Investigation of in vitro parameters and in vivo fertility of rabbit spermatozoa after chilled storage.

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ARTICLE IN PRESS

ANIREP-4966; No. of Pages 9

Animal Reproduction Science xxx (2014) xxx–xxx

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Investigation of in vitro parameters and in vivo fertility of rabbit spermatozoa after chilled storage D. Johinke ∗ , S.P. de Graaf, R. Bathgate Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia

a r t i c l e

i n f o

Article history: Received 17 December 2013 Received in revised form 25 April 2014 Accepted 26 April 2014 Available online xxx

Keywords: Rabbit Buck Semen Chilled storage Flow cytometry AI

a b s t r a c t Artificial insemination (AI) programmes in the rabbit meat industry require improved longevity of spermatozoa stored in vitro. Two studies evaluated the effects of storage temperature and extender on in vitro quality and fertility of rabbit spermatozoa over 96 h of chilled storage. In Experiment 1, three ejaculates were collected from each of five bucks and diluted 1:10 in either Extender A or B, and then divided further for storage at 5 ◦ C or 15 ◦ C. Sperm motility (MOT) was assessed by CASA at 0, 24, 48, 72 and 96 h of storage. Viability, acrosome integrity, mitochondrial membrane potential (MMP), oxidative stress and DNA integrity of the two best extenders were assessed by flow cytometry. Extender B at 15 ◦ C gave significantly higher values of MOT and MMP from 24 and 72 h, respectively. At 96 h, viability, acrosome and DNA integrity were best maintained at 15 ◦ C (P < 0.05). In contrast, storage at 5 ◦ C resulted in lower oxidative stress from 72 h. In Experiment 2, a pilot study examined fertility rates of does inseminated with spermatozoa diluted in Extender B and stored at 5 ◦ C or 15 ◦ C. Sixty seven multiparous does were inseminated with spermatozoa stored for 0 h (n = 12; control), 48 h (n = 26) or 72 h (n = 29). Kindling rates and litter sizes for does inseminated with semen stored for 48 h at 5 ◦ C or 15 ◦ C and 72 h at 5 ◦ C were similar (P > 0.05) to those of the controls; kindling rate dropped following insemination with spermatozoa held at 15 ◦ C for 72 h, though litter size did not. © 2014 Elsevier B.V. All rights reserved.

1. Introduction With the global demand for rabbit meat steadily increasing, commercial rabbit breeding has become more dependent on artificial insemination (AI) rather than tra˜ 1998; Rosato ditional natural mating (López and Alvarino, et al., 2012; Rosato and Iaffaldano, 2011). Cryopreservation techniques for this species remain suboptimal and the use of frozen-thawed rabbit spermatozoa generally result

∗ Corresponding author at: Faculty of Veterinary Science, The University of Sydney, Room 344, RMC Gunn Building B19, NSW 2006, Australia. Tel.: +61 2 9351 5832; fax: +61 2 9351 3957. E-mail addresses: [email protected], [email protected] (D. Johinke).

in lower rates of fertility than those achieved with fresh spermatozoa (Mocé et al., 2010; Mocé and Vicente, 2009; Rosato and Iaffaldano, 2011). Accordingly, AI is mostly performed with fresh diluted semen collected on-site and generally stored for no longer than 18 h (Nagy et al., 2002; Roca et al., 2000). In some regions, the fertilising ability of rabbit spermatozoa has been preserved for up to 48 h using chilled storage techniques (López-Gatius et al., 2005; Roca et al., 2000; Rosato and Iaffaldano, 2011), thus enabling the transportation of superior genetics between nearby farms. However, this is an insufficient length of time for successful transport of genes to more distant locations. The improvement of liquid storage protocols would facilitate the widespread transport of rabbit semen to more remote regions and encourage the routine use of artificial insemination programmes on commercial rabbit farms,

http://dx.doi.org/10.1016/j.anireprosci.2014.04.014 0378-4320/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Johinke, D., et al., Investigation of in vitro parameters and in vivo fertility of rabbit spermatozoa after chilled storage. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.04.014

G Model ANIREP-4966; No. of Pages 9

ARTICLE IN PRESS D. Johinke et al. / Animal Reproduction Science xxx (2014) xxx–xxx

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thereby resulting in faster genetic gain and improved growth rates. Previous studies have suggested that the motility and viability of rabbit spermatozoa can be preserved for 48 h to 96 h when stored at 5 ◦ C or 15 ◦ C in Tris-citric acidglucose based extenders (Roca et al., 2000; Rosato et al., 2012; Rosato and Iaffaldano, 2011). Similarly, Tris-based solid (gel-supplemented) extenders have prolonged in vitro viability of rabbit spermatozoa for up to 72 h when stored at 5 ◦ C (Nagy et al., 2002) or 15 ◦ C (Echegaray-Torres et al., 2004). However, in many of these studies the fertilising capacity of the chilled spermatozoa was only maintained for 48 h post-collection. Although sperm motility and viability are regularly used to predict the fertility potential of rabbit spermatozoa following different storage conditions, these parameters do not provide sufficient detail regarding the deterioration of sperm quality over time. The evaluation of additional in vitro parameters, such as DNA integrity, oxidative stress and functional mitochondrial status may offer better insights into the potential reproductive performance of rabbit sperm during chilled storage (Gillan et al., 2005; Kasimanickam et al., 2012; Lavara et al., 2005). The objective of the first study was to evaluate the effect of three Tris-based extenders on the motility, viability, acrosome integrity, mitochondrial function, ROS production and DNA integrity of rabbit spermatozoa during chilled storage at 5 ◦ C or 15 ◦ C for 96 h. In the second study, a smallscale pilot study was conducted to evaluate the fertilising ability of rabbit spermatozoa diluted in the best performing extender from Study 1, following storage at 5 ◦ C or 15 ◦ C for 0 h, 48 h and 72 h. 2. Materials and methods 2.1. Experimental design The procedures described herein were approved by the University of Sydney Animal Ethics Committee. All of the chemicals, unless otherwise stated, were sourced from Sigma–Aldrich (Castle Hill, NSW, Australia). Two experiments were conducted to assess the in vitro quality and fertility of rabbit spermatozoa following chilled storage. In Experiment 1, semen was collected from five mature rabbit bucks (three ejaculates/buck) housed at the Faculty of Veterinary Science, The University of Sydney, Australia. Each ejaculate was diluted with one of three Tris-citric acidglucose based extenders and split into two parts; one for storage at 5 ◦ C and one for storage at 15 ◦ C. In Experiment 2, the fertilising ability of chilled rabbit spermatozoa diluted in a Tris-citric acid-glucose based extender was evaluated by a small-scale field trial conducted at a commercial rabbitry in NSW, Australia. Pooled semen was diluted 1:10 with the best performing extender from Experiment 1 and subdivided into two equal parts for storage at 5 ◦ C or 15 ◦ C. Artificial inseminations were performed at 0 h, 48 h and 72 h post semen collection. 2.2. Animals and semen collection Animals were housed with a light cycle of 12 h light/dark and room temperature of 22–24 ◦ C. All rabbits were fed

a commercial diet with food and water provided ad libitum. Semen was collected from mature hybrid rabbit bucks (Flemish Giant × Crusader × New Zealand White) using a teaser doe and artificial vagina and if present, the gel plug was removed. Immediately following collection, ejaculates were placed in a water bath at 37 ◦ C until evaluation. Semen quality (colour, volume, concentration and motility) was assessed for each ejaculate. Only ejaculates that were white, >200 ␮L in volume, ≥300 × 106 cells/mL in concentration and with ≥70% motile spermatozoa were used for the experiments.

2.3. Experiment 1: effect of diluent and storage temperature on the in vitro quality of chilled rabbit spermatozoa The objective of this experiment was to test the suitability of different Tris-citric acid-glucose based extenders for the chilled storage of rabbit spermatozoa. Each selected ejaculate was divided into two equal aliquots and diluted 1:10 with one of the following extenders; Extender A (313.79 mM Tris, 103.07 mM citric acid·H2 O, 33.3 mM glucose and 80 mg/L kanamycin, adjusted to a pH of 6.9 and osmolarity of 336 (Roca et al., 2000)) or Extender B (250.04 mM Tris, 79.76 mM citric acid·H2 O, 69.38 mM glucose, 75.0 IU streptomycin and 166.2 IU GPenicillin, adjusted to a pH of 7.14 and osmolarity of 299 (Boiti et al., 2005)). The diluted semen was then further subdivided into two parts and stored at 5 ◦ C or 15 ◦ C for 96 h. Assessment of spermatozoa quality was determined at 0 h, 24 h, 48 h, 72 h and 96 h of storage. All diluted samples were evaluated for sperm motility kinematics and assessed by flow cytometry for acrosome integrity, viability, mitochondrial function, level of oxidative stress and DNA integrity. Sample aliquots destined for assessment by flow cytometry were further diluted 1:1 with the corresponding extender (final dilution factor of 1:20) prior to fluorescent staining. The generated data were analysed using the software CELLQuest (Becton–Dickinson).

2.4. Experiment 2: effect of storage temperature on the fertility of chilled rabbit spermatozoa To test the fertilising capacity of the chilled rabbit spermatozoa, semen was diluted in Extender B as it was found to be more efficient at preserving in vitro quality during the 96 h of chilled storage in Experiment 1. Ejaculates were collected from seven mature bucks on-site and initially assessed for semen quality (colour, volume, concentration and motility). The semen was then pooled, diluted 1:10 with Extender B and divided into two equal subsamples for storage at 5 ◦ C or 15 ◦ C for 72 h. A group of 12 does was inseminated at 0 h (fresh spermatozoa) in order to serve as a control. The remaining 55 does were divided into four groups and inseminated with semen stored at 5 ◦ C or 15 ◦ C at 48 h and 72 h after semen collection.




2.5. Evaluation of spermatozoa 2.5.1. Sperm motility and kinematics Sperm motility was evaluated by computer-assisted sperm analysis (CASA; HTM-IVOS v. 12; Hamilton-Thorne, Beverly, MA, USA). CASA was set up as follows: frame rate – 60 Hz; minimum contrast – 50; low and high static size gates – 0.35–3.40; low and high intensity gates – 0.75–1.70; low and high elongation gates – 0.0–70; default cell size – 7 pixels; default cell intensity – 70. Sample aliquots (5.5 ␮L) were placed on slides (Cell Vu, Millennium Sciences Inc., NY, USA; pre-warmed to 37 ◦ C) and a cover slip was added. Motility characteristics were determined by the assessment of at least three randomly selected microscopic fields so that a minimum of 300 cells per aliquot were analysed. Further dilution of the samples was unnecessary. The following kinetic parameters were recorded: percentage of motile spermatozoa (MOT); percentage of progressively motile spermatozoa (pMOT); average velocity path (VAP, ␮m/s); straight line velocity (VSL, ␮m/s); curvilinear velocity (VCL, ␮m/s); linearity (LIN; VSL/VCL); and straightness (STR; VSL/VAP), based on CASA factory settings for rabbit. 2.5.2. Viability and acrosome integrity Viability and acrosome integrity were assessed using propidium iodide (PI; Invitrogen, Mount Waverley, VIC, Australia) and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) modified dual fluorescent staining technique (Kershaw-Young and Maxwell, 2011). Aliquots of the extended spermatozoa were mixed with PI and FITCPNA to give final staining concentrations of 12 ␮M and 4 ␮g/mL, respectively. Following an incubation of 5 min at 37 ◦ C, semen samples were analysed in a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). A minimum of 10,000 events were recorded. Green fluorescence (FITC-PNA) was detected using fluorescence detector (FL)-1 and red fluorescence (PI) was detected in FL-3.The cells were classified as: viable spermatozoa with intact acrosome (PI− PNA− ); viable spermatozoa with damaged acrosome (PI− PNA+ ); spermatozoa with damaged membrane and intact acrosome (PI+ PNA− ); or spermatozoa with damaged membrane and damaged acrosome (PI+ PNA+ ). 2.5.3. Mitochondrial function Mitochondrial membrane potential (MMP) was assessed using Rhodamine 123 (R123) counterstained with PI (Invitrogen), adapted from Underwood et al. (2009). A final concentration of 0.5 ␮g/mL R123 was added to the semen sample aliquot and incubated at 37 ◦ C for 5 min. An aliquot of PI was then added to give a final staining concentration of 12 ␮M, followed by an additional 5 min of incubation at 37 ◦ C, before analysis by flow cytometry. A minimum of 10,000 events were acquired with cells recorded as: viable spermatozoa with non-functioning mitochondria (PI− R123− ); viable spermatozoa with functioning mitochondria (PI− R123+ ); spermatozoa with damaged membrane and non-functioning mitochondria (PI+ R123− ); or spermatozoa with damaged membrane and functioning mitochondria (PI+ R123+ ). Cell populations were segregated as described by Khromova et al. (2012)

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and Troiano et al. (1998). Spermatozoa which emitted a green fluorescence (R123+ ) were considered to have high MMP (hMMP) and were detected on FL-1. 2.5.4. Presence of oxidative stress The production of the reactive oxygen species (ROS) hydrogen peroxide (H2 O2 ) was detected using 2,7dichlorodihydrofluorescein diacetate (H2 DCFDA; Invitrogen) and PI fluorescent staining techniques, modified from Kim et al. (2011). Aliquots of semen samples (250 ␮L) were mixed with 200 ␮M H2 DCFDA (prepared in dimethyl sulfoxide; DMSO) and incubated at 37 ◦ C for 55 min. Propidium iodide was added to a final concentration of 12 ␮M and incubated for a further 5 min for a total incubation time of 60 min. A minimum of 10,000 cells were analysed using the FACScan flow cytometer (Becton–Dickinson) with green fluorescence (H2 DCFDA) detected in FL-1. Cells were differentiated into populations as per Kim et al. (2011) and expressed as viable spermatozoa with low intracellular H2 O2 (PI− H2 DCFDA− ); viable spermatozoa with high intracellular H2 O2 (PI− H2 DCFDA+ ); spermatozoa with damaged membrane and low intracellular H2 O2 (PI+ H2 DCFDA− ); or spermatozoa with damaged membrane and high intracellular H2 O2 (PI+ H2 DCFDA+ ). 2.5.5. DNA integrity Sperm DNA integrity was analysed using the sperm chromatin structure assay (SCSA) described by Evenson and Jost (2001). Briefly, aliquots of stored semen (200 ␮L) were mixed with 400 ␮L of acid-detergent solution (0.1% Triton-X 100, 0.08 M HCl and 0.15 M NaCl). After 30 s, 1.2 mL of acridine orange (AO) staining solution (6 ␮g/mL AO, 0.037 M citric acid, 0.126 M Na2 HPO4 , 1 mM disodium ethylenediaminetetraacetic acid [EDTA] and 0.15 M NaCl, pH 6.0; final stain concentration of 4 ␮g/mL) was added. Each sample was run on the flow cytometer for 3 min prior to reading in order to allow sufficient time for AO equilibration in the sample. A minimum of 5000 gated cells were read for each sample at a flow rate of no greater than 300 cells per second. Cell populations were segregated into the green-fluorescing double-stranded DNA (intact) and the red-fluorescing cells-outside-main-population (COMP) denatured DNA, as outlined by Evenson and Jost (2001). 2.6. Artificial insemination A total of 67 does were inseminated with diluted rabbit spermatozoa. Oestrus was synchronised with a subcutaneous injection of 0.1 mL of PMSG (Folligon, Intervet, Australia) 48 h prior to AI. To induce ovulation, 0.02 mg of GnRH analogue (Fertagyl, Intervet, Australia) was injected intramuscularly at the time of insemination. Inseminations were performed 11 days after parturition using a dose rate of 0.5 mL of diluted semen containing 15 × 106 total spermatozoa. Fecundity rates and litter sizes were recorded at time of kindling. 2.7. Statistical analyses Data in Experiment 1 were analysed using a General Linear Mixed Model in Genstat (Version 14, VSN




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Fig. 1. Motility characteristics determined by CASA for (a) total motility (tMOT) and (b) progressive motility (pMOT) of rabbit spermatozoa diluted with Extender A (䊉) or Extender B () and stored at 5 ◦ C (white, dashed line) or 15 ◦ C (black, solid line) for 96 h. Data are mean ± SEM. Means marked with an asterisk are significantly different within each time point (P < 0.05).

International Ltd., Hemel Hempstead, UK). The effects of extender type, storage temperature, incubation time and their interactions were included in the model. Replication via ejaculate and individual was incorporated into the block structure. Results were expressed as the model-derived mean ± standard error of the mean (SEM). A General Linear Mixed Model was also used in Experiment 2, including the effects of storage temperature, insemination time and their interactions. For all analyses a P value of 0.05).

3. Results

3.1.2. Viability and acrosome integrity There was an effect (P < 0.05) of storage temperature, but not of extender, on the sperm membrane and acrosome integrities of the chilled rabbit spermatozoa. At 24 h of storage, a temperature of 15 ◦ C preserved a higher percentage of viable spermatozoa with intact acrosome than a storage temperature of 5 ◦ C; this significance continued for the remaining timepoints assessed. Furthermore, samples diluted with Extender A and stored at 5 ◦ C displayed inferior viability/acrosome integrity than the 15 ◦ C-stored treatment groups from 48 h, 72 h and 96 h (P < 0.05) while samples diluted in Extender B and stored at 15 ◦ C reported the highest percentage of viable, acrosomeintact spermatozoa throughout the 96 h incubation period (Fig. 2a). The effect of storage time was also significant, with viability and acrosome integrity decreasing at each time point regardless of temperature and extender.

3.1. Experiment 1: effect of diluent and storage temperature on the in vitro quality of chilled rabbit spermatozoa 3.1.1. Sperm motility and kinematics The effects of semen extender and storage temperature on the motility parameters of chilled rabbit spermatozoa over 96 h are presented in Fig. 1. Total sperm motility did not differ between the different semen extenders or storage temperatures immediately after dilution (0 h; P > 0.05). At 24 h of storage, the tMOT was higher for sperm diluted in Extender B and stored at 15 ◦ C than those stored at 5 ◦ C in the same extender, and lowest in samples stored in Extender A (Fig. 1a, P < 0.05). This pattern of difference was maintained at all subsequent stages of incubation. For spermatozoa diluted in Extender A, MOT was significantly higher in samples stored at 5 ◦ C compared to 15 ◦ C from 48 h. Initially, pMOT was significantly greater in samples stored in Extender A (Fig. 1b). However, this difference did not continue following chilled storage as samples stored in Extender B demonstrated a higher pMOT after 24, 48 and 72 h of incubation (P < 0.05). The effects of semen extender and storage temperature on sperm velocity (VAP, VSL, VCL) and motion quality (LIN,

3.1.3. Mitochondrial function The significant effects of semen extender and storage temperature on the hMMP of the diluted spermatozoa are presented in Fig. 2b. Mitochondrial activity decreased over time for all treatment groups, irrespective of extender or storage temperature (P < 0.001). While no significant differences in the level of functioning mitochondria were observed between treatments immediately following




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Table 1 Motility characteristics determined by CASA for average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR) and linearity (LIN) of rabbit spermatozoa following chilled storage for 96 h. Data are mean ± SEM. Different superscripts within each column indicate significant difference (P < 0.05). Time point (h)

Diluent

Temperature (◦ C)

Sperm parameter

0

Extender A

5 15 5 15

71 73 70 70

± ± ± ±

1.8a 2.5a 1.4a 1.7a

50 48 49 46

± ± ± ±

1.1a 1.6ab 1.1ab 0.9bc

132 139 128 129

± ± ± ±

3.3ab 5.1a 2.3abcd 2.8abc

40 36 40 37

± ± ± ±

1.2ab 1.5bcde 1.1a 0.9abcd

72 67 71 67

± ± ± ±

1.4cdef 1.8h 1.6efgh 1.2gh

5 15 5 15

60 57 56 55

± ± ± ±

2.7b 2.8bc 2.1bc 2.2bc

40 40 41 42

± ± ± ±

1.6def 1.6def 1.3de 1.3cd

121 115 115 109

± ± ± ±

4.7bcde 4.5cdefgh 3.8cdefgh 3.9efgh

34 35 37 40

± ± ± ±

1.3cdefghi 1.3cdefg 1.3abc 1.4a

68 72 75 78

± ± ± ±

2.3fgh 2.3def 1.8abcde 1.3ab

5 15 5 15

51 46 51 45

± ± ± ±

2.4cd 2.1def 1.8cd 1.4def

36 33 37 35

± ± ± ±

1.0fghi 1.0ijk 1.1fghi 0.9ghij

113 108 115 102

± ± ± ±

5.2defgh 3.7efgh 3.1cdefgh 3.7gh

33 31 33 36

± ± ± ±

1.3efghi 0.8ijk 1.2efghij 1.3cdef

72 73 74 78

± ± ± ±

1.9cdef 1.8bcde 1.5bcde 1.5a

5 15 5 15

48 42 55 50

± ± ± ±

3.8de 3.2ef 4.2bc 2.8cd

34 31 40 37

± ± ± ±

2.3ghij 1.9jk 2.9def 2.1fgh

118 102 128 117

± ± ± ±

8.4bcdef 7.8fgh 10.3abcd 5.7bcdefg

30 32 32 33

± ± ± ±

1.0jk 1.4hijk 1.7ghij 1.1fghij

73 75 73 74

± ± ± ±

1.4cde 1.5abcd 1.4cde 1.6abcde

5 15 5 15

47 39 50 47

± ± ± ±

3.9de 1.9f 3.4cd 3.1de

33 29 38 35

± ± ± ±

2.4hijk 1.3k 2.4efg 2.2ghij

121 100 120 108

± ± ± ±

10.9bcde 5.6h 9.2bcde 7.2efgh

29 30 33 34

± ± ± ±

1.2k 1.2jk 1.6fghij 1.3defghi

71 74 73 77

± ± ± ±

1.2defg 1.5abcde 1.9cde 1.4abc

VAP (␮m/s)

Extender B 24

Extender A Extender B

48

n Extender A Extender B

72


96


dilution, samples diluted in Extender B and stored at 15 ◦ C displayed superior levels of hMMP (P < 0.05) compared to samples stored in the same extender at 5 ◦ C (48 h, 72 h, 96 h) or Extender A at 5 ◦ C (24 h, 72 h, 96 h) or 15 ◦ C (48 h, 72 h, 96 h). There were no significant differences in the hMMP of samples stored at 5 ◦ C during the incubation period, regardless of storage extender.

3.1.4. Presence of oxidative stress The proportion of viable spermatozoa with low intracellular H2 O2 decreased with storage time for all treatments (P < 0.05). Though there was a significant difference between samples diluted in Extender A at 15 ◦ C and those diluted in Extender B at 5 ◦ C immediately following dilution (92 ± 1.1% vs. 90 ± 1.2%, respectively), the percentage of viable spermatozoa with low intracellular H2 O2 levels was similar (P > 0.05) between the different treatment groups at 24 h and 48 h (Fig. 2c). Following an incubation of 72 h and 96 h, spermatozoa stored at 5 ◦ C demonstrated a significantly higher percentage of viable spermatozoa with low intracellular H2 O2 than those stored at 15 ◦ C, irrespective of extender type.

3.1.5. DNA integrity No differences were observed in the percentage of spermatozoa with non-denatured DNA during the first 72 h of incubation, regardless of treatment (P > 0.05, Fig. 2d). However, at 96 h spermatozoa diluted in Extender B and stored at 5 ◦ C displayed significantly lower DNA integrity (74 ± 2.0%) than the samples stored in Extender B at 15 ◦ C (81 ± 1.5%) or Extender A at 5 ◦ C (80 ± 1.5%) and 15 ◦ C (81 ± 1.8%).

VSL (␮m/s)

VCL (␮m/s)

LIN (%)

STR (%)

3.2. Experiment 2: effect of storage temperature on the fertility of chilled rabbit spermatozoa The percentages of does kindling and average litter size for each group are presented in Table 2. Overall, no significant interactions between storage temperature and incubation time were observed between does inseminated with fresh or chilled-stored sperm. Moreover, fertility for spermatozoa stored at 5 ◦ C or 15 ◦ C for 48 h, or stored at 5 ◦ C for 96 h was similar to that of spermatozoa from the control (fresh) group (P > 0.05). Although the rate of kindling achieved with the diluted spermatozoa stored at 15 ◦ C for 96 h was lower than that reported for the control group (P < 0.05), the average litter size was not significantly different.

4. Discussion In the present study, Tris-based extenders successfully maintained in vitro sperm characteristics during long-term chilled storage of rabbit semen over 96 h. Although sperm quality continuously decreased over storage time for all treatments, our results indicated that Extender B was more appropriate for retaining the in vitro sperm function during 96 h of storage. Moreover, the fertilising capacity of spermatozoa diluted in Extender B was preserved for 48 h at 15 ◦ C and 72 h at 5 ◦ C in the pilot study, suggesting that this extender may play an important role in the preservation of chilled rabbit sperm for use in commercial AI programmes. Sperm motility is often considered to be the most important parameter when assessing the potential reproductive performance of stored spermatozoa as it is believed to have a high correlation to the in vivo fertility of


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Fig. 2. Percentages of (a) viable spermatozoa with intact acrosome, (b) viable spermatozoa with high mitochondria membrane potential (hMMP), (c) viable spermatozoa with low levels of intracellular H2 O2 , and (d) spermatozoa with non-denatured DNA after dilution with Extender A (䊉) or Extender B () and stored at 5 ◦ C (white, dashed line) or 15 ◦ C (black, solid line) for 96 h. Data are mean ± SEM. Means marked with an asterisk are significantly different within each time point (P < 0.05). Table 2 Fertility and prolificacy rates of rabbit does after insemination with fresh semen or semen stored at 5 ◦ C or 15 ◦ C for 48 h or 72 h. Different superscripts within the same column indicate a significant difference (P 0.05). Length of incubation (h)

Storage temperature (◦ C)

No. of does inseminated

No. of does kindled (%)

0 48

37 5 15 5 15

12 12 14 15 14

8 5 9 5 2

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spermatozoa (Castellini, 2008; Hagen et al., 2010; Lavara et al., 2005; Mahadevan et al., 1997). Additionally, previous studies have shown that other motility parameters, such as VSL and LIN, may also be useful predictors of male in vitro

± ± ± ± ±

0.1 (66.7)a 0.2 (41.7)ab 0.1 (64.3)a 0.1 (33.3)ab 0.1 (14.3)b

Average litter size 7.3 5.2 8.2 4.8 4.5

± ± ± ± ±

1.1ab 0.9ab 1.3a 0.7ab 0.5b

fertility (Aitken et al., 1982; Liu et al., 1991; Milligan et al., 1980). Specifically, Liu et al. (1991) reported that linearity was one of the few variables in logistic regression that was significantly correlated to in vitro fertilisation (IVF) rates




in humans. Similarly, the VSL was found to be significant, furthering the hypothesis that the straight line speed may be utilised to predict the likelihood of spermatozoa contacting an oocyte. In this study, spermatozoa diluted in Extender B was found to preserve the highest percentages of tMOT, pMOT, VSL and LIN after 96 h compared to sperm diluted in Extender A, regardless of storage temperature, suggesting that dilution in Extender B may improve the fertility potential of chilled spermatozoa in vitro. Although the ingredients of each extender are similar, they do vary with respect to component concentration, antibiotic content, pH and osmotic pressure. Interestingly, the concentration of glucose in Extender B is more than twice that present in Extenders A or B. Mammalian spermatozoa require an intracellular energy source, often obtained through the consumption of different sugars such as glucose, sucrose, mannose, fructose or lactose, for the metabolic maintenance of sperm motility and flagellum movement patterns (Gadea, 2003; Ponglowhapan et al., 2004). To date, there have been few studies investigating the metabolism of glucose by rabbit spermatozoa during chilled storage. However, in both human and canine spermatozoa it has been observed that motility was improved when higher levels of glucose were used as the energy substrate (Iguer-ouada and Verstegen, 2001; Mahadevan et al., 1997). As such, the lower percentages of MOT reported for rabbit spermatozoa diluted in Extender A in our study may be due to a more rapid depletion of the energy source necessary for preserving sperm motility over time. Damage to the sperm membrane is generally caused by the in vitro processing of semen during which spermatozoa are often cooled to low temperatures and stored for extended periods of time. During this chilled storage, oxidative stress plays an important role in the decline of sperm quality (Bucak and Tekin, 2007). While a small amount of ROS are necessary for the initiation of many critical sperm functions, the high levels of ROS generated by spermatozoa during chilling and storage can significantly reduce both viability and fertility over time (Awda et al., 2009; Castellini et al., 2003). Importantly, there is little published information discussing the effect of ROS production on the quality of stored rabbit spermatozoa. In this experiment, the high levels of intracellular H2 O2 recorded for spermatozoa stored at 15 ◦ C indicated an increased likelihood of peroxidative damage in rabbit sperm following short-term storage at this temperature. Hydrogen peroxide (H2 O2 ) is regarded as one of the most toxic ROS for most species as it can lead to the rapid depletion of the ATP energy source and necrosis of cells (Aitken, 1995; Awda et al., 2009). In many species, peroxidative damage has been associated with decreases in sperm motility and viability (Kim et al., 2011; Leahy et al., 2010; Paulenz et al., 2002). In contrast, our results illustrate a relationship whereby the spermatozoa with high levels of intracellular H2 O2 also exhibit a significantly greater proportion of viable, acrosome-intact sperm. To our knowledge, this is the first time that this parameter has been assessed in liquid-stored rabbit sperm. Nonetheless, our findings are in accord with a study by Baumber et al. (2000) in which it was observed that an increase in ROS in horse spermatozoa did not result in an evident decrease in viability or

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acrosome integrity. These differences between species may be accounted for by a unique susceptibility of spermatozoa to oxidative stress, or perhaps in the antioxidant capacity of the sperm to offer protection from peroxidative damage. Further investigation is required. The process of cooling spermatozoa for long-term storage can lead to the disarrangement of phospholipids within the membrane (Partyka et al., 2010; Paulenz et al., 2002) and ultimately result in cold-shock. This in turn can lead to a loss of internal acrosome contents and subsequent inability of the acrosome to react at an appropriate time so as to facilitate fertilisation (Esteves et al., 2007; Partyka et al., 2010). In general, rabbit spermatozoa are considered to be more resistant to cold-shock than other domestic species, such as ram and bull, due to a high cholesterol:phospholipid ratio (Bailey et al., 2000; Mocé and Vicente, 2009) and as such, are able to better withstand cooling to lower temperatures. However, this study found that there was a significant difference between the storage temperatures, with a temperature of 5 ◦ C resulting in a lower proportion of intact membranes and acrosomes over time – typical indicators of cold shock damage. Although experiments to investigate the use of Tris-based extenders for preserving rabbit sperm viability are limited, these results are congruous with previous studies (Boiti et al., 2005; Castellini, 1996; Roca et al., 2000; Sariozkan et al., 2012) suggesting that a higher storage temperature may reduce the adverse effects of cold-shock during short-term chilled storage. Perhaps the detrimental effects associated with reduced temperatures could be alleviated with the addition of egg yolk or other protective substrates to the extender and thus permit successful storage at 5 ◦ C. In several previous studies, a positive correlation between sperm motility and high mitochondrial activity has been reported (Hallap et al., 2005; Hua et al., 2006; O’Connell et al., 2002; Padrik et al., 2010). In this experiment we observed a similar trend whereby a significant interaction between semen extender and storage temperature revealed that spermatozoa that had been diluted in Extender B and stored at 15 ◦ C exhibited both a higher TM and more functional mitochondria than Extender A at either storage temperature. As mitochondria provide sperm cells with the ATP energy necessary for the maintenance of flagellum movement (Padrik et al., 2010), it stands to reason that sperm motility is somewhat dependent on mitochondrial function and a positive correlation should exist between these two parameters. Sperm DNA integrity is of vital importance for embryo development as it determines the paternal genetic contribution to the female oocyte following fertilisation (Evenson and Jost, 2001; Partyka et al., 2010). Previous studies on human spermatozoa have reported that fertilisation by way of in vitro or in vivo methods is more likely to fail if the percentage of COMP-denatured DNA in sperm surpasses 30%; as such, this value is used by clinicians as the threshold measurement for predicting sub/infertility using SCSA technology (Evenson and Jost, 2001; Larson et al., 2000). Our results show that a high fertility potential (>74% non-denatured DNA) was maintained over time suggesting that neither semen extender nor storage temperature had a negative impact on the DNA integrity or fertilising




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capacity of the rabbit spermatozoa following 96 h of chilled storage. In Experiment 2, there were no significant differences in the rates of kindling and average litter sizes in does inseminated with sperm that had been stored for 48 h compared to those inseminated with control (fresh) sperm, regardless of storage temperature. Due to the small sample size and limitations of the statistical analyses in the pilot study, it is difficult to accurately assess the fertilising capacity of the stored semen; nonetheless these results suggest that the fertilising ability of the chilled rabbit sperm was preserved during the first 48 h of storage at 15 ◦ C. The overall rate of kindling for does inseminated with spermatozoa after 72 h of storage was significantly lower than those recorded at the previous storage times. It should be noted that the fertility rate achieved in does inseminated at 72 h with spermatozoa stored at 5 ◦ C tended to be higher than spermatozoa stored at 15 ◦ C for the same length of time, an opposite effect of temperature than that observed at the 48 h time point. In Study 1, the in vitro analyses indicated that a storage temperature of 15 ◦ C was more beneficial for the preservation of sperm quality after 96 h of chilled storage in all assessed parameters except ROS production levels; in contrast, the results of the second study suggested that the fertilising ability of rabbit spermatozoa stored at this temperature had deteriorated after 48 h, compared with spermatozoa stored at 5 ◦ C. Interestingly, in Experiment 1 we found that there was a significantly lower incidence of oxidative stress in samples held at 5 ◦ C than 15 ◦ C at 72 h and 96 h. This may be a result of a slowed metabolic function at the lower temperature and may go some way to explain the significant drop in fertility observed for does inseminated with spermatozoa stored at 15 ◦ C for 72 h compared to 48 h in Experiment 2. We hypothesise that the level of intracellular H2 O2 plays an important role in the preservation of sperm fertilising ability. Indeed, this parameter may provide a useful in vitro indicator of the potential reproductive performance of rabbit spermatozoa during long-term chilled storage. 5. Conclusion The best sperm quality, when considering in vitro assessment of total sperm motility, viability, acrosome integrity, mitochondria function and DNA integrity was obtained after storage in Extender B at 15 ◦ C, but lower ROS production was found after storage at 5 ◦ C. Similarly, after 72 h of storage, field fertility of rabbit spermatozoa was higher following incubation at 5 ◦ C compared to 15 ◦ C. This correlation between in vitro ROS production and field fertility will be investigated further. Conflict of interest statement The authors have no conflicts of interest to declare. Acknowledgements This work was supported by the Rural Industries Research and Development Council of Australia. The

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In vitro capacitation of ejaculated rabbit spermatozoa.

Capacitation of rabbit spermatozoa in vitro.

The Effect of Sperm Concentration and Storage Vessel on Quercetin-Supplemented Rabbit Semen During Chilled Storage.

The effect of caproic acid, handling technique and storage times on the fertility of fowl spermatozoa.

The yield of chromosomal aberrations in rabbit lymphocytes after irradiation in vitro and in vivo.

Microbial changes in vacuum-packed chilled pork during storage.

Semen characterization, seasonality of production, and in vitro sperm quality after chilled storage and cryopreservation in the king penguin (Aptenodytes patagonicus).

Metabolism of boar spermatozoa before, during preparation for, and after storage in liquid nitrogen.

phosphatidylcholine ratio in human spermatozoa and erythrocytes as a useful fertility marker.

Identification of spectrin and calmodulin in rabbit spermiogenesis and spermatozoa.

Expression of miRNAs in Bull Spermatozoa Correlates with Fertility Rates.

In vitro propagation of Lilium testaceum and structural investigation of the storage β-1,4-glucomannan.

Quality of chilled and cold-stored (5 °C) canine spermatozoa submitted to different rapid cooling rates.

Head morphology of ram spermatozoa is associated with their ability to migrate in vitro and correlates with fertility.

Fertility and post-thawing characteristics of deep frozen boar spermatozoa.

Definition of novel electrochemotherapy parameters and validation of their in vitro and in vivo effectiveness.

Comparison of in vitro and in vivo cell cycle parameters of lymphoid cells in pig spleens.

Fertility Preservation in Cancer Patients: In Vivo and In Vitro Options.

Phagocytosis of spermatozoa by the rabbit vagina.

Relationship between sperm apoptosis and bull fertility: in vivo and in vitro studies.

Acute toxicity of methyl isocyanate in rabbit: in vitro and in vivo effects on rabbit erythrocyte membrane.

Quality assessment of rainbow trout (Oncorhynchus mykiss) fillets during super chilling and chilled storage.

Strain, stress and stretch of peripheral nerve. Rabbit experiments in vitro and in vivo.

The binding characteristics of cholinergic sites in rabbit spermatozoa.