Acrosin activity is a suitable indicator of boar semen preservation at 17 °C when increasing environmental temperature and radiation

Theriogenology 80 (2013) 234–247

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Acrosin activity is a suitable indicator of boar semen preservation at 17
C when increasing environmental temperature and radiation E. Pinart a, *, M. Yeste b, M. Puigmulé a, X. Barrera c, S. Bonet a a

Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, Girona, Spain b Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra (Barcelona), Spain c Semen Cardona, S.L., Cardona (Barcelona), Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 September 2012 Received in revised form 4 March 2013 Accepted 3 April 2013

The effect of increasing environmental temperature and radiation on the sperm quality and the field fertility of refrigerated seminal doses from AI boars (N ¼ 30) was analyzed throughout four experimental months (from March through June). In each experimental month, analyses of sperm quality were performed at days 0, 1, 3, 5, 7, and 9 of refrigeration of seminal doses; pregnancy rate and litter size were evaluated using double monospermic inseminations of multiparous female animals using seminal doses at Days 1 to 2 and Days 3 to 4 of refrigeration. Sperm quality was assessed from the evaluation of conventional parameters of sperm concentration, sperm motility, sperm morphology, and sperm viability, and capacitation parameters of membrane lipid disorder, intracellular calcium content, and acrosin activity. Results showed that sperm quality of boar seminal doses was negatively affected by increasing temperature and radiation, which resulted in significantly decreased sperm motility and viability, acrosin activity, pregnancy rate, and litter size, and significantly increased intracellular calcium levels in the trials performed in June. In any experimental month, aging of refrigerated doses was associated with the progressive increase of intracellular calcium levels and inactivation of acrosin, that began from Day 5 of storage in the trials performed in March and April, from Day 3 in those of May, and from Day 0 in those of June. Among the sperm parameters analyzed, only acrosin activity exhibited a clearly differentiated pattern in association with increasing temperature and radiation, and a significant correlation with pregnancy rate and litter size. These results highlighted the potential role of acrosin activity as an indicator of boar sperm preservation at 17
C in boars. Ó 2013 Elsevier Inc. All rights reserved.

Keywords: Boar Seminal doses Environmental factors Sperm quality Sperm capacitation Field fertility

1. Introduction The domestic boar has been traditionally considered a nonseasonal breeder, even though the sperm quality and libido are not constant throughout the year. Despite the fact that it is generally accepted that high temperatures and long day length are inversely correlated with sperm quality [1,2] and with fertility and prolificacy [3], it is not clear * Corresponding author. Tel.: þ34 972 418366; fax: þ34 972 418300. E-mail address: [email protected] (E. Pinart). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.04.001

how increasing mean temperature and radiation alter the reproductive capacity of boars. In the porcine industry artificial insemination (AI) of female animals is usually performed using diluted seminal doses kept in refrigeration at 15
C to 17
C for at least 24 hours. These seminal doses are diluted in commercial extenders that provide an energy source, ensure a proper pH and osmotic pressure, protect spermatozoa against thermal shock, and inhibit bacterial growth [4–6]. Prolonging storage time might increase the economic benefit and production efficiency, making the choice of ejaculates

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All chemicals were obtained from Sigma-Aldrich Química, S.A. (Madrid, Spain) unless otherwise indicated.

Girona, Spain) to be packed into 90-mL commercial doses at a concentration of 3  109 spermatozoa per dose. Monospermic commercial doses were then refrigerated at 17
C, and one dose per boar per collection was sent to our laboratory in a heat-insulating recipient at 17
C, whereas the other doses were kept at 17
C and used in AI programs; refrigerator temperature was controlled regularly with a thermometer installed inside it. To analyze the effect of refrigeration on field fertility the AI seminal doses obtained from each single boar ejaculate were separated into two equivalent portions: one portion was used for AI after 1 to 2 days of refrigeration, and the other portion was used after 3 to 4 days. Lab doses were also kept in refrigeration at 17
C for 9 days, in a refrigerator temperature-controlled periodically by a thermometer installed inside it. The analysis of sperm quality of seminal doses was performed the same collection day (Day 0) and at Days 1, 3, 5, 7, and 9 of refrigeration, for the evaluation of sperm concentration, sperm motility, sperm morphology, sperm viability, and the analysis of membrane lipid disorder, intracellular calcium content, and acrosin activity, as indicators of capacitation status of spermatozoa. The same schedule was repeated throughout the four experimental months (March, April, May, and June), characterized by the increase of environmental radiation and minimum and maximum temperature of the farm where boars are placed. Data from cumulative solar radiation and from minimum, maximum, and mean temperatures throughout the experimental period were provided by the Servei Meterològic de Catalunya, and the means were used for further statistical analyses. During the experimental period a progressive increase of mean cumulative solar radiation from 172.1 W/m2 in March to 292.9 W/m2 in June was recorded, and of minimum (3.6
C– 12.8
C), maximum (13.9
C–25.9
C), and mean temperature (7.5
C–18.9
C); detailed variations of mean solar radiation and mean temperature throughout the experimental period are given in Figure 1.

2.2. Sperm samples

2.3. pH

The study was performed using 30 sexually mature 2year-old Piétrain boars of the same genetic line (Semen Cardona, Cardona, Spain), which had been previously used in artificial insemination (AI) programs. All boars produced ejaculates of high sperm quality, with more than 80% of spermatozoa without morphoabnormalities, 80% total motility, 60% progressive motility, 80% sperm viability, and 80% nonreacted acrosomes (data not shown). Before and throughout the four experimental months, the boars were kept in the same husbandry conditions, i.e., lodged in the same pens, fed according to standard protocols, and provided with water ad libitum, and subjected to a semen collection rhythm of twice per week following the guidelines established by the Animal Welfare Directive of the Autonomous Government of Catalonia. Ejaculates were obtained by the same expert technician with the gloved-hand technique. The sperm-rich fraction of each ejaculate was filtered to remove the gel, prediluted 2:1 (vol/vol) in an extra–long-term extender (Duragen; Magapor, S.L., Zaragoza, Spain) at 37
C inside a sterile collecting recipient, and transported to the AI center (BioGirona, S.L.,

Measurement of pH of seminal doses was performed in triplicate using a digital pH meter (Hanna

2.1. Material

360 340

20

Cumulative solar radiation Mean temperature

18

320 16

300 280

14

260 12

240 220

10

200 8

180 160

Mean Temperature (ºC)

2. Materials and methods

2 Cumulative solar radiation (W/m )

and extenders a major focus of concern for the swinebreeding industry [7,8]. The selection criteria of ejaculates are based on the sperm quality immediately after semen collection, whereas the extender selection is totally empirical depending on the estimated time of storage of refrigerated doses or the distribution distance [7]. Therefore, an extended range of commercial diluents have been developed for optimal preservation of boar semen at 15
C to 17
C at short-term (3–4 days), medium-term (5–6 days), long-term (7–8 days), and extra–long-term (nine or more days) [8]. Nevertheless, despite the type of extender used, most inseminations are performed before 3 days after semen collection, because of the decrease in litter size from the fourth day of refrigeration with any extender type [9,10]. Conventional semen evaluation for AI usually includes the measure of seminal volume and sperm concentration, and the percentage of progressive motile and morphologically normal spermatozoa [6,11]. Several authors suggest that this information, though important, does not accurately predict the fertility of seminal doses kept in refrigeration [6,11,12]. Different studies state the importance of assessing the sperm functionality in a semen sample to better predict its fertilizing ability [13,14]. In the present approach we analyze whether changes in environmental temperature and radiation affect the sperm quality of seminal doses throughout 9 days of refrigeration. This approach has been performed on the basis of conventional and capacitation sperm parameters, and on field fertility trials.

6 1 0

10 0 March

April

May

J u ne

Month Fig. 1. Measurements (mean  SEM) of photoperiod (cumulative solar radiation, W/m2) and mean temperature (
C) during the experimental period.

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HI-254 pH/ORP Meter; Hanna Instruments, S.L., Eibar, Spain). For each month and day of refrigeration, pH is expressed as the mean  SEM (N ¼ 30).

2.6. Sperm morphology To assess sperm morphology, 5 mL of each seminal dose was placed on a slide and mounted with a coverslip. Slides were then incubated for 30 minutes in 100% humidity at 25
C to immobilize spermatozoa. Sperm morphology was analyzed at magnification 200 using a negative phasecontrast objective coupled to an Olympus BX41 microscope (Olympus Europe GmbH), and equipped with the software SCA Production (Sperm Class Analyzer Production, 2010; Microptic, S.L.). For each boar and day of refrigeration, 100 spermatozoa from three different replicates were classified as normal, with proximal or distal droplet, or aberrant. Because the percentage of spermatozoa with proximal or distal droplet and the percentage of aberrant spermatozoa was maintained at less than 5% in all trials, for each month and day of refrigeration the sperm morphology is expressed as the percentage of normal spermatozoa (mean  SEM; N ¼ 30).

2.4. Sperm concentration Sperm concentration was assessed by measuring the absorbance at 546 nm in a Minitub SDM5 spectrophotometer (Minitub Ibérica, S.L., La Selva del Camp, Spain). Three replicates per sample and day of refrigeration were measured. For each month and day of refrigeration, the sperm concentration is expressed as the number of spermatozoa  106/mL (mean  SEM; N ¼ 30). 2.5. Sperm motility Sperm cells were preheated at 37
C for 20 minutes and then a 20-mL droplet was mounted in a Makler chamber (Sefi Medical Instruments, Haifa, Israel). The sperm motility was examined at magnification 100 using a negative phase-contrast objective coupled to an Olympus BX41 microscope (Olympus Europe GmbH, Hamburg, Germany), and equipped with the software SCA 5 (Sperm Class Analyzer 5, 2010, Microptic, S.L., Barcelona, Spain). A minimum of three replicates per ejaculate with 1000 spermatozoa in each replicate were analyzed. Twenty-five consecutive digitalized frames were acquired in each field, and two motility parameters were assessed: total motility and progressive motility (spermatozoa showing more than 45% of straightness). For each month and day of refrigeration, the sperm motility is expressed as the percentage of total motile spermatozoa (Fig. 2) and the percentage of progressive motile spermatozoa (Fig. 3) (mean  SEM; N ¼ 30).

2.7. Flow cytometry Information about flow cytometry analyses is given according to the recommendations of the International Society for Advancement of Cytometry [15]. These analyses were conducted to evaluate sperm viability, membrane lipid disorder, and intracellular calcium levels. The sperm concentration in each treatment was adjusted to 1  106 spermatozoa per mL1 in a final volume of 0.5 mL, and three measures per sample and male were performed for each flow cytometric assay. Spermatozoa were then stained with the appropriate combinations of fluorochromes, following the protocols described herein. Samples were evaluated using a Cell Laboratory QuantaSC cytometer (Beckman Coulter; Fullerton,

March April May June

100

a

90

a

a a a

% Total motile spermatozoa

80

a b

b

c

70

c c

60

c d d

d, e

50

e 40 30 20 10

f

f

f

0 0

1

2

3

4

5

6

7

8

9

Storage time at 17 ºC (Days) Fig. 2. Percentage of total motile spermatozoa of semen collected in March, April, May, and June after a 9-day period of storage at 17
C. Different letters (a–f) indicate significant differences (P < 0.05) among months and throughout storage time at 17
C (i.e., month by days of storage).

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237

March April May June

80

70

% PMOT spermatozoa

60

a

a a a

50

a a

a

b

b

40

b c

c, d

30

20

d

d d e

f g

10

h

h

0 0

1

2

3

4

5

6

7

h 8

9

Storage time at 17 ºC (Days) Fig. 3. Percentage of progressive motile (PMOT) spermatozoa (VAP > 45 mm/s1) of semen collected in March, April, May, and June after a 9-day period of storage at 17
C. Different letters (a–d) indicate significant differences (P < 0.05) among months and throughout storage time at 17
C (i.e., month by days of storage). VAP, average velocity of sperm cells.

CA, USA; Serial number AL300087; technical specification available at: https://www.beckmancoulter.com/wsrportal/ ajax/downloadDocument/721742AD.pdf?autonomyId=TP_ DOC_32032&documentName=721742AD.pdf). This instrument, which had not been altered in the original configuration provided by the manufacturer, was equipped with two light sources: an arch-discharge lamp and an argon ion laser (488 nm) set at a power of 22 mW. In our case, only the single-line visible light (488 nm) from argon laser was used to perform our analyses. Cell diameter and volume was directly measured with the Cell Lab Quanta SC cytometer employing the Coulter principle for volume assessment, which is based on measuring changes in electrical resistance produced by nonconductive particles suspended in an electrolyte solution. This system has thus forward scatter replaced by electronic volume (EV). Furthermore, the EV channel was calibrated using 10-mm Flow-Check fluorospheres (Beckman Coulter) by positioning this size bead in channel 200 on the volume scale. Optical filters were also original and they were FL1, FL2, and FL3. In this system, the optical characteristics for these filters were: FL1 (green fluorescence): Dichroic/ Splitter, dichoric long pass: 550 nm, band-pass filter: 525 nm, detection width 505 nm to 545 nm; FL2 (orange fluorescence): dichoric long pass: 600 nm, band-pass filter: 575 nm, detection width: 560 nm to 590 nm; and FL3 (red fluorescence): low pass filter: 670, detection width: 670  30 nm. Signals were logarithmically amplified and photomultiplier settings were adjusted to particular staining methods. FL1 was used to detect green fluorescence (SYBR14, YO-PRO-1 [YP1], and Fluo3-acetomethoxy ester [Fluo-3-AM]), and FL3 was used to detect propidium iodide (PI) and Merocyanine 540 (M540; Fluka).

Sheath fluid flow rate was set at 4.17 mL per minute in all analyses, and EV and side scatter (SS) were recorded in a linear mode (in EV vs. SS dot plots) for a minimum of 10,000 events per replicate. The analyzer threshold was adjusted on the EV channel to exclude subcellular debris (particles with diameter <7 mm) and cell aggregates (particles with diameter >12 mm). Therefore, the spermspecific events, which usually appeared in a typically L-shaped scatter profile, were positively gated on the basis of EV and SS distributions, and the others were gated out. In some protocols compensation was used to minimize spillover of green fluorescence into the red channel. Information on the events was collected in list-mode data files. These generated files were then analyzed using Cell Lab Quanta SC MPL Analysis Software (version 1.0; Beckman Coulter) to quantify the dot-plot sperm populations (FL1 vs. FL3) and to analyze the cytometric histograms. 2.7.1. Sperm viability (SYBR14 and PI) Sperm viability was assessed by checking the membrane integrity using the dual fluorescent probe of SYBR14 and PI (LIVE/DEAD Sperm Viability kit, Molecular Probes, Invitrogen; L-7011). SYBR14 is a membrane-permeable dye, which stains the head of spermatozoa in green (viable spermatozoa). Propidium iodide is a membraneimpermeable dye that only penetrates through a disrupted plasma membrane, staining the sperm heads in red (nonviable spermatozoa). Propidium iodide displaces or quenches SYBR14 fluorescence [16]. Briefly, the staining procedure was [17]: (1) the samples were diluted with Beltsville thawing solution to a concentration of 10  106 spermatozoa per mL; (2) aliquots of 500 mL from diluted samples were stained with 0.5 mL of SYBR14 working

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E. Pinart et al. / Theriogenology 80 (2013) 234–247

100

95

a

% Viable spermatozoa

a a

a

90

a

a

a a

a, b b

a, b b

85

b, c

c 80

75

March April May June

70

10

d

0 0

1

2

3

4

5

6

7

8

9

Storage time at 17 ºC (Days) Fig. 4. Percentage of viable spermatozoa of semen collected in March, April, May, and June after a 9-day period of storage at 17
C. Different letters (a–d) indicate significant differences (P < 0.05) among months and throughout storage time at 17
C (i.e., month by days of storage).

solution (final concentration of 100 mM) for 10 minutes at 37
C in darkness; and then (3) counterstained with 2.5 mL of PI (2.4 mM) for 5 minutes before the cytometric flow analysis. Viable spermatozoa exhibited positive staining for SYBR14 and negative staining for PI (SYBR14þ/IP). For each experimental month and day of refrigeration, sperm viability was assessed in triplicate. Results are expressed as the percentage of viable spermatozoa (mean  SEM; N ¼ 30) (Fig. 4). 2.7.2. Membrane lipid disorder (M540 and YP1) Capacitation involves a wide array of changes, including a packaging decrease of membrane phospholipids, making the sperm plasma membrane less stable, and an increase of intracellular calcium levels [17]. For this reason, the present study assessed changes in sperm capacitation using these two markers. Membrane lipid disorder was determined using M540 (Fluka, 63876) and YP1 (Molecular Probes, Invitrogen; Y3603) following the staining procedure described by Januskauskas et al. [18]. Sperm samples were incubated for 10 minutes at 37
C with 1.3 mL of M540 working solution (1 mM) and 0.5 mL of YP1 working solution (1 mM). Merocyanine 540 increases its orange fluorescence in the presence of decreasing phospholipid membrane packaging [19]. Therefore, this staining technique leads to the identification of four different types of spermatozoa [17]: (1) nonviable spermatozoa with low membrane lipid disorder (M540/YP1þ); (2) nonviable spermatozoa with high membrane lipid disorder (M540þ/YP1); (3) viable spermatozoa with low membrane lipid disorder (M540/YP1); and (4) viable spermatozoa with high membrane lipid disorder (M540þ/YP1). For each month and day of refrigeration the percentage of viable spermatozoa with high membrane lipid disorder of each semen dose was calculated as the mean of three measures performed. Results are

expressed as the percentage of viable spermatozoa with high membrane fluidity (Fig. 5) (mean  SEM; N ¼ 30). 2.7.3. Determination of intracellular calcium levels (Fluo-3-AM and PI) Intracellular calcium levels were determined using Fluo3-AM (Molecular probes, Invitrogen, F-1241) fluorochrome, which increases its fluorescence when bound to Ca2þ [20]. The staining procedure was briefly: 0.5 mL of Fluo-3-AM was added in 500 mL of diluted sperm aliquots for 5 minutes at 37
C and in darkness. Then 2.5 mL of PI was added and incubated for 5 minutes in the same conditions; after incubation, the percentage of viable (PI) spermatozoa stained with Fluo-3-AM (Fluo3þ/PI) was measured. Results are expressed as the mean percentage of spermatozoa Fluo3þ/PI  SEM (N ¼ 30) (Fig. 6). For each month and day of refrigeration, the intracellular calcium levels of spermatozoa were inferred from the geometric mean (GeoMean) of the Fluo-3-AM fluorescence intensity of each viable spermatozoa collected in FL1 from a sample (Fluo3þ) [17]. Results are expressed as the mean intracellular calcium levels of spermatozoa (GeoMean Fluo3þ; mean  SEM; N ¼ 30) (Fig. 7). 2.8. Acrosin activity Sperm acrosin activity was determined spectrophotometrically according to the method modified by Langlois et al. [21] that has been adapted in our lab [22]. For each sample, 250 mL containing 5  106 spermatozoa were centrifuged at 1000  g for 30 minutes over 500 mL of 11% Ficoll to separate the spermatozoa from the extender. The sperm pellet was subsequently suspended in 1 mL of a buffer solution containing detergent (Triton X-100) and 23 mM of N-a-benzoyl-DL-arginine l-nitroanilide hydrochloride substrate. Hydrolyzed N-a-benzoyl-DL-arginine

E. Pinart et al. / Theriogenology 80 (2013) 234–247

239

March April May June

+

Membrane lipid disorder (% spermatozoa M540 )

20

15

e d, e d

10

d c, d

c, d

5

a b b

c

c, a

a

a

a

a, b

a, b

b

a

b

0 0

1

2

3

4

5

6

7

8

9

Storage time at 17 ºC (Days) Fig. 5. Percentage of spermatozoa with membrane lipid disorder of seminal doses stored at 17
C for a 9-day period in March, April, May, and June. Different letters (a–e) indicate significant differences (P < 0.05) among months and throughout storage time at 17
C (i.e., month by days of storage). M540þ, positive stain for merocyanine 540.

l-nitroanilide hydrochloride by acrosin released the chromoforic product 4-nitroanilin that was detected at 410 nM on a SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) after 3 hours of incubation at room temperature. Total acrosin activity (mUI acrosin per 106 spermatozoa) was calculated using the formula described in Langlois et al. [21]. For each month and day of collection results are expressed as the mean  SEM (N ¼ 30) (Fig. 8).

2.9. Field fertility The sows used in the fertility trials were multiparous (sow parity, 5–8 times), fed with an adjusted diet (2.2 kg/ day), consisting of a basal diet and 0.4% premix specific for gestating sows (P462N; TecnoVit), and housed in pens on a commercial pig farm in Girona (Spain). In each experimental month, and according to the insemination programming system of production all in-all out, the

60

March April May June

55 50 45

f d a, d

+

% Spermatozoa Fluo3 /PI

-

a, d

40

a

b

b

30

a

a

35

b

b

b

b e

25

e

20

d

d

15

c

c

c

10

c, d c

5 0 0

1

2

3

4

5

6

7

8

9

Storage time at 17 ºC (Days) Fig. 6. Percentage of Fluo3þ/PI spermatozoa of seminal doses stored at 17
C for a 9-day period in March, April, May, and June. Different letters (a-e) indicate significant differences (P < 0.05) among months and throughout storage time at 17
C (i.e., month by days of storage). Fluo3þ/PI, positively stained for Fluo-3-acetomethoxy ester and negatively stained for propidium iodide.

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E. Pinart et al. / Theriogenology 80 (2013) 234–247

70

Intracellular calcium levels (GeoMean Fluo3 )

65

March April May June

60 55

e d, e d

50

d

45 40 35

25 20

a

a

a, b b

a, b

a a

a a

a a

30

b

b

b

b

c

c

c

c

15

c

10 5 0 0

1

2

3

4

5

6

7

8

9

Storage time at 17 ºC (Days) Fig. 7. Intracellular calcium levels of spermatozoa, assessed as geometric mean (GeoMean) of green fluorescence collected in FL1 (Fluo3þ), of seminal doses stored at 17
C for a 9-day period in March, April, May, and June. Different letters (a–e) indicate significant differences (P < 0.05) among months and throughout storage time at 17
C (i.e., month by days of storage).

number of weaned sows were equally divided into two groups: one group of sows (N ¼ 900) was inseminated with seminal doses kept in refrigeration for 1 and 2 days (Days 1–2), and the second group of sows (N ¼ 900) was inseminated with seminal doses kept in refrigeration for 3 and 4 days (Days 3–4). All sows were weaned at 21 days, and ovulation was then hormonally synchronized using 1250 IU of equine chorionic gonadotropin 24 hours after weaning (eCG;

Folligon; Intervet, S.A., Salamanca, Spain), followed by 750 IU of human chorionic gonadotrophin 72 hours later (hCG; Veterin Corion; Divasa, Farmavic, S.A., Barcelona, Spain). The route of administration was im in the side of the neck. After 24 hours of administration of eCG, estrous detection was carried out by the same expert technician in all trials, with the standing reflex being tested once a day. Sows were artificially inseminated twice per estrous using monospermic seminal doses from the same boar, within

90

a

a

a a

a

80

a

a

a a

a

6

Acrosin activity (µUI acrosin per 10 spz)

85

a, b a, b

75

b c 70

c

b, c

c, d c, d

65

c, d

d 60 55

March April May June

50

e

0 0

1

2

3

4

5

6

7

8

9

Storage time at 17 ºC (Days) Fig. 8. Sperm acrosin activity (mUI per 106 spermatozoa) of semen samples collected in March, April, May, and June after a 9-day period of storage at 17
C. Different letters (a–e) indicate significant differences (P < 0.05) among months and throughout storage time at 17
C (i.e., month by days of storage). spz, spermatozoa.

E. Pinart et al. / Theriogenology 80 (2013) 234–247

an interval of 24 hours. Artificial inseminations were carried out using a conventional AI method with a cervical catheter (Magapor, S.L.) lubricated with mineral oil (Fertilube; Magapor, S.L.), with the sows being kept in normal farm conditions. Seminal doses used in AI were maintained in refrigeration. At insemination time, mean values of ambient parameters in each experimental month were: 5.3
C of mean temperature, 9.1
C of maximum temperature, 0.8
C of minimum temperature, and 140.5 W/m2 of mean radiation in March; 15.1
C of mean temperature, 22.6
C of maximum temperature, 9.1
C of minimum temperature, and 232.5 W/m2 of mean radiation in April; 14.8
C of mean temperature, 21.1
C of maximum temperature, 9.6
C of minimum temperature, and 256.3 W/m2 of mean radiation in May; and 17.9
C of mean temperature, 25.0
C of maximum temperature, 12.4
C of minimum temperature, and 272.8 W/m2 of mean radiation in June. The field fertility was determined according to the diagnosis of pregnancy using ultrasonography with an Echoscan T-100 scanner (Importvet, S.A., Barcelona, Spain) at 60 days after insemination. Fertility of the male animals is expressed as the pregnancy rate using seminal doses maintained in refrigeration for 1 to 2 days and 3 to 4 days (mean  SEM; N ¼ 30). Litter size was measured at parturition, counting the total number of piglets born per litter and the number of live and dead piglets per litter (mean  SEM; N ¼ 30). 2.10. Statistical analyses Statistical analyses were performed using SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA) and Origin Pro 8.0 software (OriginLab Corp., Northampton, MA, USA). Sperm quality and function parameters (i.e., sperm concentration, sperm motility, sperm morphology, sperm viability, membrane lipid disorder intracellular calcium levels, and acrosin activity) were considered dependent variables, and each seminal sample from each boar at each relevant month was treated as a biological replicate. All the variables were first tested for normality (Kolmogorov–Smirnov test) and homocedasticity (Levene test). Further, data on percentages were recalculated using the arcsine square root () transformation, and a repeated measures ANOVA (in which the refrigeration time was the intrasubject factor, the month was the intersubject factor, and each sperm parameter was the dependent variable) was performed for determining changes in refrigerated doses among extraction months (i.e., March, April, May, and June). A post hoc Sidak test was used for multiple comparisons. Correlations between sperm parameters (sperm concentration, sperm motility, sperm morphology, sperm viability, membrane lipid disorder, intracellular calcium levels, and acrosin activity) and pregnancy rate and litter size were calculated using the Pearson correlation. Finally, sperm parameters or field fertility parameters were also correlated with environmental conditions (mean, maximal, and minimal temperatures, and mean solar radiation). In all these cases, pregnancy rates (NRR60d) were previously transformed using logit transformation (logit ¼ ln (NRR60d/

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1  NRR60d), the log-odds being used for subsequent calculations (i.e., Pearson correlation). In all statistical analyses, the significance level was set at 5%. Results are expressed as mean  SEM. 3. Results 3.1. Sperm concentration and pH In the present approach, the sperm concentration and pH were used, respectively, as estimators of sperm agglutination and bacterial proliferation during the storage period. Data showed nonsignificant differences among months in the sperm concentration of the seminal doses throughout the 9 days of refrigeration (P > 0.05), thus indicating a lack of sperm agglutination of the seminal doses throughout the 9 days of refrigeration in any of the evaluated experimental months. Similarly, pH of seminal doses did not differ significantly among experimental months (P > 0.05) or among refrigeration days (P > 0.05) with mean values of 7.33  0.21. pH stability was indicative of absence of bacterial proliferation in seminal doses during the 9 days of storage in any experimental month. 3.2. Sperm morphology Sperm morphology did not differ significantly among experimental months (P > 0.05) or throughout the storage period (P > 0.05). In each experimental month, mean percentages of normal spermatozoa were 92.2  3.9% in March, 92.2  3.1% in April, 92.9 1.7% in May, and 94.2  1.7% in June. In each storage day, mean percentages of this parameter were: 94.1  1.6% at Day 0, 93.6  2.3% at Day 1, 91.6  3.9% at Day 3, 91.3  3.4% at Day 5, 91.3  3.9% at Day 7, and 91.6  3.9% at Day 9. The analysis of sperm morphology using phase contrast microscopy did not show signs of bacterial contamination throughout the refrigeration period in any of the sperm doses analyzed. Analysis of correlation indicated that the sperm morphology of seminal doses was not correlated with increasing environmental temperature or mean radiation (Table 1). 3.3. Sperm motility Changes in the percentage of total motile spermatozoa throughout the 9 days of refrigeration in each of the experimental months are represented in Figure 2. At Day 0, the percentage of total motile spermatozoa was statistically similar in all months; however, differences existed in the trend of this parameter throughout the refrigeration period according to the experimental month. Therefore, in March the percentage of total motile spermatozoa maintained constant high values from Day 1 to 7 of refrigeration, but it decreased significantly at Day 9 (P < 0.05). The percentage of total motile spermatozoa significantly decreased at Day 7 of refrigeration in April (P < 0.05) and at Day 5 in May (P < 0.05); in both experimental months, a further significant decrease of this parameter was observed at Day 9

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Table 1 Correlations between temperature, photoperiod, and sperm parameters. Sperm parameter

Viable spermatozoa, % Spermatozoa with high membrane fluidity (M540þ/PI), % Spermatozoa with high Ca2þ levels, % Intracellular Ca2þ levels (GeoMean F1) Acrosin activity (mUI acrosin per 106 spermatozoa) Total motile spermatozoa, % Progressive motile spermatozoa, % Morphologically normal spermatozoa, %

Temperature

Mean radiation

Mean

Maximum

Minimum

0.30* 0.31* 0.41** 0.34* 0.40** 0.09 0.29* 0.17

0.31* 0.35* 0.38** 0.30* 0.30** 0.02 0.29* 0.15

0.33* 0.39* 0.46** 0.45** 0.51*** 0.13 0.31* 0.17

0.22 0.33* 0.29* 0.32* 0.32* 0.16 0.21 0.19

No superscript symbols indicate no significant correlation. Superscript symbols indicate significant differences as follows: * P < 0.05; ** P < 0.01; *** P < 0.005. Abbreviations: GeoMean, geometric mean; M540þ/PI, positively stained for Fluo-3-acetomethoxy ester and negatively stained for propidium iodide.

(P < 0.05). In June, the percentage of total motile spermatozoa decreased significantly at Day 1 of refrigeration (P < 0.05) and again at Day 5 (P < 0.05). The pattern of variation of the percentage of progressive motile spermatozoa during the refrigeration period of March, April, May, and June is shown in Figure 3. The percentage of progressive motile spermatozoa did not differ significantly among months at Day 0 (P > 0.05). Nevertheless, as for total motile spermatozoa, this parameter showed a different trend throughout the refrigeration period according to the experimental month. Therefore, in March, the percentage of progressive motile spermatozoa decreased significantly at Day 3 and at Day 9 (P < 0.05), whereas in April it decreased at Days 7 and 9 of refrigeration (P < 0.05). In May, a significant decrease of this parameter was only observed at Day 5 of refrigeration (P < 0.05), whereas in June it dropped significantly at Day 1 (P < 0.05), Day 3 (P < 0.05), and Day 5 (P < 0.05), reaching values near zero. The analysis of sperm motility using phase contrast microscopy did not show signs of bacterial proliferation throughout the refrigeration period in any of the sperm doses analyzed. The analysis of correlation indicated that the percentage of total motile spermatozoa was not correlated with increasing temperature or radiation. In contrast, the percentage of progressive motile spermatozoa was inversely correlated with minimum, maximum, and mean temperatures, but not with mean radiation (Table 1). 3.4. Sperm viability The percentage of viable spermatozoa (SYBR14þ/PI) did not differ among months at Day 0, despite that significant differences existed among trials in the trend of this parameter (Fig. 4). In March and May, the sperm viability remained constant from Day 1 to Day 7 of refrigeration, but it decreased significantly at Day 9 (P < 0.05). In April, the percentage of viable spermatozoa remained without significant variations throughout the nine storage days. The sperm viability of the trial performed in June maintained high values from Day 1 to Day 5, but it decreased significantly at Day 7 (P < 0.05) and at Day 9 (P < 0.05) of refrigeration. At the end of the trial the sperm viability was significantly lower in June than in March (P < 0.05), April (P < 0.05), and May (P < 0.05).

Correlation analysis indicated that sperm viability was negatively correlated with increasing minimum, maximum, and mean temperature, and no significant correlation was found with increasing mean radiation (Table 1). 3.5. Membrane lipid disorder At Day 0 the percentage of spermatozoa with high membrane lipid disorder (M540þ/YP1) was significantly lower in March and April than in May (P < 0.05) and June (P < 0.05). Moreover, throughout the storage days this sperm parameter exhibited a different variation pattern depending on the month (Fig. 5). Therefore, in March the percentage of spermatozoa with high membrane fluidity was low at Days 1 and 3 of refrigeration, but it increased significantly at Day 5 (P < 0.05) and again at Day 7 (P < 0.05). In April, the percentage of spermatozoa with high membrane lipid disorder was maintained without change until Day 5, but it increased significantly at Day 7 (P < 0.05) and Day 9 (P < 0.05). The percentage of spermatozoa with high membrane lipid disorder was low and constant from Day 1 to 7 of refrigeration in the experiment performed in May, but it increased significantly at Day 9 (P < 0.05). In June, this parameter maintained low values only at Day 1, and it increased significantly at Day 3 (P < 0.05), Day 7 (P < 0.05), and Day 9 (P < 0.05) of storage. At the end of refrigeration period, the frequency of spermatozoa with high membrane lipid disorder was significantly lower in the trial performed in March and significantly higher in the trial performed in June (P < 0.05). Analyses of correlation indicated that the percentage of spermatozoa with high membrane lipid disorder showed a significant and positive correlation with increasing minimum, maximum, and mean temperatures, and with increasing mean radiation (Table 1). In contrast, membrane lipid disorder was significantly and negatively correlated with the percentage of total motile spermatozoa (r ¼ 0.61; P < 0.005) and the percentage of progressive motile spermatozoa (r ¼ 0.75; P < 0.005). 3.6. Intracellular calcium levels At Day 0 the percentage of viable spermatozoa stained with Fluo-3-AM did not differ between March and April, but it was significantly higher in May and in June (P < 0.05)

E. Pinart et al. / Theriogenology 80 (2013) 234–247

(Fig. 6). Moreover, throughout the storage period, the percentage of Fluo3þ/PI spermatozoa was significantly lower in the trials performed in March and April than in those performed in May (P < 0.05) and June (P < 0.05). In March and April, this parameter did not differ from Day 1 to Day 5 of storage, but it increased significantly at Day 7 (P < 0.05) and at Day 9 (P < 0.05). In May, the percentage of Fluo3þ/PI spermatozoa remained constant until Day 7, but it increased significantly at Day 9 (P < 0.05). In June, this parameter did not differ from Day 1 to Day 5 of refrigeration but it increased significantly at Day 7 (P < 0.05) and again at Day 9 (P < 0.05). Correlation analyses indicated that the percentage of Fluo3þ/PI spermatozoa was significantly and positively correlated with increasing environmental temperature and mean radiation (Table 1). Conversely, this parameter correlated negatively and significantly with the percentage of total (r ¼ 0.37; P < 0.01) and progressive (r ¼ 0.76; P < 0.005) motile spermatozoa. Finally, as expected, intracellular calcium levels were positively and significantly correlated with lipid disorder of sperm membrane (r ¼ 0.69; P < 0.005). At Day 0 the levels of intracellular calcium were statistically lower in April and higher in June (Fig. 7). Despite that the levels of intracellular calcium of spermatozoa were significantly lower in April than in March (P < 0.05), this sperm parameter showed a similar pattern in both trials. Therefore, it remained constant until Day 7 of storage, but it increased significantly at Day 9 (P < 0.05). In May, the intracellular calcium levels increased significantly at Day 3 (P < 0.05) and Day 7 (P < 0.05). In June, intracellular calcium levels did not differ between Day 0 and Day 5 of refrigeration, but they increased significantly at Day 7 (P < 0.05) and at Day 9 (P < 0.05). At the end of the refrigeration period, the levels of intracellular calcium were significantly lower in the trials performed in March and April than in the trials performed in May (P < 0.05) and June (P < 0.05). Correlation analyses showed that the intracellular calcium levels were positively correlated with increasing minimum, maximum, and mean temperatures and also with increasing mean radiation (Table 1). Intracellular calcium levels correlated negatively with the percentage of total (r ¼ 0.73; P < 0.005) and progressive (r ¼ 0.69; P < 0.005) motile spermatozoa, and positively with the membrane lipid disorder (r ¼ 0.76; P < 0.005). 3.7. Acrosin activity At Day 0 sperm acrosin activity did not differ among March, April, and May, but it was significantly lower in June (P < 0.05) (Fig. 8). Differences were not found in the acrosin activity throughout the storage days between March and April. In contrast, in May and June, the acrosin activity decreased significantly at Day 5 (P < 0.05) and at Day 9 (P < 0.05) of refrigeration. Analyses of correlation demonstrated that the acrosin activity was negatively correlated with increasing environmental temperature and mean radiation (Table 1). Acrosin activity correlated positively with the percentage of total (r ¼ 0.39; P < 0.01) and progressive (r ¼ 0.71;

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Table 2 Fertility and prolificacy rates of boars involved in this study per month. Month Day (AI)

Fertility rate (NRR60d)

March

89.7 90.2 87.8 87.5 85.5 84.9 83.3 83.6

April May June

1–2 3–4 1–2 3–4 1–2 3–4 1–2 3–4

       

2.0 2.2 1.8 1.9 1.7 1.8 1.6 1.7

Litter size

Piglets born Alive

13.6 13.5 13.4 13.2 13.0 12.9 11.9 11.2

       

0.5 0.6 0.4 0.5 0.6 0.4 0.5a 0.6a

12.7 12.5 12.3 12.2 12.1 11.8 10.9 10.2

       

Dead 0.4 0.5 0.5 0.5 0.4 0.4 0.3a 0.4a

0.9 1.0 1.1 1.0 0.9 1.1 1.0 1.0

       

0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Results are expressed as mean  SEM (N ¼ 30). Abbreviation: NRR60d, non return rate at 60 days post-insemination. a Values statistically different among months (P < 0.05).

P < 0.005) motile spermatozoa, and negatively with the sperm membrane lipid disorder (r ¼ 0.52; P < 0.005), the percentage of spermatozoa with high levels of intracellular calcium (r ¼ 0.89; P < 0.005), and the intracellular calcium levels of spermatozoa (r ¼ 0.82; P < 0.005). 3.8. Field fertility Data from field fertility obtained in AI trials performed during the different experimental months are detailed in Table 2. Data showed a progressive and significant decrease in pregnancy rate and litter size from March through June (P < 0.05). Compared with March, in June the decrease in pregnancy rate was 7% in the inseminations performed at Days 1 to 2 and at Days 3 to 4 of refrigeration; the decrease in litter size was 12.5% in the inseminations performed at Days 1 to 2, and 17% in those performed at Days 3 to 4 of refrigeration. In May, decreases in pregnancy rate and litter size were not significant, being respectively, 5% and 4.5% at Days 1 to 2 of refrigeration, and 6% and 4.5% at Days 3 to 4. Interestingly, the decrease in litter size was because of a decrease in the number of piglets born alive, with the number of piglets born dead unaffected. Another interesting finding was that within the same experimental month the pregnancy rate and litter size of refrigerated seminal doses did not differ significantly between Days 1 to 2 and Days 3 to 4. Correlation analyses with environmental parameters indicated that pregnancy rate of seminal doses was negatively correlated with increasing minimum, maximum, and mean temperatures, but it did not correlate with increasing mean radiation (Table 3). Correlation analyses with sperm parameters showed that pregnancy rate was positively correlated with sperm viability and acrosin activity (Table 4). Litter size was negatively correlated with increasing environmental temperature, and with mean radiation (Table 3). Analyses of correlation with sperm parameters showed a significant positive correlation with sperm viability (P < 0.05), and a significant negative correlation with intracellular calcium levels (P < 0.05) (Table 4). The number of dead piglets per litter did not show any significant correlation with environmental parameters (Table 2) or sperm quality parameters (Table 3), with the exception of acrosin activity (P < 0.05).

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Table 3 Correlations between temperature, photoperiod, pregnancy rate, and litter size. Parameters

Temperature

Mean radiation

Mean

Maximum

Minimum

Pregnancy rate (NRR60d) Litter size (total piglets born) Piglets born alive Piglets born died

0.71**

0.69*

0.73**

0.48

0.67*

0.70*

0.65*

0.59*

0.76** 0.48

0.63* 0.40

0.54 0.47

0.65* 0.55

No superscript symbol indicates no significant correlation. At insemination time, mean values of ambient parameters in each experimental month were: in March, 5.3
C (mean temperature), 9.1
C (maximum temperature), 0.8
C (minimum temperature), and 140.5 W/m2 (mean radiation); in April, 15.1
C (mean temperature), 22.6
C (maximum temperature), 9.1
C (minimum temperature), and 232.5 W/m2 (mean radiation); in May, 14.8
C (mean temperature), 21.1
C (maximum temperature), 9.6
C (minimum temperature), and 256.3 W/m2 (mean radiation); and in June, 17.9
C (mean temperature), 25.0
C (maximum temperature), 12.4
C (minimum temperature), and 272.8 W/m2 (mean radiation). Superscript symbols indicate significant differences as follows: * P < 0.05; ** P < 0.01. Abbreviation: NRR60d, non return rate at 60 days post-insemination.

The number of piglets born alive per litter was negatively correlated with increasing maximum and mean temperatures, and with mean radiation (Table 3). Surprisingly, the number of live piglets per litter was not correlated with any of the assessed sperm parameters (Table 4). 4. Discussion In the present study we analyzed the effect of increasing environmental temperature and radiation on sperm quality, pregnancy rate, and litter size in refrigerated doses from boars. Results indicate that, in our conditions, sperm capacitation parameters (i.e., membrane lipid disorder, percentage of Fluo3þ/PI spermatozoa, and intracellular levels of calcium) and acrosin activity, but not conventional parameters of sperm quality (i.e., sperm viability, sperm morphology, and sperm motility), show significant

differences among experimental months after semen dilution at Day 0. These results agree with several previous studies which highlight the low predictive value of conventional sperm parameters [11,13,23]. In the trials performed in May and June we found disturbances in intracellular calcium levels and acrosin activity of great significance from Day 3 in May and from Day 0 in June. This finding is a reflection of the deleterious effect of increasing mean temperature and radiation, above 18
C and 290 W/m2, respectively, on testicular function and epididymal maturation [24]. Taking into account that in boars spermatogenesis lasts 40 to 41 days and epididymal maturation lasts 9 to 10 days [25–27], alterations observed in May are mainly attributed to epididymal disturbances, whereas alterations observed in June are the reflection of testicular and epididymal dysfunctions. Impaired sperm quality of the ejaculates as a result of increased temperature and day length have been extensively reported, despite discrepant reports on the degree of alteration of sperm parameters depending on the breed and the geographic area [1,25–28]. As it has been previously reported for short-term refrigeration [29], extra–long-term refrigeration does not alter the sperm morphology of seminal doses from Piétrain boars. In previous studies, we also failed to find any significant effect of increasing temperature and day length on the sperm morphology of purebred [1,2] and crossbred [25] boar ejaculates. Lack of alterations in sperm morphology throughout the storage period highlights the low predictive value of this parameter because of subtle defects in spermatozoa are not usually detected in routine assays [13]. Few data on the effect of seasonality on the sperm viability of refrigerated seminal doses exist. In our approach, the membrane integrity fails at Day 9 of storage in the trials performed in March, April, and May, and at Day 7 in the trials performed in June. Reduced longevity of spermatozoa in the refrigerated doses of June is attributed to the negative effect of increasing temperatures but not of increasing radiation. Nevertheless, lack of significant alterations in sperm viability throughout the refrigeration period, together with lack of pH variation and bacterial proliferation,

Table 4 Correlations between pregnancy rate, litter size, and sperm parameters. Sperm parameters

Pregnancy rate

Viable spermatozoa, % Spermatozoa with high membrane fluidity (M540þ/PI), % Spermatozoa with high Ca2þ levels, % Intracellular Ca2þ levels (GeoMean F1) Acrosin activity (mUI acrosin per 106 spermatozoa) Total motile spermatozoa, % Progessive motile spermatozoa, % Morphologically normal spermatozoa, %

0.64* 0.44 0.39 0.43 0.69** 0.38 0.30 0.36

Piglets born Total

Alive

Dead

0.60* 0.37 0.42 0.59* 0.71** 0.34 0.39 0.31

0.43 0.32 0.44 0.48 0.41 0.28 0.35 0.28

0.41 0.36 0.47 0.53 0.64* 0.31 0.24 0.32

No superscript symbol indicates no significant correlation. At insemination time, mean values of ambient parameters in each experimental month were: in March, 5.3
C (mean temperature), 9.1
C (maximum temperature), 0.8
C (minimum temperature), and 140.5 W/m2 (mean radiation); in April 15.1
C (mean temperature), 22.6
C (maximum temperature), 9.1
C (minimum temperature), and 232.5 W/m2 (mean radiation); in May, 14.8
C (mean temperature), 21.1
C (maximum temperature), 9.6
C (minimum temperature), and 256.3 W/m2 (mean radiation); and in June, 17.9
C (mean temperature), 25.0
C (maximum temperature), 12.4
C (minimum temperature), and 272.8 W/m2 (mean radiation). Superscript symbols indicate significant differences as follows: * P < 0.05; ** P < 0.01. Abbreviations: GeoMean, geometric mean; M540þ/PI, positively stained for Fluo-3-acetomethoxy ester and negatively stained for propidium iodide.

E. Pinart et al. / Theriogenology 80 (2013) 234–247

agree with the high preserving capacities of extra–longterm extenders, as extensively described [7,10,30,31]. In contrast, short-term extenders result in a decrease of sperm viability from Day 3 of refrigeration [29]. We also found that sperm membrane integrity appears more closely related with pregnancy rate of seminal doses at Days 1 to 2 and Days 3 to 4 of refrigeration than does sperm motility. This closer correlation of membrane integrity than sperm motility with fertility has been described in other reports using similar procedures, i.e., fluorochrome staining and count of large numbers of sperm cells [14,18,32]. In contrast, Gadea et al. [8] did not find any significant correlation between membrane integrity and fertility. Increasing temperature but not radiation alters the sperm motility of refrigerated doses. Therefore, in the trials performed in March, April, and May, total and progressive sperm motility is preserved until Day 5 of refrigeration, and then it declines progressively; in contrast, in the trial performed in June the decline begins at Day 1, reaching values near zero at Day 5 of storage. Decreased motility in in vitro-stored spermatozoa has been attributed to lipid peroxidation of spermatozoa, which leads to altered phosphorylation of axonemal proteins and/or reduced ATP production [6]. Some authors have also reported a significant boar effect with regard to the preservation of sperm motility [5,6] and sperm viability [10,31] during the conservation period. Nevertheless, in our approach, the boars used were the same throughout all of the experimental months and the alteration in the degree of sperm motility was similar among them, thus indicating a clear negative effect of increasing mean temperature above 18
C in sperm motility of refrigerated doses that manifest at Day 1 of storage. The preservation of sperm motility until Day 5 in the trials performed in March, April, and May, when the effect of increasing temperature is not significant, agrees with other studies analyzing the preserving capacities of extra–longterm extenders [5,30]. Sperm motility has been used as an indicator of active metabolism and membrane integrity, and of the fertilizing capacity of spermatozoa diluted in commercial extenders [5,12]. Nevertheless, in our approach, the percentage of total and progressive motile spermatozoa at Days 1 to 2 and Days 3 to 4 of refrigeration does not show any significant correlation with pregnancy rate or litter size, even considering the significant drop in this parameter in the trials performed in June. Few correlations between sperm kinematics and in vivo fertility of ejaculates [28] and of refrigerated seminal doses [5,12,33] have also been reported in boars. In refrigerated doses, Sutkeviciene et al. [33] have stated that only total sperm motility correlates with nonreturn rates after 7 days of storage. Other studies have determined that sperm motility is a useful indicator of sperm fertilizing capacity in vivo only when using lowconcentration sperm doses [34,35]. Taken together, all these results highlight the importance of design of a robust experiment using large number of boars and different sperm concentrations of seminal doses to clearly establish the predictive role of sperm motility in refrigerated doses. Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract to

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obtain the ability to penetrate and fertilize the egg [19]. Capacitation is a complex molecular process that results in changes in calcium concentration, protein phosphorylation, acrosin activation, and membrane rearrangement [22]. Finally, capacitated spermatozoa accomplish the acrosome reaction in response to zona pellucida recognition [36]. Although capacitation naturally occurs in the female reproductive tract, it can also be induced in vitro using specific media and physical conditions [22]. Moreover, handling procedures, such as sperm dilution and refrigeration, have been frequently associated with capacitationlike processes that result in decreased fertility outcomes when increasing the storage period [12,19]. Nevertheless, detailed studies have not been performed to clearly state the effect of refrigeration on sperm capacitation. In the present approach we have performed a comprehensive assay of the effects of extra–long-term refrigeration on sperm capacitation using three different tests: membrane lipid disorder, percentage of Fluo3þ/PI spermatozoa and calcium content of spermatozoa, and acrosin activity [22]. Interestingly, increasing temperature and radiation did not affect the membrane lipid disorder of boar spermatozoa at Day 0 of refrigeration, but it resulted in an increased percentage of Fluo3þ/PI spermatozoa, which was 2.4-fold in May and 3.85-fold in June, and decreased acrosin activity of 0.85-fold in June compared with March and April. In our assay, the percentage of Fluo3þ/PI spermatozoa has been revealed as the most suitable indicator of sperm capacitation status at Day 0 of refrigeration, because it shows a different pattern of variation according to environmental temperature and radiation. Using this sperm parameter as an indicator we have found that at Day 0, the seminal doses obtained in March and April were characterized by a low percentage of capacitated spermatozoa, less than 15%, but it increased to 30% in May and to 37% in June. The higher percentage of capacitated spermatozoa immediately after semen dilution could be associated with impaired activity of accessory glands induced by increasing mean temperature and radiation, which results in an abnormal content of seminal plasma proteins [1,3], and with disturbances in testicular and epididymal function [24], as previously discussed. Capacitation-like changes of spermatozoa also increase throughout the storage period, the effect of refrigeration being more significant in the trials performed in May and especially in June. Nevertheless, sperm capacitation throughout the refrigeration period shows a different pattern to that described in boars during in vitro capacitation [22] or cryopreservation [37]. In vitro assays have indicated that sperm capacitation leads to a 9-fold increase of the percentage of spermatozoa with high membrane lipid disorder, and a 1.4-fold increase of the percentage of Fluo3þ/PI spermatozoa, and a 2.25-fold increase of acrosin activity [22]. During the refrigeration period, these changes are of lower intensity for membrane lipid disorder and of higher intensity for calcium levels, whereas acrosin activity decreased rather than increased. Increased intracellular calcium levels in refrigerated spermatozoa do not correlate with pregnancy rates but they correlate negatively with litter size by reducing the number of piglets born alive.

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To our knowledge, this is the first report correlating intracellular calcium levels of refrigerated spermatozoa with litter size. The biological significance of this finding is not clear, so further studies using large numbers of boars are needed to better establish the potential predictive role of calcium levels in terms of pregnancy rates and litter size of seminal doses. Acrosin is very sensitive to increasing environmental temperature, as indicated by the significant drop in its activity in early June immediately after semen dilution at Day 0, probably as a result of disturbances in spermatogenesis and epididymal maturation caused by increasing temperatures from May, which could result in either decreased acrosin concentration [24] and/or defective proacrosin activation [38]. Other studies have also noted the presence of structural alterations on the acrosome membrane of ejaculated spermatozoa from boars housed in extreme photoperiods [28]. The acrosin activity remained constant throughout all the refrigeration periods in the assays performed in March and April, but it was reduced by 25% from Day 1 in those performed in May and June. In dogs, the acrosin activity of frozen/thawed spermatozoa is 3-fold higher than in ejaculated spermatozoa as a result of acrosin activation [39]. These authors suggested that acrosin activation in frozen/thawed spermatozoa occurs as a result of the increase of intracellular pH, which is induced by small fractures in the plasma membrane and/or in the outer acrosomal membrane [40]. In our approach, acrosin activity correlated with pregnancy rate of refrigerated seminal doses from boars. These results are in agreement with some studies performed in men and dogs stating the potential role of sperm acrosin activity as an indicator of fertilization ability of spermatozoa in vivo and in vitro [21,40]. Moreover, it is worth noting that in boars acrosin activity is also positively correlated with litter size. In ejaculates [39] and commercial seminal doses [34] significant correlations between acrosomal status and litter size have been previously reported. The alterations observed in refrigerated doses from March and April are only attributed to the effect of storage, whereas the alterations in refrigerated doses from May and June are attributed to the effect of storage and the effect of increasing temperature and radiation on testicular and epididymal function. Moreover, our results indicate that extra–long-term refrigeration does not result in premature sperm capacitation, rather, it results in a progressive loss of sperm function that occurs faster when temperature and radiation are increased. In contrast, premature sperm capacitation has been extensively reported for seminal doses diluted in shortterm extenders [4,29,30], especially in summer [3]. This different effect of short-term and long-term extenders is attributed to differences in their composition [4]. Despite that the exact composition of several extenders is unknown, short-term extenders usually contain bicarbonate which acts as a sperm capacitation inductor, by destabilizing the plasma membrane [3], whereas long-term extenders can include albumin, EDTA or HEPES, which prevent premature capacitation by chelating ions [9].

The pregnancy rate of seminal doses at Days 1 to 2 and Days 3 to 4 of refrigeration is inversely correlated with increasing temperature, whereas litter size is inversely correlated with temperature and radiation; both parameters which are lower in June. Some studies have demonstrated that extreme artificial light regimes affect the reproductive performance of boars in terms of fertility but they did not alter prolificacy of boar ejaculates [28], whereas others have reported a reduced farrowing rate of refrigerated seminal doses in summer [3]. Interestingly, pregnancy rate and litter size at Days 1 to 2 and Days 3 to 4 did not correlate with the storage period in any experimental month. In the trials performed in March and April, in which increasing temperature did not have any significant effect on pregnancy rate and litter size, the values obtained for both parameters in the double inseminations performed at Days 3 to 4 of storage were 90.2% of fertility and 13.5 piglets born in March, and 87.5% of fertility and 13.2 piglets born in April. These results are similar to those obtained in crossbred boars, also using double inseminations with seminal doses diluted in a long-term extender and stored for 4 to 5 days, with a fertility rate of 86.1% and a prolificacy of 13.4 piglets born [7]. Differences between both studies in male fertility could be attributed to the sperm concentration of seminal doses, that was of 3  109 spermatozoa per mL in our approach and of 2.5  109 spermatozoa per mL in the approach by Haugan et al. [7]. In contrast, other studies using seminal doses diluted in short-term extenders and one single AI have described a significant reduction of male fertility and prolificacy from Day 1 to Day 3 of refrigeration [9]. 4.1. Conclusions The preservation at 17
C and pregnancy rate and litter size of seminal doses from boars are negatively affected by increasing temperature above 18
C and radiation above 290 W/m2, being lower in June than in March, April, and May. It is important to note that in refrigerated doses, the degree of alteration in sperm parameters is dependent on the sperm quality immediately after semen dilution at Day 0, and that the pregnancy rate and litter size do not differ between Days 1 to 2 and Days 3 to 4 in any experimental month. Nevertheless, additional assays are needed to determine variations in field fertility with seminal doses stored for more than 5 days. Acrosin activity is the only sperm parameter analyzed that exhibited a clearly differentiated pattern in association with increasing temperature and radiation. This specific trend, together with its significant correlation with pregnancy rate and litter size of seminal doses between Days 1 to 4 of storage, makes acrosin activity a suitable indicator of preservation capacity of seminal doses at 17
C. Another important finding of our approach is that in extra–long-term refrigeration conditions, aging does not result in the capacitation of spermatozoa in any experimental month, rather it results in a progressive loss of sperm functionality, because of the increase in intracellular calcium levels and the inactivation of acrosin. To our knowledge, this is the first report describing the acrosin inactivation of long-term refrigerated spermatozoa.

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