation provided more competent sperm than did the Percoll separation. A significant (P < .01) interaction was found between the control, D -5, and D +27 semen samples and their separation fractions (Ap, Bp, As, and Bs; Table 3) for embryonic development. Moreover, among the different fractions, the developmental score for the control was lower (P < .01) for both the Bp and Bs fractions when compared to the Ap and As fractions, while for semen from the D -5 samples, only the Bp developmental score was lower (P < .01) than the others. The developmental score for semen from the D +27 sample was lowest for the Bp fraction but was not different (P > .05) from the Ap, As, or Bs fractions. The developmental scores from the A fractions (Ap and As) decreased significantly (P < .01) from the control bull semen to the D -5 sample and then was further decreased to the D +27 sample. However, the control bull and D -5 Bs fractions resulted in greater development (P < .01) than all other B fraction semen samples.
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The overall blastocyst rates for the control semen for the Percoll and swim-up separations were not different (P > .05), nor were they different from the D -5 swim-up–separated semen samples (Figure 1). But the blastocyst rates were lower (P < .05) for the D -5 Percoll preparations, while for semen from the D +27 sample, there was no blastocyst formation for the Percoll separation; however, there was a 4.2% rate for the swim-up separation (Figure 1). There was a significant interaction for semen samples relative to the semen separation fraction (P < .01). The control bull semen separation fraction A used for IVF resulted in the highest blastocyst development (Figure 2). The D -5 fractions were not different from each other (P > .05), but the A fractions resulted in a higher blastocyst rate (P < .05) than the control fraction B. Blastocyst rates for both D +27 samples were lowest (P < .05).
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A positive correlation was found between the percentage of morphologically normal sperm heads and the embryonic developmental score (r = 0.32, P < .01), while a negative correlation was found between the developmental score and the percentage of pyriform sperm (r = -0.31, P < .01), diadems (r = -0.20, P < .01), apical vacuoles (r = -0.18, P < .01), random vacuoles (r = -0.30, P < .01), decapitated sperm (r = -0.21, P < .01), and severely abnormal sperm (r = -0.29, P < .01) in the semen sample.
Experiment II: Heparin Effectiveness
Descriptive data on the percentages of individual abnormalities for
experiment II are shown in Table
4.
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The cleavage rate was significantly (P < .01) affected by the bull from which the semen sample was taken, the semen sample collection day, and the heparin concentration. A lower cleavage rate (P < .01) was found for bull I (23 ± 2%; highest responding bull) than for bull II (33 ± 2%; lowest responding bull), while the cleavage rates for the D +23 and D +34 samples were lower (P < .01) than for the semen from the D -5 sample (Figure 3). Furthermore, the overall cleavage rate was higher when heparin concentrations of 10 µg/mL (42 ± 2.2%) were used than when heparin concentrations of 0.1 µg/mL (17 ± 0.7%) and 1.0 µg/mL (24.8 ± 2.3%; P < .01) were used. There was no interaction between the bull from which the semen sample was taken, the semen sample collection day, and the heparin concentration; however, the interaction between the bull from which the semen sample was taken and the heparin concentration approached significance (P < .08; Figure 4). For bull I, the cleavage rate doubled when the heparin concentrations increased from 0.1 to 1.0 µg/mL and almost tripled with a 100-fold increase in the heparin concentration from 0.1 to 10 µg/mL. For bull II, there was no difference (P > .05) in the cleavage rates when the heparin concentrations were increased from 0.1 to 1.0 µg/mL, but cleavage rates doubled when the heparin concentration was increased to 10 µg/mL.
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In contrast to the cleavage rates, the embryonic developmental scores between the 2 bulls were not different, but there was a significant difference (P < .05) between the developmental score for semen from the D -5 sample when compared to the D +23 and D +34 samples (Figure 3). The increasing heparin concentrations resulted in no difference in the developmental score from 0.1 µg/mL (1.4 ± 0.06) to 1.0 µg/mL (1.5 ± 0.06), with a significant increase (P < .01) at 10 µg/mL (2.1 ± 0.06) that corresponded with the increase in overall cleavage rate from 18% to 42% as the heparin concentration increased from 0.1 to 10 µg/mL. There was no significant interaction (P > .05) between the bulls from which the semen samples had been taken, the semen sample collection days, and the heparin concentrations when developmental scores were tested. The rate of blastocyst formation based on the percentage of cleaved embryos was not different (P > .05) for the main effects of the bull from which the semen sample was taken, the semen sample collection, and the heparin concentration.
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Discussion |
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Separation fraction (A vs B) effects were greatest on cleavage only in the control bull (Figure 1), suggesting that there are bull differences under routine conditions and that deleterious impacts occurred due to thermal insult. Other studies conducted with cattle have shown that the donor of the semen influences the outcome of both IVF and IVC (Parrish et al, 1986; Shi et al, 1990; Shamsuddin et al, 1993; Rodriquez-Martinez et al, 1997; Ward et al, 2003). Our higher cleavage rate for the swim-up method compared to the Percoll separation was in agreement with the findings of Parrish et al (1995). They associated the higher rate with a difference in the ability of spermatozoa to penetrate ova in vitro after separation by swim-up procedures when compared to Percoll separation. Moreover, hyperactivation of spermatozoa permits an enhanced penetration of the zona pellucida (Stauss et al, 1995).
Development With Sperm Separation
We found no difference in the development rates when only the normal
(As and Ap) fractions of each separation method were
compared, which supports the findings of Parrish et al
(1995). But for the
Bs and Bp separation fractions, lower developmental
scores and developmental rates were detected for the Percoll separation, while
the scores and rates for the swim-up method were not different. Fractionation
with the separation procedures showed effective separation with the Percoll
separation technique in the control and D -5 semen samples in contrast to no
change in cleavage for the swim-up separation and the D +27 Percoll
separation. Developmental data mimic these changes
(Table 3). Somfai et al
(2002) reported that both the
swim-up and Percoll methods improved the proportion of live spermatozoa used
for IVF but that the developmental rate was higher with sperm from a Percoll
separation than with sperm from a swim-up separation. These results imply that
Percoll separation is more efficient for partitioning high-quality, live,
acrosome-intact spermatozoa from damaged or dead spermatozoa and reflect the
decrease in the developmental rate for the Bp fraction from control
bull and D -5 semen. In support, Brandeis and Manuel
(1993) found that the swim-up
method separated a higher percentage of motile spermatozoa than a Percoll
separation but that the Percoll method yielded a higher percentage of
spermatozoa with intact acrosomes. Furthermore, Somfai et al
(2002) indicated that the
results with Percoll separation were more uniform and resulted in decreased
variances between semen samples of different quality. In contrast, we found a
significant decrease in developmental rates as the percentages of
morphologically abnormal sperm per sample increased (D +27) for both Percoll
and swim-up separations, regardless of whether A or B fractions were used. The
differences may be due to the type of semen samples used. Somfai et al
(2002) used semen samples of
high quality, whereas we evaluated semen of high quality as well as semen with
high proportions of abnormalities. The fraction differences were evident only
with control bull semen and the D -5 Percoll separations and were lost with
the other samples (Table 3).
This suggests that a decrease in the efficiency of both sperm separation
methods will bring about an increase the percentage of high-quality
spermatozoa for IVF procedures. In support, Chen et al
(1995) demonstrated that the
Percoll gradient was effective in removing debris and other contaminants but
that it resulted in a low rate of sperm recovery for low-quality semen
samples. They reported that the percentage of immotile sperm after a Percoll
separation was higher than for the swim-up method when both methods were used
to prepare abnormal semen samples for IVF. Moreover, Ng et al
(1992) found that the swim-up
method was superior to the Percoll method for the selection of normal
spermatozoa, which corresponds with our results
(Figure 1). The percentage of
dead spermatozoa with swollen or damaged acrosomes was higher after swim-up
procedures than after Percoll separations
(Somfai et al, 2002), showing
the inability of the swim-up method to separate gross abnormalities from the A
fraction. Moreover, Correa and Zavos
(1996) reported that the
percentage of intact sperm recovered from either preparation method was lower
than the percentage of motile sperm. This provides evidence that spermatozoa
with primary abnormalities have enough motility to participate in the swim-up
process and therefore be present in the recovery fraction without clear
evidence that there was any potential to presumable participation during
IVF.
With the marked decrease in the developmental rates (Table 3) and blastocyst formation rates (Figure 1), the actual participation of abnormal spermatozoa (D +27) in IVF is questionable, and fertilization may have been initiated by the small number of normal spermatozoa present. There is evidence that grossly misshapen spermatozoa can become involved in the fertilization process once they gain access to the oolemma (Burruel et al, 1996). According to Ward et al (2003), IVF results were not affected when high concentrations of presumable normal spermatozoa were used, but when low concentrations of normal spermatozoa were used, the effect was more prominent. However, Thundathil et al (1999) reported that abnormal spermatozoa that penetrate the oocyte can be involved in the fertilization process, but the resulting zygotes may be less competent, thus explaining the reductions observed in the embryonic developmental score in the current study. Moreover, spermatozoa with the pyriform defect apparently have normal acrosomes and normal motility and would be expected to penetrate the zona pellucida and enter the ooplasm. However, Thundathil et al (1999) reported that the percentage of pyriform spermatozoa that bound to and penetrated the zona and subsequently gained access to the ooplasm was lower than that of normal-shaped sperm, which is supported by the negative correlation found in this study (r = -0.31), as evidenced by developmental scores.
Heparin
Heparin was tested to assess its impact on cleavage and early embryonic
development. The overall cleavage rates more than doubled (18%–42%) when
the heparin concentration was increased from 0.1 to 10 µg/mL. The same
effect was observed within each bull
(Figure 4), despite a lower
cleavage and developmental score for the samples with high percentages of
abnormal spermatozoa present (Figure
3). The nonresponding bull (bull II) had no response to heparin
concentrations until given the high dose, while the responding bull (bull I)
had a linear increase in cleavage from heparin concentrations of 0.1–10
µg/mL. With regard to fertilization, these results suggest that increasing
heparin levels provided a mechanism by which to partially overcome the
deleterious effect of morphologically abnormal spermatozoa that were induced
by thermal insulation. Saeki et al
(1995) examined the
fertilization rates of individual bulls at different heparin concentrations of
1.0, 10, and 100 µg/mL and found that the maximum fertilization rate
occurred at 10 µg/mL. The percentage of oocytes fertilized by sperm was
heparin dose-dependent and had a maximum response when heparin concentrations
of 10 µg/mL were added to the fertilization medium
(Parrish et al, 1988).
Chamberland et al (2000)
concluded that a heparin concentration of 10 µg/mL in the IVF medium was
necessary to induce the physiological changes associated with capacitation and
the subsequent increase in sperm motility. Spermatozoa must undergo
capacitation in order to bind to the zona and undergo the acrosome reaction.
Mendes et al (2003) reported
that the addition of heparin to the fertilization media improved the cleavage
rate and embryonic development, regardless of which sperm separation method
was used.
Our results indicate the possibility that the normal physiological and biochemical events associated with cryopreservation and capacitation are hindered in abnormal spermatozoa and therefore decrease the effectiveness of heparin as a capacitation agent. In support, Kot and Handel (1987) suggested that the interaction between abnormal spermatozoa and the oocyte may be different from that between normal spermatozoa and the oocyte, resulting in decreased penetration or fertilization and subsequent impairment of embryonic development. Although differences in cleavage rates existed between bulls and the intermediate heparin level appeared to equalize cleavage between the bulls (Figure 4), subsequent development did not differ, which suggests that the detrimental effects of scrotal insulation on sperm quality are delayed. Moreover, the failure of normal embryonic development correlates with the results of experiment I, in which the cleavage rate was lower when a semen sample with a high percentage of abnormal spermatozoa was used for IVF, and may be related to uncompensable seminal traits (Saacke et al, 1994) that cannot be minimized by increased sperm concentrations or increased heparin concentrations. However, this effect seems to be eliminated after initial cleavage. Thus, the effect of abnormal spermatozoa on IVF was manifested either prior to or during the early stages of embryonic development, as indicated by the differences in cleavage rates, but not during blastocyst formation. Also, an increase in the heparin concentration could partially overcome deficiencies, which suggests that morphologically abnormal spermatozoa undergo capacitation despite possible structural changes to the plasma membrane.
Interference with the normal scrotal thermoregulation of bulls (Lunstra and Coulter, 1997) caused an increase in the percentage of morphologically abnormal spermatozoa and resulted in deleterious effects on embryonic development. In addition, aberrant spermatogenesis may affect the normal development of the structure of the sperm plasma membrane, making it more susceptible to structural changes. Fraser et al (1995) suggested that the functional integrity of the sperm plasma membrane influences the physiological status of the spermatozoa and that the destabilization of the plasma membrane leads to premature capacitation.
In conclusion, in contrast to semen with high populations of normal spermatozoa when semen samples with a high percentage of abnormal spermatozoa are used for IVF, the outcome of swim-up and/or Percoll separation methods might be insufficient to allow successful embryonic development. Our data show that both separation methods for abnormal sperm populations were inadequate in their ability to provide potentially competent sperm for IVF. Furthermore, the types of abnormalities induced by scrotal insulation could be partially overcome by an increase in the heparin concentration, which indicates that morphologically abnormal spermatozoa may undergo capacitation despite possible structural changes to the plasma membrane.
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