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Motility and Fertility Evaluation of Thawed Frozen Stallion Semen After 24 Hours of Cooled Storage
Journal Pre-proof Motility and fertility evaluation of thawed frozen stallion semen after 24 hours of cooled storage Melissa J. Prell, Patrick M. McCue, Paula D. Moffett, James K. Graham PII:
S0737-0806(20)30074-5
DOI:
https://doi.org/10.1016/j.jevs.2020.102983
Reference:
YJEVS 102983
To appear in:
Journal of Equine Veterinary Science
Received Date: 26 October 2018 Revised Date:
27 February 2020
Accepted Date: 27 February 2020
Please cite this article as: Prell MJ, McCue PM, Moffett PD, Graham JK, Motility and fertility evaluation of thawed frozen stallion semen after 24 hours of cooled storage, Journal of Equine Veterinary Science (2020), doi: https://doi.org/10.1016/j.jevs.2020.102983. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Inc. All rights reserved.
Motility and fertility evaluation of thawed frozen stallion semen after 24 hours of cooled storage Melissa J. Prella, Patrick M. McCuea, Paula D. Moffettb, James K. Grahamb a
a
Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
Key Words: Stallion, semen, frozen, cooled-storage Corresponding Author:
Patrick M. McCue Equine Reproduction Laboratory Colorado State University 3101 Rampart Road Fort Collins, CO 80521 USA [email protected]
Abstract: Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were: 1) evaluate the longevity of frozen stallion semen once it had been thawed, extended and maintained at 5° C for 48 hours in a passive cooling container, and 2) determine fertility potential of frozen semen that had been thawed, extended and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE® minus cryoprotectant at a concentration of 50 million total sperm/mL. The remainder of the ejaculate was frozen in CryoMax LE® extender at a concentration of 200 million total sperm/mL. Semen was thawed using 1 of 3 thawing protocols, and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE ®minus cryoprotectant and cooled to 5° C. Sperm motility was evaluated at 24 and 48 hours. Eight mares were inseminated over two estrous cycles using frozen semen that had been thawed, extended in INRA96 and cooled for 24 hours. There was no difference in progressive motility at 24 or 48 hours of cooled-storage postthaw between the 3 thawing protocols. An overall per cycle pregnancy rate of 56 % (9/16 cycles) was achieved using frozen-thawed semen that had been extended and cooled for 24 hours. In summary, frozen stallion sperm was thawed, extended and cooled to 5°C for 24 hours and still maintained adequate (> 30 %) sperm motility and fertility.
1. Introduction Artificial insemination is a common procedure in equine clinical practice and a majority of breed associations, with the notable exception of Thoroughbreds, allow the use of artificial insemination [1-3]. Semen collected from stallions can be extended and used for fresh insemination, extended and stored at 5 to 8° C for 24 to 48 hours prior to insemination, or cryopreserved and stored indefinitely in liquid nitrogen [4,5].
Advantages of cryopreservation of stallion semen include preservation of genetic material for future use, ‘insurance’ against unanticipated loss of a stallion, ability to breed mares when the stallion is unavailable for collection, and ability to ship frozen semen anywhere in the world [6-8]. Potential disadvantages of cryopreservation of stallion semen include the fact that spermatozoa from some stallions do not exhibit adequate post-thaw motility, pregnancy rates following insemination with frozen semen is generally lower than rates associated with fresh or cooled-stored semen, and more intensive mare management is required when using frozen semen [9]. In addition, specialized equipment and training are needed for the transport, storage, handling and evaluation of frozen semen [10]. As a consequence, not all equine breeding operations are enamored with the use of frozen stallion semen and prefer to breed mares only with fresh or cooled semen. The objectives of this study were to 1) evaluate the longevity of frozen stallion semen once it had been thawed, extended and maintained at 5° C for 48 hours in a passive cooling container and 2) determine fertility potential of frozen semen that had been thawed, extended and used to inseminate mares after 24 hours of cooled storage. The hypotheses were that thawed semen, when properly extended and cooled, would maintain motility for at least 24 hours and that it would be possible to obtain pregnancies after insemination of frozen-thawed semen extended and stored at 5° C for 24 hours. 2. Materials and Methods 2.1 Semen collection and processing A total of 8 ejaculates from 5 Quarter Horse stallions were collected using a Colorado model artificial vagina. One ejaculate was collected from each of 5 stallions; three of the stallions had a second ejaculate collected. Initial semen analysis consisted of measurements of semen volume, sperm concentration and sperm motility. Volume was measured in a graduated cylinder; sperm concentration was determined using a NucleoCounter® (ChemoMetec, Allerod, Denmark) [9]; total and progressive sperm motility were evaluated by computer assisted semen analysis (CASA; SpermVision®; MOFA, Verona, WI) [12]. Each ejaculate was divided, and aliquots were cooled in both a commercial semen extender (INRA96; IMV Technologies, Maple Grove, MN) and a commercial freezing extender minus cryoprotectant (CryoMax LE®; Animal Reproduction Systems, Chino, CA) at a concentration of 50 million total sperm/mL to serve as non-cryopreserved controls. The remainder of the ejaculate was extended to a 1:1 ratio with a commercial extender (E-Z Mixin®-BF; Animal Reproduction Systems, Chino, CA) and divided into 50 mL conical tubes. A 3 mL volume of a centrifugation cushion (Maxifreeze; IMV Technologies, Maple Grove, MN) was gently deposited in the bottom of each conical tube to enhance sperm recovery. The loaded conical tubes were subsequently centrifuged at 1000 x g for 25 minutes. The supernatant and centrifugation cushion were removed
and the sperm pellet was re-suspended and extended to a concentration of 200 million total sperm/mL using a commercial freezing extender (CryoMax LE®; Animal Reproduction Systems, Chino, CA). The extended semen was loaded into 0.5 mL straws and cooled to a temperature of 5° C over a 30 minute time period in a refrigerator. Straws were then suspended 3 cm over liquid nitrogen vapor for 15 minutes before being plunged into liquid nitrogen. Frozen straws were subsequently transferred into liquid nitrogen tanks for later use. 2.2 Treatment groups Control Group C1 - non-cryopreserved semen samples diluted in INRA96 Control Group C2 - non-cryopreserved semen samples diluted in CryoMax LE® minus cryoprotectant Group 1 - straws of frozen semen were thawed in a 37° C water bath for 30 seconds Group 2 - straws were thawed in a 37° C water bath for 30 seconds, followed by centrifugation at 400 x g for 10 minutes Group 3 - straws were thawed in a 37° C water bath for 12 seconds The thawing method for Group 1 is the standard protocol used in our laboratory for frozen semen. The protocol used in Group 2 was designed to remove the freezing extender prior to dilution and eliminate potential adverse effects of the cryoprotectants. The Group 3 protocol was designed to expose the frozen straws to a water bath for enough time to reach a temperature of 5° C, but not allow the spermatozoa to warm enough to potentially incur damage to their plasma membranes by going through liquidcrystalline to gel-state phase transitions. After thawing, each sample was split into two aliquots and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE® minus cryoprotectant and cooled to 5° C in a commercial passive cooling container (Equitainer; Hamilton Biovet, Ipswich, MA). Total and progressive motility were subsequently assessed at both 24 and 48 hours. 2.3 Fertility trial Eight Quarter Horse mares, ranging from 6 to 13 years of age and of unknown parity, were used in a preliminary breeding trial over two consecutive estrous cycles to evaluate the fertility potential of frozen semen that had been thawed, extended and cooled for 24 hours. All mares had exhibited normal reproductive cycles, but no uterine cultures, cytology or biopsy samples were collected prior to insemination. Breeding management on the first estrous cycle was designed to mimic the timed ovulation and insemination timing normally used when breeding mares with frozen semen at our facility, with the exception that mares were only examined once per day. Breeding management on the second cycle was designed to mimic the timed ovulation and insemination timing normally used when breeding mares with cooled-transported semen at our facility. Mares were only examined once per day and all inseminations
were into the uterine body. Ultrasound pregnancy examinations were performed 14 days post-ovulation. Frozen-thawed semen from one stallion was used to inseminate all 8 mares during the first estrous cycle, while frozen-thawed semen from a second stallion was used to inseminate all 8 mares during the second estrous cycle. Stallion selection was based on post-thaw motility after 24 hours of cooled storage and availability of sufficient doses of frozen semen to complete the breeding trial. Cycle 1 Protocol. Mares were examined once per day by transrectal ultrasonography at approximately 0900 hrs. Once a follicle ≥ 35 mm was detected in the presence of uterine edema, a single 500 µg intramuscular dose of histrelin (Doc Lanes Veterinary Pharmacy, Lexington, KY)) was administered at 2000 hrs that night. The next morning at approximately 0900 hrs, a dose of frozen semen, consisting of 8 straws at 100 million total sperm per straw, was thawed, extended to a concentration of 50 million total sperm/mL in INRA96 (16 mls total volume) and placed in a passive cooling system for 24 hours. The following morning at approximately 0900 hrs, mares were re-examined by ultrasound and inseminated. The apriori expectation was that mares should ovulate an average of 40 hours after receiving histrelin, which would equate to approximately 1200 hrs, or about 3 hours after being inseminated with frozen-cooled semen. Cycle 2 Protocol. Mares were examined once per day at approximately 0900 hrs. Once a follicle ≥ 35 mm was detected in the presence of uterine edema, a single 500 µg intramuscular dose of histrelin was immediately administered and a dose of frozen semen, consisting of 8 straws at 100 million total sperm per straw, was thawed, extended in INRA96 and placed in a passive cooling system for 24 hours. The following morning at approximately 0900 hrs, mares were re-examined by ultrasound and inseminated. The apriori expectation was that mares should ovulate approximately 40 hours after receiving histrelin, which would equate to approximately 0100 hrs, or about 16 hours after being inseminated with frozen-cooled semen. 2.5 Statistical Analysis Data were analyzed by univariate, repeated-measures analysis of variance using the General Linear Model (GLM) procedure of SAS Institute, with p-value set at <0.05. Data are presented as mean ± SD. 3. Results 3.1 Motility assessment Total and progressive sperm motility were higher (p<0.05) at 24 and 48 hours for noncryopreserved semen samples extended in INRA96 (C1) as compared to all frozenthawed semen samples (Groups 1, 2 and 3) extended in INRA96 (Table 1). Total and progressive sperm motilities were lower at 24 hours of cooled storage for frozen semen samples thawed in a 37° C water bath for 12 seconds (Group 3) and subsequently extended in INRA96 than semen samples in Groups 1 and 2.
Progressive sperm motility was lower for Group 1 and Group 3 samples stored at 5° C in CryoMax LE® minus cryoprotectant for 24 hours compared to the non-cryopreserved control (Table 1). A significant difference was also noted in total and progressive motility in Group 3 samples as compared to the non-cryopreserved control and in Group 2 versus Group 3 at 48 hours of cooled storage. Table 1. Sperm motility parameters of cooled controls and frozen-cooled treatment groups maintained in INRA96 or CryoMax LE® minus cryoprotectant (CPA). The non-cryopreserved control samples were maintained at 5º C and evaluated at 24 and 48 hours. Semen samples for Groups 1, 2 and 3 were cryopreserved, thawed as described, diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE® minus cryoprotectant and cooled to 5° C in a commercial passive cooling container and evaluated at 24 and 48 hours.
CryoMax LE® minus CPA
INRA96 Group NonCryopreserved Controls
Motility 24 h
Motility 48 h
a
a
Motility 24 h
Motility 48 h a
64.3 ± 13.1 /53.8 ± 15.4a
56.1 ± 15.8ab /45.9 ± 16.5a
77.1 ± 6.9 /71.3 ± 8.6a
75.4 ± 9.2 /71.3 ± 10.3a
50.3 ± 9.6b /45.0 ± 12.0b
51.6 ± 12.8b 41.5 ± 10.4bc /45.6 ± 12.5b /29.5 ± 10.6b
35.5 ± 10.8ac /25.5 ± 10.9ab
48.6 ± 10.1b /38.9 ± 10.8b
47.0 ± 13.9b 49.9 ± 10.9ab /37.5 ± 14.2b /38.6 ± 10.9ab
50.1 ± 12.7b /34.9 ± 11.5a
41.5 ± 6.5b /34.8 ± 8.8b
32.8 ± 12.3c /21.6 ±10.2b
(Total/Progressive)
Group 1 Thawed at 37°C for 30 sec (Total/Progressive)
Group 2 Thawed at 37°C for 30 sec and centrifuged (Total/Progressive)
Group 3 Thawed at 37°C for 12 sec (Total/Progressive)
32.8 ± 6.6c /28.6 ± 7.3c
32.3 ± 8.5c /26.5 ± 9.8b
3.2 Fertility trial In the first estrous cycle, 4 of 8 mares (50 %) became pregnant after insemination with frozen semen that had been thawed at 37° C, extended in INRA96 and cooled for 24 hours (Table 2). Ovulation was detected in 5 of the 8 mares prior to insemination and only one of the 5 became pregnant. In the second estrous cycle, 5 of the 8 mares (63 %) became pregnant after insemination with frozen semen that had been thawed, extended and cooled. Only one mare in this group ovulated before insemination and she was determined to be pregnant at day 14. The pregnancy rate for the two cycles combined was 56.3 % (9 for 16). Table 2. Follicle size, edema score (range 0 to 3) on the day of histrelin administration, post-thaw sperm motility and pregnancy status at 14 days post-ovulation for the eight mares in Cycle 1 and Cycle 2. Mares in Cycle 1 were with inseminated with frozen-
thawed stored semen from one stallion, while mares in Cycle 2 were inseminated with semen from a second stallion. Breeding Cycle
Follicle size (mm)
Edema score
Total Motility (%)
Progressive Motility (%)
Pregnancy Status (d 14)
Comments
Cycle 1 (Histrelin at 2000 hrs) Cycle 2 (Histrelin at 0900 hrs)
39.1 ± 2.5
1.4 ± 0.6
27.3 ± 7.0
19.9 ± 5.8
4/8 (50 %)
5 mares ovulated prior to AI
42.9 ± 4.2
1.6 ± 0.6
41.3 ± 7.3
34.1 ± 7.3
5/8 (62.5 %)
1 mare ovulated prior to AI
4. Discussion This is the first study to demonstrate that it is possible to thaw a dose frozen stallion semen, dilute it in a commercial extender and cool it to 5° C in a passive cooling system for 24 hours and maintain acceptable (i.e. > 30 %) sperm motility and fertility potential. Historically it has been assumed that frozen stallion sperm would not survive for a prolonged period after thawing. The current study suggests that dilution in an appropriate extender after thawing and storage at 5° C for at least 24 hours is possible. Comparison of sperm motility after 24 hours of cooled storage for three semen thawing protocols indicated that there was no advantage in centrifugation after thawing or in thawing for only 12 seconds, which was determined to be the time for thawed semen to reach 5° C. Consequently, the standard semen thawing protocol of immersion in a 37° C water bath for 30 seconds was selected for the breeding trial. Two semen extenders were selected for dilution of frozen semen after thawing. INRA96 is a commercially available extender commonly used for cooled-storage of stallion semen [13]. The second extender (CryoMax LE® minus cryoprotectant) was selected because it was the same extender used for initial cryopreservation [14], but without the cryoprotectants. The hypothesis was that use of the same extender base would decrease adverse issues associated with dramatic changes in osmolality, pH or other factors. However, this extender without cryoprotectants is not commercially available and therefore not likely to be used in clinical practice. Sperm motility results after 24 and 48 hours of cooled storage in CryoMax LE® minus cryoprotectant in this study were more varied than those in which thawed semen was diluted in INRA96. Consequently, INRA96 was selected as the extender for cooled-storage for the breeding trial. The standard protocol used for management of mares being bred with one dose of frozen semen at our facility is to administer a GnRH agonist, such as histrelin or deslorelin, at 2000 hrs (8:00 pm), perform ultrasound examinations every 6 hours and thaw semen and inseminate the mare using a deep-horn protocol as soon as ovulation is detected [15]. In our clinical program, ovulation is detected in a majority of mares approximately 40 hours after histrelin treatment, with some mares ovulating prior to 40 hours and some ovulating after 40 hours [16]. The goal of Cycle 1 in the fertility trial was to mimic this protocol, albeit with mare examinations performed only once per day, and inseminate mares in the
uterine body with the thawed-cooled semen close to the anticipated ovulation. In the current study, 4 of the 8 mares became pregnant using this protocol. However, ovulation was detected prior to insemination 5 of the 8 mares in Cycle 1 and only one of those 5 mares became pregnant. A different ovulation induction protocol is used at our facility for managing mares being bred with cooled-transported semen. Mares are administered a GnRH agonist at the time semen is ordered and confirmed (approximately 0800 hr) and the mare is inseminated the next day after semen is delivered by overnight courier (approximately 1100 hrs). A majority of mares ovulate later that night. Consequently, the goal of Cycle 2 in the current study was to mimic events in which a dose of frozen semen would be ordered in the morning from the facility storing the frozen semen. Semen would subsequently be thawed, extended and then cooled to 5° C for approximately 24 hours during shipment to the mare facility, and the mare inseminated the next day. In this scenario, there is inevitably a longer interval from insemination to ovulation than in the previous breeding management protocol. In the current study, 5 of the 8 mares became pregnant using this more conservative protocol and only 1 mare ovulated prior to insemination. Another goal of the project was to determine if mares could be effectively managed using the frozen-cooled protocol by performing ultrasound examinations only once per day instead of 4 times per day. In that regard, the overall pregnancy rate of 56.3 % (9 pregnancies out of 16 cycles) suggests that the one ultrasound examination per day management protocol was successful. It is clearly recognized that the limited breeding trial conducted in this study offers only preliminary data and should be interpreted accordingly. The sample size was low (n=8) and mares were only bred over two estrous cycles. Ideally, a much larger number of mares would be used and several additional control groups would be included, such as insemination of mares with non-cryopreserved semen stored at 5° C for 24 hours and insemination of mares with cryopreserved semen thawed and inseminated immediately. In addition, pregnancy data was only obtained from frozen-thawed semen of two stallions. It is likely that this protocol may be successful for some stallions, but not for others. It is our opinion that traditional cooled-transported semen (i.e. non-cryopreserved) should remain the primary option, if available, for breeding mares located a distance from the stallion. However, the technique evaluated in this study would allow an equine breeding facility to thaw frozen semen, dilute in a commercial extender and transport the extended semen overnight to another facility for insemination the following day. This may enable veterinarians to utilize frozen semen without the usual added cost, equipment, expertise and mare management issues. In summary, results of this study suggest that it is possible to obtain pregnancies using a standard dose of frozen semen (800 million total spermatozoa) that had been thawed at 37° C for 30 seconds, diluted to a concentration of 50 million spermatozoa per ml in a
commercial semen extender (INRA96) and cooled to 5° C in a passive cooling system and stored for 24 hours before insemination. References [1] Bowen JM. Historical perspectives of artificial insemination. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 4th ed, Ames, Iowa: Wiley-Blackwell; 2011, p. 1261-1267. [2] Conboy HS. Management of stallion in artificial insemination. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 4th ed, Ames, Iowa: Wiley-Blackwell; 2011, p. 1198-207. [3] Ray MA, Grimes PW. The determinants of breeding regulation in the horse industry: an empirical analysis. Journal Socio-Economics 1991;20:169-80. [4] Samper JC. Artificial insemination with fresh and cooled semen. In: Samper JC, editor. Equine breeding management and artificial insemination. 2nd ed. St. Louis: Saunders Elsevier; 2009, p 165-174. [5] Sanchez R, I Gomez, JC Samper. Artificial insemination with frozen semen. In: Samper JC, editor. Equine breeding management and artificial insemination. 2nd ed. St. Louis: Saunders Elsevier; 2009, p 175-184. [6] Amann RP, Pickett BW. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J Eq Vet Sci 1987;7:145-73. [7] Loomis PR. The equine frozen semen industry. Anim Reprod Sci 2001;68:191200. [8] Barbacini S. Breeding with frozen semen. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 4th ed, Ames, Iowa: Wiley-Blackwell; 2011, p. 2987-2993. [9] Squires, EL, Pickett, BW, Graham, JK, Vanderwall, DK, McCue, PM, Bruemmer, JE. Cooled and frozen stallion semen. In: Animal Reproduction and Biotechnology Laboratory, Bulletin No. 9. Fort Collins, Colorado:Colorado State University; 1999, p. 39-41. [10] Loomis PR, Squires EL. Frozen semen management in equine breeding programs. Theriogenology 2005;64:480-91. [11] Johansson CS, Matsson FC, Lehn-Jensen H, Nielson JM, Petersen MM. Equine spermatozoa viability comparing the NucleoCounter SP-100 and the eosin-nigrosin stain. Anim Reprod Sci 2008;23:24-5. [12] Dascanio JJ. Computer‐Assisted Sperm Analysis. In: Dascanio JJ, McCue PM, editors. Equine reproductive procedures. Ames, Iowa: Wiley-Blackwell; 2014, p. 368-72. [13] Vidament M, Magistrini M, Le Foll Y, Levillain N, Yvon JM, Duchamp G, Blesbois E. Temperatures from 4 to 15° C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h or more in INRA96 extender. Theriogenology 2012;78:297-307. [14] Moffett P, Fors G, Graham JK. Cryopreservation of stallion sperm in freezing diluents containing glycerol alone or a combination of cryoprotectants. J Eq Vet Sci 2016;43:S69.
[15] McCue PM, RA Ferris. Induction of a timed ovulation. In: Formulary and Protocols in Equine Reproduction. 2nd ed. Fort Collins, Colorado: Colorado State University; 2018, p.29. [16] McCue PM. Breeding management of the mare. In: Techniques in Equine Reproduction. 6th ed. Chino, California: Animal Reproduction Systems; 2018, p. 128131.
Acknowledgements None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of this article.
Highlights Stallion semen was cryopreserved and later thawed, extended in a commercial cooling extender and maintained at 5° C for 48 hours Progressive motility ranged from 22 % to 39 % after 48 hours of cooled storage A pregnancy rate of 56 % (9 pregnancies over 16 estrous cycles) was obtained during a limited breeding trial
Ethical Statement The authors adhered to ethical standards in regard to scientific principles and animal welfare..
Conflict of Interest Declarations of interest: none
S0737-0806(20)30074-5
DOI:
https://doi.org/10.1016/j.jevs.2020.102983
Reference:
YJEVS 102983
To appear in:
Journal of Equine Veterinary Science
Received Date: 26 October 2018 Revised Date:
27 February 2020
Accepted Date: 27 February 2020
Please cite this article as: Prell MJ, McCue PM, Moffett PD, Graham JK, Motility and fertility evaluation of thawed frozen stallion semen after 24 hours of cooled storage, Journal of Equine Veterinary Science (2020), doi: https://doi.org/10.1016/j.jevs.2020.102983. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Inc. All rights reserved.
Motility and fertility evaluation of thawed frozen stallion semen after 24 hours of cooled storage Melissa J. Prella, Patrick M. McCuea, Paula D. Moffettb, James K. Grahamb a
a
Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
Key Words: Stallion, semen, frozen, cooled-storage Corresponding Author:
Patrick M. McCue Equine Reproduction Laboratory Colorado State University 3101 Rampart Road Fort Collins, CO 80521 USA [email protected]
Abstract: Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were: 1) evaluate the longevity of frozen stallion semen once it had been thawed, extended and maintained at 5° C for 48 hours in a passive cooling container, and 2) determine fertility potential of frozen semen that had been thawed, extended and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE® minus cryoprotectant at a concentration of 50 million total sperm/mL. The remainder of the ejaculate was frozen in CryoMax LE® extender at a concentration of 200 million total sperm/mL. Semen was thawed using 1 of 3 thawing protocols, and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE ®minus cryoprotectant and cooled to 5° C. Sperm motility was evaluated at 24 and 48 hours. Eight mares were inseminated over two estrous cycles using frozen semen that had been thawed, extended in INRA96 and cooled for 24 hours. There was no difference in progressive motility at 24 or 48 hours of cooled-storage postthaw between the 3 thawing protocols. An overall per cycle pregnancy rate of 56 % (9/16 cycles) was achieved using frozen-thawed semen that had been extended and cooled for 24 hours. In summary, frozen stallion sperm was thawed, extended and cooled to 5°C for 24 hours and still maintained adequate (> 30 %) sperm motility and fertility.
1. Introduction Artificial insemination is a common procedure in equine clinical practice and a majority of breed associations, with the notable exception of Thoroughbreds, allow the use of artificial insemination [1-3]. Semen collected from stallions can be extended and used for fresh insemination, extended and stored at 5 to 8° C for 24 to 48 hours prior to insemination, or cryopreserved and stored indefinitely in liquid nitrogen [4,5].
Advantages of cryopreservation of stallion semen include preservation of genetic material for future use, ‘insurance’ against unanticipated loss of a stallion, ability to breed mares when the stallion is unavailable for collection, and ability to ship frozen semen anywhere in the world [6-8]. Potential disadvantages of cryopreservation of stallion semen include the fact that spermatozoa from some stallions do not exhibit adequate post-thaw motility, pregnancy rates following insemination with frozen semen is generally lower than rates associated with fresh or cooled-stored semen, and more intensive mare management is required when using frozen semen [9]. In addition, specialized equipment and training are needed for the transport, storage, handling and evaluation of frozen semen [10]. As a consequence, not all equine breeding operations are enamored with the use of frozen stallion semen and prefer to breed mares only with fresh or cooled semen. The objectives of this study were to 1) evaluate the longevity of frozen stallion semen once it had been thawed, extended and maintained at 5° C for 48 hours in a passive cooling container and 2) determine fertility potential of frozen semen that had been thawed, extended and used to inseminate mares after 24 hours of cooled storage. The hypotheses were that thawed semen, when properly extended and cooled, would maintain motility for at least 24 hours and that it would be possible to obtain pregnancies after insemination of frozen-thawed semen extended and stored at 5° C for 24 hours. 2. Materials and Methods 2.1 Semen collection and processing A total of 8 ejaculates from 5 Quarter Horse stallions were collected using a Colorado model artificial vagina. One ejaculate was collected from each of 5 stallions; three of the stallions had a second ejaculate collected. Initial semen analysis consisted of measurements of semen volume, sperm concentration and sperm motility. Volume was measured in a graduated cylinder; sperm concentration was determined using a NucleoCounter® (ChemoMetec, Allerod, Denmark) [9]; total and progressive sperm motility were evaluated by computer assisted semen analysis (CASA; SpermVision®; MOFA, Verona, WI) [12]. Each ejaculate was divided, and aliquots were cooled in both a commercial semen extender (INRA96; IMV Technologies, Maple Grove, MN) and a commercial freezing extender minus cryoprotectant (CryoMax LE®; Animal Reproduction Systems, Chino, CA) at a concentration of 50 million total sperm/mL to serve as non-cryopreserved controls. The remainder of the ejaculate was extended to a 1:1 ratio with a commercial extender (E-Z Mixin®-BF; Animal Reproduction Systems, Chino, CA) and divided into 50 mL conical tubes. A 3 mL volume of a centrifugation cushion (Maxifreeze; IMV Technologies, Maple Grove, MN) was gently deposited in the bottom of each conical tube to enhance sperm recovery. The loaded conical tubes were subsequently centrifuged at 1000 x g for 25 minutes. The supernatant and centrifugation cushion were removed
and the sperm pellet was re-suspended and extended to a concentration of 200 million total sperm/mL using a commercial freezing extender (CryoMax LE®; Animal Reproduction Systems, Chino, CA). The extended semen was loaded into 0.5 mL straws and cooled to a temperature of 5° C over a 30 minute time period in a refrigerator. Straws were then suspended 3 cm over liquid nitrogen vapor for 15 minutes before being plunged into liquid nitrogen. Frozen straws were subsequently transferred into liquid nitrogen tanks for later use. 2.2 Treatment groups Control Group C1 - non-cryopreserved semen samples diluted in INRA96 Control Group C2 - non-cryopreserved semen samples diluted in CryoMax LE® minus cryoprotectant Group 1 - straws of frozen semen were thawed in a 37° C water bath for 30 seconds Group 2 - straws were thawed in a 37° C water bath for 30 seconds, followed by centrifugation at 400 x g for 10 minutes Group 3 - straws were thawed in a 37° C water bath for 12 seconds The thawing method for Group 1 is the standard protocol used in our laboratory for frozen semen. The protocol used in Group 2 was designed to remove the freezing extender prior to dilution and eliminate potential adverse effects of the cryoprotectants. The Group 3 protocol was designed to expose the frozen straws to a water bath for enough time to reach a temperature of 5° C, but not allow the spermatozoa to warm enough to potentially incur damage to their plasma membranes by going through liquidcrystalline to gel-state phase transitions. After thawing, each sample was split into two aliquots and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE® minus cryoprotectant and cooled to 5° C in a commercial passive cooling container (Equitainer; Hamilton Biovet, Ipswich, MA). Total and progressive motility were subsequently assessed at both 24 and 48 hours. 2.3 Fertility trial Eight Quarter Horse mares, ranging from 6 to 13 years of age and of unknown parity, were used in a preliminary breeding trial over two consecutive estrous cycles to evaluate the fertility potential of frozen semen that had been thawed, extended and cooled for 24 hours. All mares had exhibited normal reproductive cycles, but no uterine cultures, cytology or biopsy samples were collected prior to insemination. Breeding management on the first estrous cycle was designed to mimic the timed ovulation and insemination timing normally used when breeding mares with frozen semen at our facility, with the exception that mares were only examined once per day. Breeding management on the second cycle was designed to mimic the timed ovulation and insemination timing normally used when breeding mares with cooled-transported semen at our facility. Mares were only examined once per day and all inseminations
were into the uterine body. Ultrasound pregnancy examinations were performed 14 days post-ovulation. Frozen-thawed semen from one stallion was used to inseminate all 8 mares during the first estrous cycle, while frozen-thawed semen from a second stallion was used to inseminate all 8 mares during the second estrous cycle. Stallion selection was based on post-thaw motility after 24 hours of cooled storage and availability of sufficient doses of frozen semen to complete the breeding trial. Cycle 1 Protocol. Mares were examined once per day by transrectal ultrasonography at approximately 0900 hrs. Once a follicle ≥ 35 mm was detected in the presence of uterine edema, a single 500 µg intramuscular dose of histrelin (Doc Lanes Veterinary Pharmacy, Lexington, KY)) was administered at 2000 hrs that night. The next morning at approximately 0900 hrs, a dose of frozen semen, consisting of 8 straws at 100 million total sperm per straw, was thawed, extended to a concentration of 50 million total sperm/mL in INRA96 (16 mls total volume) and placed in a passive cooling system for 24 hours. The following morning at approximately 0900 hrs, mares were re-examined by ultrasound and inseminated. The apriori expectation was that mares should ovulate an average of 40 hours after receiving histrelin, which would equate to approximately 1200 hrs, or about 3 hours after being inseminated with frozen-cooled semen. Cycle 2 Protocol. Mares were examined once per day at approximately 0900 hrs. Once a follicle ≥ 35 mm was detected in the presence of uterine edema, a single 500 µg intramuscular dose of histrelin was immediately administered and a dose of frozen semen, consisting of 8 straws at 100 million total sperm per straw, was thawed, extended in INRA96 and placed in a passive cooling system for 24 hours. The following morning at approximately 0900 hrs, mares were re-examined by ultrasound and inseminated. The apriori expectation was that mares should ovulate approximately 40 hours after receiving histrelin, which would equate to approximately 0100 hrs, or about 16 hours after being inseminated with frozen-cooled semen. 2.5 Statistical Analysis Data were analyzed by univariate, repeated-measures analysis of variance using the General Linear Model (GLM) procedure of SAS Institute, with p-value set at <0.05. Data are presented as mean ± SD. 3. Results 3.1 Motility assessment Total and progressive sperm motility were higher (p<0.05) at 24 and 48 hours for noncryopreserved semen samples extended in INRA96 (C1) as compared to all frozenthawed semen samples (Groups 1, 2 and 3) extended in INRA96 (Table 1). Total and progressive sperm motilities were lower at 24 hours of cooled storage for frozen semen samples thawed in a 37° C water bath for 12 seconds (Group 3) and subsequently extended in INRA96 than semen samples in Groups 1 and 2.
Progressive sperm motility was lower for Group 1 and Group 3 samples stored at 5° C in CryoMax LE® minus cryoprotectant for 24 hours compared to the non-cryopreserved control (Table 1). A significant difference was also noted in total and progressive motility in Group 3 samples as compared to the non-cryopreserved control and in Group 2 versus Group 3 at 48 hours of cooled storage. Table 1. Sperm motility parameters of cooled controls and frozen-cooled treatment groups maintained in INRA96 or CryoMax LE® minus cryoprotectant (CPA). The non-cryopreserved control samples were maintained at 5º C and evaluated at 24 and 48 hours. Semen samples for Groups 1, 2 and 3 were cryopreserved, thawed as described, diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE® minus cryoprotectant and cooled to 5° C in a commercial passive cooling container and evaluated at 24 and 48 hours.
CryoMax LE® minus CPA
INRA96 Group NonCryopreserved Controls
Motility 24 h
Motility 48 h
a
a
Motility 24 h
Motility 48 h a
64.3 ± 13.1 /53.8 ± 15.4a
56.1 ± 15.8ab /45.9 ± 16.5a
77.1 ± 6.9 /71.3 ± 8.6a
75.4 ± 9.2 /71.3 ± 10.3a
50.3 ± 9.6b /45.0 ± 12.0b
51.6 ± 12.8b 41.5 ± 10.4bc /45.6 ± 12.5b /29.5 ± 10.6b
35.5 ± 10.8ac /25.5 ± 10.9ab
48.6 ± 10.1b /38.9 ± 10.8b
47.0 ± 13.9b 49.9 ± 10.9ab /37.5 ± 14.2b /38.6 ± 10.9ab
50.1 ± 12.7b /34.9 ± 11.5a
41.5 ± 6.5b /34.8 ± 8.8b
32.8 ± 12.3c /21.6 ±10.2b
(Total/Progressive)
Group 1 Thawed at 37°C for 30 sec (Total/Progressive)
Group 2 Thawed at 37°C for 30 sec and centrifuged (Total/Progressive)
Group 3 Thawed at 37°C for 12 sec (Total/Progressive)
32.8 ± 6.6c /28.6 ± 7.3c
32.3 ± 8.5c /26.5 ± 9.8b
3.2 Fertility trial In the first estrous cycle, 4 of 8 mares (50 %) became pregnant after insemination with frozen semen that had been thawed at 37° C, extended in INRA96 and cooled for 24 hours (Table 2). Ovulation was detected in 5 of the 8 mares prior to insemination and only one of the 5 became pregnant. In the second estrous cycle, 5 of the 8 mares (63 %) became pregnant after insemination with frozen semen that had been thawed, extended and cooled. Only one mare in this group ovulated before insemination and she was determined to be pregnant at day 14. The pregnancy rate for the two cycles combined was 56.3 % (9 for 16). Table 2. Follicle size, edema score (range 0 to 3) on the day of histrelin administration, post-thaw sperm motility and pregnancy status at 14 days post-ovulation for the eight mares in Cycle 1 and Cycle 2. Mares in Cycle 1 were with inseminated with frozen-
thawed stored semen from one stallion, while mares in Cycle 2 were inseminated with semen from a second stallion. Breeding Cycle
Follicle size (mm)
Edema score
Total Motility (%)
Progressive Motility (%)
Pregnancy Status (d 14)
Comments
Cycle 1 (Histrelin at 2000 hrs) Cycle 2 (Histrelin at 0900 hrs)
39.1 ± 2.5
1.4 ± 0.6
27.3 ± 7.0
19.9 ± 5.8
4/8 (50 %)
5 mares ovulated prior to AI
42.9 ± 4.2
1.6 ± 0.6
41.3 ± 7.3
34.1 ± 7.3
5/8 (62.5 %)
1 mare ovulated prior to AI
4. Discussion This is the first study to demonstrate that it is possible to thaw a dose frozen stallion semen, dilute it in a commercial extender and cool it to 5° C in a passive cooling system for 24 hours and maintain acceptable (i.e. > 30 %) sperm motility and fertility potential. Historically it has been assumed that frozen stallion sperm would not survive for a prolonged period after thawing. The current study suggests that dilution in an appropriate extender after thawing and storage at 5° C for at least 24 hours is possible. Comparison of sperm motility after 24 hours of cooled storage for three semen thawing protocols indicated that there was no advantage in centrifugation after thawing or in thawing for only 12 seconds, which was determined to be the time for thawed semen to reach 5° C. Consequently, the standard semen thawing protocol of immersion in a 37° C water bath for 30 seconds was selected for the breeding trial. Two semen extenders were selected for dilution of frozen semen after thawing. INRA96 is a commercially available extender commonly used for cooled-storage of stallion semen [13]. The second extender (CryoMax LE® minus cryoprotectant) was selected because it was the same extender used for initial cryopreservation [14], but without the cryoprotectants. The hypothesis was that use of the same extender base would decrease adverse issues associated with dramatic changes in osmolality, pH or other factors. However, this extender without cryoprotectants is not commercially available and therefore not likely to be used in clinical practice. Sperm motility results after 24 and 48 hours of cooled storage in CryoMax LE® minus cryoprotectant in this study were more varied than those in which thawed semen was diluted in INRA96. Consequently, INRA96 was selected as the extender for cooled-storage for the breeding trial. The standard protocol used for management of mares being bred with one dose of frozen semen at our facility is to administer a GnRH agonist, such as histrelin or deslorelin, at 2000 hrs (8:00 pm), perform ultrasound examinations every 6 hours and thaw semen and inseminate the mare using a deep-horn protocol as soon as ovulation is detected [15]. In our clinical program, ovulation is detected in a majority of mares approximately 40 hours after histrelin treatment, with some mares ovulating prior to 40 hours and some ovulating after 40 hours [16]. The goal of Cycle 1 in the fertility trial was to mimic this protocol, albeit with mare examinations performed only once per day, and inseminate mares in the
uterine body with the thawed-cooled semen close to the anticipated ovulation. In the current study, 4 of the 8 mares became pregnant using this protocol. However, ovulation was detected prior to insemination 5 of the 8 mares in Cycle 1 and only one of those 5 mares became pregnant. A different ovulation induction protocol is used at our facility for managing mares being bred with cooled-transported semen. Mares are administered a GnRH agonist at the time semen is ordered and confirmed (approximately 0800 hr) and the mare is inseminated the next day after semen is delivered by overnight courier (approximately 1100 hrs). A majority of mares ovulate later that night. Consequently, the goal of Cycle 2 in the current study was to mimic events in which a dose of frozen semen would be ordered in the morning from the facility storing the frozen semen. Semen would subsequently be thawed, extended and then cooled to 5° C for approximately 24 hours during shipment to the mare facility, and the mare inseminated the next day. In this scenario, there is inevitably a longer interval from insemination to ovulation than in the previous breeding management protocol. In the current study, 5 of the 8 mares became pregnant using this more conservative protocol and only 1 mare ovulated prior to insemination. Another goal of the project was to determine if mares could be effectively managed using the frozen-cooled protocol by performing ultrasound examinations only once per day instead of 4 times per day. In that regard, the overall pregnancy rate of 56.3 % (9 pregnancies out of 16 cycles) suggests that the one ultrasound examination per day management protocol was successful. It is clearly recognized that the limited breeding trial conducted in this study offers only preliminary data and should be interpreted accordingly. The sample size was low (n=8) and mares were only bred over two estrous cycles. Ideally, a much larger number of mares would be used and several additional control groups would be included, such as insemination of mares with non-cryopreserved semen stored at 5° C for 24 hours and insemination of mares with cryopreserved semen thawed and inseminated immediately. In addition, pregnancy data was only obtained from frozen-thawed semen of two stallions. It is likely that this protocol may be successful for some stallions, but not for others. It is our opinion that traditional cooled-transported semen (i.e. non-cryopreserved) should remain the primary option, if available, for breeding mares located a distance from the stallion. However, the technique evaluated in this study would allow an equine breeding facility to thaw frozen semen, dilute in a commercial extender and transport the extended semen overnight to another facility for insemination the following day. This may enable veterinarians to utilize frozen semen without the usual added cost, equipment, expertise and mare management issues. In summary, results of this study suggest that it is possible to obtain pregnancies using a standard dose of frozen semen (800 million total spermatozoa) that had been thawed at 37° C for 30 seconds, diluted to a concentration of 50 million spermatozoa per ml in a
commercial semen extender (INRA96) and cooled to 5° C in a passive cooling system and stored for 24 hours before insemination. References [1] Bowen JM. Historical perspectives of artificial insemination. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 4th ed, Ames, Iowa: Wiley-Blackwell; 2011, p. 1261-1267. [2] Conboy HS. Management of stallion in artificial insemination. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 4th ed, Ames, Iowa: Wiley-Blackwell; 2011, p. 1198-207. [3] Ray MA, Grimes PW. The determinants of breeding regulation in the horse industry: an empirical analysis. Journal Socio-Economics 1991;20:169-80. [4] Samper JC. Artificial insemination with fresh and cooled semen. In: Samper JC, editor. Equine breeding management and artificial insemination. 2nd ed. St. Louis: Saunders Elsevier; 2009, p 165-174. [5] Sanchez R, I Gomez, JC Samper. Artificial insemination with frozen semen. In: Samper JC, editor. Equine breeding management and artificial insemination. 2nd ed. St. Louis: Saunders Elsevier; 2009, p 175-184. [6] Amann RP, Pickett BW. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J Eq Vet Sci 1987;7:145-73. [7] Loomis PR. The equine frozen semen industry. Anim Reprod Sci 2001;68:191200. [8] Barbacini S. Breeding with frozen semen. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 4th ed, Ames, Iowa: Wiley-Blackwell; 2011, p. 2987-2993. [9] Squires, EL, Pickett, BW, Graham, JK, Vanderwall, DK, McCue, PM, Bruemmer, JE. Cooled and frozen stallion semen. In: Animal Reproduction and Biotechnology Laboratory, Bulletin No. 9. Fort Collins, Colorado:Colorado State University; 1999, p. 39-41. [10] Loomis PR, Squires EL. Frozen semen management in equine breeding programs. Theriogenology 2005;64:480-91. [11] Johansson CS, Matsson FC, Lehn-Jensen H, Nielson JM, Petersen MM. Equine spermatozoa viability comparing the NucleoCounter SP-100 and the eosin-nigrosin stain. Anim Reprod Sci 2008;23:24-5. [12] Dascanio JJ. Computer‐Assisted Sperm Analysis. In: Dascanio JJ, McCue PM, editors. Equine reproductive procedures. Ames, Iowa: Wiley-Blackwell; 2014, p. 368-72. [13] Vidament M, Magistrini M, Le Foll Y, Levillain N, Yvon JM, Duchamp G, Blesbois E. Temperatures from 4 to 15° C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h or more in INRA96 extender. Theriogenology 2012;78:297-307. [14] Moffett P, Fors G, Graham JK. Cryopreservation of stallion sperm in freezing diluents containing glycerol alone or a combination of cryoprotectants. J Eq Vet Sci 2016;43:S69.
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Acknowledgements None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of this article.
Highlights Stallion semen was cryopreserved and later thawed, extended in a commercial cooling extender and maintained at 5° C for 48 hours Progressive motility ranged from 22 % to 39 % after 48 hours of cooled storage A pregnancy rate of 56 % (9 pregnancies over 16 estrous cycles) was obtained during a limited breeding trial
Ethical Statement The authors adhered to ethical standards in regard to scientific principles and animal welfare..
Conflict of Interest Declarations of interest: none