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The fertility of ram sperm held for 24 h at 5 °C prior to cryopreservation
Animal Reproduction Science 118 (2010) 231–235
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
The fertility of ram sperm held for 24 h at 5 ◦ C prior to cryopreservation夽 Phillip H. Purdy a,∗ , Eva Mocé b , Robert Stobart c , William J. Murdoch c , Gary E. Moss c , Brent Larson c , Shawn Ramsey d , James K. Graham e , Harvey D. Blackburn a a
USDA-ARS-NCGRP, National Animal Germplasm Program, 1111 S. Mason St., Fort Collins, CO 80521-4500, USA Instituto Valenciano de Investigaciones Agrarias-Centro de Tecnología Animal (IVIA-CITA), Polígono La Esperanza, n◦ 100, 12400-Segorbe (Castellón), Spain c Department of Animal Science, University of Wyoming, Laramie, WY 82071-3684, USA d Department of Animal Science, Texas A&M University, College Station, TX 77843-2471, USA e Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA b
a r t i c l e
i n f o
Article history: Received 5 August 2008 Received in revised form 18 May 2009 Accepted 18 June 2009 Available online 26 June 2009 Keywords: Fertility Holding time Ram Sperm Artificial insemination
a b s t r a c t Diluted ram sperm can be held for 24 h at 5 ◦ C prior to cryopreservation without impacting cryosurvival rates, however, the effects this storage has on subsequent fertility are unknown. These studies were conducted to evaluate the fertility of semen held for 24 h (to mimic shipping semen to a cryopreservation center), prior to freezing. Semen from Suffolk rams (n = 3 in experiment 1 and n = 6 in experiment 2) with initial motility of greater than 70%, were diluted to 200 × 106 sperm/mL, in one step, with a Tris–egg yolk–glycerol diluent. In experiment 1, diluted samples were cooled to 5 ◦ C over 2 h, and then divided. Sperm in one fraction were loaded into 0.5 mL straws, frozen (T0) and stored in liquid nitrogen until thawing. Sperm in the second fraction were held at 5 ◦ C for 24 h (T24) before being frozen. In experiment 2 ejaculates were collected and divided into two fractions. Sperm in one fraction were treated with cholesterol-loaded cyclodextrin (CLC) and sperm in the other served as control. Both fractions were diluted, cooled, and cryopreserved as described in experiment 1. Stage of the estrous cycle was synchronized in ewes (n = 196) using controlled internal drug releasing devices (CIDR) for 12 d and at CIDR removal each ewe was administered PMSG (500 IU in experiment 1 and 350 IU in experiment 2) immediately before insemination. Ewes were stratified by age and randomly assigned to one of the semen treatments; experiment 1: Fresh (F), T0, or T24; experiment 2: F, T24, or CLC, and inseminated laparoscopically 56 h after CIDR removal. Differences in fertility were detected between experiments, but not for treatments within experiments. Differences in fertility were also observed due to ewe age, with the 3-year-old ewes having the greatest fertility (50.7%) and 6-year-old ewes having the least fertility (9.6%; P < 0.05). Differences in the prolificacy rates due to semen treatment were also observed but differences due to ewe age were not detected. Therefore, sperm can be held at 5 ◦ C for 24 h prior to cryopreservation without altering sperm fertility. Published by Elsevier B.V.
夽 Mention of a trade name or proprietary product does not constitute a guaranty or warranty by the USDA and does not imply approval to the exclusion of other products that may be suitable. USDA, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer. All agency services are available without discrimination. ∗ Corresponding author. Tel.: +1 970 495 3258; fax: +1 970 221 1427. E-mail address: [email protected] (P.H. Purdy). 0378-4320/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.anireprosci.2009.06.014
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P.H. Purdy et al. / Animal Reproduction Science 118 (2010) 231–235
1. Introduction The sheep industry has not been able to utilize many of the assisted reproductive technologies (ART) in general and AI in particular, as other livestock industries, due to inefficiencies in collecting, freezing and inseminating frozen ram semen. Furthermore, few commercial studs routinely collect and freeze ram semen, and there is still a need to optimize cryopreservation and breeding protocols for ram semen. The USDA National Animal Germplasm Program (NAGP) is charged with developing a national repository of animal germplasm and tissue to provide genetic security and facilitate genetic understanding of livestock species (Blackburn, 2004). This task is complicated by the lack of ART infrastructure for the U.S. sheep industry and by the general inability to efficiently cryopreserve ram semen. One way to quickly improve the quality of cryopreserved ram semen would be to collect the semen where the ram resides and ship the semen to a central processing center with the expertise in cryopreserving the semen, in much the same way cooled stallion semen is currently used (Backman et al., 2004). This system would permit semen from a ram to be cryopreserved without the owner having to transport the ram, house the ram in a collection facility, or have to have the equipment for or expertise in cryopreservation. Diluted ram semen can be held at 5 ◦ C for up to 48 h prior to cryopreservation without detrimental effects on sperm physiology (Purdy, 2006). In addition, new cryopreservation technologies, such as treating ram sperm with cholesterol-loaded cyclodextrins (CLC) show promise in increasing sperm cryosurvival rates (Morrier and Bailey, 2003; Morrier et al., 2004; Mocé et al., 2009). These studies were conducted to determine the fertilizing potential of ram sperm cryopreserved 24 h after collection in conjunction with CLC treatment. 2. Materials and methods
glycerol, 15% egg yolk, 1 mg/mL streptomycin sulfate and 0.06 mg/mL benzylpenicillin; Sanchez-Partida et al., 1998) at 37 ◦ C.
2.2. Experiment 1 Diluted semen samples were cooled to 5 ◦ C over 2 h and split into two aliquots. One aliquot from each ejaculate was immediately (designated T0) loaded into 0.5 mL CBS straws (IMV Corp., Minneapolis, MN, USA) and cryopreserved as described by (Sanchez-Partida et al., 1998). The second aliquot was held for 24 h (T24) at 5 ◦ C, before being packaged into straws and cryopreserved. Sperm were frozen in liquid nitrogen vapor using a Mini Digitcool UJ400 programmable freezer (IMV Corp., Minneapolis, MN, USA). The sperm were cooled from 5 ◦ C to −5 ◦ C at 4 ◦ C/min; from −5 ◦ C to −110 ◦ C at 25 ◦ C/min; and from −110 ◦ C to −140 ◦ C at 35 ◦ C/min. After reaching −140 ◦ C the straws were plunged into liquid nitrogen for storage. Frozen straws were thawed in 37 ◦ C water for 30 s and were analyzed for the percentage of motile sperm, using a computer assisted semen analysis system (IVOS Version 10.9, Hamilton Thorne Bioscience, Beverly, MA, USA) with a 10× phase contrast objective and the following settings: 50 frames acquired, frame rate of 60 Hz, minimum contrast of 60, minimum cell size of 5 pixels, VAP (path velocity) cutoff of 20 m, progressive minimum VAP cutoff of 50 m/s, VSL (progressive velocity) cutoff of 30 m/s, static head size of 0.24–3.66, and magnification of 1.89. For each sample, an aliquot was diluted 1:3 (v/v; sample to diluent) with Tris-buffered saline (Purdy and Graham, 2004) and 5 L was placed onto a Standard Count Analysis Chamber (Spectrum Technologies, Healdsburg, CA, USA). A minimum of 500 sperm and five fields were analyzed to estimate the percentage of motile sperm from each treatment.
2.1. Semen collection and handling Adult rams and ewes were housed at the University of Wyoming (Laramie, WY, USA). Animals were fed a diet providing 100% of their nutritional needs, and provided water ad libitum. All protocols for working with the sheep were approved by the University of Wyoming Institutional Animal Care and Use Committee. Single ejaculates from sexually mature Suffolk rams were collected using electro-ejaculation (Evans and Maxwell, 1987) during the month of October and ewes were inseminated the following month. After collection, the percentage of motile sperm in each ejaculate was determined using bright field microscopy (Nasco Advanced Laboratory Microscope, Nasco Farm and Ranch Supplies, Fort Atkinson, WI, USA) at 400× magnification and 23 ◦ C to ensure that each sample had a minimum of 70% motile sperm. The volume and sperm concentration (Spermacue, IMV Corp., Minneapolis, MN, USA) were determined and ejaculates were diluted in one step (in 50 mL centrifuge tubes) to 200 × 106 sperm/mL with Tris–egg yolk–glycerol medium (300 mM Tris, 28 mM glucose, 95 mM citric acid, 5% (v/v)
2.3. Experiment 2 In experiment 2, conducted the following year, semen samples from six rams were collected and split prior to diluting the sperm. One aliquot was diluted with the Tris–egg yolk–glycerol medium to 200 × 106 sperm/mL. The second aliquot was treated with cholesterol-loaded methyl- cyclodextrin (2 mg/120 × 106 sperm or CLC (3.33 mg/200 × 106 sperm) for 15 min at 23 ◦ C and then diluted in the same manner as the first aliquot (Mocé and Graham, 2006). Both were then cooled to 5 ◦ C over 2 h and held at 5 ◦ C for 24 h before being packaged into straws and cryopreserved as described above. Straws were thawed and motility analyses conducted, as described previously. In addition, sperm plasma membrane integrity (PMI) was determined in this experiment using flow cytometry, as described by Garner et al. (1994). Briefly, the sperm were stained with SYBR-14 (to detect cells from debris) and propidium iodide (to detect cells lacking intact plasma membranes (Garner et al., 1994)). A minimum of 10,000 sperm were analyzed from each sample.
P.H. Purdy et al. / Animal Reproduction Science 118 (2010) 231–235
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Table 1 The least square means of sperm motion measurements for ram sperm held for zero (T0) or 24 h at 5 ◦ C prior to cryopreservation (experiment 1; n = 3) and for ram sperm treated with 0 mg or 3.33 mg cholesterol-loaded cyclodextrin (CL)/200 × 106 and then held for 24 h at 5 ◦ C prior to cryopreservation (experiment 2; n = 6). Experiment
Treatment
MOT
PMOT
VAP
ALH
BCF
LIN
PMI
1
T0 T24 SEM
44 46 2
29 23 2
110 109 3
6.9 7.4 0.2
33.7 28.0 1.2
51 45 2
– –
2
T24 T24 + CLC SEM
36 38 2
18 19 2
110 112 5
7.3 7.5 0.4
29.4 39.8 2.1
47 47 2
41 49 5
MOT = total motility (%); PMOT = progressive motility (%); VAP = average path velocity (m/s); ALH = lateral head amplitude; BCF = beat cross frequency; LIN = linearity (ratio of VSL:VCL); PMI = plasma membrane intact.
2.4. Fertility trials Immediately prior to insemination, fresh semen samples from the same rams were collected, and the motility and concentration determined, as described previously. This fresh semen served as a control treatment. In experiment 1, the fresh semen was diluted in Dulbecco’s PBS (171 mM NaCl, 3 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 ) to 200 × 106 sperm/mL and maintained at 37 ◦ C until inseminated (less than 2 h after collection). In experiment 2, the fresh semen was diluted in Tris-buffered saline (Purdy and Graham, 2004), as described previously. 2.5. Ewe group assignments and synchronization of estrus Western white-faced ewes (n = 97), ages 1–8 years, and body weights ranging from 90 kg to 100 kg, were used in experiment 1 and 101 ewes from the same flock were used in experiment 2. The stage of reproductive cycles of the ewes were synchronized using controlled internal drug releasing devices (CIDR; Pfizer, Australia) containing progesterone (0.3 g/CIDR). Twelve days after insertion, the CIDRs were removed and the ewes injected i.m. with 500 IU of PMSG (Pfizer, Australia) in experiment 1 and 350 IU PMSG in experiment 2. Ewes in each experiment were blocked by age and then randomly assigned to be inseminated with Fresh (F), Time 0 (T0), or Time 24 (T24) treated sperm in experiment 1, and F, T24 and CLC semen treatments in experiment 2. The ewes were inseminated laparoscopically 56 h after PMSG treatment (Donovan et al., 2004) using a single 0.5 mL semen straw per insemination, with half of the volume of each straw placed into each uterine horn. Fertility and prolificacy data were determined by the number of ewes producing lambs and the number of lambs born to each ewe, respectively. 2.6. Statistics Percentage data were arcsine transformed. Treatment differences, in post-thaw sperm motility characteristics, were determined using ANOVA (SAS Inst. Inc., Cary, NC, USA). Treatment differences in fertility were determined using Wald’s chi square. Prolificacy data was analyzed using ANOVA. Initial fertility/prolificacy models included the effects of semen treatment (F, T0, T24, CLC), ram, ewe age and all two-way interactions. However, no interactions
were significant; therefore, these analyses were removed from the model. Similarly, treatment differences due to individual rams were not significant and were also removed from the model.
3. Results Sperm motion parameters were similar for sperm cryopreserved immediately after collection and for sperm that had been held at 5 ◦ C prior to cryopreservation (Table 1; experiment 1). Furthermore, sperm motion parameters and cell viability were similar for control and CLC-treated ram sperm (Table 1; experiment 2). The overall motility (45%) and progressive motility (25.7%) observed in year 1 were greater than that measured in the second year (37 and 18.5%, respectively; P < 0.05). Fertility rates were less in experiment 1 (17%) than in experiment 2 (43%; P < 0.05) although prolificacy rates were similar (P > 0.05). However, the prolificacy rate was greater for sperm stored for 24 h at 5 ◦ C prior to cryopreservation, than for other treatments in experiment 1 (P < 0.05; Table 2). Similarly, in experiment 2, all sperm treatments (fresh sperm, sperm with or without CLC treatment, stored for 24 h at 5 ◦ C prior to cryopreservation) resulted in similar fertility and prolificacy rates (P > 0.05; Table 2). The age of the ewe affected fertility rate, with 3-year-old ewes exhibiting the greatest fertility and 6-year-old ewes exhibiting the least fertility (P < 0.05). However, prolificacy rates were similar for ewes, regardless of age (P > 0.05).
Table 2 The fertility rate (%) and prolificacy (lambs born per ewe lambing) of ram sperm that was inseminated fresh or after cryopreservation. Cryopreserved sperm were frozen immediately after reaching 5 ◦ C (T0), after incubating at 5 ◦ C for 24 h (T24), or after initial treatment with CLC followed by cooling to 5 ◦ C and incubation for 24 h prior to freezing (CLC). Experiment
Treatment
Fertility % (#ewes)
Prolificacy
1 1 1 1 2 2 2 2
Fresh T0 T24 SEM Fresh T24 CLC SEM
25.0 (27) 20.1 (35) 16.0 (35) 4.2 37.4 (33) 45.3 (29) 41.0 (36) 5.0
1.14a 1.27a 1.72b 0.08 1.45 1.20 1.48 0.07
Column means with different letters are different within an experiment at P < 0.05.
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4. Discussion Incubating sperm for short time periods, prior to cryopreservation, is beneficial for boar sperm (Kikuchi et al., 1998; Guthrie and Welch, 2005) and can be used for bull (Foote and Kaproth, 2002), stallion (Crockett et al., 2001; Backman et al., 2004) and ram (Jones and Martin, 1964; Lightfoot and Salamon, 1969; Fiser and Batra, 1984) sperm as well, although incubating sperm too long prior to cryopreservation may be detrimental. The main purpose for freezing sperm 24 h after collection, in most species including horses and sheep, would be that it allows semen to be collected on site, where the ram is located, but permit the semen to be shipped to a location with expertise in cryopreserving the sperm. In the current study, (experiment 1) holding ram sperm at 5 ◦ C for 24 h did not affect either the post thaw motility of the sperm nor their fertilizing potential, compared to sperm that were cryopreserved immediately after collection. However, current cryopreservation techniques for ram sperm necessitate surgical insemination; a technique which has limitations due to the time, expertise and costs associated with it. Treating ram sperm with CLC prior to cryopreservation improves sperm cryosurvival rates (Mocé et al., 2009) and the surviving sperm exhibit increased longevity and capacity to bind to the zona pellucida (Mocé et al., 2009). Therefore, in experiment 2, impacts of CLC-treatment on sperm incubated 24 h prior to cryopreservation as well as the fertilizing potential of these sperm were investigated. Treating the sperm with CLC tended to improve post-thaw sperm motility and plasma membrane integrity, which is similar to previous studies (Morrier et al., 2004; Mocé et al., 2009). The fertility achieved in experiment 1 was only about half that achieved in experiment 2; which is similar to fertility rates reported for cryopreserved ram sperm (Langford et al., 1979; Hill et al., 1998; Gillan et al., 1999; Donovan et al., 2004). The reason for the fertility differences between the two experiments is not known. They are most likely due to different estrous synchronization regimens (PMSG dose in experiment 1 was 500 IU and was adjusted to 350 IU in experiment 2 because of the lesser fertility achieved in the first experiment), but could also be due to different inseminators performing the inseminations in the different experiments, or to ram differences, as the rams used in each experiment were different and were not selected for previous cryosurvival or fertility rate. Maintaining sperm at lesser temperatures prior to cryopreservation decreases prolificacy in pigs (Guthrie and Welch, 2005), but results are mixed in sheep (Langford et al., 1979; Donovan et al., 2004). In the current experiments, holding ram sperm for 24 h prior to cryopreservation did not affect either the fertility rate or prolificacy, compared to sperm frozen immediately or to fresh sperm. The greatest fertility rates were achieved by 3-year-old ewes in this study, which agrees with Dickerson and Glimp (1975) who demonstrated that the peak fertility for Rambouillet ewes, the major genetic influence of the ewes inseminated in this study, is 3–4 years of age. The fertility rates achieved indicate that ram sperm can be cryopreserved 24 h after collection without affecting
sperm quality or fertilizing capacity. This means that semen can be collected and sent to a facility specializing in sperm cryopreservation, which should produce the best quality product for use in the industry or for preserving germplasm at a gene bank. 5. Implications The sheep industry has not effectively used artificial insemination due to the difficulty in inseminating cryopreserved ram sperm. However, being able to collect, hold and freeze semen increases the opportunity for this industry to utilize selected genetics for storage at resource centers as well as in commercial artificial insemination programs. Acknowledgements The technical assistance with the artificial insemination and animal handling by, J.D. Sexton, Doug Predmore, Dave Moore, Laura McCormick, Carrie Welsh, Scott Spiller and Glen Erickson is gratefully acknowledged. Eva Mocé was supported by a post-doctoral scholarship from Secretaría de Estado de Educación y Universidades y Fondo Social Europeo (Ref. EX 2003-1028). References Backman, T., Bruemmer, J.E., Graham, J.K., Squires, E.L., 2004. Pregnancy rates of mares inseminated with semen cooled for 18 h and then frozen. J. Anim. Sci. 82, 690–694. Blackburn, H.D., 2004. Development of national animal genetic resource programs. Reprod. Fertil. Dev. 16, 27–32. Crockett, E.C., Graham, J.K., Bruemmer, J.E., Squires, E.L., 2001. Effect of cooling equine spermatozoa before freezing on post-thaw motility: preliminary results. Theriogenology 55, 793–803. Dickerson, G.E., Glimp, H.A., 1975. Breed and age effects on lamb production of ewes. J. Anim. Sci. 40, 397–408. Donovan, A., Hanrahan, J.P., Kummen, E., Duffy, P., Boland, M.P., 2004. Fertility in the ewe following cervical insemination with fresh or frozen-thawed semen at a natural or synchronized oestrus. Anim. Reprod. Sci. 84, 359–368. Evans, G., Maxwell, W.M.C., 1987. Salamon’s Artificial Insemination of Sheep and Goats. Butterworths, Wellington, New Zealand. Fiser, P.S., Batra, T.R., 1984. Effect of equilibration time at 5 ◦ C and photoperiod on survival of ram spermatozoa frozen in straws. Can. J. Anim. Sci. 64, 777–780. Foote, R.H., Kaproth, M.T., 2002. Large batch freeing of bull semen: effect of time of freezing and fructose on fertility. J. Dairy Sci. 85, 453–456. Garner, D.L., Johnson, L.A., Yue, S.T., Roth, B.L., Haugland, R.P., 1994. Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. J. Androl. 15, 620–629. Gillan, L., Skovgold, K., Watson, P.F., Evans, G., Maxwell, W.M.C., 1999. Fate and functional integrity of fresh and frozen-thawed ram spermatozoa following intrauterine insemination. Reprod. Fertil. Dev. 11, 309–315. Guthrie, H.D., Welch, G.R., 2005. Impact of storage prior to cryopreservation on plasma membrane function and fertility of boar sperm. Theriogenology 63, 396–410. Hill, J.R., Thompson, J.A., Perkins, N.R., 1998. Factors affecting pregnancy rates following laparoscopic insemination of 28,447 Merino ewes under commercial conditions: a survey. Theriogenology 49, 697–709. Jones, R.C., Martin, I.C.A., 1964. Deep-freezing ram spermatozoa: the effects of milk, yolk-citrate and synthetic diluents containing sugar. J. Reprod. Fertil. 10, 413–423. Kikuchi, K., Nagai, T., Kashiwazaki, N., Ikeda, H., Noguchi, J., Shimada, A., Soloy, E., Kaneko, H., 1998. Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididimides stored at 4 ◦ C. Theriogenology 50, 615–623. Langford, G.A., Marcus, G.J., Hackett, A.J., Ainsworth, L., Wolynetz, M.S., Peters, H.F., 1979. A comparison of fresh and frozen semen in the insemination of confined sheep. Can. J. Anim. Sci. 59, 685–691.
P.H. Purdy et al. / Animal Reproduction Science 118 (2010) 231–235 Lightfoot, R.J., Salamon, S., 1969. Freezing ram spermatozoa by the pellet method II. The effects of method of dilution, dilution rate, glycerol concentration, and duration of storage at 5 ◦ C prior to freezing on survival of spermatozoa. Aust. J. Biol. Sci. 22, 1547–1560. Mocé, E., Graham, J.K., 2006. Cholesterol-loaded cyclodextrins added to fresh bull ejaculates improve sperm cryosurvival. J. Anim. Sci. 84, 826–833. Mocé, E., Purdy, P.H., Graham, J.K., 2010. Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival. Anim. Reprod. Sci. 118, 236–247. Morrier, A., Bailey, J.L., 2003. Cholesterol loaded methyl--cyclodextrin protects ram sperm during cryopreservation and cold-shock. In: Proceedings of the 5th International Conference Boar Semen Preservation, Doorwerth, The Netherlands, p. P16B.
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Morrier, A., Theriault, M., Castonguay, F., Bailey, J.L., 2004. Effect of cholesterol loaded methyl--cyclodextrin on ram sperm during cryopreservation, cold–shock and artificial insemination. In: Proc. Soc. Study of Reproduction Mtg., Vancouver, BC, Canada. Biology of Reproduction, Madison, WI, p. 239. Purdy, P.H., 2006. The post-thaw quality of ram sperm held for 0 to 48 hr at 5 ◦ C prior to cryopreservation. Anim. Reprod. Sci. 93, 114–123. Purdy, P.H., Graham, J.K., 2004. Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm. Cryobiology 48, 36–45. Sanchez-Partida, L.G., Setchell, B.P., Maxwell, W.M.C., 1998. Effect of compatible solutes and diluent composition on the post-thaw motility of ram sperm. Reprod. Fertil. Dev. 10, 347–357. SAS Institute Inc., 1985. SAS User’s Guide: Statistics. SAS Institute Inc., Cary, NC.
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
The fertility of ram sperm held for 24 h at 5 ◦ C prior to cryopreservation夽 Phillip H. Purdy a,∗ , Eva Mocé b , Robert Stobart c , William J. Murdoch c , Gary E. Moss c , Brent Larson c , Shawn Ramsey d , James K. Graham e , Harvey D. Blackburn a a
USDA-ARS-NCGRP, National Animal Germplasm Program, 1111 S. Mason St., Fort Collins, CO 80521-4500, USA Instituto Valenciano de Investigaciones Agrarias-Centro de Tecnología Animal (IVIA-CITA), Polígono La Esperanza, n◦ 100, 12400-Segorbe (Castellón), Spain c Department of Animal Science, University of Wyoming, Laramie, WY 82071-3684, USA d Department of Animal Science, Texas A&M University, College Station, TX 77843-2471, USA e Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA b
a r t i c l e
i n f o
Article history: Received 5 August 2008 Received in revised form 18 May 2009 Accepted 18 June 2009 Available online 26 June 2009 Keywords: Fertility Holding time Ram Sperm Artificial insemination
a b s t r a c t Diluted ram sperm can be held for 24 h at 5 ◦ C prior to cryopreservation without impacting cryosurvival rates, however, the effects this storage has on subsequent fertility are unknown. These studies were conducted to evaluate the fertility of semen held for 24 h (to mimic shipping semen to a cryopreservation center), prior to freezing. Semen from Suffolk rams (n = 3 in experiment 1 and n = 6 in experiment 2) with initial motility of greater than 70%, were diluted to 200 × 106 sperm/mL, in one step, with a Tris–egg yolk–glycerol diluent. In experiment 1, diluted samples were cooled to 5 ◦ C over 2 h, and then divided. Sperm in one fraction were loaded into 0.5 mL straws, frozen (T0) and stored in liquid nitrogen until thawing. Sperm in the second fraction were held at 5 ◦ C for 24 h (T24) before being frozen. In experiment 2 ejaculates were collected and divided into two fractions. Sperm in one fraction were treated with cholesterol-loaded cyclodextrin (CLC) and sperm in the other served as control. Both fractions were diluted, cooled, and cryopreserved as described in experiment 1. Stage of the estrous cycle was synchronized in ewes (n = 196) using controlled internal drug releasing devices (CIDR) for 12 d and at CIDR removal each ewe was administered PMSG (500 IU in experiment 1 and 350 IU in experiment 2) immediately before insemination. Ewes were stratified by age and randomly assigned to one of the semen treatments; experiment 1: Fresh (F), T0, or T24; experiment 2: F, T24, or CLC, and inseminated laparoscopically 56 h after CIDR removal. Differences in fertility were detected between experiments, but not for treatments within experiments. Differences in fertility were also observed due to ewe age, with the 3-year-old ewes having the greatest fertility (50.7%) and 6-year-old ewes having the least fertility (9.6%; P < 0.05). Differences in the prolificacy rates due to semen treatment were also observed but differences due to ewe age were not detected. Therefore, sperm can be held at 5 ◦ C for 24 h prior to cryopreservation without altering sperm fertility. Published by Elsevier B.V.
夽 Mention of a trade name or proprietary product does not constitute a guaranty or warranty by the USDA and does not imply approval to the exclusion of other products that may be suitable. USDA, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer. All agency services are available without discrimination. ∗ Corresponding author. Tel.: +1 970 495 3258; fax: +1 970 221 1427. E-mail address: [email protected] (P.H. Purdy). 0378-4320/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.anireprosci.2009.06.014
232
P.H. Purdy et al. / Animal Reproduction Science 118 (2010) 231–235
1. Introduction The sheep industry has not been able to utilize many of the assisted reproductive technologies (ART) in general and AI in particular, as other livestock industries, due to inefficiencies in collecting, freezing and inseminating frozen ram semen. Furthermore, few commercial studs routinely collect and freeze ram semen, and there is still a need to optimize cryopreservation and breeding protocols for ram semen. The USDA National Animal Germplasm Program (NAGP) is charged with developing a national repository of animal germplasm and tissue to provide genetic security and facilitate genetic understanding of livestock species (Blackburn, 2004). This task is complicated by the lack of ART infrastructure for the U.S. sheep industry and by the general inability to efficiently cryopreserve ram semen. One way to quickly improve the quality of cryopreserved ram semen would be to collect the semen where the ram resides and ship the semen to a central processing center with the expertise in cryopreserving the semen, in much the same way cooled stallion semen is currently used (Backman et al., 2004). This system would permit semen from a ram to be cryopreserved without the owner having to transport the ram, house the ram in a collection facility, or have to have the equipment for or expertise in cryopreservation. Diluted ram semen can be held at 5 ◦ C for up to 48 h prior to cryopreservation without detrimental effects on sperm physiology (Purdy, 2006). In addition, new cryopreservation technologies, such as treating ram sperm with cholesterol-loaded cyclodextrins (CLC) show promise in increasing sperm cryosurvival rates (Morrier and Bailey, 2003; Morrier et al., 2004; Mocé et al., 2009). These studies were conducted to determine the fertilizing potential of ram sperm cryopreserved 24 h after collection in conjunction with CLC treatment. 2. Materials and methods
glycerol, 15% egg yolk, 1 mg/mL streptomycin sulfate and 0.06 mg/mL benzylpenicillin; Sanchez-Partida et al., 1998) at 37 ◦ C.
2.2. Experiment 1 Diluted semen samples were cooled to 5 ◦ C over 2 h and split into two aliquots. One aliquot from each ejaculate was immediately (designated T0) loaded into 0.5 mL CBS straws (IMV Corp., Minneapolis, MN, USA) and cryopreserved as described by (Sanchez-Partida et al., 1998). The second aliquot was held for 24 h (T24) at 5 ◦ C, before being packaged into straws and cryopreserved. Sperm were frozen in liquid nitrogen vapor using a Mini Digitcool UJ400 programmable freezer (IMV Corp., Minneapolis, MN, USA). The sperm were cooled from 5 ◦ C to −5 ◦ C at 4 ◦ C/min; from −5 ◦ C to −110 ◦ C at 25 ◦ C/min; and from −110 ◦ C to −140 ◦ C at 35 ◦ C/min. After reaching −140 ◦ C the straws were plunged into liquid nitrogen for storage. Frozen straws were thawed in 37 ◦ C water for 30 s and were analyzed for the percentage of motile sperm, using a computer assisted semen analysis system (IVOS Version 10.9, Hamilton Thorne Bioscience, Beverly, MA, USA) with a 10× phase contrast objective and the following settings: 50 frames acquired, frame rate of 60 Hz, minimum contrast of 60, minimum cell size of 5 pixels, VAP (path velocity) cutoff of 20 m, progressive minimum VAP cutoff of 50 m/s, VSL (progressive velocity) cutoff of 30 m/s, static head size of 0.24–3.66, and magnification of 1.89. For each sample, an aliquot was diluted 1:3 (v/v; sample to diluent) with Tris-buffered saline (Purdy and Graham, 2004) and 5 L was placed onto a Standard Count Analysis Chamber (Spectrum Technologies, Healdsburg, CA, USA). A minimum of 500 sperm and five fields were analyzed to estimate the percentage of motile sperm from each treatment.
2.1. Semen collection and handling Adult rams and ewes were housed at the University of Wyoming (Laramie, WY, USA). Animals were fed a diet providing 100% of their nutritional needs, and provided water ad libitum. All protocols for working with the sheep were approved by the University of Wyoming Institutional Animal Care and Use Committee. Single ejaculates from sexually mature Suffolk rams were collected using electro-ejaculation (Evans and Maxwell, 1987) during the month of October and ewes were inseminated the following month. After collection, the percentage of motile sperm in each ejaculate was determined using bright field microscopy (Nasco Advanced Laboratory Microscope, Nasco Farm and Ranch Supplies, Fort Atkinson, WI, USA) at 400× magnification and 23 ◦ C to ensure that each sample had a minimum of 70% motile sperm. The volume and sperm concentration (Spermacue, IMV Corp., Minneapolis, MN, USA) were determined and ejaculates were diluted in one step (in 50 mL centrifuge tubes) to 200 × 106 sperm/mL with Tris–egg yolk–glycerol medium (300 mM Tris, 28 mM glucose, 95 mM citric acid, 5% (v/v)
2.3. Experiment 2 In experiment 2, conducted the following year, semen samples from six rams were collected and split prior to diluting the sperm. One aliquot was diluted with the Tris–egg yolk–glycerol medium to 200 × 106 sperm/mL. The second aliquot was treated with cholesterol-loaded methyl- cyclodextrin (2 mg/120 × 106 sperm or CLC (3.33 mg/200 × 106 sperm) for 15 min at 23 ◦ C and then diluted in the same manner as the first aliquot (Mocé and Graham, 2006). Both were then cooled to 5 ◦ C over 2 h and held at 5 ◦ C for 24 h before being packaged into straws and cryopreserved as described above. Straws were thawed and motility analyses conducted, as described previously. In addition, sperm plasma membrane integrity (PMI) was determined in this experiment using flow cytometry, as described by Garner et al. (1994). Briefly, the sperm were stained with SYBR-14 (to detect cells from debris) and propidium iodide (to detect cells lacking intact plasma membranes (Garner et al., 1994)). A minimum of 10,000 sperm were analyzed from each sample.
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Table 1 The least square means of sperm motion measurements for ram sperm held for zero (T0) or 24 h at 5 ◦ C prior to cryopreservation (experiment 1; n = 3) and for ram sperm treated with 0 mg or 3.33 mg cholesterol-loaded cyclodextrin (CL)/200 × 106 and then held for 24 h at 5 ◦ C prior to cryopreservation (experiment 2; n = 6). Experiment
Treatment
MOT
PMOT
VAP
ALH
BCF
LIN
PMI
1
T0 T24 SEM
44 46 2
29 23 2
110 109 3
6.9 7.4 0.2
33.7 28.0 1.2
51 45 2
– –
2
T24 T24 + CLC SEM
36 38 2
18 19 2
110 112 5
7.3 7.5 0.4
29.4 39.8 2.1
47 47 2
41 49 5
MOT = total motility (%); PMOT = progressive motility (%); VAP = average path velocity (m/s); ALH = lateral head amplitude; BCF = beat cross frequency; LIN = linearity (ratio of VSL:VCL); PMI = plasma membrane intact.
2.4. Fertility trials Immediately prior to insemination, fresh semen samples from the same rams were collected, and the motility and concentration determined, as described previously. This fresh semen served as a control treatment. In experiment 1, the fresh semen was diluted in Dulbecco’s PBS (171 mM NaCl, 3 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 ) to 200 × 106 sperm/mL and maintained at 37 ◦ C until inseminated (less than 2 h after collection). In experiment 2, the fresh semen was diluted in Tris-buffered saline (Purdy and Graham, 2004), as described previously. 2.5. Ewe group assignments and synchronization of estrus Western white-faced ewes (n = 97), ages 1–8 years, and body weights ranging from 90 kg to 100 kg, were used in experiment 1 and 101 ewes from the same flock were used in experiment 2. The stage of reproductive cycles of the ewes were synchronized using controlled internal drug releasing devices (CIDR; Pfizer, Australia) containing progesterone (0.3 g/CIDR). Twelve days after insertion, the CIDRs were removed and the ewes injected i.m. with 500 IU of PMSG (Pfizer, Australia) in experiment 1 and 350 IU PMSG in experiment 2. Ewes in each experiment were blocked by age and then randomly assigned to be inseminated with Fresh (F), Time 0 (T0), or Time 24 (T24) treated sperm in experiment 1, and F, T24 and CLC semen treatments in experiment 2. The ewes were inseminated laparoscopically 56 h after PMSG treatment (Donovan et al., 2004) using a single 0.5 mL semen straw per insemination, with half of the volume of each straw placed into each uterine horn. Fertility and prolificacy data were determined by the number of ewes producing lambs and the number of lambs born to each ewe, respectively. 2.6. Statistics Percentage data were arcsine transformed. Treatment differences, in post-thaw sperm motility characteristics, were determined using ANOVA (SAS Inst. Inc., Cary, NC, USA). Treatment differences in fertility were determined using Wald’s chi square. Prolificacy data was analyzed using ANOVA. Initial fertility/prolificacy models included the effects of semen treatment (F, T0, T24, CLC), ram, ewe age and all two-way interactions. However, no interactions
were significant; therefore, these analyses were removed from the model. Similarly, treatment differences due to individual rams were not significant and were also removed from the model.
3. Results Sperm motion parameters were similar for sperm cryopreserved immediately after collection and for sperm that had been held at 5 ◦ C prior to cryopreservation (Table 1; experiment 1). Furthermore, sperm motion parameters and cell viability were similar for control and CLC-treated ram sperm (Table 1; experiment 2). The overall motility (45%) and progressive motility (25.7%) observed in year 1 were greater than that measured in the second year (37 and 18.5%, respectively; P < 0.05). Fertility rates were less in experiment 1 (17%) than in experiment 2 (43%; P < 0.05) although prolificacy rates were similar (P > 0.05). However, the prolificacy rate was greater for sperm stored for 24 h at 5 ◦ C prior to cryopreservation, than for other treatments in experiment 1 (P < 0.05; Table 2). Similarly, in experiment 2, all sperm treatments (fresh sperm, sperm with or without CLC treatment, stored for 24 h at 5 ◦ C prior to cryopreservation) resulted in similar fertility and prolificacy rates (P > 0.05; Table 2). The age of the ewe affected fertility rate, with 3-year-old ewes exhibiting the greatest fertility and 6-year-old ewes exhibiting the least fertility (P < 0.05). However, prolificacy rates were similar for ewes, regardless of age (P > 0.05).
Table 2 The fertility rate (%) and prolificacy (lambs born per ewe lambing) of ram sperm that was inseminated fresh or after cryopreservation. Cryopreserved sperm were frozen immediately after reaching 5 ◦ C (T0), after incubating at 5 ◦ C for 24 h (T24), or after initial treatment with CLC followed by cooling to 5 ◦ C and incubation for 24 h prior to freezing (CLC). Experiment
Treatment
Fertility % (#ewes)
Prolificacy
1 1 1 1 2 2 2 2
Fresh T0 T24 SEM Fresh T24 CLC SEM
25.0 (27) 20.1 (35) 16.0 (35) 4.2 37.4 (33) 45.3 (29) 41.0 (36) 5.0
1.14a 1.27a 1.72b 0.08 1.45 1.20 1.48 0.07
Column means with different letters are different within an experiment at P < 0.05.
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4. Discussion Incubating sperm for short time periods, prior to cryopreservation, is beneficial for boar sperm (Kikuchi et al., 1998; Guthrie and Welch, 2005) and can be used for bull (Foote and Kaproth, 2002), stallion (Crockett et al., 2001; Backman et al., 2004) and ram (Jones and Martin, 1964; Lightfoot and Salamon, 1969; Fiser and Batra, 1984) sperm as well, although incubating sperm too long prior to cryopreservation may be detrimental. The main purpose for freezing sperm 24 h after collection, in most species including horses and sheep, would be that it allows semen to be collected on site, where the ram is located, but permit the semen to be shipped to a location with expertise in cryopreserving the sperm. In the current study, (experiment 1) holding ram sperm at 5 ◦ C for 24 h did not affect either the post thaw motility of the sperm nor their fertilizing potential, compared to sperm that were cryopreserved immediately after collection. However, current cryopreservation techniques for ram sperm necessitate surgical insemination; a technique which has limitations due to the time, expertise and costs associated with it. Treating ram sperm with CLC prior to cryopreservation improves sperm cryosurvival rates (Mocé et al., 2009) and the surviving sperm exhibit increased longevity and capacity to bind to the zona pellucida (Mocé et al., 2009). Therefore, in experiment 2, impacts of CLC-treatment on sperm incubated 24 h prior to cryopreservation as well as the fertilizing potential of these sperm were investigated. Treating the sperm with CLC tended to improve post-thaw sperm motility and plasma membrane integrity, which is similar to previous studies (Morrier et al., 2004; Mocé et al., 2009). The fertility achieved in experiment 1 was only about half that achieved in experiment 2; which is similar to fertility rates reported for cryopreserved ram sperm (Langford et al., 1979; Hill et al., 1998; Gillan et al., 1999; Donovan et al., 2004). The reason for the fertility differences between the two experiments is not known. They are most likely due to different estrous synchronization regimens (PMSG dose in experiment 1 was 500 IU and was adjusted to 350 IU in experiment 2 because of the lesser fertility achieved in the first experiment), but could also be due to different inseminators performing the inseminations in the different experiments, or to ram differences, as the rams used in each experiment were different and were not selected for previous cryosurvival or fertility rate. Maintaining sperm at lesser temperatures prior to cryopreservation decreases prolificacy in pigs (Guthrie and Welch, 2005), but results are mixed in sheep (Langford et al., 1979; Donovan et al., 2004). In the current experiments, holding ram sperm for 24 h prior to cryopreservation did not affect either the fertility rate or prolificacy, compared to sperm frozen immediately or to fresh sperm. The greatest fertility rates were achieved by 3-year-old ewes in this study, which agrees with Dickerson and Glimp (1975) who demonstrated that the peak fertility for Rambouillet ewes, the major genetic influence of the ewes inseminated in this study, is 3–4 years of age. The fertility rates achieved indicate that ram sperm can be cryopreserved 24 h after collection without affecting
sperm quality or fertilizing capacity. This means that semen can be collected and sent to a facility specializing in sperm cryopreservation, which should produce the best quality product for use in the industry or for preserving germplasm at a gene bank. 5. Implications The sheep industry has not effectively used artificial insemination due to the difficulty in inseminating cryopreserved ram sperm. However, being able to collect, hold and freeze semen increases the opportunity for this industry to utilize selected genetics for storage at resource centers as well as in commercial artificial insemination programs. Acknowledgements The technical assistance with the artificial insemination and animal handling by, J.D. Sexton, Doug Predmore, Dave Moore, Laura McCormick, Carrie Welsh, Scott Spiller and Glen Erickson is gratefully acknowledged. Eva Mocé was supported by a post-doctoral scholarship from Secretaría de Estado de Educación y Universidades y Fondo Social Europeo (Ref. EX 2003-1028). References Backman, T., Bruemmer, J.E., Graham, J.K., Squires, E.L., 2004. Pregnancy rates of mares inseminated with semen cooled for 18 h and then frozen. J. Anim. Sci. 82, 690–694. Blackburn, H.D., 2004. Development of national animal genetic resource programs. Reprod. Fertil. Dev. 16, 27–32. Crockett, E.C., Graham, J.K., Bruemmer, J.E., Squires, E.L., 2001. Effect of cooling equine spermatozoa before freezing on post-thaw motility: preliminary results. Theriogenology 55, 793–803. Dickerson, G.E., Glimp, H.A., 1975. Breed and age effects on lamb production of ewes. J. Anim. Sci. 40, 397–408. Donovan, A., Hanrahan, J.P., Kummen, E., Duffy, P., Boland, M.P., 2004. Fertility in the ewe following cervical insemination with fresh or frozen-thawed semen at a natural or synchronized oestrus. Anim. Reprod. Sci. 84, 359–368. Evans, G., Maxwell, W.M.C., 1987. Salamon’s Artificial Insemination of Sheep and Goats. Butterworths, Wellington, New Zealand. Fiser, P.S., Batra, T.R., 1984. Effect of equilibration time at 5 ◦ C and photoperiod on survival of ram spermatozoa frozen in straws. Can. J. Anim. Sci. 64, 777–780. Foote, R.H., Kaproth, M.T., 2002. Large batch freeing of bull semen: effect of time of freezing and fructose on fertility. J. Dairy Sci. 85, 453–456. Garner, D.L., Johnson, L.A., Yue, S.T., Roth, B.L., Haugland, R.P., 1994. Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. J. Androl. 15, 620–629. Gillan, L., Skovgold, K., Watson, P.F., Evans, G., Maxwell, W.M.C., 1999. Fate and functional integrity of fresh and frozen-thawed ram spermatozoa following intrauterine insemination. Reprod. Fertil. Dev. 11, 309–315. Guthrie, H.D., Welch, G.R., 2005. Impact of storage prior to cryopreservation on plasma membrane function and fertility of boar sperm. Theriogenology 63, 396–410. Hill, J.R., Thompson, J.A., Perkins, N.R., 1998. Factors affecting pregnancy rates following laparoscopic insemination of 28,447 Merino ewes under commercial conditions: a survey. Theriogenology 49, 697–709. Jones, R.C., Martin, I.C.A., 1964. Deep-freezing ram spermatozoa: the effects of milk, yolk-citrate and synthetic diluents containing sugar. J. Reprod. Fertil. 10, 413–423. Kikuchi, K., Nagai, T., Kashiwazaki, N., Ikeda, H., Noguchi, J., Shimada, A., Soloy, E., Kaneko, H., 1998. Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididimides stored at 4 ◦ C. Theriogenology 50, 615–623. Langford, G.A., Marcus, G.J., Hackett, A.J., Ainsworth, L., Wolynetz, M.S., Peters, H.F., 1979. A comparison of fresh and frozen semen in the insemination of confined sheep. Can. J. Anim. Sci. 59, 685–691.
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