Freezing of Stallion Semen: In Vitro Evaluation of Motility and Acrosin Activity in Sperm Cells Cryopreserved Using Different Semen Extenders

Abstract

The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was fractioned into two equal parts and resuspended in an 11% lactose solution in a 1:1 proportion, where it remained for 20 minutes at room temperature. The semen was centrifuged at 600 g for 10 minutes, and after the second centrifugation, each pellet received the freezing extender (Merck or Zorlesco) and was loaded into 4 mL straws. Each straw was placed in liquid nitrogen vapor steam for 15 minutes and further immersion in liquid nitrogen at −196°C for long-term storage. After thawing, semen samples were initially evaluated for sperm motility, both total and progressive, and acrosin activity. Moreover, semen was incubated at 37°C and further assessed at 60 and 120 minutes in a thermoresistance test (TRT) for sperm motility and acrosin activity. Immediately after thawing, both progressive and total motility, and acrosin activity were lower (p < 0.05) in thawed semen than in fresh semen. During the TRT, total sperm motility and acrosin activity after 60 minutes were lower (p < 0.05) than those obtained after thawing. Similarly, total sperm motility and acrosin activity were lower (p < 0.05) after 120 minutes than at 60 minutes of the TRT. The analysis of motility and acrosin activity allowed the conclusion that both extenders have a similar capacity to preserve the integrity of sperm cells subject to freezing and thawing.

Introduction

Semen cryopreservation, coupled with artificial insemination (AI) and embryo transfer, constitutes a relevant tool for dissemination and preservation of superior genetic resources for undetermined periods of time.¹ Despite the intensification of research aiming to improve horse semen cryopreservation,^2,3 there are significant limitations to this technology. Therefore, it limits its efficient use and under large-scale applications.^4,5

Since the first report in the last century,⁶ the leading issue of semen cryopreservation is the low motility and viability of sperm cells after thawing.^1,7 This fact was further reinforced by authors^8,9 who observed a decrease in sperm motility between 20% and 30% after thawing of equine semen pellets. Even the advent of semen straws¹⁰ for semen storage did not confer full protection to sperm cells.^1,11

During cryopreservation sperm cells are exposed to several stress conditions that may alter their fertilizing potential.^3,12 This diminishment of sperm cell viability is exerted at varying nonphysiological temperatures, and ice crystals formation that may compromise cell viability^13,14 due to plasma membrane damage.^3,7 Until now, the most critical goal aiming for improvement in semen cryopreservation continues to focus on increasing sperm cell survival.¹⁵

Due to the above facts, semen extenders must be formulated by substances that lower such cryoinjuries in sperm cells due to freezing and thawing.^12,16 Therefore, the development of chemically defined extenders has been a major priority for researchers throughout the world.⁴ In general, the extenders are available with the most common being BotuCrio, INRA, and Lactose-EDTA^1,17; however, it is virtually impossible to draw valid conclusions by comparing data from two or more reports about different extenders.¹⁶

Additionally, it is still known that the freezing process leads to capacitation-like changes in the sperm cell,¹⁸ also called cryocapacitation. This event makes the sperm more likely to undergo the acrosome reaction, decreasing its viability time.¹⁷ From all available semen and sperm parameters that can be used to estimate its viability, motility, and acrosin activity, analysis has gained broader attention due to their more accurate estimation of sperm integrity.^11,19

Since the precise fertilizing potential of sperm cells cannot be performed in clinics and reproduction centers,²⁰ other more sophisticated analysis has been experimentally described and adopted in semen analysis protocols, such as the sperm chromatin assay, terminal deoxynucleotidyl transferase-mediated nick-end labeling, single-cell gel electrophoresis, and the sperm chromatin dispersion test.^21,22 However, the adoption of such analyses in routine andrology exams is limited due to dependency upon sophisticated equipment.²³

In contrast to other expensive tests mentioned above, the measurement of acrosin activity does not require sophisticated equipment.^19,24 Acrosin is a proteolytic enzyme contained in the sperm acrosome^24,25 that has an essential function during the process of fertilization.^25,26 The acrosin activity was also initially described in the year 1975²⁷ and further measured in equine sperm cells by other authors.^11,19 Moreover, its activity is directly related to acrosome integrity.²⁷

In light of these facts, the objective of our study was to evaluate the efficiency of different semen extenders on the cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters to predict the fertility of cryopreserved semen.

Materials and Methods

The semen of two Hanoverian stallions (6–10 years old) under a semen collection regimen was used for semen cryopreservation using glycerol. Semen collection was performed twice a week with an artificial vagina warmed at 42°C, using a phantom or mares in estrus for stimulation of the stallion.

Ejaculates (n = 20; 10 from each stallion) were initially filtered to discard the gel, and the semen volume was measured using a scaled tube. Immediately after, the fresh semen was used for analysis of motility, concentration, supravital color, and sperm morphology, according to Colégio Brasileiro de Reprodução Animal (CBRA).²⁸ Sperm cells were also evaluated for acrosin activity as previously described by Vieira and Klug.¹¹

The semen was extended 1:1 (semen: semen extender) in 11% lactose solution and kept at room temperature for 20 minutes.¹⁰ Immediately after, the semen was centrifuged at 600 g for 10 minutes. After discarding the supernatant, each pellet was rediluted with the semen extender above and centrifuged at 600 g for 10 minutes.

The pellets were diluted according to each freezing semen extender, Merck or Zorlesco, to obtain a concentration of 200 × 10⁶ viable sperm cells/mL. The composition of both Merck and Zorlesco extenders are described below (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/bio). The freezing semen extenders were formulated with 25 mL of Merck or Zorlesco semen extender +50 mL of 11% lactose solution +20 mL of egg yolk +0.8 mL of Orvus-Paste ((Equex STM, Cincinnati, OH); Procter & Gamble, Cincinnati, OH) +5 mL of glycerol.

The semen was loaded into 4 mL straws, as previously described¹⁰ and placed in liquid nitrogen vapor for 15 minutes before immersion and stored at −196°C in cryogenic devices. After thawing at 50°C for 40 seconds, semen samples were initially evaluated for sperm motility and acrosin activity. Immediately after, the semen was incubated at 37°C for 60 and 120 minutes in a thermoresistance test (TRT) and evaluated for sperm motility and acrosin activity.

Data were analyzed for the normality condition by the Shapiro–Wilk test. Data with collected percentages were subject to arcsine transformation and analysis of variance. Mean comparisons were carried out with the Tukey–Kramer test, with a significance level of 5%.

Results

Although not used for statistical analysis, data of sperm motility, acrosome alterations, and acrosin activity are presented as percentages in all tables in parentheses immediately below the data subject to arcsine transformation.

The progressive sperm motility did not differ between the fresh semen and prefrozen samples, independent of extender choice (Table 1). Both fresh and prefrozen semen showed greater motility (p < 0.05) than thawed semen, independent of semen extender choice and stallion.

Table 1.

Mean Values ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} $$\bar x$$ \end{document} ± SD), Displays the Data Transformed by Arcsine √ and the Percentages in Parenthesis, of Sperm Cell Progressive Motility in Fresh Semen, Prefrozen, and Post-Thawing

		Prefrozen		Post-thawing
Animal	Fresh semen	Lactose-Merck diluent	Lactose-Zorlesco diluent	Lactose-Merck diluent	Lactose-Zorlesco diluent
1	0.72 ± 0.06^A (66.0 ± 5.1)	0.75 ± 0.09^A (68.0 ± 6.7)	0.75 ± 0.010^A (68.5 ± 74)	0.45 ± 0.006^B (43.5 ± 5.7)	0.44 ± 0.08^B (42.5 ± 7.5)
2	0.76 ± 0.11^A (69.0 ± 8.4)	0.77 ± 0.07^A (70.0 ± 5.2)	0.75 ± 0.08^A (68.0 ± 6.3)	0.45 ± 0.008^B (43.5 ± 7.4)	0.46 ± 0.09^B (45.0 ± 8.8)
Total (1 + 2)	0.74 ± 0.09^A (67.5 ± 6.9)	0.76 ± 0.08^A (69.0 ± 5.9)	0.75 ± 0.09^A (68.2 ± 6.7)	0.45 ± 0.07^B (43.5 ± 6.5)	0.45 ± 0.09^B (43.7 ± 8.0)

Different uppercase letters on same line denotes statistical difference (p < 0.05).

The total sperm motility did not differ between fresh semen, and that resuspended in semen extenders after two steps of centrifugation, irrespective of semen extender choice (Table 2). Both fresh semen and prefrozen samples displayed greater motility (p < 0.05) than those after thawing, independent of semen extender choice and stallion.

Table 2.

Mean Values ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} $$\bar x$$ \end{document} ± SD), Displays the Data Transformed by Arcsine √ and the Percentages in Parenthesis, of Sperm Cell Total Motility in Fresh Semen, Prefrozen, and Post-Thawing

		Prefrozen		Post-thawing
Animal	Fresh semen	Lactose-Merck diluent	Lactose-Zorlesco diluent	Lactose-Merck diluent	Lactose-Zorlesco diluent
1	1.06 ± 0.07^A (87.5 ± 3.5)	0.98 ± 0.09^A (83.0 ± 4.8)	0.98 ± 0.10^A (83.0 ± 5.8)	0.57 ± 0.04^B (54.0 ± 3.9)	0.57 ± 0.06^B (54.0 ± 5.6)
2	0.96 ± 0.07^A (82.0 ± 4.8)	1.00 ± 0.09^A (84.0 ± 5.1)	0.98 ± 0.10^A (83.0 ± 5.8)	0.60 ± 0.08^B (56.5 ± 7.0)	0.60 ± 0.08^B (57.0 ± 7.1)
Total (1 + 2)	1.01 ± 0.09^A (84.7 ± 4.9)	0.99 ± 0.09^A (83.5 ± 4.8)	0.98 ± 0.09^A (83.0 ± 5.7)	0.58 ± 0.06^B (55.2 ± 5.7)	(0.59 ± 0.07)^B (55.5 ± 6.4)

Different uppercase letters in same row denotes statistical difference (p < 0.05).

It is possible to note in Table 3 that the data of acrosome alterations in fresh semen did not differ (p > 0.05) between stallions. Moreover, no correlation was found (p > 0.05) between such alterations and acrosin activity between fresh semen (r = 0.28) and thawed semen with Lactose-Merck (r = 0.37) and Lactose-Zorlesco (r = 0.29) semen extenders.

Table 3.

Mean Values ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} $$\bar x$$ \end{document} ± SD), Displays the Data Transformed by Arcsine √ and the Percentages in Parenthesis, of Acrosome Alterations and Acrosin Activity in Fresh Semen, Prefrozen, and Post-Thawing

		Acrosin activity
			Thawed semen
Animal	Acrosome Fresh semen	Fresh semen	Lactose-Merck diluent	Lactose-Zorlesco diluent
1	0.02 ± 0.01^a (2.14 ± 1.1)	1.13 ± 0.08^A (90.21 ± 3.6)	(1.02 ± 0.09)^B (84.95 ± 5.1)	1.01 ± 0.09^B (84.35 ± 5.3)
2	0.02 ± 0.01^a (2.90 ± 1.3)	1.16 ± 0.05^A (91.65 ± 2.2)	(1.05 ± 0.05)^B (86.95 ± 2.6)	10.06 ± 0.06^B (87.10 ± 3.2)
Total	0.02 ± 0.01 (2.52 ± 1.2)	1.14 ± 0.07^A (90.0 ± 3.0)	1.03 + 0.07^B (85.9 ± 4.1)	1.03 ± 0.07^B (85.7 ± 4.5)

Different uppercase letters in same row and lowercase letters in the same column denotes statistical difference (p < 0.05).

The acrosin activity in fresh semen, independent of the stallion and semen extender choice, was greater (p < 0.05) than thawed semen (Table 3).

During the TRT, and irrespective of semen extender choice, both total sperm motility and acrosin activity immediately after thawing were higher (p < 0.05) than those after 60 and 120 minutes of the TRT. Accordingly, the values of both parameters after 60 minutes of the TRT were greater (p < 0.05) than those obtained after 120 minutes of the TRT (Table 4).

Table 4.

Mean Values ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} $$\bar x$$ \end{document} ± SD), Displays the Data Transformed by Arcsine √ and the Percentages in Parenthesis, of Sperm Cell Total Motility and Acrosin Activity During a Thermoresistance Test

	Diluent
	Lactose-Merck		Lactose-Zorlesco
Minutes	Motility	Acrosin	Motility	Acrosin
0	0.53 ± 0.06^a (51.7 ± 5.6)	1.08 ± 0.06^a (88.3 ± 3.1)	0.54 ± 0.07^a (54.0% ± 5.9)	1.08 ± 0.06^a (88.1 ± 3.0)
60	0.30 ± 0.07^b (32.2 ± 7.1)	0.84 ± 0.05^b (74.6 ± 3.8)	0.27 ± 0.05^b (30.7 ± 5.9)	0.87 ± 0.07^b (76.8 ± 4.4)
120	0.12 ± 0.07^c (14.7 ± 6.9)	0.57 ± 0.04^c (54.0 ± 3.9)	0.18 ± 0.10^c (12.5 ± 5.2)	0.55 ± 0.06^c (52.9 ± 5.1)

Different lowercase letters in same column denotes statistical difference (p < 0.05).

Discussion

When developing the project that led to this experiment, the performance of the Merck semen extender was already known for equine semen cryopreservation that was initially used by Martin et al.¹⁰ However, the Zorlesco semen extender, initially formulated by Gottardi et al.²⁹ for cryopreservation of swine semen, had not been tested for cryopreservation of equine semen. However, the initial hypothesis was that sperm motility would be diminished after thawing, irrespective of semen extender choice. This assumption is based on results previously observed by many groups.^{8–10,13,22,30–32} The same reasoning was adopted for acrosin activity, and the findings are in agreement with the initial hypothesis based on results by other authors.^11,19

This expectation was based on the fact that both semen extenders had been formulated with the same cryoprotectant, and more importantly, no cryoprotectant described to date can provide full protection against cryoinjuries to sperm cells during freezing and thawing. The results cited in this study are in agreement with the hypothesis above due to the diminished sperm motility and acrosin activity after thawing. This occurrence was probably due to several types of cryoinjuries during both freezing and thawing, as outlined by various researchers.^{3,7,13–15,19,22}

On the other hand, it is paramount to outline that the semen must maintain a minimum quality necessary for its use in AI, since it has been²⁸ established that after thawing, the semen must display a minimum motility of 30%. Thus, it is possible to state that, irrespective of semen extender choice and stallion, the semen exhibited immediately after thawing, the conditions to be used for AI according to minimum requirements by CBRA.²⁸ Since the results of acrosin activity were similar to those obtained by other authors,^11,19 it is possible to suggest that both freezing and thawing did not produce acrosome damage that may affect the fertilizing potential of sperm cells.

The TRT generates an acceptable in vitro setting that allows estimating, at least in theory, the probability of the sperm cell from a good semen sample to reach the site of fertilization. During the TRT and even after 60 minutes, the semen after thawing still displayed the minimum acceptable motility, according to CBRA.²⁸ Based on this single sperm parameter for evaluation and the fact that sperm transport in the mare reproductive tract is relatively fast,^33,34 it can be suggested that both semen extenders can be efficiently used for equine semen cryopreservation.

The sperm plasma membrane undergoes metabolic changes with the semen extender, and its integrity can be compromised by cryopreservation, thus altering both changes in sperm motility and the activity of acrosome enzymes.³⁵ A noticeable sharp decrease in both sperm motility and acrosin activity immediately after thawing and during the TRT,¹⁹ is suggestive that the semen quality is lowered accordingly.

For acrosin activity, data described in this study showed a significant reduction of its activity during the TRT. Different from motility, there is not an established minimum activity level for acrosin in fresh or thawed semen. In contrast, acrosin activity is correlated with acrosome activity.²⁶ The work described here did not find a correlation between acrosome alterations and acrosin activity, although it is inappropriate to state that sperm fertilizing ability was not compromised during the TRT.

It is also pertinent to highlight that the efficiency of both freezing and thawing described here may be due to an appropriate semen extender formulation, as previously suggested by several reports.^{12,16,36–38} Moreover, based on further work by several groups,^12,39–42 it possible to state that both semen extenders were composed of reagents that maintained sperm cell metabolism, pH, and osmolality under physiological levels, diminished microbial growth, and protected sperm cells from thermal damage. Therefore, after sperm motility and acrosin activity evaluations, the results described here allow the conclusion that both semen extenders showed potential for conservation of sperm cell integrity after the freezing and thawing processes.

Footnotes

Animal Welfare Statement

This research was performed after evaluation and approval of the Ethics Committee of the Faculdade Pio Décimo, Aracaju-Se, Brazil (Protocol: 01/17).

Acknowledgment

The authors would like to acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for fellowship support during the study.

Author Disclosure Statement

The authors declare that they have no conflicts of interest and are available to provide any clarification.

References

Alvarenga

, Papa

, Neto

. Advances in stallion semen cryopreservation. Vet Clin North Am Equine Pract, 2016; 32:521–530.

Samper

, Plough

. Techniques for the insemination of low doses of stallion sperm. Reprod Domest Anim, 2010; 45:35–39.

Santos

MAM

, Gradela

, Moraes

, et al. Sperm characteristics in fresh and cryopreserved semen of Nordestino stallions breed. Pesqui Veta Brasil, 2015; 35:925–932.

Papa

, Felício

, Melo-Oña

, et al. Replacing egg yolk with soybean lecithin in the cryopreservation of stallion semen. Anim Reprod Sci, 2011; 129:73–77.

Batellier

, Vidament

, Fauquant

, et al. Advances in cooled semen technology. Anim Reprod Sci, 2001; 68:181–190.

Smith

, Polge

. Survival of spermatozoa at low temperatures. Nature, 1950; 166:668–669.

Sieme

, Oldenhof

, Wolkers

. Sperm Membrane Behaviour during Cooling and Cryopreservation. Reprod Domest Anim, 2016; 50:20–26.

Merckt

, Krause

. Experiments with deep-frozen equine semen using the so-called pellet method. Dtsch Tierarztl Wochenschr, 1966; 73:267–268.

Nagase

, Soejima

, Tomizuka

, et al. Studies on the freezing of stallion semen. II. Factors affecting survival rates of stallion spermatozoa after freezing and thawing and results of a fertility trial. J Anim Reprod, 1966; 12:52–57.

10.

Martin

, Klug

, Günzel

. Centrifugation of stallion semen and its storage in large volume straws. J Reprod Fertil, 1979; 27:47–51.

11.

Vieira

, Klug

. Acrosin activity as influenced by the freezing technique in equine semen. Rev Bras Reprod Anim, 1982; 6:15–20.

12.

, Zheng

, Luo

, et al. Cryopreservation of stallion spermatozoa using different cryoprotectants and combinations of cryoprotectants. Anim Reprod Sci, 2015; 163:75–81.

13.

Holt

. Fundamental aspects of sperm cryobiology: The importance of species and individual differences. Theriogenology, 2000; 53:47–58.

14.

Watson

. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci, 2000; 60:481–492.

15.

Sieme

, Harrison

, Petrunkina

. Cryobiological determinants of frozen semen quality, with special reference to stallion. Anim Reprod Sci, 2008; 107:276–292.

16.

Amann

, Pickett

. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J Equine Vet Sci, 1987; 7:145–173.

17.

Fayrer-Hosken

, Abreu-Barbosa

, Heusner

, et al. Cryopreservation of stallion spermatozoa with INRA96 and glycerol. J Equine Vet Sci, 2008; 28:672–676.

18.

Ellington

, Ball

, Blue

BJ et al

. Capacitation-like membrane changes and prolonged viability in vitro of equine spermatozoa cultured with uterine tube epithelial cells. Am J Vet Res, 1993; 54:1505–1510.

19.

Ferreira-Silva

, Rocha

, Ferreira

, et al. Freezing of stallion semen: 1. In vitro evaluation of motility and acrosin activity in sperm cells cryopreserved under different glycerol concentrations. Pferdeheilkunde, 2018; 34:51–56.

20.

López-Fernández

, Crespo

, Arroyo

, et al. Dynamics of sperm DNA fragmentation in domestic animals: II. The stallion. Theriogenology, 2007; 68:1240–1250.

21.

Gosálvez

, Fernández

, Goyanes

, et al. Analysis of DNA fragmentation in spermatozoa using the sperm chromatin dispersion (SCD) test. Biotechnol, 2006; 1:38–51.

22.

Ferreira

, Ferreira-Silva

, Rocha

, et al. Variable inter-assay estimation of sperm DNA fragmentation in stallions classified as good and bad semen freezers. Cryo Lett, 2018; 39:67–71.

23.

Enciso

, López-Fernández

, Fernández

, et al. A new method to analyze boar sperm DNA fragmentation under bright-field or fluorescence microscopy. Theriogenology, 2006; 65:308–316.

24.

Penn

, Gledhill

, Darżynkiewicz

. Modification of the gelatin substrate procedure for demonstration of acrosomal proteolytic activity. J Histochem Cytochem, 1972; 20:499–506.

25.

Ball

, Fagnan

, Dobrinski

. Determination of acrosin amidase activity in equine spermatozoa. Theriogenology, 1997; 48:1191–1198.

26.

Bedford

. Ultrastructural changes in the sperm head during fertilization in the rabbit. Am J Anat, 1968; 123:329–357.

27.

Wendt

, Leidl

, Schill

. Methodological studies on gelatinolysis by sperm. Fortsch Fertilitätsforschung, 1975:181–188. German.

28.

Colégio Brasileiro de Reprodução Animal. Manual for andrological examination and evaluation of animal semen. 3rd ed. Belo Horizonte; 2013:104.

29.

Gottardi

, Brunel

, Zanelli

. New dilution media for artificial insemination in the pig. In: 9th International Congress on Animal Reproduction and Artificial Insemination, Madrid. 1980:49–53.

30.

Bader

, Mahler

. Freezing and insemination experiments with stallion semen using the pellet method. Zuchthygiene, 1968; 3:6–13. German.

31.

Blobel

, Klug

. Freezing-seed transmission in the Stallion, an experiment under practical conditions of the barn. Berl Münch Tierärztl Wschr, 1975; 24:465–468. German.

32.

Watson

. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fertil Dev, 1995; 7:871–891.

33.

Bader

, Krause

. An investigation of sperm migration into the oviducts of the mare. J Reprod Fertil, 1982; 32:59–64.

34.

Fiala

, Jobim

MIM

, Katila

, et al. Sperm distribution in the oviduct and uterus of mares within two hours after artificial insemination. Pferdeheilkunde, 2008; 24:96–98.

35.

Lagares

, Petzoldt

, Sieme

, et al. Assessing equine sperm-membrane integrity. Andrologia, 2000; 32:163–167.

36.

Squires

, Keith

, Graham

. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology, 2004; 62:1056–1065.

37.

Moore

, Squires

, Graham

. Effect of seminal plasma on the cryopreservation of equine spermatozoa. Theriogenology, 2005; 63:2372–2381.

38.

Oldenhof

, Bigalk

, Hettel

, et al. Stallion sperm cryopreservation using various permeating agents: Interplay between concentration and cooling rate. Biopreserv Biobank, 2017; 15:422–431.

39.

Blanchard

, Varner

, Schumacher

, et al. Semen preservation. In Blanchard

, Varner

, Love

, Brinsko

, Rigby

, Schumacher

, eds. Manual of Equine Reproduction, 2nd ed. Philadelphia, PA: Elsevier-Mosby; 2003:165–177.

40.

Pickett

, Amann

. Cryopreservation of semen. In: Mckinnon

, Voss

(eds). Equine Reproduction. Philadelphia, PA: Lea & Febiger;, 1993:769–789.

41.

Brinsko

, Crockett

, Squires

. Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. Theriogenology, 2000; 54:129–136.

42.

Ball

, Vo

. Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. J Androl, 2001; 22:1061–1069.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.05 MB