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Comparison of pregnancy outcome in mares among methods used to evaluate and select spermatozoa for insemination
Animal Reproduction Science 69 (2002) 211–222
Comparison of pregnancy outcome in mares among methods used to evaluate and select spermatozoa for insemination G.J. Nie∗,1 , J.G.W. Wenzel, K.E. Johnson Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, 146 McAdory Hall, AL 36849-5522, USA Received 21 December 2000; received in revised form 25 September 2001; accepted 12 October 2001
Abstract An artificial insemination dose for mares consisting of 500 million progressively motile spermatozoa is considered “standard” by most clinicians. However, little information is available directly comparing pregnancy outcome among methods of evaluating and selecting spermatozoa for insemination. The objective of this study was to determine if the method of spermatozoal evaluation and selection influences fertility as measured by pregnancy outcome. Mares were inseminated with 100 or 500 million spermatozoa that were selected for progressive motility, normal morphology, hypoosmotic swelling or absolute number regardless for evaluation method or quality. Thirty-two breeding cycles were tested for each treatment group and at each spermatozoal dose. Pregnancy outcomes were 44 and 41%, 55 and 41%, 39 and 31%, and 45 and 41%, for the 100 and 500 million progressively motile, morphologically normal, hypoosmotic swelling positive and absolute number treatment groups, respectively. Pregnancy outcome did not differ among methods of spermatozoal evaluation and selection for artificial insemination in the 100 (P = 0.52) or 500 (P = 0.78) million spermatozoa groups. Also the total number of spermatozoa and the absolute number of progressively motile, morphologically normal or hypoosmotic swelling positive spermatozoa inseminated, were not closely associated with pregnancy outcome in the 100 (P = 0.24, 0.29, 0.33 and 0.38, respectively) or 500 (P = 0.20, 0.84, 0.50 and 0.74, respectively) million spermatozoa groups. In this study, we found that the method of spermatozoal evaluation did not offer an advantage for pregnancy when used to select spermatozoa for insemination at the doses tested. These results were
∗ Corresponding author. Present address: Rood and Riddle Equine Hospital, 2150 Georgetown Road, P.O. Box 12070, Lexington, KY 40580, USA. E-mail address: [email protected] (G.J. Nie). 1 Tel.: +1-859-233-0371; fax: +1-859-255-5367.
0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 8 0 - 4
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surprising, as we expected there would be differences among the evaluation methods. Instead, we found that evaluating spermatozoa offered no advantage for pregnancy over simply inseminating with a specified number of spermatozoa not selected for any particular characteristic under the conditions of our experiment. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Artificial insemination; Progressive motility; Morphology; Hypoosmotic swelling; Equine
1. Introduction Reports during the 1970s and early 1980s suggested that an artificial insemination (AI) dose for mares consist of 500 million progressively motile spermatozoa (Voss and Pickett, 1976; Householder et al., 1981). In the two decades, since those reports were published an industry-wide emphasis has been placed on selecting insemination doses for motility characteristics. Today, an inseminate is typically selected for progressive motility and a dose of 500 million progressively motile spermatozoa is considered standard by most clinicians in order to maximize pregnancy rates (Samper, 1997; Metcalf, 2000). Motility is certainly a quick and easy spermatozoal characteristic to evaluate. However, motility characteristics are poorly correlated with fertility (Jasko, 1992; Jasko et al., 1992). Questions raised by the audience at a recent national symposium on stallion reproduction challenged our acceptance as an industry of motility as the best means of selecting spermatozoa for insemination (Stallion Reproduction Symposium, ACT/SFT/AAEP Baltimore, MD, 1998). Some members of the audience suggested that perhaps we should be selecting spermatozoa by other means (e.g. morphology) to optimize stallion factor fertility. In a recent study, Scott et al. (2000) found that over 90% of spermatozoa recovered from the uterotubal junction (UTJ) of mares were morphologically normal following insemination of those mares with doses containing as many as 85% abnormal cells. The researchers concluded that morphology, as well as motility should be considered when selecting an inseminate for mares. Another method of spermatozoal evaluation that may also offer some advantage in selecting insemination doses for mares is the hypoosmotic swelling (HOS) test. By assessing plasma membrane functional integrity and spermatozoal fertilizing capacity, the HOS test is a useful adjunct to existing spermatozoal assays in human reproduction (Jeyendran et al., 1992). Consequently, results of the HOS test may provide a better means of selecting stallion spermatozoa for insemination. Currently, we are unaware of any reports directly comparing fertility results among methods of evaluating and selecting spermatozoa for insemination. Progressive motility seems to have been established as the standard method of spermatozoal selection for insemination without valid comparisons with other methods. The objective of our study was to determine whether the spermatozoal evaluation and selection method influences fertility under conditions that would be encountered in routine breeding operations throughout the industry. We compared fertility results when the insemination dose was selected by routine analysis methods, including progressive motility morphology, HOS, and if no-evaluation method were used.
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2. Materials and methods A total of 39 mares and five stallions of various light-horse breeds (Equus caballus), ranging in age from 3 to 20 years and weighing 400–550 kg were used in this study. Horses were maintained in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (First Revised Edition, January 1999). All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee at Auburn University (IACUC Protocol No. 0211-R-2209). Mares were housed in groups of 4–8 in large paddocks, while stallions were individually housed in smaller paddocks. All horses were fed a commercial concentrate ration (12% protein) and costal bermuda grass hay for maintenance of body condition. The entire study was conducted as two separate experiments in successive years. Seven mares and one stallion were replaced between Experiments 1 and 2 due to medical and/or management circumstances. Protocols were identical in each experiment with the exception of total spermatozoal number selected for the AI dose. In Experiments 1 and 2, 100 and 500 million spermatozoa, respectively, were used as a selected insemination dose. The spermatozoa used for insemination were selected for progressive motility, normal morphology, HOS or absolute number without regard for evaluation method or quality. In each experiment, 32 mares were randomly and equally distributed among four stallions. The eight mares assigned to each stallion were bred in each of four AI/semen-evaluation treatment groups, which consisted of progressive motility, morphology, HOS and absolute number. This ensured 32 breeding cycles per stallion and per treatment group in each experiment. Cycles were successive and the treatment order was randomized for each mare. Mares were bred in each AI/semen-evaluation treatment group using spermatozoa from the assigned stallion for that experiment. All mares had ovulated at least once early in the breeding season and prior to entering the study. Mares and stallions were screened for breeding soundness using routine techniques (Dascanio et al., 1997a,b; Hurtgen, 1992). As mares entered estrus, the reproductive tract was examined every other day via palpation and ultrasonography for follicles >30 mm in diameter. When follicles reached 30–35 mm mares were examined daily. All examinations were conducted in the morning between 6 and 8 a.m. When a ≥35 mm follicle was detected, the mare was given 2500 units of human chorionic gonadotropin (Novarel® , Ferring Pharmaceuticals, Tarrytown, NY), intravenously, to induce ovulation. Each mare was inseminated once per cycle, the following morning approximately 24 h following ovulation induction. On the morning of insemination the assigned stallion was collected using an artificial vagina. Equal volumes of semen and prewarmed (37 ◦ C) extender were mixed within 2 min of collection. The extender used in this study consisted of non-fat-dry-milk-solid (2.4 g) and glucose (4.9 g) dissolved in sterile, de-ionized water to a total volume of 100 ml. The extender also included 1 mg/ml of ticarcillin (Ticar® , SmithKline Beecham Pharmaceuticals, Philadelphia, PA). Spermatozoal concentration of the undiluted semen was determined using a densimeter (Animal Reproduction Systems, Chino, CA). The 1:1 extended aliquot was then further diluted with extender to a final concentration of 30 million spermatozoa/ml. Extended spermatozoa were examined for motility, morphology and HOS parameters. During analysis the reservoir of extended spermatozoa was placed in a dark cabinet which had a constant temperature of 25–27 ◦ C.
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Motility parameters were evaluated within minutes of collection using a computer-aided spermatozoal analysis system (CASA; HTM-C, Hamilton Thorne Reasearch, Beverly, MA). A 4.5 l drop of extended spermatozoa was placed on a pre-warmed (37 ◦ C) glass slide and covered with a 22 mm × 22 mm cover slip. Slide temperature was maintained with a stage warmer during evaluation. A minimum of five fields and 500 spermatozoa were analyzed from each sample using the CASA system. Analysis setup included frames acquired (30), frame rate (60 Hz), straightness threshold (80%), low path velocity(VAP) cutoff (5.0 m/s), medium path velocity (VAP) cutoff (25.0 m/s), low progressive velocity (VSL) cutoff (11.0 m/s), and slow cells motile (no). Insemination doses of 100 or 500 million progressively motile spermatozoa were calculated from this measure of motility. Spermatozoal morphology was evaluated using an eosin–nigrosin morphology stain (Lane Mfg, Denver, CO). Equal volumes of raw semen and stain were smeared and air dried on glass slides. Slides were examined for spermatozoal morphology using bright-field microscopy (1000×) to evaluate 100 cells. Spermatozoa were classified as normal or abnormal. Insemination doses of 100 or 500 million morphologically normal spermatozoa were calculated from this measure of morphology. Spermatozoa were evaluated for HOS using the procedures described by Nie and Wenzel (2001). A 100 ml volume of the extended spermatozoal sample was added to 1.0 ml of a prewarmed (37 ◦ C) 100 millionmole sucrose solution. The mixture was incubated at 37 ◦ C for 30 min in a 1.5 ml micro-centrifuge tube. Following incubation, a small drop of sample was placed on a microscope slide and cover-slipped for evaluation. Samples were examined using phase contrast microscopy (400×) to evaluate 100 spermatozoa for evidence of swelling and curling (HOS+) changes. Insemination doses of 100 or 500 million HOS+ spermatozoa were calculated from this measure of hypoosmotic swelling. Mares were inseminated using routine procedures (Metcalf, 2000). Inseminations were performed following spermatozoal evaluation by the assigned selection method and within approximately 10, 15, 20 or 45 min after a collection for the absolute number, motility, morphology and HOS AI/semen-evaluation treatment groups, respectively. The total insemination volume for all treatment groups and the total number of spermatozoa inseminated for all treatment groups except the absolute number group were dependent on ejaculate quality that day. Mares were inseminated with either 100 (Experiment 1) or 500 (Experiment 2) million spermatozoa that were progressively motile, morphologically normal or HOS+. In the case of the absolute number treatment group, mares were inseminated with either 100 (Experiment 1) or 500 (Experiment 2) million total spermatozoa regardless of quality. All mares were provided routine post-breeding management standard for our unit, including daily examinations through the detection of ovulation. Pregnancy examinations were conducted at 14 days following ovulation using transrectal ultrasonography. Pregnancy outcome at 14 days was considered the endpoint for each treatment. Following the pregnancy examination each mare was given a luteolytic dose of cloprostenol (Estrumate® , Bayer Animal Health, Shawnee Mission, KS) to bring her into estrus for the next assigned treatment cycle. Proportions of pregnancies versus non-pregnancies were contrasted with logistic regression using the maximum-likelihood method (SAS-PROC CATMOD, SAS Institute, Cary, NC). Models were constructed using forward stepwise selection. In the first analysis, semen evaluation methods were contrasted as a categorical variable; in the second, the effect on
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Table 1 Characteristics of stallion spermatozoa used to inseminate maresa Parameter
Insemination volume (ml) TSN (×106 ) Progressive motility (%) Normal morphology (%) HOS+ (%)
Insemination data Experiment 1 cycles
Experiment 2 cycles
5.2 ± 0.18 155 ± 5.37 51.8 ± 1.04 70.6 ± 0.90 66.7 ± 1.14
26.2 ± 1.34 796.8 ± 42.0 51.1 ± 1.13 74.2 ± 1.27 68.3 ± 1.37
a Insemination data for Experiment 1 cycles (n = 125) and Experiment 2 cycles (n = 128) are means (±S.E.M.). TSN, total spermatozoal number inseminated. HOS+, percentage of spermatozoa inseminated which were positive for hypoosmotic swelling.
pregnancy of the absolute number of spermatozoa within each evaluation method was assessed. Tukey’s multiple comparison test was used to identify differences among the evaluation methods for the total spermatozoal number inseminated and the stallions for inseminate quality and total spermatozoal number inseminated.
3. Results The total number of breeding cycles in Experiments 1 and 2 were 125 and 128, respectively. Due to an unexpected loss of one stallion, three cycles assigned to that stallion
Fig. 1. The mean percentage of progressively motile, morphologically normal and HOS+ spermatozoa by stallion in Experiment 1 (100 million spermatozoal dose) of the study. Differences include, motility: different superscripts are different (P < 0.001); morphology: different superscripts are different (P < 0.05); HOS+: a vs. c, d (P < 0.001); b, c vs. d (P < 0.001).
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Fig. 2. The mean percentage of progressively motile, morphologically normal and HOS+ spermatozoa by stallion in Experiment 2 (500 million spermatozoal dose) of the study. Differences include motility: a vs. c, d and b vs. c and c vs. d (P < 0.05); morphology: a vs. b, c and b vs. c (P < 0.05); HOS+: a vs. c, d (P < 0.01) and c vs. d (P < 0.001).
were not completed in Experiment 1. A replacement stallion was used in Experiment 2. Specific insemination data are presented in Table 1. Inseminate quality by stallion and the mean total number of spermatozoa inseminated by stallion or treatment group are depicted in Figs. 1–6. Overall pregnancy outcomes for Experiments 1 and 2 were 45.6 and 38.3%,
Fig. 3. The mean total spermatozoal number inseminated in each Al/semen-evaluation treatment group in Experiment 1(100 million spermatozoal dose) of the study. Differences include, a vs. b, e (P < 0.01); b vs. c, e (P < 0.01); c vs. d, e (P < 0.05) and d vs. e (P < 0.001).
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Fig. 4. The mean total spermatozoal number inseminated in each Al/semen-evaluation treatment 311 group in Experiment 2 (500 million spermatozoal dose) of the study. Differences include, a vs. b (P < 0.05).
respectively. Pregnancy results for the individual treatment groups are depicted in Figs. 7–9. Pregnancy outcome was not different among methods of spermatozoal evaluation and selection for insemination in Experiment 1 (P = 0.52) or Experiment 2 (P = 0.78). Also the total number of spermatozoa and the absolute number of progressively motile,
Fig. 5. The mean total spermatozoal number inseminated for each stallion in Experiment 1 (100 million spermatozoal dose) of the study. Differences include, a vs. b (P < 0.01).
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Fig. 6. The mean total spermatozoal number inseminated for each stallion in Experiment 2 (500 million spermatozoal dose) of the study. Differences include, a vs. b (P < 0.05).
morphologically normal or HOS+ spermatozoa inseminated (Table 1) were not closely associated with pregnancy outcome in the experiments where 100 (P = 0.24, 0.29, 0.33 and 0.38, respectively) or 500 (P = 0.20, 0.84, 0.50 and 0.74, respectively) million spermatozoa were used.
Fig. 7. Pregnancy outcomes for the Al/semen-evaluation treatment groups in Experiment 1 (100 million spermatozoal dose) and Experiment 2 (500 million spermatozoal dose) of the study.
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Fig. 8. Pregnancy outcomes for the Al/semen-evaluation treatment groups within each stallion for Experiment 1 (100 million spermatozoal dose) of the study.
Fig. 9. Pregnancy outcomes for the Al/semen-evaluation treatment groups within each stallion for Experiment 2 (500 million spermatozoal dose) of the study.
4. Discussion In this study, the effect on pregnancy outcome of the method used to evaluate and select spermatozoa for artificial insemination was compared under conditions that would be encountered in routine breeding operations throughout the industry. None of the semen evaluation methods had an advantage for pregnancy outcome when used to select
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spermatozoa for insemination at either the 100 or 500 million dose. The results were surprising, as differences among evaluation methods were expected, particularly for the group in which only an absolute number of spermatozoa were inseminated. Instead evaluating spermatozoa offered no advantage for pregnancy over simply inseminating spermatozoa not selected for any particular characteristic, at least under the conditions of this study. These results were found in two independent experiments conducted in separate years. Having evaluated all ejaculates by each evaluation method, the total number of spermatozoa inseminated, which reflected the selection criteria used in these experiments, was known for every breeding cycle. Using this continuum of inseminated spermatozoa, it was possible to determine the association between each spermatozoal evaluation method and pregnancy outcome. It was interesting that the absolute number of spermatozoa inseminated was not associated with pregnancy outcome. The total number of spermatozoa inseminated ranged from a low of 100 million to well over a billion in Experiment 2 (Figs. 3, 4 and 6). This indicated, at the doses tested in this study, that adding more spermatozoa to the inseminate, whether selected for quality or not, would not mean the mare was more likely to become pregnant. Though, the pregnancy results were not statistically different among the AI/semenevaluation treatment groups in either experiment, the HOS+ group had the lowest absolute number of pregnancies in both years (Fig. 7). This may have occurred due to the incubation time required for the HOS test. The extended semen was held at room temperature for approximately 45 min after collection before being inseminated when HOS was used to select the spermatozoa. Holding time was not standardized for all AI/semen-evaluation treatment groups because the intent was to make comparisons between methods as applied under field conditions. Spermatozoa would most likely be inseminated shortly after evaluation under field conditions regardless of the method used. Methods requiring a shorter evaluation time naturally mean the spermatozoa would be inseminated sooner. Without question, evaluation methods are important for assessing and monitoring specific spermatozoal characteristics. Many of the characteristics related to form or function of spermatozoa would also logically seem to be important for fertility, though, we may not currently be able to determine a strong association. Individual spermatozoal assays may evaluate one or more of these characteristics. However, no currently available method, which can be easily and quickly applied, appears to be capable of assessing a combination of spermatozoal characteristics that would provide an advantage for inseminate selection. Under field conditions this would seem to be quite logical, as there are three factors involved in any fertility equation involving horses: the mare, the stallion and management practices. Any of these can negatively influence the results. Spermatozoal evaluation methods cannot possibly predict what influence the mare or management factors will have in the fertility equation. Nevertheless, in the present study, where the mare factor was randomized and the management factor was standardized as much as possible, the method of spermatozoal evaluation and selection still did not offer an advantage for pregnancy outcome. Though, determining progressive motility is traditionally the means by which a “standard” dose of spermatozoa is selected for insemination of mares, results of the present study did not indicate that this method offers any advantage for a positive pregnancy outcome as compared with the other methods considered in the present study. Also, the absolute number
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of spermatozoa inseminated did not suggest any greater likelihood for a mare to be pregnant or not pregnant. Nevertheless, it seems logical to surmise that at some point the absolute number of spermatozoa inseminated would fall below a minimum threshold and fertility would decline. If this is the case then there is likely a minimum number of spermatozoa required for each individual stallion to achieve an optimum fertility outcome in a given group of mares. The absolute number of spermatozoa would seem to be a more critical determinant of pregnancy outcome than spermatozoal quality, at least when selecting an inseminate by the methods used to assess spermatozoal quality for the dose ranges used in this study. Perhaps the number of spermatozoa used in the present study for each individual stallion exceeded the optimum fertility threshold, described by Saacke (1983), for all the evaluation methods, thus, making an association with fertility non-detectable. Reducing the number of spermatozoa selected by a given evaluation method below the threshold on the dose response curve for optimum fertility results would theoretically allow an association to be identified. The results of the present study would suggest reproductive management practices in the equine industry pertaining to semen evaluation and selection for insemination could be altered significantly without influencing fertility. However, it is likely that the threshold value for optimum fertility will vary among stallions and mare groups. Though surprising and undoubtedly contentious for some, results of the present study should not be dismissed. Instead we would encourage independent investigations to further evaluate results.
Acknowledgements This study was funded by grants from the Birmingham Racing Commission and the Auburn University College of Veterinary Medicine Animal Health and Disease Research Program. The authors wish to acknowledge the ERC technicians and all of the veterinary students who participated in this study. References Dascanio, J.J., Parker, N.A., Purswell, B.J., Digrassie, W.A., Bailey, T.L., Ley, W.B., Bowen, J.M., 1997a. Diagnostic procedures in mare reproduction: basic evaluation. Compend. Contin. Ed. Pract. Vet. 19, 980–985. Dascanio, J.J., Parker, N.A., Ley, W.B., Bailey, T.L., Purswell, B.J., Bowen, J.M., Digrassie, W.A., 1997b. Diagnostic procedures in mare reproduction: uterine evaluation, hysteroscopy, oviductal patency, and scintigraphy. Compend. Contin. Ed. Pract. Vet. 19, 1069–1076. Householder, D.D., Pickett, B.W., Voss, J.L., Olar, T.T., 1981. Effect of extender, number of spermatozoa and hCG on equine fertility. J. Eq. Vet. Sci. 1, 9–13. Hurtgen, J.P., 1992. Evaluation of the stallion for breeding soundness. Vet. Clin. N. Am. Eq. Pract. 8, 149–165. Jasko, D.J., 1992. Evaluation of stallion semen. Vet. Clin. N. Am. Eq. Pract. 8, 129–148. Jasko, D.J., Little, T.V., Lein, D.H., Foote, R.H., 1992. Comparison of spermatozoal movement and semen characteristics with fertility in stallions: 64 cases (1987–1988). J. Am. Vet. Med. Assoc. 200, 979–985. Metcalf, E.S., 2000. Insemination and breeding management. In: Samper, J.C. (Ed.), Equine Breeding Management and Artificial Insemination. WB Saunders Company, Philadelphia, pp. 179–194. Nie, G.J., Wenzel, J.G.W., 2001. Adaptation of the hypoosmotic swelling test to assess functional integrity of stallion spermatozoal plasma membranes. Theriogenology 55, 1005–1018.
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Saacke, R.G., 1983. Semen quality in relation to semen preservation. J. Dairy Sci. 66, 2635–2644. Samper, J.C., 1997. Techniques for artificial insemination. In: Youngquist, R.S. (Ed.), Current Therapy in Large Animal Theriogenology. WB Saunders Company, Philadelphia, pp. 36–42. Scott, M.A., Liu, I.K.M., Overstreet, J.W., Enders, A.C., 2000. The structural morphology and epithelial association of spermatozoa at the uterotubal junction: a descriptive study of equine spermatozoa in situ using scanning electron microscopy. J. Reprod. Fertil., Suppl. 56, 415–421. Voss, J.L., Pickett, B.W., 1976. Reproductive management of the broodmare. Colorado St. Univ. Exper. St. Gen. Ser. Bul. 961, 1–29.
Comparison of pregnancy outcome in mares among methods used to evaluate and select spermatozoa for insemination G.J. Nie∗,1 , J.G.W. Wenzel, K.E. Johnson Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, 146 McAdory Hall, AL 36849-5522, USA Received 21 December 2000; received in revised form 25 September 2001; accepted 12 October 2001
Abstract An artificial insemination dose for mares consisting of 500 million progressively motile spermatozoa is considered “standard” by most clinicians. However, little information is available directly comparing pregnancy outcome among methods of evaluating and selecting spermatozoa for insemination. The objective of this study was to determine if the method of spermatozoal evaluation and selection influences fertility as measured by pregnancy outcome. Mares were inseminated with 100 or 500 million spermatozoa that were selected for progressive motility, normal morphology, hypoosmotic swelling or absolute number regardless for evaluation method or quality. Thirty-two breeding cycles were tested for each treatment group and at each spermatozoal dose. Pregnancy outcomes were 44 and 41%, 55 and 41%, 39 and 31%, and 45 and 41%, for the 100 and 500 million progressively motile, morphologically normal, hypoosmotic swelling positive and absolute number treatment groups, respectively. Pregnancy outcome did not differ among methods of spermatozoal evaluation and selection for artificial insemination in the 100 (P = 0.52) or 500 (P = 0.78) million spermatozoa groups. Also the total number of spermatozoa and the absolute number of progressively motile, morphologically normal or hypoosmotic swelling positive spermatozoa inseminated, were not closely associated with pregnancy outcome in the 100 (P = 0.24, 0.29, 0.33 and 0.38, respectively) or 500 (P = 0.20, 0.84, 0.50 and 0.74, respectively) million spermatozoa groups. In this study, we found that the method of spermatozoal evaluation did not offer an advantage for pregnancy when used to select spermatozoa for insemination at the doses tested. These results were
∗ Corresponding author. Present address: Rood and Riddle Equine Hospital, 2150 Georgetown Road, P.O. Box 12070, Lexington, KY 40580, USA. E-mail address: [email protected] (G.J. Nie). 1 Tel.: +1-859-233-0371; fax: +1-859-255-5367.
0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 8 0 - 4
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surprising, as we expected there would be differences among the evaluation methods. Instead, we found that evaluating spermatozoa offered no advantage for pregnancy over simply inseminating with a specified number of spermatozoa not selected for any particular characteristic under the conditions of our experiment. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Artificial insemination; Progressive motility; Morphology; Hypoosmotic swelling; Equine
1. Introduction Reports during the 1970s and early 1980s suggested that an artificial insemination (AI) dose for mares consist of 500 million progressively motile spermatozoa (Voss and Pickett, 1976; Householder et al., 1981). In the two decades, since those reports were published an industry-wide emphasis has been placed on selecting insemination doses for motility characteristics. Today, an inseminate is typically selected for progressive motility and a dose of 500 million progressively motile spermatozoa is considered standard by most clinicians in order to maximize pregnancy rates (Samper, 1997; Metcalf, 2000). Motility is certainly a quick and easy spermatozoal characteristic to evaluate. However, motility characteristics are poorly correlated with fertility (Jasko, 1992; Jasko et al., 1992). Questions raised by the audience at a recent national symposium on stallion reproduction challenged our acceptance as an industry of motility as the best means of selecting spermatozoa for insemination (Stallion Reproduction Symposium, ACT/SFT/AAEP Baltimore, MD, 1998). Some members of the audience suggested that perhaps we should be selecting spermatozoa by other means (e.g. morphology) to optimize stallion factor fertility. In a recent study, Scott et al. (2000) found that over 90% of spermatozoa recovered from the uterotubal junction (UTJ) of mares were morphologically normal following insemination of those mares with doses containing as many as 85% abnormal cells. The researchers concluded that morphology, as well as motility should be considered when selecting an inseminate for mares. Another method of spermatozoal evaluation that may also offer some advantage in selecting insemination doses for mares is the hypoosmotic swelling (HOS) test. By assessing plasma membrane functional integrity and spermatozoal fertilizing capacity, the HOS test is a useful adjunct to existing spermatozoal assays in human reproduction (Jeyendran et al., 1992). Consequently, results of the HOS test may provide a better means of selecting stallion spermatozoa for insemination. Currently, we are unaware of any reports directly comparing fertility results among methods of evaluating and selecting spermatozoa for insemination. Progressive motility seems to have been established as the standard method of spermatozoal selection for insemination without valid comparisons with other methods. The objective of our study was to determine whether the spermatozoal evaluation and selection method influences fertility under conditions that would be encountered in routine breeding operations throughout the industry. We compared fertility results when the insemination dose was selected by routine analysis methods, including progressive motility morphology, HOS, and if no-evaluation method were used.
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2. Materials and methods A total of 39 mares and five stallions of various light-horse breeds (Equus caballus), ranging in age from 3 to 20 years and weighing 400–550 kg were used in this study. Horses were maintained in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (First Revised Edition, January 1999). All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee at Auburn University (IACUC Protocol No. 0211-R-2209). Mares were housed in groups of 4–8 in large paddocks, while stallions were individually housed in smaller paddocks. All horses were fed a commercial concentrate ration (12% protein) and costal bermuda grass hay for maintenance of body condition. The entire study was conducted as two separate experiments in successive years. Seven mares and one stallion were replaced between Experiments 1 and 2 due to medical and/or management circumstances. Protocols were identical in each experiment with the exception of total spermatozoal number selected for the AI dose. In Experiments 1 and 2, 100 and 500 million spermatozoa, respectively, were used as a selected insemination dose. The spermatozoa used for insemination were selected for progressive motility, normal morphology, HOS or absolute number without regard for evaluation method or quality. In each experiment, 32 mares were randomly and equally distributed among four stallions. The eight mares assigned to each stallion were bred in each of four AI/semen-evaluation treatment groups, which consisted of progressive motility, morphology, HOS and absolute number. This ensured 32 breeding cycles per stallion and per treatment group in each experiment. Cycles were successive and the treatment order was randomized for each mare. Mares were bred in each AI/semen-evaluation treatment group using spermatozoa from the assigned stallion for that experiment. All mares had ovulated at least once early in the breeding season and prior to entering the study. Mares and stallions were screened for breeding soundness using routine techniques (Dascanio et al., 1997a,b; Hurtgen, 1992). As mares entered estrus, the reproductive tract was examined every other day via palpation and ultrasonography for follicles >30 mm in diameter. When follicles reached 30–35 mm mares were examined daily. All examinations were conducted in the morning between 6 and 8 a.m. When a ≥35 mm follicle was detected, the mare was given 2500 units of human chorionic gonadotropin (Novarel® , Ferring Pharmaceuticals, Tarrytown, NY), intravenously, to induce ovulation. Each mare was inseminated once per cycle, the following morning approximately 24 h following ovulation induction. On the morning of insemination the assigned stallion was collected using an artificial vagina. Equal volumes of semen and prewarmed (37 ◦ C) extender were mixed within 2 min of collection. The extender used in this study consisted of non-fat-dry-milk-solid (2.4 g) and glucose (4.9 g) dissolved in sterile, de-ionized water to a total volume of 100 ml. The extender also included 1 mg/ml of ticarcillin (Ticar® , SmithKline Beecham Pharmaceuticals, Philadelphia, PA). Spermatozoal concentration of the undiluted semen was determined using a densimeter (Animal Reproduction Systems, Chino, CA). The 1:1 extended aliquot was then further diluted with extender to a final concentration of 30 million spermatozoa/ml. Extended spermatozoa were examined for motility, morphology and HOS parameters. During analysis the reservoir of extended spermatozoa was placed in a dark cabinet which had a constant temperature of 25–27 ◦ C.
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Motility parameters were evaluated within minutes of collection using a computer-aided spermatozoal analysis system (CASA; HTM-C, Hamilton Thorne Reasearch, Beverly, MA). A 4.5 l drop of extended spermatozoa was placed on a pre-warmed (37 ◦ C) glass slide and covered with a 22 mm × 22 mm cover slip. Slide temperature was maintained with a stage warmer during evaluation. A minimum of five fields and 500 spermatozoa were analyzed from each sample using the CASA system. Analysis setup included frames acquired (30), frame rate (60 Hz), straightness threshold (80%), low path velocity(VAP) cutoff (5.0 m/s), medium path velocity (VAP) cutoff (25.0 m/s), low progressive velocity (VSL) cutoff (11.0 m/s), and slow cells motile (no). Insemination doses of 100 or 500 million progressively motile spermatozoa were calculated from this measure of motility. Spermatozoal morphology was evaluated using an eosin–nigrosin morphology stain (Lane Mfg, Denver, CO). Equal volumes of raw semen and stain were smeared and air dried on glass slides. Slides were examined for spermatozoal morphology using bright-field microscopy (1000×) to evaluate 100 cells. Spermatozoa were classified as normal or abnormal. Insemination doses of 100 or 500 million morphologically normal spermatozoa were calculated from this measure of morphology. Spermatozoa were evaluated for HOS using the procedures described by Nie and Wenzel (2001). A 100 ml volume of the extended spermatozoal sample was added to 1.0 ml of a prewarmed (37 ◦ C) 100 millionmole sucrose solution. The mixture was incubated at 37 ◦ C for 30 min in a 1.5 ml micro-centrifuge tube. Following incubation, a small drop of sample was placed on a microscope slide and cover-slipped for evaluation. Samples were examined using phase contrast microscopy (400×) to evaluate 100 spermatozoa for evidence of swelling and curling (HOS+) changes. Insemination doses of 100 or 500 million HOS+ spermatozoa were calculated from this measure of hypoosmotic swelling. Mares were inseminated using routine procedures (Metcalf, 2000). Inseminations were performed following spermatozoal evaluation by the assigned selection method and within approximately 10, 15, 20 or 45 min after a collection for the absolute number, motility, morphology and HOS AI/semen-evaluation treatment groups, respectively. The total insemination volume for all treatment groups and the total number of spermatozoa inseminated for all treatment groups except the absolute number group were dependent on ejaculate quality that day. Mares were inseminated with either 100 (Experiment 1) or 500 (Experiment 2) million spermatozoa that were progressively motile, morphologically normal or HOS+. In the case of the absolute number treatment group, mares were inseminated with either 100 (Experiment 1) or 500 (Experiment 2) million total spermatozoa regardless of quality. All mares were provided routine post-breeding management standard for our unit, including daily examinations through the detection of ovulation. Pregnancy examinations were conducted at 14 days following ovulation using transrectal ultrasonography. Pregnancy outcome at 14 days was considered the endpoint for each treatment. Following the pregnancy examination each mare was given a luteolytic dose of cloprostenol (Estrumate® , Bayer Animal Health, Shawnee Mission, KS) to bring her into estrus for the next assigned treatment cycle. Proportions of pregnancies versus non-pregnancies were contrasted with logistic regression using the maximum-likelihood method (SAS-PROC CATMOD, SAS Institute, Cary, NC). Models were constructed using forward stepwise selection. In the first analysis, semen evaluation methods were contrasted as a categorical variable; in the second, the effect on
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Table 1 Characteristics of stallion spermatozoa used to inseminate maresa Parameter
Insemination volume (ml) TSN (×106 ) Progressive motility (%) Normal morphology (%) HOS+ (%)
Insemination data Experiment 1 cycles
Experiment 2 cycles
5.2 ± 0.18 155 ± 5.37 51.8 ± 1.04 70.6 ± 0.90 66.7 ± 1.14
26.2 ± 1.34 796.8 ± 42.0 51.1 ± 1.13 74.2 ± 1.27 68.3 ± 1.37
a Insemination data for Experiment 1 cycles (n = 125) and Experiment 2 cycles (n = 128) are means (±S.E.M.). TSN, total spermatozoal number inseminated. HOS+, percentage of spermatozoa inseminated which were positive for hypoosmotic swelling.
pregnancy of the absolute number of spermatozoa within each evaluation method was assessed. Tukey’s multiple comparison test was used to identify differences among the evaluation methods for the total spermatozoal number inseminated and the stallions for inseminate quality and total spermatozoal number inseminated.
3. Results The total number of breeding cycles in Experiments 1 and 2 were 125 and 128, respectively. Due to an unexpected loss of one stallion, three cycles assigned to that stallion
Fig. 1. The mean percentage of progressively motile, morphologically normal and HOS+ spermatozoa by stallion in Experiment 1 (100 million spermatozoal dose) of the study. Differences include, motility: different superscripts are different (P < 0.001); morphology: different superscripts are different (P < 0.05); HOS+: a vs. c, d (P < 0.001); b, c vs. d (P < 0.001).
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Fig. 2. The mean percentage of progressively motile, morphologically normal and HOS+ spermatozoa by stallion in Experiment 2 (500 million spermatozoal dose) of the study. Differences include motility: a vs. c, d and b vs. c and c vs. d (P < 0.05); morphology: a vs. b, c and b vs. c (P < 0.05); HOS+: a vs. c, d (P < 0.01) and c vs. d (P < 0.001).
were not completed in Experiment 1. A replacement stallion was used in Experiment 2. Specific insemination data are presented in Table 1. Inseminate quality by stallion and the mean total number of spermatozoa inseminated by stallion or treatment group are depicted in Figs. 1–6. Overall pregnancy outcomes for Experiments 1 and 2 were 45.6 and 38.3%,
Fig. 3. The mean total spermatozoal number inseminated in each Al/semen-evaluation treatment group in Experiment 1(100 million spermatozoal dose) of the study. Differences include, a vs. b, e (P < 0.01); b vs. c, e (P < 0.01); c vs. d, e (P < 0.05) and d vs. e (P < 0.001).
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Fig. 4. The mean total spermatozoal number inseminated in each Al/semen-evaluation treatment 311 group in Experiment 2 (500 million spermatozoal dose) of the study. Differences include, a vs. b (P < 0.05).
respectively. Pregnancy results for the individual treatment groups are depicted in Figs. 7–9. Pregnancy outcome was not different among methods of spermatozoal evaluation and selection for insemination in Experiment 1 (P = 0.52) or Experiment 2 (P = 0.78). Also the total number of spermatozoa and the absolute number of progressively motile,
Fig. 5. The mean total spermatozoal number inseminated for each stallion in Experiment 1 (100 million spermatozoal dose) of the study. Differences include, a vs. b (P < 0.01).
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Fig. 6. The mean total spermatozoal number inseminated for each stallion in Experiment 2 (500 million spermatozoal dose) of the study. Differences include, a vs. b (P < 0.05).
morphologically normal or HOS+ spermatozoa inseminated (Table 1) were not closely associated with pregnancy outcome in the experiments where 100 (P = 0.24, 0.29, 0.33 and 0.38, respectively) or 500 (P = 0.20, 0.84, 0.50 and 0.74, respectively) million spermatozoa were used.
Fig. 7. Pregnancy outcomes for the Al/semen-evaluation treatment groups in Experiment 1 (100 million spermatozoal dose) and Experiment 2 (500 million spermatozoal dose) of the study.
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Fig. 8. Pregnancy outcomes for the Al/semen-evaluation treatment groups within each stallion for Experiment 1 (100 million spermatozoal dose) of the study.
Fig. 9. Pregnancy outcomes for the Al/semen-evaluation treatment groups within each stallion for Experiment 2 (500 million spermatozoal dose) of the study.
4. Discussion In this study, the effect on pregnancy outcome of the method used to evaluate and select spermatozoa for artificial insemination was compared under conditions that would be encountered in routine breeding operations throughout the industry. None of the semen evaluation methods had an advantage for pregnancy outcome when used to select
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spermatozoa for insemination at either the 100 or 500 million dose. The results were surprising, as differences among evaluation methods were expected, particularly for the group in which only an absolute number of spermatozoa were inseminated. Instead evaluating spermatozoa offered no advantage for pregnancy over simply inseminating spermatozoa not selected for any particular characteristic, at least under the conditions of this study. These results were found in two independent experiments conducted in separate years. Having evaluated all ejaculates by each evaluation method, the total number of spermatozoa inseminated, which reflected the selection criteria used in these experiments, was known for every breeding cycle. Using this continuum of inseminated spermatozoa, it was possible to determine the association between each spermatozoal evaluation method and pregnancy outcome. It was interesting that the absolute number of spermatozoa inseminated was not associated with pregnancy outcome. The total number of spermatozoa inseminated ranged from a low of 100 million to well over a billion in Experiment 2 (Figs. 3, 4 and 6). This indicated, at the doses tested in this study, that adding more spermatozoa to the inseminate, whether selected for quality or not, would not mean the mare was more likely to become pregnant. Though, the pregnancy results were not statistically different among the AI/semenevaluation treatment groups in either experiment, the HOS+ group had the lowest absolute number of pregnancies in both years (Fig. 7). This may have occurred due to the incubation time required for the HOS test. The extended semen was held at room temperature for approximately 45 min after collection before being inseminated when HOS was used to select the spermatozoa. Holding time was not standardized for all AI/semen-evaluation treatment groups because the intent was to make comparisons between methods as applied under field conditions. Spermatozoa would most likely be inseminated shortly after evaluation under field conditions regardless of the method used. Methods requiring a shorter evaluation time naturally mean the spermatozoa would be inseminated sooner. Without question, evaluation methods are important for assessing and monitoring specific spermatozoal characteristics. Many of the characteristics related to form or function of spermatozoa would also logically seem to be important for fertility, though, we may not currently be able to determine a strong association. Individual spermatozoal assays may evaluate one or more of these characteristics. However, no currently available method, which can be easily and quickly applied, appears to be capable of assessing a combination of spermatozoal characteristics that would provide an advantage for inseminate selection. Under field conditions this would seem to be quite logical, as there are three factors involved in any fertility equation involving horses: the mare, the stallion and management practices. Any of these can negatively influence the results. Spermatozoal evaluation methods cannot possibly predict what influence the mare or management factors will have in the fertility equation. Nevertheless, in the present study, where the mare factor was randomized and the management factor was standardized as much as possible, the method of spermatozoal evaluation and selection still did not offer an advantage for pregnancy outcome. Though, determining progressive motility is traditionally the means by which a “standard” dose of spermatozoa is selected for insemination of mares, results of the present study did not indicate that this method offers any advantage for a positive pregnancy outcome as compared with the other methods considered in the present study. Also, the absolute number
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of spermatozoa inseminated did not suggest any greater likelihood for a mare to be pregnant or not pregnant. Nevertheless, it seems logical to surmise that at some point the absolute number of spermatozoa inseminated would fall below a minimum threshold and fertility would decline. If this is the case then there is likely a minimum number of spermatozoa required for each individual stallion to achieve an optimum fertility outcome in a given group of mares. The absolute number of spermatozoa would seem to be a more critical determinant of pregnancy outcome than spermatozoal quality, at least when selecting an inseminate by the methods used to assess spermatozoal quality for the dose ranges used in this study. Perhaps the number of spermatozoa used in the present study for each individual stallion exceeded the optimum fertility threshold, described by Saacke (1983), for all the evaluation methods, thus, making an association with fertility non-detectable. Reducing the number of spermatozoa selected by a given evaluation method below the threshold on the dose response curve for optimum fertility results would theoretically allow an association to be identified. The results of the present study would suggest reproductive management practices in the equine industry pertaining to semen evaluation and selection for insemination could be altered significantly without influencing fertility. However, it is likely that the threshold value for optimum fertility will vary among stallions and mare groups. Though surprising and undoubtedly contentious for some, results of the present study should not be dismissed. Instead we would encourage independent investigations to further evaluate results.
Acknowledgements This study was funded by grants from the Birmingham Racing Commission and the Auburn University College of Veterinary Medicine Animal Health and Disease Research Program. The authors wish to acknowledge the ERC technicians and all of the veterinary students who participated in this study. References Dascanio, J.J., Parker, N.A., Purswell, B.J., Digrassie, W.A., Bailey, T.L., Ley, W.B., Bowen, J.M., 1997a. Diagnostic procedures in mare reproduction: basic evaluation. Compend. Contin. Ed. Pract. Vet. 19, 980–985. Dascanio, J.J., Parker, N.A., Ley, W.B., Bailey, T.L., Purswell, B.J., Bowen, J.M., Digrassie, W.A., 1997b. Diagnostic procedures in mare reproduction: uterine evaluation, hysteroscopy, oviductal patency, and scintigraphy. Compend. Contin. Ed. Pract. Vet. 19, 1069–1076. Householder, D.D., Pickett, B.W., Voss, J.L., Olar, T.T., 1981. Effect of extender, number of spermatozoa and hCG on equine fertility. J. Eq. Vet. Sci. 1, 9–13. Hurtgen, J.P., 1992. Evaluation of the stallion for breeding soundness. Vet. Clin. N. Am. Eq. Pract. 8, 149–165. Jasko, D.J., 1992. Evaluation of stallion semen. Vet. Clin. N. Am. Eq. Pract. 8, 129–148. Jasko, D.J., Little, T.V., Lein, D.H., Foote, R.H., 1992. Comparison of spermatozoal movement and semen characteristics with fertility in stallions: 64 cases (1987–1988). J. Am. Vet. Med. Assoc. 200, 979–985. Metcalf, E.S., 2000. Insemination and breeding management. In: Samper, J.C. (Ed.), Equine Breeding Management and Artificial Insemination. WB Saunders Company, Philadelphia, pp. 179–194. Nie, G.J., Wenzel, J.G.W., 2001. Adaptation of the hypoosmotic swelling test to assess functional integrity of stallion spermatozoal plasma membranes. Theriogenology 55, 1005–1018.
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