- Email: [email protected]
Fixed-time insemination with frozen semen in mares: is it suitable for poorly fertile stallions?
Theriogenology 83 (2015) 1389–1393
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Fixed-time insemination with frozen semen in mares: is it suitable for poorly fertile stallions? Bruno Ribeiro Avanzi a, Renata dos Santos Ramos a, Gustavo Henrique Marques Araujo b, Eduardo Gorzoni Fioratti a, Luzia Aparecida Trinca c, José Antonio Dell’Aqua Jr a, Cely Marini Melo e Oña d, Fabíola Soares Zahn a, Ian Martin a, Marco Antonio Alvarenga a, Frederico Ozanam Papa a, * a
Department of Animal Reproduction and Veterinary Radiology, Faculdade de Medicina Veterinária e Zootecnia (FMVZ) – Universidade Estadual Paulista (UNESP), Botucatu, São Paulo, Brazil b Camilo Castelo Branco University, Descalvado, São Paulo, Brazil c Department of Biostatistics - UNESP, Botucatu, São Paulo, Brazil d Veterinary School of Goiás Federal University, Goiânia, Goiás, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 November 2013 Received in revised form 27 June 2014 Accepted 3 July 2014
The purpose of the present study was to compare two protocols for equine frozen semen programs using either postovulation insemination or fixed-time insemination (FT), evaluating both pregnancy rates and intrauterine fluid (IUF) accumulation after artificial insemination with semen obtained from either highly or poorly fertile stallions. Six ejaculates from two stallions (n ¼ 12) were processed. After thawing, semen samples were evaluated by computerized semen analysis. Fifteen mares (30 cycles) were inseminated with frozen semen from highly fertile stallion A, and 14 mares (28 cycles) were inseminated with frozen semen from poorly fertile stallion B. Ovulations were induced with 1 mg (intramuscular) of deslorelin acetate after the observation of a greater than 35 mm follicle and uterine edema. In postovulation insemination group, mares were inseminated once with 800 106 total sperm in a maximum 6-hour interval after ovulation. In FT group, mares were inseminated twice with 400 106 total sperm, 24 and 40 hours after induction. Mares were ultrasonographically examined for IUF accumulation 24 hours and for pregnancy diagnosis 14 days after the last insemination. Although IUF accumulation was more evident in mares inseminated once postovulation, pregnancy rates were similar for both protocols, regardless of the stallion, although a significant effect of the stallion was observed. These results indicated that FTs may be used for both highly and poorly fertile stallions as a practical tool to help spreading the use of frozen semen in equine reproduction programs. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Horse Artificial insemination Frozen semen Fertility
1. Introduction Artificial insemination with cryopreserved semen has established an important role in the utilization of superior stallions in breeding programs. As a consequence of the * Corresponding author. Tel./fax: þ5514 38802119. E-mail address: [email protected] (F.O. Papa). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.07.007
improvement of cooled semen shipping techniques, horse breeders realized that it is much more worthful to transport the semen to the mares than bringing the mares to the stallions. More recently, the logistical limitations (time, distance, and shipping delays) associated with cooled semen and the added benefits of frozen semen have led breeders to look toward frozen semen as a practical and efficient method for stallion semen shipping and storage [1].
1390
B.R. Avanzi et al. / Theriogenology 83 (2015) 1389–1393
The use of frozen semen is not fully encouraged yet in some breeds, and there is still some skepticism about it. Some of the impediments to the widespread commercial application of equine frozen semen include greater costs for reproductive management of mares inseminated with frozen semen; the need for experienced professionals for semen processing; lower fertility with frozen semen than with fresh or liquid-cooled semen for many stallions; and lack of customized protocols and quality standards leading to variable post-thaw semen quality after storage [1–3]. Two procedures are currently adopted for inseminations using frozen semen in mares. In the first one, mares are inseminated immediately before or after ovulation, which requires multiple examinations, involving considerable effort and costs. This allows mares to be inseminated only once per cycle, avoiding the waste of semen in those cases in which the progress of ovulation is unusual [3]. As an alternative, a simple, cost-effective, timed insemination protocol involving two frozen semen inseminations at 24 and 40 hours after administration of an ovulation inducing agent (such as hCG or Deslorelin) to a mare with a mature preovulatory follicle has been adopted for managing mares as a way to decrease labor and associated costs. In this protocol, the induced ovulation typically occurs between the two inseminations. This technique allows mares to be examined once daily (during daytime) until ovulation is confirmed, similar to the management of mares inseminated with liquid-cooled semen [1,4,5]. The main disadvantage of this technique is the possibility of semen waste and, also, some practitioners believe that a more severe postinsemination inflammatory response is likely to occur [3]. A review on frozen semen management in equine breeding programs [6] pointed out the need for controlled experiments to evaluate the efficacy of frozen semen artificial insemination (AI) protocols, as most of the available information comes from clinical data. Thus, the purpose of this study was to compare two protocols for equine frozen semen programs using either postovulation (or “conventional”) AI technique or timed insemination technique, evaluating both pregnancy rates and intrauterine fluid (IUF) accumulation after AI with semen obtained from either highly or poorly fertile stallions.
2. Material and methods Semen samples were obtained from two stallions with different reproductive records in frozen semen insemination programs: one Westfalen stallion (highly fertile, A) and one Mangalarga Marchador stallion (poorly fertile, B). Six ejaculates from each stallion were processed. Semen samples were immediately evaluated by CASA (HTM-IVOS 12; Hamilton-Thorne Research, Danvers, MA). Setup parameters are presented on Table 1, and only those presenting at least 60% total motility were cryopreserved, according to the technique described by Monteiro et al. [7]. For centrifugation purposes, a skim milk–based extender (BotuSemen, Botupharma, Botucatu, Brazil) was used at the concentration of 1:1 (v:v). For freezing purposes, pellets were diluted in an egg yolk–based extender (Botu-Crio
Table 1 Hamilton Thorne motion analyzer (HTMA) – IVOS 12 settings for seminal evaluation. Parameter
Adjust
Number of frames Minimum contrast Minimum cell size Contrast to static cells Straightness Minimum average path velocity to progressive cells Average path velocity cutoff Straight line velocity cutoff to slow cells Static head size Static head intensity Static head elongation Magnification Slow counted as motile Temperature
30 60 pixels 3 pixels 30 pixels 80% 70 mm/s 30 mm/s 20 mm/s 0.62–2.98 pixels 0.24–1.19 100–0 1.95 No 37 C
Botupharma, Botucatu, Brazil) at a final concentration of 200 million viable sperm per milliliter. Frozen samples were thawed as described by Dell’Aqua Jr. et al. [8] and evaluated by CASA according to the specifications described by Monteiro et al. [7]. A minimum of 1000 cells were examined per sample. The plasma membrane integrity was evaluated under epifluorescence microscope using the fluorescent probes carboxyfluorescein diacetate and propidium iodide; a minimum of 200 cells were examined per sample. Fifty-eight cycles from 29 mares were used for inseminations and were randomly distributed between stallions (A, n ¼ 30 cycles or B, n ¼ 28 cycles) and groups (postovulation AI, n ¼ 29 or fixed-time AI, n ¼ 29) in the first cycle. All the mares included in this experiment were clinically normal, without any record of reproductive failure or disturb. No maiden nor foaled mares were used. In the second cycle, each mare was inseminated using the same stallion but different AI method, so that they could be considered as control of themselves when AI methods were compared. Fifteen mares (30 cycles) were inseminated with stallion A frozen semen, and 14 mares (28 cycles) were inseminated with stallion B frozen semen. Mares were examined daily by rectal palpation and transrectal ultrasonography for estrus monitoring and uterine evaluation. When one follicle reached 35 mm and uterine edema was detected, ovulation was induced with 1 mg (intramuscular) of deslorelin acetate. Mares were monitored every 6 hours, starting 18 hours after the induction of ovulation, until ovulation was detected. This procedure was adopted for both groups, so that the moment of ovulations could be recorded. In postovulation insemination (PO) group, a single AI was performed with 800 106 total sperm (8 0.5-mL straws containing 100 106 each; 4 mL final insemination volume) in a maximum 6-hour interval after ovulation (mares were examined every 6 hours until ovulation). In fixed-time insemination (FT) group, two inseminations with 400 106 total sperm (4 0.5-mL straws containing 100 106 each; 2 mL final insemination volume) were performed: 24 hours after deslorelin injection and 40 hours after deslorelin injection. All inseminations were
B.R. Avanzi et al. / Theriogenology 83 (2015) 1389–1393
performed in the tip of the uterine horn ipsilateral to the ovulatory follicle using a flexible pipette (Minitube do Brasil Ltda, Porto Alegre-RS, Brazil). Twenty-four hours after the last AI, mares were examined ultrasonographically for IUF accumulation and any evidence of free fluid inside the uterine lumen (>0.3 cm) was considered positive. Pregnancy diagnosis was done by transrectal ultrasonography 14 days after the last insemination. All statistic tests were performed using SAS 9.1. computer program (SAS Institute Inc., Cary, NC). Frozen-thawed semen characteristics from stallions were compared by ANOVA. Logistic regression analysis (Proc GENMOD) was used to evaluate pregnancy rates and IUF accumulation between stallions and AI methods. Additionally, ANOVA was used to evaluate differences in the moment of ovulation in both groups. Differences were considered significant when P < 0.05. 3. Results and discussion After thawing, sperm kinetic parameters were significantly different between stallions A and B but there was no influence of the stallion on membrane integrity (Table 2). A significant effect of the stallion on pregnancy rates was also observed, but the technique of insemination did not influence pregnancy results, considering both average results and each stallion separately (Table 3). The use of a single PO protocol stimulated IUF accumulation (as observed at 24 hours after the last insemination) in a significantly higher percentage of mares when compared with FT protocol, in which mares were inseminated twice, at 24 and 40 hours after the induction of ovulation (58.6% [17/29] vs. 34.0% [10/29], respectively; P < 0.05). The average interval between administration of deslorelin acetate and ovulation was similar in both groups (40.9 hours), and in 87.9% (51/58) of cycles, mares ovulated between 30 and 48 hours after induction. According to the results obtained in sperm motion and pregnancy rates, differences in the resistance of sperm to freezing–thawing procedures between the two stallions are evident. This was not a surprise and agrees with a previous report that demonstrated the influence of the
Table 2 Mean values and standard deviations of post-thaw seminal parameters of samples obtained from stallions A and B. Parameter
Stallion A
Total motility (%) Progressive motility (%) Average path velocity (mm/s) Straight line velocity (mm/s) Curvilinear velocity (mm/s) Amplitude of lateral head displacement (mm) Beat-cross frequency (Hz) Straightness (%) Linearity (%) Rapid sperm (%) Plasma membrane integrity (%)
77.3 42.1 99.1 79.3 181.1 6.7
3.40a 3.18a 3.34a 3.25a 7.34a 0.16a
Stallion B 70.5 27.0 86.2 67.3 165.7 7.0
1.38b 1.41b 2.48b 1.75b 5.32b 0.19b
33.9 79.6 44.7 65.3 34.0
1.07a 1.13a 0.49a 4.07a 3.16
32.3 78.0 41.5 47.0 31.7
1.21b 0.89b 0.84b 2.97b 4.08
a,b Values followed by different letters in a line indicate significant difference (P < 0.05).
1391
Table 3 Pregnancy rates of mares submitted to a single post-ovulation insemination (PO) or two fixed-time inseminations (FT) with post-thaw sperm obtained from two different stallions (A and B). Stallion
PO
FT
Total
A B Total
46.7% (7/15) 35.7% (5/14) 41.4% (12/29)
80.0% (12/15) 21.4% (3/14) 51.7% (15/29)
63.3% (19/30)a 28.6% (8/28)b 46.6% (27/58)
POs were performed in a 6-hour interval after ovulations and FTs were performed at 24 hours and 40 hours after induction of ovulations. a,b Values followed by different letters in a column indicate significant difference (P < 0.05).
stallion breed on sperm resistance to freezing procedures, in which semen obtained from Mangalarga Marchador stallions was more susceptible to freezing damage when compared with semen obtained from jumping horse breeds [9]. According to Samper [10], this variation among stallions is one of the most important barriers for frozen semen use on a commercial basis. In this experiment, there was no effect of the stallion on post-thaw sperm membrane integrity. This may be because of the small population of sperm evaluated by epifluorescence microscopy, when compared with samples analyzed by CASA or flow cytometry [11]. Regardless of the insemination protocol, pregnancy rates obtained in the present study were within the range described by others [12–16]. One of the goals of the present study was to verify the suitability of FT protocol for frozen semen programs involving poorly fertile stallions. According to Samper [10], it is unlikely that inseminators are able to assess the quality of frozen semen when inseminating a mare. Semen quality is often suboptimal, but reproductive management may increase the probability of obtaining pregnancy. Sperm longevity in the female reproductive tract determines the maximum interval between insemination and ovulation. When sperm reach the uterus, they migrate into the oviduct and bind to the oviductal epithelium [17]. In mares, this reservoir of oviductal sperm is established inside the isthmus after coitus, and the sperm are gradually released so that they can participate in oocyte fertilization. This interaction between sperm and oviduct promotes a selection for morphologically and biochemically intact sperm [18], thus extending the viability and fertilizing capacity of sperm cell population [19–21]. Some reports demonstrated differences among stallions on the capacity to form a sperm reservoir in the oviduct, so that the population of motile sperm in the oviduct after insemination may be smaller for some subfertile stallions [18,22]. Additionally, semen freezing protocols may also lead to sublethal sperm damage and impair the formation of adequate sperm reservoir in the oviduct, thus shortening frozen semen viability after insemination [18]. According to Samper and Bader [10,17], cryopreserved sperm may need more time to reach the oviduct. Most authors in equine reproduction agree that mares to be inseminated with frozen semen should be inseminated as close as possible to ovulation. Therefore, it was a concern that inseminations using semen obtained from poorly fertile stallion on FT protocol could be ineffective because of the association
1392
B.R. Avanzi et al. / Theriogenology 83 (2015) 1389–1393
between sperm fertility and longevity. However, there was no significant difference between protocols for both stallions in the present study. The ideal moment and number of inseminations per cycle are still not completely determined [23]; some authors reported higher pregnancy rates after multiple inseminations with frozen semen when compared with a single insemination [16,24], while others did not observe such effect [5,15]. According to Sieme et al. [14], the beneficial effect of multiple ovulations would be due to the higher probability that one of the inseminations would occur close to ovulation, thus confirming the importance of the inseminationto-ovulation interval. Loomis and Squires [6], however, reported field data in which the frequency of insemination had no effect on pregnancy rates. Another study demonstrated similar pregnancy rates for one or two inseminations per cycle, although preovulation inseminations were significantly more effective than POs [25]. The influence of the moment of insemination on pregnancy rates is another debatable point. Some authors reported no effect of the moment of insemination [6,26], while others affirm that a single preovulatory insemination or both pre- and post-ovulatory inseminations would result in higher pregnancy rates when compared with single POs [23]. Some studies demonstrated that POs lead to a greater number of embryonic deaths, although this was more evident when inseminations were carried out more than 6 hours after ovulation. [27,28]. The problem with trying to establish ideal patterns for insemination protocols on the basis of different experiment results is that each experiment was conducted under different conditions (e.g., number of mares, number and quality of donor stallions, insemination dose, and technique). This may explain the contrasting results presented on reports. The FT protocol used in the present study was designed so that mares that ovulate 18 to 52 hours after administration of the ovulatory agent will have had spermatozoa deposited in the reproductive tract within an interval from 12 hours before ovulation to 6 hours after ovulation [6]. In the present study, most mares ovulated 30 to 48 hours after induction, which may suggest that the first insemination (24 hours) could be ineffective. In a retrospective study [29], mares treated with hCG ovulated from less than 12 hours to more than 48 hours after induction, and 75% of mares ovulated 24 to 48 hours after induction. The presence of uterine edema and a greater than 35 mm follicle at the moment of induction may be a reliable criterion to predict the moment of ovulation, which shall occur 36 to 42 hours after induction in more than 95% of mares [30]. In the present study, both criteria were adopted, which may explain the high concentration of ovulations in a narrow time. Although the uterine infusion of any kind of fluid can cause endometrial inflammation, it is known that spermatozoa themselves initiate the strongest polymorphonuclear (PMN) response [31], and this may be the cause of stronger inflammatory responses observed after inseminations with frozen semen, as it is used in high
sperm concentrations when compared with fresh or cooled semen [32]. On the other hand, Fiala et al. [33] reported high PMN numbers in uterine lavage fluid and endometrial biopsies immediately after insemination (2–4 hours) for mares inseminated with higher sperm concentrations but lower PMN numbers at 24 hours for the highest sperm concentration. It is possible that the sperm concentration (200 106/ mL) used in the present study may have contributed to the IUF accumulation in mares inseminated once after ovulation. In addition, it is known that uterine contractility is necessary for IUF elimination and that, when inseminations are performed after ovulation, the decreasing estrogen levels may interfere with this mechanism [34]. Some authors have reported that, in mares, persistent postmating endometritis, IUF or exaggerated uterine edema may dramatically reduce the possibility of pregnancy [35,36]. More recent studies, however, have shown that detection of postinsemination IUF was associated with positive cytology but not with bacteriology [37] or even that the presence of greater than 1 cm or equal of IUF in estrous mares is not well correlated with PMN-positive cytology of uterine samples [38]. The incidence of IUF accumulation in the present study was higher than that observed by Barbacini et al. [39], but here, any detectable amount of fluid was considered positive, while those authors considered positive for IUF accumulation only when at least 2 cm were found. 3.1. Conclusions Under the circumstances in which it was conducted, the present study demonstrated that the use of a simplified insemination protocol for mares using frozen semen was efficient and brought satisfactory pregnancy results for both highly and poorly fertile stallions. The decision of either examining mares frequently and inseminating once after ovulation or adopting a protocol that allows mares to be examined less frequently will depend on the evaluation of semen availability and veterinary expenses. Although it cannot be assumed that all stallions with either high or low fertility will behave as the ones used in this study, presented data support the choice for a more practical insemination protocol and may contribute to the spreading of frozen semen utilization in equine reproduction. Acknowledgment The authors acknowledge Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP) for financial support. References [1] Loomis PR, Graham JK. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 2008;105:119–28. [2] Katila T. Effect of the inseminate and site of insemination on the uterus and pregnancy rates of mares. Anim Reprod Sci 2005;89:31–8. [3] Samper JC, Estrada AJ, Mckinnon AO. Insemination with frozen semen. In: Samper JC, Pycock JF, Mckinnon AO, editors. Current therapy in equine reproduction. First edition. St. Louis: Saunders; 2007. p. 285–8.
B.R. Avanzi et al. / Theriogenology 83 (2015) 1389–1393 [4] Metcalf ES. The efficient use of equine cryopreserved semen. Theriogenology 2007;68:423–8. [5] Squires EL, Barbacini S, Necchi D, Reger HP, Bruemmer JE. Simplified strategy for insemination of mares with frozen semen. Proc Ann Conv Am Ass Equine Pract 2003;49:314–8. [6] Loomis PR, Squires EL. Frozen semen management in equine breeding programs. Theriogenology 2005;64:480–91. [7] Monteiro GA, Papa FO, Zahn FS, Dellaqua Jr JA, Melo CM, Maziero RRD, et al. Cryopreservation and fertility of ejaculated and epididymal stallion sperm. Anim Reprod Sci 2011;127:197–201. [8] Dell’aqua Jr JA, Papa FO, Zahn FS. Effects of warming rate on sperm parameters and of insemination site and dose on the fertility of equine frozen semen. Anim Reprod Sci 2002;68:344–6. [9] Alvarenga MA, Papa FO, Buratini Jr J. The effect of breed spermatic parameters over equine semen freezability. Theriogenology 2000;54:29–136. [10] Samper JC. Management and fertility of mares bred with frozen semen. Anim Reprod Sci 2001;68:219–28. [11] Varner DD. Developments in stallion semen evaluation. Theriogenology 2008;70:448–62. [12] Barbacini S, Loomis P, Squires EL. The effect of sperm number and frequency of insemination on pregnancy rates of mares inseminated with frozen-thawed spermatozoa. Anim Reprod Sci 2005;89:203–5. [13] Loomis PR. The equine frozen semen industry. Anim Reprod Sci 2001;68:191–200. [14] Sieme H, Schäfer T, Stout TAE, Klug E, Waberski D. The effects of different insemination regimes on fertility in mares. Theriogenology 2003;60:1153–64. [15] Squires EL, Reger HP, Maclellan LJ, Bruemmer JE. Effect of time of insemination and site of insemination on pregnancy rates with frozen semen. Theriogenology 2002;58:655–8. [16] Vidament M, Magistrini M, Palmer E, Clement F. Equine artificial insemination in French National Studs. Reprod Domest Anim 1999;(Suppl 6):61–6. [17] Bader H. An investigation of sperm migration into the oviducts of the mare. J Reprod Fertil Suppl 1982;32:59–64. [18] Ellington JE, Samper JC, Jones AE, Oliver SA, Burnett KM, Wright RW. In vitro interactions of cryopreserved stallion spermatozoa and oviduct (uterine tube) epithelial cells or their secretory products. Anim Reprod Sci 1999;56:51–65. [19] Dobrinski I, Thomas PGA, Ball BA. Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal ephithelial cells and zonae pellucidae in vitro. J Androl 1995;16:536–42. [20] Ellington JE, Ball BA, Blue BJ, Wilker CE. Capacitation-like membrane changes and prolonged viability in vitro of equine spermatozoa cultured with uterine tube epithelial cells. Am J Vet Res 1993;54:1505–10. [21] Thomas PGA, Ball BA, Miller PG, Brinsko SP, Southwood L. A subpopulation of morphologically normal, motile spermatozoa attach to equine oviductal epithelial cell monolayers. Biol Reprod 1994;51:303–9. [22] Scott MA, Liu IK, Overstreet JW. Sperm transport to the oviducts: abnormalities and their clinical implications. Proc Ann Conv Am Ass Equine Pract 1995;41:1–2.
1393
[23] Samper JC, Hankins K. Breeding mares with frozen semen in private practice. Proc Ann Conv Am Ass Equine Pract 2001;47:314–8. [24] Vidament M, Dupere AM, Julienne P, Evain A, Noue P, Palmer E. Equine frozen semen freezability and fertility results. Theriogenology 1997;48:907–17. [25] Metcalf ES. Optimizing pregnancy rates using frozen-thawed equine semen. Anim Reprod Sci 2005;89:297–9. [26] Barbacini S, Gulden P, Marchi V, Zavaglia G. Incidence of embryonic loss in mares inseminated before or after ovulation. Equine Vet Educ 1999;1:108–12. [27] Koskinen E, Lindeberg H, Kuntsi H, Ruotsalainen L, Katila T. Fertility of mares after postovulatory insemination. J Vet Med A 1990;37:77–80. [28] Woods J, Bergfeld DR, Ginther OJ. Effects of time of insemination relative to ovulation on pregnancy rate and embryonic loss rate in mares. Equine Vet J 1990;22:410–5. [29] Barbacini S, Zavaglia G, Gulden P, Marchi V, Necchi D. Retrospective study on the efficacy of hCG in an equine artificial insemination programme using frozen semen. Equine Vet Educ 2000;2:404–8. [30] Samper JC. Ultrasonographic appearance and the pattern of uterine edema to time ovulation in mares. Proc Ann Conv Am Ass Equine Pract 1997;43:41–3. [31] Katila T. Post-mating inflammatory responses of the uterus. Reprod Domest Anim 2012;47(Suppl 5):31–41. [32] Kotilainen T, Huhtinen M, Katila T. Sperm-induced leucocytosis in the equine uterus. Theriogenology 1994;41:629–36. [33] Fiala SM, Pimentel CA, Mattos AL, Gregory RM, Mattos RC. Effect of sperm numbers and concentration on sperm transport and uterine inflammatory response in the mare. Theriogenology 2007;67: 556–62. [34] Nikolakopoulos E, Watson ED. Uterine contractility is necessary for the clearance of intrauterine fluid but not bacteria after bacterial infusion in the mare. Theriogenology 1999;52:413–23. [35] Troedsson MHT, Steiger BN, Ibrahim NM, Foster DN, Crabo BG. Mechanism of sperm-induced endometritis in the mare. Biol Reprod 1995;52(Suppl 1):507. [36] Troedsson MHT. Uterine clearance and resistance to persistent endometritis in the mare. Theriogenology 1999;52:461–71. [37] LeBlanc MM, Burleson MD, Riddle WT, Hendricks KEM. Endometrial pathogens are associated with different ultrasonographic and endometrial cytology findings in Thoroughbred mares. Pferdeheilkunde 2012;28:86. [38] De Borba EVC, Camozzato GC, Malschitzky E, Bustamante-Filho IC, Martins AA, Mattos RC, et al. Is the presence of uterine fluid a reliable indicator of endometrial inflammation? Pferdeheilkunde 2012;28:27–9. [39] Barbacini S, Necchi D, Zavaglia G, Squires EL. Retrospective study of the incidence of post-insemination uterine fluid in mares inseminated with frozen-thawed semen. J Equine Vet Sci 2003; 23:493–6.
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Fixed-time insemination with frozen semen in mares: is it suitable for poorly fertile stallions? Bruno Ribeiro Avanzi a, Renata dos Santos Ramos a, Gustavo Henrique Marques Araujo b, Eduardo Gorzoni Fioratti a, Luzia Aparecida Trinca c, José Antonio Dell’Aqua Jr a, Cely Marini Melo e Oña d, Fabíola Soares Zahn a, Ian Martin a, Marco Antonio Alvarenga a, Frederico Ozanam Papa a, * a
Department of Animal Reproduction and Veterinary Radiology, Faculdade de Medicina Veterinária e Zootecnia (FMVZ) – Universidade Estadual Paulista (UNESP), Botucatu, São Paulo, Brazil b Camilo Castelo Branco University, Descalvado, São Paulo, Brazil c Department of Biostatistics - UNESP, Botucatu, São Paulo, Brazil d Veterinary School of Goiás Federal University, Goiânia, Goiás, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 November 2013 Received in revised form 27 June 2014 Accepted 3 July 2014
The purpose of the present study was to compare two protocols for equine frozen semen programs using either postovulation insemination or fixed-time insemination (FT), evaluating both pregnancy rates and intrauterine fluid (IUF) accumulation after artificial insemination with semen obtained from either highly or poorly fertile stallions. Six ejaculates from two stallions (n ¼ 12) were processed. After thawing, semen samples were evaluated by computerized semen analysis. Fifteen mares (30 cycles) were inseminated with frozen semen from highly fertile stallion A, and 14 mares (28 cycles) were inseminated with frozen semen from poorly fertile stallion B. Ovulations were induced with 1 mg (intramuscular) of deslorelin acetate after the observation of a greater than 35 mm follicle and uterine edema. In postovulation insemination group, mares were inseminated once with 800 106 total sperm in a maximum 6-hour interval after ovulation. In FT group, mares were inseminated twice with 400 106 total sperm, 24 and 40 hours after induction. Mares were ultrasonographically examined for IUF accumulation 24 hours and for pregnancy diagnosis 14 days after the last insemination. Although IUF accumulation was more evident in mares inseminated once postovulation, pregnancy rates were similar for both protocols, regardless of the stallion, although a significant effect of the stallion was observed. These results indicated that FTs may be used for both highly and poorly fertile stallions as a practical tool to help spreading the use of frozen semen in equine reproduction programs. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Horse Artificial insemination Frozen semen Fertility
1. Introduction Artificial insemination with cryopreserved semen has established an important role in the utilization of superior stallions in breeding programs. As a consequence of the * Corresponding author. Tel./fax: þ5514 38802119. E-mail address: [email protected] (F.O. Papa). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.07.007
improvement of cooled semen shipping techniques, horse breeders realized that it is much more worthful to transport the semen to the mares than bringing the mares to the stallions. More recently, the logistical limitations (time, distance, and shipping delays) associated with cooled semen and the added benefits of frozen semen have led breeders to look toward frozen semen as a practical and efficient method for stallion semen shipping and storage [1].
1390
B.R. Avanzi et al. / Theriogenology 83 (2015) 1389–1393
The use of frozen semen is not fully encouraged yet in some breeds, and there is still some skepticism about it. Some of the impediments to the widespread commercial application of equine frozen semen include greater costs for reproductive management of mares inseminated with frozen semen; the need for experienced professionals for semen processing; lower fertility with frozen semen than with fresh or liquid-cooled semen for many stallions; and lack of customized protocols and quality standards leading to variable post-thaw semen quality after storage [1–3]. Two procedures are currently adopted for inseminations using frozen semen in mares. In the first one, mares are inseminated immediately before or after ovulation, which requires multiple examinations, involving considerable effort and costs. This allows mares to be inseminated only once per cycle, avoiding the waste of semen in those cases in which the progress of ovulation is unusual [3]. As an alternative, a simple, cost-effective, timed insemination protocol involving two frozen semen inseminations at 24 and 40 hours after administration of an ovulation inducing agent (such as hCG or Deslorelin) to a mare with a mature preovulatory follicle has been adopted for managing mares as a way to decrease labor and associated costs. In this protocol, the induced ovulation typically occurs between the two inseminations. This technique allows mares to be examined once daily (during daytime) until ovulation is confirmed, similar to the management of mares inseminated with liquid-cooled semen [1,4,5]. The main disadvantage of this technique is the possibility of semen waste and, also, some practitioners believe that a more severe postinsemination inflammatory response is likely to occur [3]. A review on frozen semen management in equine breeding programs [6] pointed out the need for controlled experiments to evaluate the efficacy of frozen semen artificial insemination (AI) protocols, as most of the available information comes from clinical data. Thus, the purpose of this study was to compare two protocols for equine frozen semen programs using either postovulation (or “conventional”) AI technique or timed insemination technique, evaluating both pregnancy rates and intrauterine fluid (IUF) accumulation after AI with semen obtained from either highly or poorly fertile stallions.
2. Material and methods Semen samples were obtained from two stallions with different reproductive records in frozen semen insemination programs: one Westfalen stallion (highly fertile, A) and one Mangalarga Marchador stallion (poorly fertile, B). Six ejaculates from each stallion were processed. Semen samples were immediately evaluated by CASA (HTM-IVOS 12; Hamilton-Thorne Research, Danvers, MA). Setup parameters are presented on Table 1, and only those presenting at least 60% total motility were cryopreserved, according to the technique described by Monteiro et al. [7]. For centrifugation purposes, a skim milk–based extender (BotuSemen, Botupharma, Botucatu, Brazil) was used at the concentration of 1:1 (v:v). For freezing purposes, pellets were diluted in an egg yolk–based extender (Botu-Crio
Table 1 Hamilton Thorne motion analyzer (HTMA) – IVOS 12 settings for seminal evaluation. Parameter
Adjust
Number of frames Minimum contrast Minimum cell size Contrast to static cells Straightness Minimum average path velocity to progressive cells Average path velocity cutoff Straight line velocity cutoff to slow cells Static head size Static head intensity Static head elongation Magnification Slow counted as motile Temperature
30 60 pixels 3 pixels 30 pixels 80% 70 mm/s 30 mm/s 20 mm/s 0.62–2.98 pixels 0.24–1.19 100–0 1.95 No 37 C
Botupharma, Botucatu, Brazil) at a final concentration of 200 million viable sperm per milliliter. Frozen samples were thawed as described by Dell’Aqua Jr. et al. [8] and evaluated by CASA according to the specifications described by Monteiro et al. [7]. A minimum of 1000 cells were examined per sample. The plasma membrane integrity was evaluated under epifluorescence microscope using the fluorescent probes carboxyfluorescein diacetate and propidium iodide; a minimum of 200 cells were examined per sample. Fifty-eight cycles from 29 mares were used for inseminations and were randomly distributed between stallions (A, n ¼ 30 cycles or B, n ¼ 28 cycles) and groups (postovulation AI, n ¼ 29 or fixed-time AI, n ¼ 29) in the first cycle. All the mares included in this experiment were clinically normal, without any record of reproductive failure or disturb. No maiden nor foaled mares were used. In the second cycle, each mare was inseminated using the same stallion but different AI method, so that they could be considered as control of themselves when AI methods were compared. Fifteen mares (30 cycles) were inseminated with stallion A frozen semen, and 14 mares (28 cycles) were inseminated with stallion B frozen semen. Mares were examined daily by rectal palpation and transrectal ultrasonography for estrus monitoring and uterine evaluation. When one follicle reached 35 mm and uterine edema was detected, ovulation was induced with 1 mg (intramuscular) of deslorelin acetate. Mares were monitored every 6 hours, starting 18 hours after the induction of ovulation, until ovulation was detected. This procedure was adopted for both groups, so that the moment of ovulations could be recorded. In postovulation insemination (PO) group, a single AI was performed with 800 106 total sperm (8 0.5-mL straws containing 100 106 each; 4 mL final insemination volume) in a maximum 6-hour interval after ovulation (mares were examined every 6 hours until ovulation). In fixed-time insemination (FT) group, two inseminations with 400 106 total sperm (4 0.5-mL straws containing 100 106 each; 2 mL final insemination volume) were performed: 24 hours after deslorelin injection and 40 hours after deslorelin injection. All inseminations were
B.R. Avanzi et al. / Theriogenology 83 (2015) 1389–1393
performed in the tip of the uterine horn ipsilateral to the ovulatory follicle using a flexible pipette (Minitube do Brasil Ltda, Porto Alegre-RS, Brazil). Twenty-four hours after the last AI, mares were examined ultrasonographically for IUF accumulation and any evidence of free fluid inside the uterine lumen (>0.3 cm) was considered positive. Pregnancy diagnosis was done by transrectal ultrasonography 14 days after the last insemination. All statistic tests were performed using SAS 9.1. computer program (SAS Institute Inc., Cary, NC). Frozen-thawed semen characteristics from stallions were compared by ANOVA. Logistic regression analysis (Proc GENMOD) was used to evaluate pregnancy rates and IUF accumulation between stallions and AI methods. Additionally, ANOVA was used to evaluate differences in the moment of ovulation in both groups. Differences were considered significant when P < 0.05. 3. Results and discussion After thawing, sperm kinetic parameters were significantly different between stallions A and B but there was no influence of the stallion on membrane integrity (Table 2). A significant effect of the stallion on pregnancy rates was also observed, but the technique of insemination did not influence pregnancy results, considering both average results and each stallion separately (Table 3). The use of a single PO protocol stimulated IUF accumulation (as observed at 24 hours after the last insemination) in a significantly higher percentage of mares when compared with FT protocol, in which mares were inseminated twice, at 24 and 40 hours after the induction of ovulation (58.6% [17/29] vs. 34.0% [10/29], respectively; P < 0.05). The average interval between administration of deslorelin acetate and ovulation was similar in both groups (40.9 hours), and in 87.9% (51/58) of cycles, mares ovulated between 30 and 48 hours after induction. According to the results obtained in sperm motion and pregnancy rates, differences in the resistance of sperm to freezing–thawing procedures between the two stallions are evident. This was not a surprise and agrees with a previous report that demonstrated the influence of the
Table 2 Mean values and standard deviations of post-thaw seminal parameters of samples obtained from stallions A and B. Parameter
Stallion A
Total motility (%) Progressive motility (%) Average path velocity (mm/s) Straight line velocity (mm/s) Curvilinear velocity (mm/s) Amplitude of lateral head displacement (mm) Beat-cross frequency (Hz) Straightness (%) Linearity (%) Rapid sperm (%) Plasma membrane integrity (%)
77.3 42.1 99.1 79.3 181.1 6.7
3.40a 3.18a 3.34a 3.25a 7.34a 0.16a
Stallion B 70.5 27.0 86.2 67.3 165.7 7.0
1.38b 1.41b 2.48b 1.75b 5.32b 0.19b
33.9 79.6 44.7 65.3 34.0
1.07a 1.13a 0.49a 4.07a 3.16
32.3 78.0 41.5 47.0 31.7
1.21b 0.89b 0.84b 2.97b 4.08
a,b Values followed by different letters in a line indicate significant difference (P < 0.05).
1391
Table 3 Pregnancy rates of mares submitted to a single post-ovulation insemination (PO) or two fixed-time inseminations (FT) with post-thaw sperm obtained from two different stallions (A and B). Stallion
PO
FT
Total
A B Total
46.7% (7/15) 35.7% (5/14) 41.4% (12/29)
80.0% (12/15) 21.4% (3/14) 51.7% (15/29)
63.3% (19/30)a 28.6% (8/28)b 46.6% (27/58)
POs were performed in a 6-hour interval after ovulations and FTs were performed at 24 hours and 40 hours after induction of ovulations. a,b Values followed by different letters in a column indicate significant difference (P < 0.05).
stallion breed on sperm resistance to freezing procedures, in which semen obtained from Mangalarga Marchador stallions was more susceptible to freezing damage when compared with semen obtained from jumping horse breeds [9]. According to Samper [10], this variation among stallions is one of the most important barriers for frozen semen use on a commercial basis. In this experiment, there was no effect of the stallion on post-thaw sperm membrane integrity. This may be because of the small population of sperm evaluated by epifluorescence microscopy, when compared with samples analyzed by CASA or flow cytometry [11]. Regardless of the insemination protocol, pregnancy rates obtained in the present study were within the range described by others [12–16]. One of the goals of the present study was to verify the suitability of FT protocol for frozen semen programs involving poorly fertile stallions. According to Samper [10], it is unlikely that inseminators are able to assess the quality of frozen semen when inseminating a mare. Semen quality is often suboptimal, but reproductive management may increase the probability of obtaining pregnancy. Sperm longevity in the female reproductive tract determines the maximum interval between insemination and ovulation. When sperm reach the uterus, they migrate into the oviduct and bind to the oviductal epithelium [17]. In mares, this reservoir of oviductal sperm is established inside the isthmus after coitus, and the sperm are gradually released so that they can participate in oocyte fertilization. This interaction between sperm and oviduct promotes a selection for morphologically and biochemically intact sperm [18], thus extending the viability and fertilizing capacity of sperm cell population [19–21]. Some reports demonstrated differences among stallions on the capacity to form a sperm reservoir in the oviduct, so that the population of motile sperm in the oviduct after insemination may be smaller for some subfertile stallions [18,22]. Additionally, semen freezing protocols may also lead to sublethal sperm damage and impair the formation of adequate sperm reservoir in the oviduct, thus shortening frozen semen viability after insemination [18]. According to Samper and Bader [10,17], cryopreserved sperm may need more time to reach the oviduct. Most authors in equine reproduction agree that mares to be inseminated with frozen semen should be inseminated as close as possible to ovulation. Therefore, it was a concern that inseminations using semen obtained from poorly fertile stallion on FT protocol could be ineffective because of the association
1392
B.R. Avanzi et al. / Theriogenology 83 (2015) 1389–1393
between sperm fertility and longevity. However, there was no significant difference between protocols for both stallions in the present study. The ideal moment and number of inseminations per cycle are still not completely determined [23]; some authors reported higher pregnancy rates after multiple inseminations with frozen semen when compared with a single insemination [16,24], while others did not observe such effect [5,15]. According to Sieme et al. [14], the beneficial effect of multiple ovulations would be due to the higher probability that one of the inseminations would occur close to ovulation, thus confirming the importance of the inseminationto-ovulation interval. Loomis and Squires [6], however, reported field data in which the frequency of insemination had no effect on pregnancy rates. Another study demonstrated similar pregnancy rates for one or two inseminations per cycle, although preovulation inseminations were significantly more effective than POs [25]. The influence of the moment of insemination on pregnancy rates is another debatable point. Some authors reported no effect of the moment of insemination [6,26], while others affirm that a single preovulatory insemination or both pre- and post-ovulatory inseminations would result in higher pregnancy rates when compared with single POs [23]. Some studies demonstrated that POs lead to a greater number of embryonic deaths, although this was more evident when inseminations were carried out more than 6 hours after ovulation. [27,28]. The problem with trying to establish ideal patterns for insemination protocols on the basis of different experiment results is that each experiment was conducted under different conditions (e.g., number of mares, number and quality of donor stallions, insemination dose, and technique). This may explain the contrasting results presented on reports. The FT protocol used in the present study was designed so that mares that ovulate 18 to 52 hours after administration of the ovulatory agent will have had spermatozoa deposited in the reproductive tract within an interval from 12 hours before ovulation to 6 hours after ovulation [6]. In the present study, most mares ovulated 30 to 48 hours after induction, which may suggest that the first insemination (24 hours) could be ineffective. In a retrospective study [29], mares treated with hCG ovulated from less than 12 hours to more than 48 hours after induction, and 75% of mares ovulated 24 to 48 hours after induction. The presence of uterine edema and a greater than 35 mm follicle at the moment of induction may be a reliable criterion to predict the moment of ovulation, which shall occur 36 to 42 hours after induction in more than 95% of mares [30]. In the present study, both criteria were adopted, which may explain the high concentration of ovulations in a narrow time. Although the uterine infusion of any kind of fluid can cause endometrial inflammation, it is known that spermatozoa themselves initiate the strongest polymorphonuclear (PMN) response [31], and this may be the cause of stronger inflammatory responses observed after inseminations with frozen semen, as it is used in high
sperm concentrations when compared with fresh or cooled semen [32]. On the other hand, Fiala et al. [33] reported high PMN numbers in uterine lavage fluid and endometrial biopsies immediately after insemination (2–4 hours) for mares inseminated with higher sperm concentrations but lower PMN numbers at 24 hours for the highest sperm concentration. It is possible that the sperm concentration (200 106/ mL) used in the present study may have contributed to the IUF accumulation in mares inseminated once after ovulation. In addition, it is known that uterine contractility is necessary for IUF elimination and that, when inseminations are performed after ovulation, the decreasing estrogen levels may interfere with this mechanism [34]. Some authors have reported that, in mares, persistent postmating endometritis, IUF or exaggerated uterine edema may dramatically reduce the possibility of pregnancy [35,36]. More recent studies, however, have shown that detection of postinsemination IUF was associated with positive cytology but not with bacteriology [37] or even that the presence of greater than 1 cm or equal of IUF in estrous mares is not well correlated with PMN-positive cytology of uterine samples [38]. The incidence of IUF accumulation in the present study was higher than that observed by Barbacini et al. [39], but here, any detectable amount of fluid was considered positive, while those authors considered positive for IUF accumulation only when at least 2 cm were found. 3.1. Conclusions Under the circumstances in which it was conducted, the present study demonstrated that the use of a simplified insemination protocol for mares using frozen semen was efficient and brought satisfactory pregnancy results for both highly and poorly fertile stallions. The decision of either examining mares frequently and inseminating once after ovulation or adopting a protocol that allows mares to be examined less frequently will depend on the evaluation of semen availability and veterinary expenses. Although it cannot be assumed that all stallions with either high or low fertility will behave as the ones used in this study, presented data support the choice for a more practical insemination protocol and may contribute to the spreading of frozen semen utilization in equine reproduction. Acknowledgment The authors acknowledge Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP) for financial support. References [1] Loomis PR, Graham JK. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 2008;105:119–28. [2] Katila T. Effect of the inseminate and site of insemination on the uterus and pregnancy rates of mares. Anim Reprod Sci 2005;89:31–8. [3] Samper JC, Estrada AJ, Mckinnon AO. Insemination with frozen semen. In: Samper JC, Pycock JF, Mckinnon AO, editors. Current therapy in equine reproduction. First edition. St. Louis: Saunders; 2007. p. 285–8.
B.R. Avanzi et al. / Theriogenology 83 (2015) 1389–1393 [4] Metcalf ES. The efficient use of equine cryopreserved semen. Theriogenology 2007;68:423–8. [5] Squires EL, Barbacini S, Necchi D, Reger HP, Bruemmer JE. Simplified strategy for insemination of mares with frozen semen. Proc Ann Conv Am Ass Equine Pract 2003;49:314–8. [6] Loomis PR, Squires EL. Frozen semen management in equine breeding programs. Theriogenology 2005;64:480–91. [7] Monteiro GA, Papa FO, Zahn FS, Dellaqua Jr JA, Melo CM, Maziero RRD, et al. Cryopreservation and fertility of ejaculated and epididymal stallion sperm. Anim Reprod Sci 2011;127:197–201. [8] Dell’aqua Jr JA, Papa FO, Zahn FS. Effects of warming rate on sperm parameters and of insemination site and dose on the fertility of equine frozen semen. Anim Reprod Sci 2002;68:344–6. [9] Alvarenga MA, Papa FO, Buratini Jr J. The effect of breed spermatic parameters over equine semen freezability. Theriogenology 2000;54:29–136. [10] Samper JC. Management and fertility of mares bred with frozen semen. Anim Reprod Sci 2001;68:219–28. [11] Varner DD. Developments in stallion semen evaluation. Theriogenology 2008;70:448–62. [12] Barbacini S, Loomis P, Squires EL. The effect of sperm number and frequency of insemination on pregnancy rates of mares inseminated with frozen-thawed spermatozoa. Anim Reprod Sci 2005;89:203–5. [13] Loomis PR. The equine frozen semen industry. Anim Reprod Sci 2001;68:191–200. [14] Sieme H, Schäfer T, Stout TAE, Klug E, Waberski D. The effects of different insemination regimes on fertility in mares. Theriogenology 2003;60:1153–64. [15] Squires EL, Reger HP, Maclellan LJ, Bruemmer JE. Effect of time of insemination and site of insemination on pregnancy rates with frozen semen. Theriogenology 2002;58:655–8. [16] Vidament M, Magistrini M, Palmer E, Clement F. Equine artificial insemination in French National Studs. Reprod Domest Anim 1999;(Suppl 6):61–6. [17] Bader H. An investigation of sperm migration into the oviducts of the mare. J Reprod Fertil Suppl 1982;32:59–64. [18] Ellington JE, Samper JC, Jones AE, Oliver SA, Burnett KM, Wright RW. In vitro interactions of cryopreserved stallion spermatozoa and oviduct (uterine tube) epithelial cells or their secretory products. Anim Reprod Sci 1999;56:51–65. [19] Dobrinski I, Thomas PGA, Ball BA. Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal ephithelial cells and zonae pellucidae in vitro. J Androl 1995;16:536–42. [20] Ellington JE, Ball BA, Blue BJ, Wilker CE. Capacitation-like membrane changes and prolonged viability in vitro of equine spermatozoa cultured with uterine tube epithelial cells. Am J Vet Res 1993;54:1505–10. [21] Thomas PGA, Ball BA, Miller PG, Brinsko SP, Southwood L. A subpopulation of morphologically normal, motile spermatozoa attach to equine oviductal epithelial cell monolayers. Biol Reprod 1994;51:303–9. [22] Scott MA, Liu IK, Overstreet JW. Sperm transport to the oviducts: abnormalities and their clinical implications. Proc Ann Conv Am Ass Equine Pract 1995;41:1–2.
1393
[23] Samper JC, Hankins K. Breeding mares with frozen semen in private practice. Proc Ann Conv Am Ass Equine Pract 2001;47:314–8. [24] Vidament M, Dupere AM, Julienne P, Evain A, Noue P, Palmer E. Equine frozen semen freezability and fertility results. Theriogenology 1997;48:907–17. [25] Metcalf ES. Optimizing pregnancy rates using frozen-thawed equine semen. Anim Reprod Sci 2005;89:297–9. [26] Barbacini S, Gulden P, Marchi V, Zavaglia G. Incidence of embryonic loss in mares inseminated before or after ovulation. Equine Vet Educ 1999;1:108–12. [27] Koskinen E, Lindeberg H, Kuntsi H, Ruotsalainen L, Katila T. Fertility of mares after postovulatory insemination. J Vet Med A 1990;37:77–80. [28] Woods J, Bergfeld DR, Ginther OJ. Effects of time of insemination relative to ovulation on pregnancy rate and embryonic loss rate in mares. Equine Vet J 1990;22:410–5. [29] Barbacini S, Zavaglia G, Gulden P, Marchi V, Necchi D. Retrospective study on the efficacy of hCG in an equine artificial insemination programme using frozen semen. Equine Vet Educ 2000;2:404–8. [30] Samper JC. Ultrasonographic appearance and the pattern of uterine edema to time ovulation in mares. Proc Ann Conv Am Ass Equine Pract 1997;43:41–3. [31] Katila T. Post-mating inflammatory responses of the uterus. Reprod Domest Anim 2012;47(Suppl 5):31–41. [32] Kotilainen T, Huhtinen M, Katila T. Sperm-induced leucocytosis in the equine uterus. Theriogenology 1994;41:629–36. [33] Fiala SM, Pimentel CA, Mattos AL, Gregory RM, Mattos RC. Effect of sperm numbers and concentration on sperm transport and uterine inflammatory response in the mare. Theriogenology 2007;67: 556–62. [34] Nikolakopoulos E, Watson ED. Uterine contractility is necessary for the clearance of intrauterine fluid but not bacteria after bacterial infusion in the mare. Theriogenology 1999;52:413–23. [35] Troedsson MHT, Steiger BN, Ibrahim NM, Foster DN, Crabo BG. Mechanism of sperm-induced endometritis in the mare. Biol Reprod 1995;52(Suppl 1):507. [36] Troedsson MHT. Uterine clearance and resistance to persistent endometritis in the mare. Theriogenology 1999;52:461–71. [37] LeBlanc MM, Burleson MD, Riddle WT, Hendricks KEM. Endometrial pathogens are associated with different ultrasonographic and endometrial cytology findings in Thoroughbred mares. Pferdeheilkunde 2012;28:86. [38] De Borba EVC, Camozzato GC, Malschitzky E, Bustamante-Filho IC, Martins AA, Mattos RC, et al. Is the presence of uterine fluid a reliable indicator of endometrial inflammation? Pferdeheilkunde 2012;28:27–9. [39] Barbacini S, Necchi D, Zavaglia G, Squires EL. Retrospective study of the incidence of post-insemination uterine fluid in mares inseminated with frozen-thawed semen. J Equine Vet Sci 2003; 23:493–6.