- Email: [email protected]
Sperm head morphometry analysis of ejaculate and dismount stallion semen samples
REPSON SCIENCE ELSEYIER
Animal Reproduction
Science 47 ( 1997) 149- 155
Sperm head morphometry analysis of ejaculate and dismount stallion semen samples C.G. Gravance a,*, Z. Champion a, I.K.M. Liu b, P.J. Casey a a Research Centre in Reproductive Medicine, Department of Obstetrics and Gynaecology, The University of Auckland, Auckland, New Zealand b School of Veterinary Medicine, Population Health and Reproduction, University of California, Davis, CA, USA Accepted 31 October
1996
Abstract The evaluation of seminal characteristics is important in the clinical detection of stallion subfertility. Conventional semen evaluation includes subjective determination of sperm concentration, motility, and gross morphology. Due to the subjectivity and variability of the manual morphology assessment, computer automated sperm morphology analyses has been developed. Computer automated sperm morphology analysis was applied in the current study to determine if the morphometric measurements of sperm heads from collected and dismount samples of the same ejaculate were similar. If the post-ejaculate dismount sample is representative of the entire ejaculate, this sample may be utilised in determining the fertility of the ejaculate. Ejaculate samples were collected from ten stallions using an artificial vagina. Post-ejaculate dismount samples of the same ejaculate were taken from the head of the penis. A thin smear of the collected and dismount samples were prepared onto microscope slides and spermatozoa were stained for 40 min in haematoxylin. At least 200 properly digitised sperm heads from each slide were analysed using computer automated sperm morphometry analysis. The mean values for length, width, width/length, area, and perimeter were recorded from each analysis of collected and dismount samples and compared by paired t-test. The coefficients of variation of each analysis was also recorded and compared between collected and dismount samples by paired t-test. No significant differences (P > 0.10) in any measurements were found between collected and dismount samples. The mean values for all stallions for collected and dismount samples were length = 5.96 p,M and 6.06 FM, width = 2.95 pM and 2.98 p,M, width/length = 0.49 and 0.49,
* Corresponding author. Research Centre in Reproductive Medicine, Department of OB/GYN, &of Unit, National Women’s Hospital, Claude Road, Epsom, Auckland, New Zealand. Tel: 64.9.630.9943 x3234; fax: 64.9.630.9858; e-mail: [email protected]. 0378-4320/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)01634-X
150
C.G. Gravance
et al./Animal Reproduction Science 47 (1997) 149-155
area= 13.31 PM* and 13.65 ~.LM*and perimeter= 15.54 PM and 15.74 PM respectively. No significant differences were detected in the coefficients of variation of sperm head measurements from collected and dismount samples. These results indicate sperm head measurements from dismount semen are representative of those of the ejaculate. Hence, sperm head measurements of dismount samples may be viably applied to studies of fertility or in cases of clinical fertility assessment. This finding will further assist in the development of normal sperm head morphometry criteria in the stallion. Clinically, a slide can be prepared in the field after natural service matings and analysed accurately and objectively by ASMA. 0 1997 Elsevier Science B.V. Keywords: Horse; ASMA; Spermatozoa-morphology
1. Introduction The evaluation of seminal characteristics is important in the clinical detection of stallion subfertility (Voss et al., 1981). An important component of the conventional semen evaluation includes the visual assessment of normal sperm morphology. In the stallion (Jasko et al., 1990) as well as a number of other species (Saake, 1972; Chandler et al., 19881, a reduction in the percentage of morphologically normal spermatozoa in the ejaculate is indicative of impaired fertility. The morphology assay is performed by visual, subjective analysis of fixed spermatozoa through a phase contrast microscope (Hafez, 1987). Abnormal sperm morphology is also correlated with reduced fertility in men (Kruger et al., 1988; Chan et al., 1989). In the clinical evaluation of normal human sperm morphology, morphometric criteria are applied to the assessment of normal sperm head morphology (World Health Organization, 1992). While sperm head dimensions have been described for fertile stallions from electron micrographs (Bielanski and Kaczmarski, 1979), and light microscopy (Nishikawa et al., 1951; Dott, 1975), such criteria have not been applied to routine sperm morphology assessment. However, visual morphology analysis has been applied to the assessment of stallion fertility for nearly 70 years (Walton and Fair, 1928; Williams and Savage, 1930). While normal sperm morphology appears to be correlated with fertility, the conventional, manual methods of type classification of stallion sperm morphology, like human sperm morphology assessment, are extremely variable (Baker and Clarke, 1987). In addition, sperm morphology criteria for normal classification are difficult to uniformly apply between technicians (Davis et al., 1995). In order to reduce the subjectivity of sperm morphology analysis, computer automated sperm morphometry analysis (ASMA) has been developed for morphometric analysis of stallion (Davis et al., 1993a; Ball and Mohammed, 1995) and human (Davis et al., 1992, Kruger et al., 1993) spermatozoa. Results of ASMA have shown that analyses of stallion sperm heads are extremely precise when standardised protocols are developed (Davis et al., 1993a). Furthermore, recent data from ASMA results of stallion spermatozoa, suggests that morphometric differences in sperm head dimension occur between fertile and subfertile stallions (Casey et al., 1997). Therefore, the clinical application of ASMA to the detection of stallion subfertility appears promising.
C.G. Grauance et al. /Animal Reproduction Science 47 (1997) 149-155
151
To further evaluate sperm morphometry and the relationship to fertility, it is necessary to collect data on large numbers of individuals. Morphometric data of spermatozoa generated from these analyses can then be correlated to ultimate fertility of the ejaculate. Sampling an ejaculate for evaluation is readily accomplished when artificial insemination (AI) is utilised, however AI is not always possible. Due to certain horse breed association by-laws restricting AI (ie thoroughbred) and other situations of natural service matings, it is not possible to get a sample prior to insemination. An alternative method of analysing the morphometric parameters of spermatozoa of a natural service mating may be by sampling spermatozoa from the semen remaining on the penis after servicing a mare. Previous studies have shown that semen characteristics from various fractions of the ejaculate are significantly different (Kosiniak, 1975). It is therefore possible that sperm characteristics of a post ejaculate sample collected from the penis may not be representative of the entire ejaculate. If the morphometric dimensions of sperm heads from a post-ejaculate dismount samples are representative of the entire ejaculate, a simple semen smear can be analysed and the results viably correlated to the fertility of the ejaculate. Not only would this be a significant finding for further studies of correlating ASMA results to fertility of the ejaculate but such a finding possess clinical relevance as well. For example, if semen collected after mating can be prepared in the field from a dismount sample, the sample can then be sent to the andrology laboratory for accurate and subjective ASMA analysis. The aim of this present study, utilising the accuracy of ASMA technology, was to determine if sperm head morphometric dimensions from dismount semen samples represent those of the population of the ejaculate.
2. Methods Semen samples were collected from ten stallions by artificial vagina (AV) and filtered to remove the gel fraction and particulate material of the ejaculate (Casey et al., 1991). Immediately after removing the AV, a sample of semen was then collected from the penis of the stallion into a 15 ml centrifuge tube (dismount sample). The collected and dismount semen samples were maintained at room temperature prior to slide preparation. Microscope slides of stallion semen were prepared for ASMA according to Davis et al. (1993a). A thin smear of collected and dismount semen from all stallions was prepared on a microscope slide by placing 7 p,l of the sample on the end of a frosted slide and dragging the drop across the slide (Zaneveld and Polakoski, 1977). Slides were air dried for a minimum of 2 h and spermatozoa stained 40 min in haematoxylin (Davis et al., 1993a). The total number of images acquired from each slide for analysis was 250. Any misdigitised sperm heads were deleted from the analysis using the edit option of the software so that at least 200 properly digitised sperm heads were analysed from each slide. Sperm heads were defined as properly digitised when the computer generated outline of the sperm head included the entire visual image of the anterior of the end of the sperm head and did not include any portion of the midpiece region. Spermatozoa were imaged through an Achromat X 60 (N.A 0.80) objective lens on
C.G. Gravance et al./Animal Reproduction Science 47 (1997) 149-155
152
an Olympus B X 40 microscope. The images were transferred to the image processing system via a Cohu 4915 CCD camera with an X 5.0 photo-ocular lens. The total image magnification on the video display was determined by stage micrometer to be X 3800. Sperm head dimensions were determined using a computer automated sperm morphometry analysis (ASMA) system (Motion Analysis Corp, Santa Rosa, CA, USA). The operation of the ASMA system has been previously described (Davis et al., 1993a; Casey et al., 1997). The mean values for length (L), width (WI, W/L, area (A) and perimeter (P) were recorded from each analysis of each slide. The mean measurements for L, W, W/L, A and P of sperm heads from collected and dismount semen samples were compared by two-sample paired t-test (NCSS, Kaysville UT, USA). The coefficients of variation (CV) of each analysis were also recorded and compared between collected and dismount samples by paired t-test (NCSS). 3. Results The mean number of properly digitised sperm heads analysed per slide was 235, therefore the ASMA system was able to properly digitise and provide head measurements on 94% of spermatozoa encountered. The mean measurements for L, W, W/L, A and P of sperm heads from collected and dismount samples of all stallions are summarised in Table 1. No significant differences (P > 0.10) in any measurements were found between sperm head dimensions of collected and dismount samples when analysed by paired t-test. The mean morphometric values and coefficients of variation (CV) for all analysis were L = 6.01 p.M (3.7), W = 2.97 pM (4.21, W/L = 0.49 (4.71, A = 13.48 JJ,M (6.8) and P = 15.64 p,M (3.3).
Table 1 Mean measurements
(FM) and coefficients
perimeter (P) for collection Treatment
Collection Dismount
and dismount
of variation
(CV) for length (L), width (W), W/L,
Measurements L (CV)
W (CV)
W/L (CV)
A (CV)
P (CV)
5.96 * (2.7) 6.06 * (5.2)
2.95 * (3.6) 2.98 * (3.6)
0.49 ’(3.8) 0.49 * (4.6)
13.31 * (5.1) 13.65 * (6.1)
15.54 * (2.8) 15.74 * (4.3)
* Values within columns not significantly
different (P > 0.10).
Table 2 Mean coefficient of variation (CV) for length (L), width (W), W/L, and dismount samples Treatment
Collection Dismount
area (A) and
samples
area (A) and perimeter (P) for collection
cv L
W
W/L
A
P
5.5 * 6.0 *
5.9 * 5.9 *
6.7 * 7.1 *
8.8 * 8.7 *
5.7 * 5.8 *
* Values within columns not significantly
different (P > 0.10).
C.G. Gravance et al./Animl
Reproduction Science 47 (1997) 149-155
80
120
70 60
g
153
100
=60
g
: 40 g34l lf 20
c 5 3 e II.
10
00 60 40 20
0
0 7
v
T-
T-
7
7
Area (uM)
7
7
.-.
r
..-
F
Perimeter (uM)
90 80 s 21 8 840 g u.
70 60 50 30 20
10 0 Length (uM)
Width (uM)
90 60 z c
70 60 50
Histooram Leaend
5 SN e 30 y
?? Collected samples ?? Dismount samples
20 10 0 ~%%Z%:x d d
d
d
W/L
Fig. 1. Histograms of mean frequencies (per analysis) of measurements for area, perimeter, width/length (W/L) of sperm heads from collected and dismount samples.
length, width and
The mean CVs of sperm head measurements for all stallions are summarised in Table 2. No significant differences (P > 0.10) in CVs of the measurements of L, W, W/L, A or P were detected between collected and dismount semen samples. The mean overall CVs for L, W, W/L A and P were 5.8%, 5.9%, 6.9%, 8.8% and 5.8% respectively. Fig. 1 shows the mean frequency, per analysis. of sperm head measurements from collected and dismount samples.
4. Discussion Previous studies of sperm head morphometry utilising similar ASMA systems have shown the systems to be accurate in the analysis of stallion (Davis et al., 1993a) and human (Davis et al., 1992) spermatozoa. In the current study, ASMA was able to properly recognise the visual outline of, and provide metric measurements for 94% of all spermatozoa encountered. Davis et al. (1992) found that the variation of repeated
154
C.G. Gravance et al./Animal Reproduction Science 47 (1997) 149-155
measurements of the same, properly digitised sperm head were < 1%. The extreme accuracy of ASMA has been utilised in the detection of differences in the sperm head dimensions of fertile and subfertile stallions (Casey et al., 1997). Additionally, ASMA has been employed to detect detrimental effects of reproductive toxins when no effect was detected by subjective type classification methods (Davis et al., 1993b). Yet, in the current study, the discerning power of ASMA was not able to detect any significant differences in L, W, W/L, A or P of sperm heads from collected or dismount samples. The overall mean values for L (6.01 PM), W (2.97 FM), W/L (0.491, A (13.48 p,M) and P (15.64 p,M) are similar to those previously described (Davis et al., 1993a). The measurements of L (5.75 FM) and W (2.93 PM) determined from electron microscopy by Bielanski and Kaczmarski (1979) are also similar to those of the current study. However, Dott (1975) and Nishikawa et al. (1951) reported larger values. In addition to similar sperm head dimensions, similar CVs were observed between sperm head measurements of collected and dismount semen samples. The morphometric measurements of sperm heads of each analysis were also found to be normally distributed (data not shown). These results indicate that not only are mean values of sperm heads similar but the distribution of values around the means are similar (Casey et al., 1997). The results of this study indicate that stallion sperm head dimensions from dismount semen represent those of the complete ejaculate. Applying these results, it is now possible to begin to correlate sperm head dimensions from semen of natural matings to fertility of the ejaculate. Additionally, these analyses can be performed by an objective and repeatable method (i.e. ASMA). Sampling of spermatozoa from the dismount sample will provide access to semen samples that would not normally be available to the researcher. Furthermore, the fertility of stallions can also be clinically analysed throughout the season and over different years by preparing dismount smears after each service The smear can then be analysed accurately and objectively by ASMA. The accuracy of this system will allow studfarms to easily and inexpensively assess and monitor the fertility of valuable stallions using an objective method.
Acknowledgements This project was supported by the California Center for Equine Health and Performance, University of California, Davis with funds provided by the Oak Tree Racing Association, the State of California satellite wagering fund and contributions from private donors.
References Baker, H.W.G. and Clarke, G.N., 1987. Sperm morphology: consistency of assessment of the same sperm by different observers. Clin. Reprod. Fertil., 5: 37-43. Ball, B.A. and Mohammed, H.O., 1995. Morphometry of stallion spermatozoa by computer assisted image analysis. Theriogenology, 44: 367-377.
C.G. Gravance et al./Aninml Reproduction Science 47 (1997) 149-155 Bielanski, W. and Kaczmarski, F., 1979. Morphology fertility. J. Reprod. Fertil., (Suppl 27): 39-45.
of spermatozoa
155
in the semen from stallions of normal
Casey, P.J., Liu, I.K.M., Stewart, D.R. and Scott, M.A., 1991. The effects of equine relaxin on sperm longevity and motility in chilled and cryopreserved semen. J. Reprod. Fertil. (Suppl.), 44: 645-646. Casey, P.J., Gravance, C.G., Davis, R.O., Chabot, D.D. and Liu, I.K.M., 1997. Morphometric differences in sperm head dimensions of fertile and subfertile stallions. Theriogenology, 47: 575-582. Chan, S.Y.W., Wang, C., Chan, S.T.H., Ho, P.C., So, W.W.K., Chan, Y.F. and Ma, H.K., 1989. Predictive value of sperm morphology and movement characteristics in the outcome of in vitro fertilization of human oocytes. J. In Vitro Fertil. Emb. Trans., 6: 142-148. Chandler, J.E., Painter, C.L., Adkinson, R.W., Memon, M.A. and Hoyt, P.G., 1988. Semen quality characteristics of dairy goats. J. Dairy Sci., 71: 163881646. Davis, R.O., Bain, D.E., Siemers, R.J., Thal, D.M., Andrew, J.B. and Gravance, C.G., 1992. Accuracy and precision of the CellFonn-Human automated sperm morphometry system. Fertil. Steril., 58: 763-769. Davis, R.O., Gravance, C.G. and Casey, P.J., 1993a. Automated morphometric analysis of stallion spermatozoa. Amer. J. Vet. Res., 54: 1808-1811. Davis, R.O., Katz, D.F., Gravance, C.G. and Osario, A.M., 1993b. Sperm morphology abnormalities among lead-exposed battery plant workers. Fertil. Steril., 60:56s (Abstract). Davis, R.O., Gravance, C.G. and Overstreet, J.W., 1995. A standardized test for the visual analysis of human sperm morphology. Fertil. Steril., 63: 1058-1063. Dott, H.M., 1975. Morphology of stallion spermatozoa. J. Reprod. Fertil., (Suppl. 23): 41-46. Hafez, E.S.E.. 1987. Semen Evaluation. In: E.S.E Hafez (Editor). Reproduction in Farm Animals, 5th edn. Lea and Febiger, Philadelphia, pp. 455-480. Jasko, D.J., Lein, D.H. and Foote, R.H., 1990. Determination of the relationship between sperm morphologic classification and fertility in stallions: 66 cases (1987-19881. J. Am. Vet. Med. Assoc., 197: 389-394. Kosiniak, K., 1975. Characteristics of the successive jets of ejaculated semen of stallions. J. Reprod. Fertil., (Suppl. 23): 59-61. Kruger, T.F., Acosta, A.A., Simmons, K.F., Swanson, R.J., Matta, J.F. and Oehninger, S., 1988. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil. Steril., 49: 112-l 17. Kruger, T.F., Tuitot, T.C., Franken, D.R., Acosta, A.A., Oehninger, SC., Menkveld, R. and Lombard, C.J., 1993. A new computerized method of reading sperm morphology (strict criteria) is as efficient as technician reading. Fertil. Steril., 59: 202-209. Nishikawa, Y., Waide, Y. and Onuma, H., 1951. Studies on AI in the horse, VI. Morphological studies on the horse’s spermatozoa. Bull. Natn. Inst. Agric. Sci., Tokyo, Series G, I: 29-36. Saake, R.G., 1972. Semen quality tests and their relationship to fertility. NAAB Proceedings of the 4th Technical Conference on Animal Reproduction and Fertility, pp. 22-28. Voss, J.L., Pickett, B.W. and Squires, E.L., 1981. Stallion spermatozoa1 morphology and motility and their relationship to fertility. J. Am. Vet. Med. Assoc., 178: 287-289. Walton, A. and Fair, K.K., 1928. Preliminary investigations on the fecundity of premium stallions. J. Agric. Sci. Cambridge, 18: 772-777. Williams, W.L. and Savage, 327-332. World Health Organization, Semen-Cervical Mucus Cambridge.
A., 1930. A study of the head length of equine spermatozoa.
Can. J. Res., 3:
1992. WHO Laboratory Manual for the Examination of Human Semen and Interaction, 3rd edn. The Press Syndicate of the University of Cambridge,
Zaneveld, L.J.D. and Polakoski, K.L., 1977. Collection and physical examination of the ejaculate. Hafez (Editor). Techniques in Human Andrology. Elsevier, Amsterdam, pp. 147-172.
In: E.S.E.
Animal Reproduction
Science 47 ( 1997) 149- 155
Sperm head morphometry analysis of ejaculate and dismount stallion semen samples C.G. Gravance a,*, Z. Champion a, I.K.M. Liu b, P.J. Casey a a Research Centre in Reproductive Medicine, Department of Obstetrics and Gynaecology, The University of Auckland, Auckland, New Zealand b School of Veterinary Medicine, Population Health and Reproduction, University of California, Davis, CA, USA Accepted 31 October
1996
Abstract The evaluation of seminal characteristics is important in the clinical detection of stallion subfertility. Conventional semen evaluation includes subjective determination of sperm concentration, motility, and gross morphology. Due to the subjectivity and variability of the manual morphology assessment, computer automated sperm morphology analyses has been developed. Computer automated sperm morphology analysis was applied in the current study to determine if the morphometric measurements of sperm heads from collected and dismount samples of the same ejaculate were similar. If the post-ejaculate dismount sample is representative of the entire ejaculate, this sample may be utilised in determining the fertility of the ejaculate. Ejaculate samples were collected from ten stallions using an artificial vagina. Post-ejaculate dismount samples of the same ejaculate were taken from the head of the penis. A thin smear of the collected and dismount samples were prepared onto microscope slides and spermatozoa were stained for 40 min in haematoxylin. At least 200 properly digitised sperm heads from each slide were analysed using computer automated sperm morphometry analysis. The mean values for length, width, width/length, area, and perimeter were recorded from each analysis of collected and dismount samples and compared by paired t-test. The coefficients of variation of each analysis was also recorded and compared between collected and dismount samples by paired t-test. No significant differences (P > 0.10) in any measurements were found between collected and dismount samples. The mean values for all stallions for collected and dismount samples were length = 5.96 p,M and 6.06 FM, width = 2.95 pM and 2.98 p,M, width/length = 0.49 and 0.49,
* Corresponding author. Research Centre in Reproductive Medicine, Department of OB/GYN, &of Unit, National Women’s Hospital, Claude Road, Epsom, Auckland, New Zealand. Tel: 64.9.630.9943 x3234; fax: 64.9.630.9858; e-mail: [email protected]. 0378-4320/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)01634-X
150
C.G. Gravance
et al./Animal Reproduction Science 47 (1997) 149-155
area= 13.31 PM* and 13.65 ~.LM*and perimeter= 15.54 PM and 15.74 PM respectively. No significant differences were detected in the coefficients of variation of sperm head measurements from collected and dismount samples. These results indicate sperm head measurements from dismount semen are representative of those of the ejaculate. Hence, sperm head measurements of dismount samples may be viably applied to studies of fertility or in cases of clinical fertility assessment. This finding will further assist in the development of normal sperm head morphometry criteria in the stallion. Clinically, a slide can be prepared in the field after natural service matings and analysed accurately and objectively by ASMA. 0 1997 Elsevier Science B.V. Keywords: Horse; ASMA; Spermatozoa-morphology
1. Introduction The evaluation of seminal characteristics is important in the clinical detection of stallion subfertility (Voss et al., 1981). An important component of the conventional semen evaluation includes the visual assessment of normal sperm morphology. In the stallion (Jasko et al., 1990) as well as a number of other species (Saake, 1972; Chandler et al., 19881, a reduction in the percentage of morphologically normal spermatozoa in the ejaculate is indicative of impaired fertility. The morphology assay is performed by visual, subjective analysis of fixed spermatozoa through a phase contrast microscope (Hafez, 1987). Abnormal sperm morphology is also correlated with reduced fertility in men (Kruger et al., 1988; Chan et al., 1989). In the clinical evaluation of normal human sperm morphology, morphometric criteria are applied to the assessment of normal sperm head morphology (World Health Organization, 1992). While sperm head dimensions have been described for fertile stallions from electron micrographs (Bielanski and Kaczmarski, 1979), and light microscopy (Nishikawa et al., 1951; Dott, 1975), such criteria have not been applied to routine sperm morphology assessment. However, visual morphology analysis has been applied to the assessment of stallion fertility for nearly 70 years (Walton and Fair, 1928; Williams and Savage, 1930). While normal sperm morphology appears to be correlated with fertility, the conventional, manual methods of type classification of stallion sperm morphology, like human sperm morphology assessment, are extremely variable (Baker and Clarke, 1987). In addition, sperm morphology criteria for normal classification are difficult to uniformly apply between technicians (Davis et al., 1995). In order to reduce the subjectivity of sperm morphology analysis, computer automated sperm morphometry analysis (ASMA) has been developed for morphometric analysis of stallion (Davis et al., 1993a; Ball and Mohammed, 1995) and human (Davis et al., 1992, Kruger et al., 1993) spermatozoa. Results of ASMA have shown that analyses of stallion sperm heads are extremely precise when standardised protocols are developed (Davis et al., 1993a). Furthermore, recent data from ASMA results of stallion spermatozoa, suggests that morphometric differences in sperm head dimension occur between fertile and subfertile stallions (Casey et al., 1997). Therefore, the clinical application of ASMA to the detection of stallion subfertility appears promising.
C.G. Grauance et al. /Animal Reproduction Science 47 (1997) 149-155
151
To further evaluate sperm morphometry and the relationship to fertility, it is necessary to collect data on large numbers of individuals. Morphometric data of spermatozoa generated from these analyses can then be correlated to ultimate fertility of the ejaculate. Sampling an ejaculate for evaluation is readily accomplished when artificial insemination (AI) is utilised, however AI is not always possible. Due to certain horse breed association by-laws restricting AI (ie thoroughbred) and other situations of natural service matings, it is not possible to get a sample prior to insemination. An alternative method of analysing the morphometric parameters of spermatozoa of a natural service mating may be by sampling spermatozoa from the semen remaining on the penis after servicing a mare. Previous studies have shown that semen characteristics from various fractions of the ejaculate are significantly different (Kosiniak, 1975). It is therefore possible that sperm characteristics of a post ejaculate sample collected from the penis may not be representative of the entire ejaculate. If the morphometric dimensions of sperm heads from a post-ejaculate dismount samples are representative of the entire ejaculate, a simple semen smear can be analysed and the results viably correlated to the fertility of the ejaculate. Not only would this be a significant finding for further studies of correlating ASMA results to fertility of the ejaculate but such a finding possess clinical relevance as well. For example, if semen collected after mating can be prepared in the field from a dismount sample, the sample can then be sent to the andrology laboratory for accurate and subjective ASMA analysis. The aim of this present study, utilising the accuracy of ASMA technology, was to determine if sperm head morphometric dimensions from dismount semen samples represent those of the population of the ejaculate.
2. Methods Semen samples were collected from ten stallions by artificial vagina (AV) and filtered to remove the gel fraction and particulate material of the ejaculate (Casey et al., 1991). Immediately after removing the AV, a sample of semen was then collected from the penis of the stallion into a 15 ml centrifuge tube (dismount sample). The collected and dismount semen samples were maintained at room temperature prior to slide preparation. Microscope slides of stallion semen were prepared for ASMA according to Davis et al. (1993a). A thin smear of collected and dismount semen from all stallions was prepared on a microscope slide by placing 7 p,l of the sample on the end of a frosted slide and dragging the drop across the slide (Zaneveld and Polakoski, 1977). Slides were air dried for a minimum of 2 h and spermatozoa stained 40 min in haematoxylin (Davis et al., 1993a). The total number of images acquired from each slide for analysis was 250. Any misdigitised sperm heads were deleted from the analysis using the edit option of the software so that at least 200 properly digitised sperm heads were analysed from each slide. Sperm heads were defined as properly digitised when the computer generated outline of the sperm head included the entire visual image of the anterior of the end of the sperm head and did not include any portion of the midpiece region. Spermatozoa were imaged through an Achromat X 60 (N.A 0.80) objective lens on
C.G. Gravance et al./Animal Reproduction Science 47 (1997) 149-155
152
an Olympus B X 40 microscope. The images were transferred to the image processing system via a Cohu 4915 CCD camera with an X 5.0 photo-ocular lens. The total image magnification on the video display was determined by stage micrometer to be X 3800. Sperm head dimensions were determined using a computer automated sperm morphometry analysis (ASMA) system (Motion Analysis Corp, Santa Rosa, CA, USA). The operation of the ASMA system has been previously described (Davis et al., 1993a; Casey et al., 1997). The mean values for length (L), width (WI, W/L, area (A) and perimeter (P) were recorded from each analysis of each slide. The mean measurements for L, W, W/L, A and P of sperm heads from collected and dismount semen samples were compared by two-sample paired t-test (NCSS, Kaysville UT, USA). The coefficients of variation (CV) of each analysis were also recorded and compared between collected and dismount samples by paired t-test (NCSS). 3. Results The mean number of properly digitised sperm heads analysed per slide was 235, therefore the ASMA system was able to properly digitise and provide head measurements on 94% of spermatozoa encountered. The mean measurements for L, W, W/L, A and P of sperm heads from collected and dismount samples of all stallions are summarised in Table 1. No significant differences (P > 0.10) in any measurements were found between sperm head dimensions of collected and dismount samples when analysed by paired t-test. The mean morphometric values and coefficients of variation (CV) for all analysis were L = 6.01 p.M (3.7), W = 2.97 pM (4.21, W/L = 0.49 (4.71, A = 13.48 JJ,M (6.8) and P = 15.64 p,M (3.3).
Table 1 Mean measurements
(FM) and coefficients
perimeter (P) for collection Treatment
Collection Dismount
and dismount
of variation
(CV) for length (L), width (W), W/L,
Measurements L (CV)
W (CV)
W/L (CV)
A (CV)
P (CV)
5.96 * (2.7) 6.06 * (5.2)
2.95 * (3.6) 2.98 * (3.6)
0.49 ’(3.8) 0.49 * (4.6)
13.31 * (5.1) 13.65 * (6.1)
15.54 * (2.8) 15.74 * (4.3)
* Values within columns not significantly
different (P > 0.10).
Table 2 Mean coefficient of variation (CV) for length (L), width (W), W/L, and dismount samples Treatment
Collection Dismount
area (A) and
samples
area (A) and perimeter (P) for collection
cv L
W
W/L
A
P
5.5 * 6.0 *
5.9 * 5.9 *
6.7 * 7.1 *
8.8 * 8.7 *
5.7 * 5.8 *
* Values within columns not significantly
different (P > 0.10).
C.G. Gravance et al./Animl
Reproduction Science 47 (1997) 149-155
80
120
70 60
g
153
100
=60
g
: 40 g34l lf 20
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7
Area (uM)
7
7
.-.
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Perimeter (uM)
90 80 s 21 8 840 g u.
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Width (uM)
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5 SN e 30 y
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d
W/L
Fig. 1. Histograms of mean frequencies (per analysis) of measurements for area, perimeter, width/length (W/L) of sperm heads from collected and dismount samples.
length, width and
The mean CVs of sperm head measurements for all stallions are summarised in Table 2. No significant differences (P > 0.10) in CVs of the measurements of L, W, W/L, A or P were detected between collected and dismount semen samples. The mean overall CVs for L, W, W/L A and P were 5.8%, 5.9%, 6.9%, 8.8% and 5.8% respectively. Fig. 1 shows the mean frequency, per analysis. of sperm head measurements from collected and dismount samples.
4. Discussion Previous studies of sperm head morphometry utilising similar ASMA systems have shown the systems to be accurate in the analysis of stallion (Davis et al., 1993a) and human (Davis et al., 1992) spermatozoa. In the current study, ASMA was able to properly recognise the visual outline of, and provide metric measurements for 94% of all spermatozoa encountered. Davis et al. (1992) found that the variation of repeated
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C.G. Gravance et al./Animal Reproduction Science 47 (1997) 149-155
measurements of the same, properly digitised sperm head were < 1%. The extreme accuracy of ASMA has been utilised in the detection of differences in the sperm head dimensions of fertile and subfertile stallions (Casey et al., 1997). Additionally, ASMA has been employed to detect detrimental effects of reproductive toxins when no effect was detected by subjective type classification methods (Davis et al., 1993b). Yet, in the current study, the discerning power of ASMA was not able to detect any significant differences in L, W, W/L, A or P of sperm heads from collected or dismount samples. The overall mean values for L (6.01 PM), W (2.97 FM), W/L (0.491, A (13.48 p,M) and P (15.64 p,M) are similar to those previously described (Davis et al., 1993a). The measurements of L (5.75 FM) and W (2.93 PM) determined from electron microscopy by Bielanski and Kaczmarski (1979) are also similar to those of the current study. However, Dott (1975) and Nishikawa et al. (1951) reported larger values. In addition to similar sperm head dimensions, similar CVs were observed between sperm head measurements of collected and dismount semen samples. The morphometric measurements of sperm heads of each analysis were also found to be normally distributed (data not shown). These results indicate that not only are mean values of sperm heads similar but the distribution of values around the means are similar (Casey et al., 1997). The results of this study indicate that stallion sperm head dimensions from dismount semen represent those of the complete ejaculate. Applying these results, it is now possible to begin to correlate sperm head dimensions from semen of natural matings to fertility of the ejaculate. Additionally, these analyses can be performed by an objective and repeatable method (i.e. ASMA). Sampling of spermatozoa from the dismount sample will provide access to semen samples that would not normally be available to the researcher. Furthermore, the fertility of stallions can also be clinically analysed throughout the season and over different years by preparing dismount smears after each service The smear can then be analysed accurately and objectively by ASMA. The accuracy of this system will allow studfarms to easily and inexpensively assess and monitor the fertility of valuable stallions using an objective method.
Acknowledgements This project was supported by the California Center for Equine Health and Performance, University of California, Davis with funds provided by the Oak Tree Racing Association, the State of California satellite wagering fund and contributions from private donors.
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