The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa

Theriogenology 67 (2007) 217–227 www.theriojournal.com

The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa S.P. de Graaf a,*, G. Evans a, L. Gillan b, M.M.P. Guerra c, W.M.C. Maxwell a, J.K. O’Brien a a

Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, The University of Sydney, Sydney, NSW 2006, Australia b Sydney IVF, Sydney, NSW 2000, Australia c Federal University of Pernambuco, Department of Veterinary Medicine, Recife, PE, Brazil Received 20 June 2006; accepted 19 July 2006

Abstract In an effort to improve the number of functional spermatozoa following sex-sorting and cryopreservation, the effects on in vitro sperm characteristics of the additives: (i) catalase (pre-sorting); (ii) cholesterol-loaded cyclodextrins (CLCs; pre-sorting); and (iii) seminal plasma (post-thawing) were investigated. For all experiments, spermatozoa (three males, n = 3 ejaculates/male) were processed using a high speed flow cytometer before cryopreservation, thawing and incubation for 6 h. Catalase had no effect (P > 0.05) on post-thaw motility characteristics (as measured by CASA) of sex-sorted ram spermatozoa, but pre-sort addition of CLCs reduced (P < 0.05) sperm quality after post-thaw incubation for 0 h (motility), 3 h (motility, average path velocity, viability and acrosome integrity) and 6 h (motility, average path and curvilinear velocity, straightness, linearity, viability and acrosome integrity). Seminal plasma had a differential effect (P < 0.001) on sex-sorted and non-sorted spermatozoa. Post-thaw supplementation of increasing levels of seminal plasma caused all motility characteristics of sex-sorted, frozen-thawed spermatozoa to decline (P < 0.05); conversely, non-sorted, frozen-thawed spermatozoa exhibited improvements (P < 0.05) in motility, viability, acrosome integrity and mitochondrial respiration. In summary, incorporation of catalase, CLCs and seminal plasma into the sorting protocol failed to improve post-thaw sperm quality and, consequently efficiency of sex-sorting of ram spermatozoa. The paradoxical effect of seminal plasma supplementation on the in vitro characteristics of ram spermatozoa provides further evidence that sex-sorting by flow cytometry produces a selected population of cells with different functions compared with non-sorted spermatozoa. # 2006 Elsevier Inc. All rights reserved. Keywords: Seminal plasma; Antioxidants; Sex-sorting; Sex preselection; Cholesterol-loaded cyclodextrins

1. Introduction The artificial insemination of sex-sorted, cryopreserved spermatozoa to produce pre-sexed offspring has

* Corresponding author. Tel.: +61 2 9351 5832; fax: +61 2 9351 3957. E-mail address: [email protected] (S.P. de Graaf). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.07.008

been reported in a number of species [1]. Despite these successes, sexed spermatozoa exhibit a higher incidence of cell damage [2], DNA fragmentation [3], premature capacitation, accelerated degradation of motility, acrosome integrity and oviduct epithelial cell association [4], and decreased velocity characteristics (stallion [5]; bull [6]; ram [7]). These changes are a result of the numerous potential stressors inherent in the sperm sorting process, including nuclear staining,

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re-warming and incubation, mechanical forces during flow cytometric passage, high dilution, exposure to intense UV laser light, electric charge, and high gravity during deceleration into the sort collection tube and reconcentration for post-sort packaging and cryopreservation [2,8]. In an attempt to minimise the effects of these stressors, research efforts have focused on methods of preparing and handling spermatozoa before, during and after passage through the flow cytometer. Studies to determine the diluent requirements, centrifugation speeds and cryopreservation methods specific to sexed spermatozoa have been undertaken (bull [9]; ram [10,11]; boar [12]; stallion [13]; dolphin [14]) with the goal of increasing the number of motile, viable and functional cells available post-sorting and postthawing. Integration of the advances made in the processing of non-sorted spermatozoa by inclusion in existing diluents of additives, such as seminal plasma [15], antioxidants [16] and cholesterol-loaded cyclodextrins (CLCs) [17] have the potential to further the goal of improved sexed sperm quality. For non-sorted spermatozoa, reports on the beneficial effect of seminal plasma on sperm quality upon its reintroduction to a previously diluted sample exist for a number of species (reviewed by Maxwell and Johnson [15]). Osmotic effects aside, this is thought to be due in part to the reconcentration of proteins, natural antioxidants and other uncharacterised beneficial components (e.g. decapacitation factors [18]) present in seminal plasma [15]. Using standard speed sorters, improvement in the quality of sex-sorted ram, boar [10] and bull [12] spermatozoa supplemented with seminal plasma has also been demonstrated, albeit using superseded technology. Supplementation of collection media with 1% seminal plasma is standard practice in modern boar sorting protocols [19], but whether a beneficial effect of seminal plasma on sperm quality exists for spermatozoa of other species separated using the current generation of high speed sperm sorters [20] is yet to be determined. However, results from experiments with highly diluted non-sorted dolphin spermatozoa (simulated sex-sorting conditions) indicate no improvement in quality with the supplementation of homologous seminal plasma [14]. The addition of antioxidants or CLCs to steps in the sexsorting process have not been recorded, but have the potential to reduce membrane damage from lipid peroxidation during dilution [21] and phase transition during cryopreservation [17], respectively. Improvement in the number of functional sexed spermatozoa following sorting and cryopreservation could greatly increase the efficiency of the sex-sorting process. As a consequence, investigation of new

methods to facilitate this improvement has substantial importance, from both a commercial and scientific standpoint. With this goal in mind, the aim of the present study was to characterise the effect of antioxidants, CLCs or seminal plasma on the in vitro quality of sex-sorted, cryopreserved ram spermatozoa. 2. Materials and methods 2.1. Experimental design Procedures herein were approved by The University of Sydney’s Animal Ethics Committee. Three experiments were conducted to assess the in vitro quality of sex-sorted ram spermatozoa. Semen from the same three rams (n = 3 ejaculates/male) was used in each experiment. In Experiment 1, semen was diluted for sex-sorting in staining media with or without catalase, before standard freezing and thawing. Experiment 2 tested the introduction of CLCs (prepared using method described by Purdy and Graham [17], see Section 2.6) into the staining medium, and the effect on post-thaw sex-sorted sperm quality. In Experiment 3a, characteristics of sex-sorted and non-sorted spermatozoa diluted with commercial thawing media [Androhep (AH), Minitube Australia, Smythes Creek, Australia], Androhep plus 40% homologous seminal plasma, or an artificial seminal plasma (ASP) were compared. In Experiment 3b, the effect of seminal plasma on the characteristics of sex-sorted and non-sorted spermatozoa after thawing were further examined, with the addition of 0, 10, 20, and 40% seminal plasma to the Androhep extension medium. 2.2. Semen collection, freezing and thawing Ejaculates were collected by artificial vagina from Merino rams (n = 3) during November 2004 (Experiment 1), October 2005 (Experiment 2) and March and April 2005 (Experiment 3a and 3b). Portions of ejaculates destined for non-sorted treatments were immediately diluted 1:4 (semen:diluent, v/v) using a tris–citrate– glucose cryoprotective diluent containing 15% egg yolk and 5% glycerol (v/v) and frozen and thawed (37 8C, 2 min) using the pellet method [22]. 2.3. Evaluation of spermatozoa 2.3.1. Motility The percentage of motile spermatozoa was assessed objectively using computer assisted sperm analysis (CASA; HTM-IVOS v. 12; Hamilton–Thorne, Beverly,

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MA, USA). Semen samples (5.5 mL, 10 to 20  106 spermatozoa/mL) were placed on slides (Cell Vu, Millennium Sciences Corp., NY, USA; pre-warmed to 37 8C) and enclosed using a 22 mm  22 mm coverslip before immediate transfer to the CASA. Motility characteristics were determined by assessment of at least three randomly selected microscopic fields (>300 spermatozoa/sample) utilising factory CASA settings (ram) at an image sampling frequency of 60 Hz. 2.3.2. Viability and acrosome integrity Assessment of sperm viability was conducted using the membrane impermeable DNA supravital stain Ethidium homodimer 1 (Eth-D1; Molecular probes, Eugene, OR, USA), and acrosome integrity simultaneously determined with fluorescein-conjugated peanut agglutinin (FITC-PNA; Sigma–Aldrich, Sydney, Australia) (modified from Szasz et al. [23]). Briefly, 12 mL aliquots of sperm suspension were combined with 10 mL of Eth-D1 [4 mM working stock in AndroHep (Minitube Australia, Smythes Creek, Australia)] pre-warmed to 37 8C and incubated for 30 s before introduction of 4 mL of FITC-PNA [10 mg/mL working stock in phosphate buffered saline (PBS; Sigma–Aldrich)] and fixation with 4 mL of PBS containing 0.5% glutaraldehyde (Sigma–Aldrich). Samples were observed under phase contrast (400 magnification, 200 cells/sample) with an Olympus BHS fluorescence microscope comprising a 520– 550 nm band pass filter and a supplementary 515 nm exciter filter. Emissions were observed through a 565 nm dichroic mirror with an additional 610 nm barrier filter. Sperm cells which displayed bright green or patchy green fluorescence were considered acrosome non-intact or damaged, respectively, whereas cells that did not stain positive for FITC-PNA in this region were regarded as acrosome intact. Cells were considered non-viable, and hence plasma membrane damaged with the display of red fluorescence while spermatozoa regarded as viable were those which excluded the membrane impermeable Eth-D1 dye. Hence, the sperm cells assessed are distributed through four categories as viable acrosome intact, viable acrosome non-intact, non-viable acrosome intact, and nonviable acrosome non-intact. Acrosome integrity in Experiment 3a was determined using the method outlined by Roth et al. [24]. 2.3.3. Mitochondrial respiration Mitochondrial activity was determined using Rhodamine 123 (R123; Molecular Probes, Eugene, OR, USA [25]). Samples (10  106 spermatozoa/mL) were

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incubated for 20 min at 37 8C with 0.2 mg R123/mL of sperm suspension (modified from Gillan [26]). Aliuqots (10 mL) were removed and transferred onto a clean slide with a 22 mm  22 mm coverslip placed over the sample. Spermatozoa were viewed on an Olympus BHS microscope fitted with phase contrast and fluorescence optics (400 magnification). The R123 emission was observed through a 565 nm dichroic mirror with an additional 610 nm barrier filter after the passage of the excitation beam through a 520– 550 nm band pass filter with supplementary 515 nm exciter filter. Spermatozoa with normal, respiring mitochondria displayed red fluorescence in the midpiece due to the accumulation of R123. Spermatozoa not accumulating the fluorochrome and therefore not fluorescing were designated as having non-respiring mitochondria. 2.3.4. Migration test The ability of spermatozoa to migrate through artificial cervical mucus [10 mg/mL sodium hyaluronate (Bioniche Australasia, Armidale, NSW, Australia) diluted with 33% ASP [27] containing 1% w/v BSA (Sigma–Aldrich)] was assessed by means of a sperm migration test modified for sex-sorted ram spermatozoa [28]. Aliquots (50 mL) containing 0.5  106 spermatozoa from each treatment were incubated (37 8C; 1 h) in capsules (BEEM; ProSciTech, Thuringawa, QLD, Australia) containing a vertically placed rectangular capillary tube (0.3 mm  0.3 mm  100 mm; Microslides; VitroCom, Mountain Lakes, NJ, USA) filled with artificial cervical mucus. The number of spermatozoa from each treatment migrating 5 mm into artificial mucus and the distance travelled by the vanguard spermatozoon were assessed for each treatment under phase contrast microscopy (200, Olympus, Tokyo, Japan). 2.4. Preparation and sex-sorting of spermatozoa Spermatozoa destined for sex-sorting were diluted to a concentration of 400  106 spermatozoa/mL with tris– citrate–fructose (TRIS; [22]) containing either 0 or 100 U/mL of Catalase (Sigma–Aldrich; Experiment 1), 0 or 6 mg CLCs (Experiment 2) or no additives (Experiment 3a and 3b), in addition to 267–311 mM Hoechst stain (H33342; Sigma–Aldrich). Samples were incubated at 34 8C and inverted every 15 min to improve staining uniformity. After 1 h, samples were removed, diluted 1:1 (sperm sample:diluent, v/v) with TRIS containing 4% (v/ v) EY and 0.002% (w/v) food dye (Warner Jenkinson Company, Inc., St. Louis, MO, USA) and filtered (35 mm, Falcon 2235; Becton Dickinson, MO, USA).

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Spermatozoa were processed using a modified high speed flow cytometer (SX MoFlo1; Dako Colorado, Fort Collins, CO, USA) operating at 40 psi with a TRIS sheath fluid running a continuous wave argon laser (Spectra Physics, Mountain View, CA, USA) at 200 mW [4,29]. Sorting gates were set to select the entire viable, oriented population to enable maximum sorting rates (6000– 13,000 spermatozoa/s). Spermatozoa were sorted and processed as described previously [29]. Briefly, spermatozoa were sorted into 10 mL tubes containing 0.25 mL of Androhep supplemented with 20% egg yolk (v/v) adjusted to pH 7.4. Six million spermatozoa were collected into each tube with an additional 0.25 mL of Androhep supplemented with 20% egg yolk (v/v) added after the initial two million spermatozoa had been collected. Sex-sorted spermatozoa were centrifuged at 750 g for 7.5 min at 21 8C. The supernatant was discarded and the remaining sperm pellet resuspended 1:4 (pellet:diluent, v/v) with the aforementioned tris–citrate– glucose cryoprotective media [22] prior to freezing by the pellet method previously described.

(Sigma–Aldrich) was dissolved in 2 mL of methanol (Sigma–Aldrich) in a separate tube. 0.45 mL of cholesterol solution was transferred to the cyclodextrin solution and stirred before solvents were removed with nitrogen gas and the CLC crystals air-dried. Similar to Experiment 1, utilising promising data from a pilot study assessing the effect of CLC concentration on the freezability of non-sorted ram spermatozoa (data not shown), ejaculates destined for sex-sorting in Experiment 2 were diluted with staining medium containing either 0 or 6 mg CLCs/mL. Following processing, freezing and subsequent thawing as described above, all samples were extended immediately (1:1; v/v; 500 mL semen:500 mL AH) and incubated (37 8C, 6 h). Motility characteristics, viability and acrosome integrity were analysed at 0, 3, and 6 h post-thaw by CASA and Eth-D1/FITC-PNA staining, respectively.

2.5. Experiment 1: effect of antioxidant (catalase) on in vitro sperm characteristics

Experiment 3 was undertaken to determine the effect of seminal plasma, added post-thaw, on both sex-sorted and non-sorted, cryopreserved ram spermatozoa. The ejaculates of each ram used for Experiment 3a were split into two parts: the first destined for sex-sorting and the second frozen without further processing to provide a non-sorted control (control). After thawing, control samples were diluted with TRIS to standardise sperm concentration with that of sex-sorted samples (20  106 spermatozoa/mL). Thawed sex-sorted and control samples were split into three groups for extension (1:1; v/ v; 133 mL semen:133 mL diluent) with: (i) Androhep (AH SP); (ii) Androhep supplemented with 40% homologous seminal plasma (AH + SP); or (iii) ASP. Seminal plasma was collected and pooled from the same three rams in the weeks prior to the experiment and prepared as described previously [30]. All samples were incubated for 6 h at 37 8C. The ability of each treatment group to migrate through cervical mucus was assessed at 0 h post-thaw, while motility characteristics and acrosome integrity were analysed at 0, 3 and 6 h post-thaw. Following from the results of Experiment 3a, further investigation to characterise the action of seminal plasma on sorted and non-sorted ram spermatozoa was warranted. Ejaculates for Experiment 3b were split into sex-sorted and control groups. Sex-sorted sperm samples were processed by flow cytometry and cryopreserved. Control sperm samples were diluted to 20  106 spermatozoa/mL and cryopreserved. Following thawing, both treatment samples were extended (1:1; v/v; 133 mL

A pilot study (data not shown) on highly diluted nonsorted ram spermatozoa (simulating the sorting process) was conducted to initially screen the potential type and concentration of antioxidant for use in the sex-sorting system. Results from this study indicated catalase to have a beneficial effect when supplemented (100 U/mL) into initial dilution media. Therefore, ejaculates from each ram used in Experiment 1 were diluted immediately (as described above) in staining media containing either 0 or 100 U/mL of catalase with sorting data (percentage orientation [spermatozoa with their flat surface of the head facing the laser beam], rate of coincidences at maximum sorting rate [related to sample wastage], and rate of sorting) recorded for each treatment group. All samples were diluted 1:1 (v/v; 500 mL semen:500 mL Androhep immediately after thawing and incubated; 37 8C, 6 h). Motility characteristics were analysed by CASA at 0, 2, 4, and 6 h postthaw. 2.6. Experiment 2: effect of cholesterol-loaded cyclodextrins on in vitro sperm characteristics The CLCs were prepared using the method described by Purdy and Graham [17]. Briefly, cholesterol (Sigma– Aldrich; 200 mg) was dissolved in 1 mL of chloroform (Sigma–Aldrich), and 1 g of methyl-b-cyclodextrin

2.7. Experiment 3a and 3b: effect of seminal plasma, added post-thaw, on in vitro sperm characteristics

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semen:133 mL diluent) in four different diluents: (i) Androhep, (ii) Androhep + 10% SP, (iii) Androhep + 20% SP and (iv) Androhep + 40% SP (seminal plasma from the same three rams). The resultant eight treatment groups per ram (control and sex-sorted  AH + final SP percentage: 0% SP, 5% SP, 10% SP and 20% SP) were incubated for 6 h at 37 8C. Aliquots were removed for assessment of motility characteristics, viability, acrosome integrity and mitochondrial activity at 0, 3 and 6 h post-thaw. 2.8. Statistical analyses All statistical analyses were conducted using GENSTAT (version 8.1, VSN International, Hemel Hempstead, UK). Sperm motility characteristics and

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sort data were analysed by general analysis of variance, utilising arc-sin transformations to attain normality where necessary. The main effects of ram, sperm type (sorted or non-sorted), experimental treatment [antioxidant level (Experiment 1), CLC level (Experiment 2) and post-thaw diluent (Experiment 3a and 3b)] and incubation time point were assessed in each ANOVA, with replication via ejaculate incorporated into the block structure. Data on dual staining for spermatozoa viability and acrosome integrity and sperm migration through cervical mucus were both analysed by Poisson regression. In Experiments 2 and 3b, only data for the percentage of viable and acrosome intact cells are presented as acrosome non-intact (viable and nonviable) populations were not altered by treatment (P > 0.05) and the effect on non-viable, acrosome intact

Fig. 1. Experiment 2: motility characteristics determined by CASA for (a) total motility, (b) beat cross frequency [BCF; —] and amplitude of lateral head displacement [ALH; – – –], (c) average path velocity [VAP; – – –], straight line velocity [VSL; - - -] and curvilinear velocity [VCL; —], and (d) straightness [STR; —] and linearity [LIN; – – –] for control (&) and CLC (~) treated sex-sorted ram spermatozoa during post-thaw incubation for 6 h at 37 8C (data are means  S.E.M.).

42  5.4 bcd 17  7.1 e 39  5.9 cd 8  5.4 e 42  5.5 bcd 43  5.8 bcd 45  1.7bcd 36  6.0cd Within row, values without common superscripts differ (P < 0.05) (data are means  S.E.M.).

44  2.9 bcd 34  8.6 d 40  5.4 cd 52  2.1 ab 46  1.4 bc 52  1.5 ab Non-sexed Sexed LIN (%)

45  2.1bcd 53  1.1ab

42  1.4bcd 59  1.3a

5.5  0.7 cd 1.4  1.0 fg 5.3  0.8 cd 0.6  0.6 g 5.4  0.7 cd 4.8  0.9 d 7.5  0.5ab 3.0  1.0ef 6.1  0.4 bcd 3.1  0.8 e 5.8  1.1 cd 6.2  0.3 bcd 7.6  0.3 ab 5.5  0.2 cd Non-sexed Sexed ALH (mm)

8.8  0.3a 6.9  0.3bc

8.8  0.4a 5.9  0.3bcd

83  11.4def 20  8.5 jk 73  11.9efg 9  5.9 k 66  12.4fgh 59  8.7 gh 106  6.4bc 49  8.8hi 92  6.0 cd 38  9.9 ij 90  5.5 cde 83  6.0 def 144  6.9a 101  7.0cd Non-sexed Sexed VAP (mms 1)

125  6.9 ab 84  2.2 def

27  5.4 35  9.4 d 31  5.8 38  9.3d 69  7.8 31  10.3d 41  7.2 57  8.0 b 54  7.0 77  2.9a 76  5.1 83  2.1 a 56  6.0 80  2.8a Non-sexed Sexed TM (%)

135  5.5a 107  4.3bc

31  5.6 d 13  7.5 ef 57  9.2 2  1.3 f

b

+SP

de

SP +SP

a

ASP

bc

SP

cd

+SP

ab

ASP

d

6h 3h

b

Samples stained for sex-sorting in the presence of CLCs exhibited a lower (P < 0.05) percentage of motile spermatozoa following incubation for 0, 3 and 6 h than samples stained in the absence of CLCs (Fig. 1a). Immediately post-thaw, all velocity characteristics (VAP, VSL, VCL) were similar (P > 0.05) between spermatozoa from CLC and control groups, though control spermatozoa displayed higher (P < 0.05) VAP at 3 h, and increased VAP and VSL at 6 h incubation than CLC treated spermatozoa (Fig. 1c). Beat cross frequency was higher (P < 0.05) in CLC spermatozoa immediately post-thaw, but similar (P > 0.05) to control spermatozoa after incubation for 3 and 6 h (Fig. 1b). Amplitude of lateral head displacement remained indistinguishable (P > 0.05) between both treatment groups through all time points of incubation (Fig. 1b). The linearity and straightness (Fig. 1d) of both CLC and control

SP

3.2. Experiment 2: effect of cholesterol-loaded cyclodextrins on in vitro sperm characteristics

0h

Flow cytometric analysis detected no differences in the percentages of correctly oriented spermatozoa, plasma membrane intact spermatozoa, coincidence rate or sort rate, between samples stained in the presence or absence of catalase. After thawing, no significant interactions were observed for any motility characteristic between ram and treatment, ram and time or treatment and time. Data were therefore pooled over ram and time point to increase the power of comparison between treatments (n = 36/treatment). Motility parameters were similar for sperm samples incubated in the presence or absence of the antioxidant catalase: total motility (TM; 75.5  2.22 and 73.4  1.89%), average path velocity (VAP; 111.3  2.96 and 111.8  3.08 mm s 1), straight line velocity (VSL; 89.3  2.04 and 90.3  2.00 mm s 1), curvilinear velocity (VCL; 192.5  5.47 and 191.5  5.85 mm s 1), amplitude of lateral head displacement (ALH; 7.2  0.18 and 7.2  0.19 mm), beat cross frequency (BCF; 34.8  0.62 and 34.7  0.67 Hz), straightness (STR; 80.3  0.79 and 80.6  0.81%), linearity (LIN; 49.0  0.84 and 49.6  0.91%), respectively.

Incubation time and post-thaw medium

3.1. Experiment 1: effect of antioxidant (catalase) on in vitro sperm characteristics

Sperm type

3. Results

Motility parameter

cells was the inverse of the viable, intact population. Mitochondrial activity and acrosome integrity (single stain) were determined by binomial logistic regression.

ASP

S.P. de Graaf et al. / Theriogenology 67 (2007) 217–227 Table 1 Experiment 3a: motility characteristics during incubation for 6 h at 37 8C of frozen-thawed sexed (sex-sorted) and non-sexed (non-sorted) ram spermatozoa extended with Androhep containing no seminal plasma ( SP), 40% seminal plasma (+SP) or artificial seminal plasma (ASP)

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spermatozoa were similar (P > 0.05) during measurement at 0 and 3 h of incubation, but higher (P < 0.05) for each characteristic in control treatments after 6 h (Fig. 1d). The percentages of viable, acrosome intact spermatozoa were comparable between CLC and control treatments immediately after thawing (73.4  3.84 and 77.9  2.78%, respectively), but following 3 h (69.4  3.32 and 81.2  2.23%) and 6 h (30.0  6.30 and

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56.1  5.82%) of incubation, control spermatozoa were superior (P < 0.05). 3.3. Experiment 3: effect of seminal plasma, added post-thaw, on in vitro sperm characteristics Motility parameters (data for VSL, VCL, BCF and STR not shown) following the post-thaw addition of

Fig. 2. Experiment 3b: motility characteristics determined by CASA for (a) total motility, (b) beat cross frequency [BCF; —] and amplitude of lateral head displacement [ALH; – – –], (c) average path velocity [VAP; – – –], straight line velocity [VSL; - - -] and curvilinear velocity [VCL; —], (d) straightness [STR; —] and linearity [LIN; – – –], and in vitro sperm quality assessed by Eth-D1/FITC-PNA for (e) viability and acrosome integrity, and R123 for (f) mitochondrial respiration for non-sorted control (&) and sex-sorted (~) ram spermatozoa supplemented with 0, 5, 10 or 20% (v/v) whole seminal plasma (data are means  S.E.M. pooled for 0, 3 and 6 h post-thaw incubation at 37 8C.).

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SP, +SP or ASP media to sex-sorted and non-sorted ram spermatozoa (Experiment 3a) are summarised in Table 1. For sorted samples, supplementation with seminal plasma (+SP) or use of artificial seminal plasma was detrimental (P < 0.05) to all velocity characteristics (0–6 h +SP; 3–6 h ASP), in addition to decreasing total motility, ALH, BCF, STR and LIN at 3 and 6 h when compared with SP sorted samples. Conversely, for non-sorted Control spermatozoa, total motility was improved (P < 0.05) by the addition of seminal plasma in comparison with SP or ASP (0, 3 and 6 h) while other motility parameters remained unaffected. A higher (P < 0.05) proportion of non-sorted spermatozoa were identified as acrosome intact (94.1  0.87 and 93.6  1.25%) when incubated in the presence of seminal plasma than when diluted in SP (91.6  1.14 and 84.3  2.2%) or ASP (88.8  1.24 and 83.9  2.3%, at 3 and 6 h, respectively). Acrosome integrity of sex-sorted spermatozoa was not affected by diluent at either time point. A comparison of sorted and non-sorted spermatozoa within the SP treatment group revealed that sorted spermatozoa demonstrated higher (P < 0.05) total motility, but lower (P < 0.05) ALH, VAP and VCL immediately post-thaw. Total motility of sorted spermatozoa remained superior (P < 0.05) after 3 h incubation, but was equivalent (P > 0.05) to non-sorted samples after 6 h. The ability to migrate through artificial cervical mucus, as measured by the number of spermatozoa penetrating to 1 cm and the distance travelled by the vanguard spermatozoon, was not affected by post-thaw medium for either sorted or non-sorted spermatozoa. However, non-sorted spermatozoa did penetrate to 1 cm in greater (P < 0.05) numbers (43.3  3.75) than sorted spermatozoa (22.0  3.46 spermatozoa). For all variables measured in Experiment 3b, there was an interaction (P < 0.001) between sperm type (sorted or non-sorted) and the percentage of seminal plasma (data pooled across incubation time points). With increasing proportions of whole seminal plasma in the post-thaw medium, motility (Fig. 2a), ALH and BCF (Fig. 2b), velocity (VAP, VCL and VSL; Fig. 2c) straightness and linearity (Fig. 2d), viability and acrosome integrity (Fig. 2e), and mitochondrial activity (Fig. 2f) of sex-sorted, frozen-thawed spermatozoa declined (P < 0.05), compared with non-sorted, frozenthawed spermatozoa (Fig. 2). Conversely, non-sorted, frozen-thawed spermatozoa exhibited a significant improvement (P < 0.05) in motility, viability, acrosome integrity and mitochondrial respiration when exposed to increasing percentages of seminal plasma whilst maintaining similar sperm kinematics.

4. Discussion Antioxidants, cholesterol-loaded cyclodextrins and seminal plasma failed to improve the efficiency of sexsorting of ram spermatozoa by flow cytometry. However, this study shed light on the functional differences between control (unsorted) and sex-sorted spermatozoa. The sex-sorting process selected a subpopulation of spermatozoa with different functional characteristics from the unsorted population, to the extent that seminal plasma was no longer capable of providing a beneficial effect after freezing and thawing, but rather was toxic. Similarly, there was some indication that additives, such as CLCs do not provide the benefits to sorted spermatozoa that have been observed in non-sorted spermatozoa. Previous studies using standard speed sorters suggested that sperm viability and motility from the ram and boar could be improved by the addition of 10% homologous seminal plasma to the staining medium [10] and that the inclusion of 10% seminal plasma in the post-sort collection medium improved post-thaw motility of boar spermatozoa [12]. Supplementation of the staining medium with seminal plasma in the present study was not undertaken as pre-sort improvements in previous studies were attributed to reduced sperm agglutination [10]. In addition, it would be impractical to provide sufficient ram seminal plasma for staining samples under commercial sorting conditions due to the low yield of seminal plasma per ram ejaculate. Post-thaw addition of seminal plasma was examined in the present study based on the improved fertility after cervical insemination and ability to penetrate cervical mucus [30] and elevated motility [31] after post-thaw addition of seminal plasma to ram and boar sperm samples, respectively. Similar to the latter studies, results herein showed that the quality of non-sorted ram spermatozoa, as assessed by motility, viability, acrosome integrity and mitochondrial activity, was significantly improved by the addition of seminal plasma. Unexpectedly, sex-sorted spermatozoa did not share this beneficial response. Indeed, contrary to the findings of Catt et al. [10] and Maxwell et al. [12] which used the older generation of flow cytometers, seminal plasma proved deleterious when added to spermatozoa processed using current sorting technology. Whereas high concentrations of seminal plasma have a detrimental effect on boar spermatozoa [12], results of Experiment 3b demonstrated the deleterious effect to be linearly dose-dependent rather than beneficial followed by an abrupt toxic effect. In addition, the decrease in motility of sorted spermatozoa was not due to viable cells

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exhibiting reduced mitochondrial activity, but rather an overall cell death. The cause of the detrimental effect of seminal plasma on sex-sorted cells, which was not seen in previous studies, may be the greatly increased pressure inside a high speed sorter (increase from 20 to 40 psi) to which spermatozoa are exposed. This pressure and the associated dilution and mechanical agitation inherent to the sorting process may alter the proteins present on the sperm surface revealing ligands and binding sites that render sexed spermatozoa more susceptible to any detrimental factors within the seminal plasma [32]. These negative factors are apparent in some studies where the addition of seminal plasma appears to be detrimental to the quality of non-sexed spermatozoa (boar, [33]; stallion, [34]). Notwithstanding, the reactions of sexsorted and non-sorted spermatozoa to seminal plasma suggested a fundamental functional difference between the two sperm types, possibly brought about by the stressors involved in flow cytometry. Contrary to the findings of Mortimer and Maxwell [35], artificial seminal plasma did not improve the postthaw quality of non-sorted spermatozoa in the current study, having the same effect to dilution with SP medium, suggesting the beneficial effects of seminal plasma, in this case, resulted from more than its ionic component. Sex-sorted spermatozoa in the present study also exhibited depressed in vitro characteristics when diluted in ASP, supporting the results of a study utilising ASP as a potential sheath fluid [36]. That CLCs did not provide a protective effect for sexsorted spermatozoa during freezing and thawing was surprising. Pilot studies showed that incorporation of CLCs into the cryopreservation protocol of non-sorted ram spermatozoa significantly increased their survival after freeze-thawing [de Graaf et al., unpublished observations], similar to findings in the bull [17,37] and stallion [38]. In the latter studies, the protective effect of CLCs was attributed to their positive influence on membrane fluidity [39], which is known to correlate positively with cryosurvival [40]. However, the effect of increasing membrane fluidity prior to high dilution and pressure remains unknown. High membrane fluidity prior to flow cytometric sorting may be detrimental to sperm survival due to the osmotic impact of high dilution. Alternatively, incorporation of CLCs into post-sorting media may prove beneficial to post-thaw quality. However, this will be difficult to achieve without changing current protocols which utilise high concentrations of egg yolk, as spermatozoa and CLCs must be co-incubated in a lipid-free medium to avoid transfer of the majority of the cholesterol, pre-loaded into the CLC, to the egg yolk [17].

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Antioxidants were tested in the staining medium, as the high dilution and mechanical stresses involved in flow cytometry cause an increase in the oxidative stress to spermatozoa [41]. In the current study, catalase had neither positive nor negative effects on the measured motility characteristics. While this is in contrast to the beneficial effect of catalase when added to ram spermatozoa during extended liquid storage [21], the lower number of dead cells (associated with elevated production of ROS) seen in the present study could have created an environment low in ROS whereby the effect of antioxidants would be less pronounced. The effect may also be species dependent as indicated by the improved pregnancy rate of cattle inseminated with sexsorted bull spermatozoa that had been processed in the presence of antioxidants [42]. In the present study, spermatozoa sex-sorted using normal protocols (i.e. diluted in media without seminal plasma) displayed significantly higher motility than non-sorted controls at 0 and 3 h post-thaw. In terms of motility, these findings are different from those of Hollinshead et al. [4] who showed sex-sorted samples to have lower motility during 4 h of incubation, using a different cryodiluent [43]. However, both studies observed diminished velocity (measured subjectively using a 0–5 forward progressive scale in [4]) for sexsorted compared with control samples, as do other studies on the kinematics of sexed spermatozoa [5–7]. Such results suggest that for certain aspects of in vitro quality, namely motility, sex-sorted spermatozoa (when not exposed to certain additives, such as seminal plasma) are superior to non-sorted spermatozoa. Further investigation of in vitro and in vivo functional status of sex-sorted spermatozoa using current methodologies is warranted to determine whether the observed improvements of in vitro survival have implications for fertility. Unfortunately, all of the supplements tested in the present study failed to improve the post-thaw quality of sex-sorted ram spermatozoa. Indeed, cholesterol-loaded cyclodextrins and seminal plasma proved detrimental. These results contrasted with those observed in nonsorted ram spermatozoa that benefited from the inclusion of seminal plasma in the post-thaw medium. In conclusion, although efforts to improve the efficiency of sex-sorting were unsuccessful, this study revealed new insights into functional differences between sexed and non-sexed spermatozoa. Acknowledgements This work was supported by XY, Inc. (Fort Collins, CO, USA). S.P. de Graaf was supported by The

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Australian Sheep CRC with a postgraduate research scholarship. Bioniche Animal Health Australasia is thanked for donation of the sodium hyaluronate for sperm migration tests. The authors thank Dr. M. Ruckholdt for operation of the MoFlo1 SX cell sorter, Ms. S. Underwood, Ms. T. Leahy, Ms. K. Heasman and Mr. A. Souter for technical assistance. References [1] Garner DL. Flow cytometric sexing of mammalian sperm. Theriogenology 2006;65:43–957. [2] Seidel Jr GE, Garner DL. Current status of sexing mammalian spermatozoa. Reproduction 2002;124:733–43. [3] Garner DL, Schenk JL, Seidel Jr GE. Chromatin stability in sexsorted sperm. J Androl 2001;22(Suppl):162. [4] Hollinshead FK, Gillan L, O’Brien JK, Evans G, Maxwell WMC. In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing. Reprod Fertil Dev 2003;15:351–9. [5] Lindsey AC, Muckle LK, Squires EL. Effects of caffeine stimulation on stallion sperm motion characteristics following 18-h storage, flow sorting, and cryopreservation. Theriogenology 2003;59:510. [6] Suh TK, Schenk JL. Pressure during flow sorting of bull sperm affects post-thaw motility characteristics. Theriogenology 2003; 59:516. [7] de Graaf SP, Gillan L, Evans G, Maxwell WMC, O’Brien JK. Effect of flow cytometric sorting and freezing diluent preparation on in vitro quality of ram spermatozoa. 15th International Congress on Animal Reproduction. Brazil: Porto Seguro; 2004. p. 477 [abstract]. [8] Maxwell WMC, Evans G, Hollinshead FK, Bathgate R, de Graaf SP, Eriksson BM, et al. Integration of sperm sexing technology into the ART toolbox. Anim Reprod Sci 2004;82–83:79–95. [9] Schenk JL, Suh TK, Cran DG, Seidel Jr GE. Cryopreservation of flow-sorted bovine spermatozoa. Theriogenology 1999;52:1375– 91. [10] Catt SL, O’Brien JK, Maxwell WMC, Evans G. Assessment of ram and boar spermatozoa during cell-sorting by flow cytometry. Reprod Domest Anim 1997;32:251–8. [11] Hollinshead FK, O’Brien JK, Maxwell WMC, Evans G. Production of lambs of predetermined sex after the insemination of ewes with low numbers of frozen-thawed sorted X- or Y-chromosomebearing spermatozoa. Reprod Fertil Dev 2002;14:503–8. [12] Maxwell WMC, Welch GR, Johnson LA. Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma. Reprod Fertil Dev 1997;8:1165–78. [13] Buchanan BR, Seidel Jr GE, McCue PM, Schenk JL, Herickhoff LA, Squires EL. Insemination of mares with low numbers of either unsexed or sexed spermatozoa. Theriogenology 2000;53: 1333–44. [14] O’Brien JK, Robeck TR. Development of sperm sexing and associated assisted reproductive technology for sex preselection of captive bottlenose dolphins (Tursiops truncatus). Reprod Fertil Dev 2006;18:319–29. [15] Maxwell WMC, Johnson LA. Physiology of spermatozoa at high dilution rates: the influence of seminal plasma. Theriogenology 1999;52:1353–62.

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