Season of collection and sperm head shape impacts expression of CARHSP and FTL from motile-rich boar sperm

Agri Gene 7 (2018) 1–6

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Season of collection and sperm head shape impacts expression of CARHSP and FTL from motile-rich boar sperm☆

MARK

L.A. Rempela,⁎, M.M. Krautkramerb, T.M. Loetherb, J.J. Parrishb, J.R. Milesa a b

USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE, USA1 Department of Animal Science, University of Wisconsin, Madison, USA

A B S T R A C T The objective of the current study was to evaluate transcript activity of motile-rich sperm collected from June (spring) or August (summer), stored as cooled-extended (ExT) or cryopreserved (FrZ), and selected for least or most sperm head shape change, using Fourier harmonic analysis techniques, between June and August. Even with the lack of an extended heat stress, motile-rich sperm transcripts were influenced by season and putatively by sperm head shape change. Transcripts that had previously been associated with seasonality of sperm collection and methylation pathway transcripts were evaluated among semen samples. Calcium-regulated heatstable protein 1-like transcript from motile-rich sperm tended (P = 0.0829) to be greater in samples collected in June in comparison to August samples. Ferritin light polypeptide transcript tended (P = 0.0838) to be greater from motile-rich sperm with least head shape change from June collection in contrast to sperm collected in August. Both transcripts have a functional role in cytoprotection and may serve to improve boar semen activity and quality during thermal stress or seasonal changes.

1. Introduction Seasonal decline in reproductive efficiency of swine during the warm months is a globally recognized phenomenon and results in significant economic loss for the pork industry. Attempts to reduce loss by controlling environmental factors have been met with limited success (St-Pierre et al., 2003; Kunavongkrit et al., 2005). Male contributions to seasonally reduced fertility have been noted as a reduction in semen quality measures including sperm count, volume, and motility (Trudeau and Sanford, 1986; Ciereszko et al., 2000; Smital, 2009). The potential exists to mitigate loss, by the use of cryopreserved semen collected from the cool months for warm season breeding as a means to temporally separate seasonal impacts on the male. Both cryopreservation and heat

stress are known to reduce semen quality in swine (Maxwell and Johnson, 1997; Cerolini et al., 2001; Barranco et al., 2013; Didion et al., 2013) making it necessary to explore the impacts of both season and semen preparation to fully assess the potential of mitigation. The exact cause of seasonal fluctuations in boar semen quality is currently unclear. Suggested factors include elevated temperatures, high humidity and photoperiod. Most likely each of these factors plays a role in the observed reductions to semen quality parameters. Heat impacts have been well studied and supported (McNitt and First, 1970; Trudeau and Sanford, 1986; Ciereszko et al., 2000). It is interesting to note that heat stress in the bull or boar affected the process of spermatogenesis at stages specific to meiosis and initiation of spermatogenesis with effects manifesting as differences in nuclear head shape of

Abbreviations: CARHSP, calcium-regulated heat-stable protein 1-like; CIB1, Calcium and integrin binding 1; ExT, cooled-extended semen; FHA, Fourier harmonic analysis; FrZ, cryopreserved semen; FTL, ferritin light polypeptide; HA, harmonic amplitude; PPC, positive PCR control; RABAC1, Rab acceptor 1; RQ, relative quantity; RPL8, Ribosomal protein L8; RPL10A, Ribosomal protein L10a; RPS20, Ribosomal protein S20; SGDC, swine genomic DAN contamination; SMCP, Sperm mitochondria-associated cysteine-rich protein; SPATA3, Spermatogenesis-associated 3 ☆ Mention of trade names is necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the same by USDA implies no approval of the product to the exclusion of others that may also be suitable. ⁎ Corresponding author at: P.O. Box 166, Clay Center, NE 68933, USA. E-mail address: [email protected] (L.A. Rempel). 1 The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual's income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA's TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportunity provider and employer. http://dx.doi.org/10.1016/j.aggene.2017.10.002 Received 13 July 2017; Received in revised form 13 October 2017; Accepted 13 October 2017 Available online 18 October 2017 2352-2151/ Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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spermatozoa, decreases in percent motile sperm, increases in morphologically abnormal sperm and reduced fertilization and embryo development in vitro (Nikkilä et al., 2013; Parrish et al., 2014; Parrish et al., 2017). However, it has been observed that semen collected during summer months (July–September) was less likely to withstand cryopreservation, autonomous of the sperm quality of the ejaculate in comparison to winter (January–March) or spring (April–June) collections from boars housed in temperature-regulated buildings (Barranco et al., 2013). In addition to heat stress, individual male fertility differences and cryopreservation techniques are associated with sperm nuclear shape differences and aberrations in DNA integrity that can contribute to early embryonic loss and reduced live-birth rate (Ostermeier et al., 2001; D'Occhio et al., 2007; Yildiz et al., 2007). Nuclear head shape is an uncompensable trait of fertility because it reflects the capacity of an ejaculate to fertilize and support embryogenesis rather than the ability to reach the oocyte (Saacke, 2008; Parrish et al., 2014). Fourier harmonic analysis is a proven, sensitive and effective measure of sperm nuclear shape (Ostermeier et al., 2001). The objective of the current study was to assess changes in transcripts from motile-rich sperm fractions collected from warm or cool seasons from boars identified as having the least or most sperm head shape change between June and August; and storage of collected semen as cooled-extended or cryopreserved.

Fig. 1. Maximum outdoor temperatures, prior to semen collection, in June (—) or August (–). The grey area represents the period of time prior to semen collection that has the greatest thermal impact upon sperm quality (i.e., 19–33 d prior). Temperatures are reported as weekly means ± SEM.

was chosen as a parameter least sensitive to differences of the two systems. Viability was determined with fluorescent staining (Live Dead Sperm Viability Kit, Molecular Probes, Eugene, OR) with SYBR-14 and propidium iodide (PI), as described by (Garner and Johnson, 1995). Briefly, ExT or FrZ semen was diluted in semen extender (40 million sperm/mL) and placed in a 37 °C water bath. Five μL SYBR-14 (40 μM in DMSO) was added and the sample was incubated for 10 min, at which point 10 μL PI (2.4 mM) was added and an additional 5 min of incubation was allowed. A 10 μL drop of stained semen was placed on a slide and observed under an epifluorescent microscope. Those cells appearing green (SYBR14) were counted as live and red (PI) as dead. A minimum of 200 sperm were counted per sample and results were expressed as percent live. Morphology was subjectively evaluated on phase contrast images of the 100 individual sperm that were identified for use in Fourier harmonic analysis (described below). The proportion of sperm with abnormal morphology including head, mid-piece and tail abnormalities, as well as retention of cytoplasmic droplets, was determined, then percent normal was reported. Sperm nuclear shape assessment by Fourier harmonic analysis was conducted via image analysis of fluorescently stained nuclei and varied by semen type. Slide preparation consisted of staining semen with Hoechst 33342 (Molecular Probes, Eugene, OR) only for ExT samples, and both Hoechst 33342 and YOYO-1 (Molecular Probes, Eugene, OR) for FrZ samples, as described by Willenburg (Willenburg, 2008). The use of YOYO-1 allowed the exclusion of membrane-compromised or dead sperm from FHA for FrZ samples. Fourier harmonic amplitudes (HA) were obtained for sperm nuclei via computer-assisted image analysis with open source software ImageJ (Rasband, 1997) as described (Ostermeier et al., 2001). Briefly, Hoechst 33342-stained sperm nuclei were identified and perimeter coordinates determined for all samples (ExT and FrZ), with dead sperm being omitted from analysis for FrZ samples. Perimeter information was evaluated with Statistical Analysis System 9.3 (SAS, 2011) and the mean harmonic amplitudes (HA) 0–5 were determined for each sample. A mean sperm nuclear shape was constructed from the perimeter data as described (Ostermeier et al., 2001). Individual boars were ranked from least to most absolute change in nuclear shape between June and August ExT semen samples. This was achieved by calculating the absolute value of the difference between June and August means for HA0-5 within boars. Boars were ranked from least to most change for each HA value. Rank values were summed by boar giving each HA equal weight. The sum of ranks was the basis

2. Materials and methods 2.1. Animals and management All protocols utilizing animals were approved by the U.S. Meat Animal Research Center Animal Care and Use Committee (EO #543831000-091-07) and were conducted in accordance to the Federation of Animal Science Societies guidelines for the care and use of agricultural animals (FASS, Guide for the Care and Use of Agricultural Animals Used in Research and Teaching, 2010). Semen samples were collected from twelve mature Duroc boars that were housed in evaporative-cooled buildings at a commercial boar stud in June and August 2014. Samples from each boar were either extended and cooled to 16 °C (ExT) or extended and frozen over liquid nitrogen and thawed (FrZ) according to standard proprietary procedure of the boar stud at time of assessment for each collection. All samples were analyzed for semen quality by the methods described below. Studies on the impact of heat stress on boar semen have shown differences in semen quality parameters, including nuclear shape change, occur from 19 to 33 days after a given heat event (Parrish et al., 2017). No temperature data from the boar stud facility was available, but a weather station located within 15 miles from the stud recorded daily temperatures (U.S. Climate Data, n.d.). Weekly maximum temperature means are reported in Fig. 1 for 8 weeks prior through time of collection as well as the 19–33 d period prior (shaded). During the 19–33 d prior to semen collection in June 2014, mean ( ± standard error) daily high temperature was 17.9 °C ( ± 1.88 °C) and the period prior to August semen collection was 25.9 °C (0.71). There were 5 days within the period prior to the August collection with recorded high temperatures of at least 29.0 °C ( ± 0.78 °C) of which, 2 days were consecutively 30.8 °C ( ± 0.25 °C). 2.2. Sperm assessment Percent motile sperm was determined with computer assisted sperm analysis (CASA), performed on a Hamilton Thorne (v.10.9i, Waltham, MA, USA) for June ExT samples, and on a Minitube Androvision® fitted with a Olympus CX41 microscope for June FrZ and August ExT and FrZ samples. Motile sperm assessment was performed on semen samples diluted 1:1 in semen extender (IMV Technologies, Maple Grove, MN) and warmed to 37 °C in a water-bath for 10 min. Total percent motile 2

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Reverse Transcriptase (Invitrogen, Carlsbad, CA) then reverse transcribed into first-strand cDNA using random hexamer primers for realtime qPCR. A custom-designed RT2 Profiler PCR Array (Qiagen, Valencia, CA; Table 1) was used to investigate putative sperm-specific gene transcripts altered by seasonal effects (Yang et al., 2010), methylation activity transcripts, and control targets. Amplified first-strand cDNA was used at a dilution of 1:250 in the final quantitative PCR reaction. All reactions were performed on a BioRad CFX384 real-time PCR instrument (Hercules, CA) under the following conditions; 95°C for 10 min followed by 40 cycles at 95°C for 15 s, 60°C for 1 min, with a final melting curve from 65 to 95 °C. All samples were void of non-transcribed swine genomic DNA contamination (SGDC; i.e., no detection of SGDC in the RT2 Profiler Assay). The positive PCR control (PPC) was consistent across all samples and averaged 21.6 ± 0.229 CT, within the acceptable parameters for PCR amplification as per manufacturer's instructions.

Fig. 2. Impact of season on sperm nuclear shape among boars showing least- and mostamount of nuclear head shape change between cooled semen collections by season. Six total boars were selected that met commercial boar stud quality standards, based on exhibiting the most (n = 3 boars) or least (n = 3 boars) amount of nuclear shape change between June and August cooled-extended semen collections. The average nuclear shape of all 12 boars from June was used as a base from which the HA0-5 were replaced with HA0-5 means from August cooled extended semen of those boars showing the least- and most-change separately. A new Cartesian graph was derived to illustrate the manner of change for these two groups from June. Boars whose semen showed a greater degree of shape change between seasons (red) had smaller nuclei on average than did those showing less change (black). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.4. Statistical analyses Semen assessment data (motility, viability, and morphology) for each semen type were evaluated by ANOVA using a mixed model in SAS (SAS, 2011). Semen collection season (spring or summer), type of semen (ExT or FrZ), and season by type interaction were considered fixed effects while boar was considered random. One boar failed to meet the boar stud's cryopreservation standards resulting in no frozen sample for August and was removed from the study, leaving 11 boars for semen quality comparisons. Real-time quantitative PCR results were analyzed using the MIXED procedure in SAS (SAS, 2011). Analyses were performed using the comparative Ct method as described (Livak and Schmittgen, 2001) with target genes normalized to the complementary PPC. The full model included the fixed effects of: change in sperm nuclear shape (least or most), type of semen (ExT or FrZ), semen collection season (spring or summer), interactions; and boar as a repeated effect. In the event that the interactions were not significant, only main effects were tested. Target genes were lowly expressed in comparison to the PPC normalization product, therefore reported data were adjusted to a relative quantity (RQ) of expression after back transformation of the leastsquare means and standard errors. Pearson correlation coefficient estimates between sperm parameters (i.e., motility, morphology, or viability) and transcripts were calculated with the CORR procedure of SAS (SAS, 2011). If a correlation was identified (P ≤ 0.10) regression analyses were performed using the MIXED procedure in SAS (SAS, 2011). The regression model for sperm parameters (i.e., morphology, viability, and motility) included the fixed effects; transcript, type of semen (ExT or FrZ), semen collection season (spring or summer), and the interaction of type by season, and boar as random effect.

for selection of boars with least- (n = 3) and most- (n = 3) change in nuclear shape (Fig. 2). Analysis of the rankings were done via a oneway ANOVA and found to be different (P < 0.001) for the least (23 ± 3.1) and most (48 ± 2.2) change in nuclear head shape. Two of the original 12 boars were excluded from selection because they did not meet the boar stud's standard operating procedure for semen collection.

2.3. Motile rich sperm, RNA isolation, and transcription Motile rich sperm was isolated from ½ of a single dose of semen (FrZ; 5 × 109 cells/dose and ExT; 3 × 109 cells/dose) as described (Yang et al., 2010) within 18 h of receiving semen from distributor. Briefly, FrZ semen was diluted with extender as per the commercial boar stud's recommendations. Extended semen, both FrZ and ExT, was incubated at 37 °C for 30 min to settle non-motile sperm and cellular debris. The upper half of semen was collected and placed into a 50 mL conical, washed with equal parts 1× RNase-free PBS (Sigma-Aldrich, St. Louis, MO), then centrifuged at 300 × g for 10 min, 18 °C. The upper half of diluted semen was collected and placed into a new conical, washed with 1 × RNase-free PBS, then centrifuged at 1200 ×g for 5 min at 18 °C at which point the motile sperm was in the formed pellet. Supernatant was removed and checked for motile sperm by microscopy before discarding. The pellet was washed 5× with 1× RNase-free PBS then centrifuged at 1200 ×g, 5 min at 18 °C, the final pellet was snap frozen in liquid N and stored at −80 °C until RNA isolation. Motile-rich sperm pellets from least- and most- nuclear head shape change both ExT and FrZ samples from June and August collections were selected for transcript analyses. Total RNA was extracted from motile rich sperm using Trizol (Life Technologies, Grand Island, NY) according to manufacturer's recommended procedures. Total RNA was quantified and purity assessed with 1 μL of each sample using an Agilent RNA 6000 Pico assay and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Purity of the sperm RNA was determined by the absence of somatic cell 18S and 28S rRNA peaks (Fig. 3). Five hundred pg total sperm RNA was amplified using the MessageBOOSTER cDNA synthesis kit (Epicentre, Madison, WI) according to manufacturer's recommendations with Superscript III

3. Results Boars with the least and most changes in nuclear head shape from June and August were identified using FHA (Table 2 and Fig. 2). Boars 1, 2, and 3 had the least head change as determined by; 1) total average rank of HA0-5 and 2) average rank of HA0, 2, and 4. Boars 4, 5, and 6 had the greatest changes in FHA measurements (Table 2). Morphology (percent normal) and percent motility were determined using CASA techniques (Table 3). Morphology of sperm was effected by preparation technique, in which ExT semen had lower (P < 0.05) percent normal in comparison to FrZ semen (Table 4). Sperm viability was greater (P < 0.0001) in ExT semen than FrZ semen (Table 4). Semen collected in June and ExT had the greatest (P ≤ 0.002) percent motility, followed by August ExT, while least percent motile sperm was derived from FrZ samples (Table 5). Of the 40 target genes (Table 1) assessed, only nine consistently produced detectable transcripts among all semen types. The nine genes 3

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Fig. 3. Representative electropherograms of sperm RNA (A) and somatic cell RNA (B) ran on an Agilent Bioanalyzer using picoChip arrays. The first peak that appears at approximately 22 s is the embedded marker; peak at approximately 25 s is derived by small RNAs, tRNAs, and 5S and 5.8S rRNA; and peaks at 40 and 45 s are 18S and 28S rRNA, respectively, of which sperm does not contain. Absence of peak disturbances from approximately 42 through 65 s indicates no genomic DNA contamination. [FU] – fluorescence, [s] – seconds.

Table 1 Gene identification for custom RT2 profiler PCR assay (CAPS13099). Gene symbol

Refseq number

Official full name

BRD4 CARHSP1 CCL5 CIB1 DEDD2 DMAP1a

NM_001204772 XM_003124608 NM_001129946 XM_005653532 XM_005655885 NM_001243640

LOC100517496 DNMT1a DNMT3Aa DNMT3Ba FTL GPR161 HSPA5

XM_003132408 NM_001032355 NM_001097437 XM_001928593 NM_001244131 XM_001926833 XM_001927795

NOP10

XM_001925450

LOC100512133 LCN5 LGALS3 TSSK2

XM_003132152 XM_003122329 NM_001097501 NM_001287413

LOC100519519 LOC100626607

XM_005657567 XM_003356757

LYZL4 MAP4K1

XM_003132131 XM_003127115

MINK1 RABAC1 RPL10A RPL8 RPS20 SESN1 SMCP

XM_005669169 NM_001031795 NM_001097477 XM_005655297 NM_001129954 XM_003121339 NM_001008685

SPAM-1 SPATA3 SPDYA SPRP LOC100518118 TESK1 TESK2 TET1a TET2a TET3a TRDMT1a B2Mb HPRT1b RPLP1b SGDCc RTCc PPCc

NM_214011 NM_001198920 NM_001025223 NM_214238 XM_005662128 XM_003121984 XM_003128044 XM_003359222 XM_003129278 XM_003125027 NM_001162885 NM_213978 NM_001032376 XM_005673878 SA_00133 SA_00104 SA_00103

Bromodomain containing 4 Calcium-regulated heat stable protein 1-like Chemokine (CeC motif) ligand 5 Calcium and integrin binding 1 (calmyrin) Death effector domain containing 2 DNA methyltransferase 1 associated protein 1 DnaJ homolog subfamily B member 8-like DNA (cytosine-5-)-methyltransferase 1 DNA (cytosine-5-)-methyltransferase 3 alpha DNA (cytosine-5-)-methyltransferase 3 beta Ferritin, light polypeptide G protein-coupled receptor 161 Heat shock 70 kDa protein 5 (glucoseregulated protein, 78 kDa) H/ACA ribonucleoprotein complex subunit 3-like Kinesin-like protein KIF15-like Lipocalin 5 Lectin, galactoside-binding, soluble, 3 Testis-specific serine/threonine-protein kinase 2-like Titin-like Activator of 90 kDa heat shock protein ATPase homolog 1-like Lysozyme-like protein 4-like Mitogen-activated protein kinase kinase kinase kinase 1 Misshapen-like kinase 1 Rab acceptor 1 (prenylated) Ribosomal protein L10a Ribosomal protein L8 Ribosomal protein S20 Sestrin-1-like Sperm mitochondria-associated cysteine-rich protein Sperm adhesion molecule 1 Spermatogenesis associated 3 Speedy homolog A (Xenopus laevis) Small proline-rich protein Stannin-like Testis-specific kinase 1 Testis-specific kinase 2 Tet methylcytosine dioxygenase 1 Tet oncogene family member 2 Tet oncogene family member 3 TRNA aspartic acid methyltransferase 1 Beta-2-microglobulin Hypoxanthine phosphoribosyltransferase 1 Ribosomal protein, large, P1 Pig genomic DNA contamination Reverse transcription control Positive PCR control

a b c

Table 2 Ranked changes from June and August collections, within each harmonic amplitude measurement (HA0-5), total average rank, and average rank for HA0, 2 and 4. Boara

HA0

HA1

HA2

HA3

HA4

HA5

Average rank

Average HA0, 2, 4

1 2 3 4 5 6 7 8 9 10b 11 12

1 3 2 7 10 8 5 6 4 12 11 9

1 10 9 3 11 5 8 2 4 12 7 6

2 5 8 10 9 4 1 12 7 11 3 6

4 1 2 6 3 10 9 11 5 12 8 7

8 1 3 11 9 12 5 2 6 7 10 4

1 5 3 7 9 11 8 2 6 12 10 4

2.83 4.17 4.50 7.33 8.50 8.33 6.00 5.83 5.33 11.00 8.17 6.00

3.67 3.00 4.33 9.33 9.33 8.00 3.67 6.67 5.67 10.00 8.00 6.33

HA – harmonic amplitude. a Boars 1–3 identified as least HA change and boars 4–6 identified as most HA change. b Excluded from study, did not meet standard collection procedures in August. Table 3 CASA assessment of boar semen collected in spring and summer from 12 boars. Boara

% Normal (June)

%Normal (August)

%Change in normal from June to August

% Motile (June)

%Motile (August)

%Change in motility from June to August

1 2 3 4 5 6 7 8 9 10b 11 12

32.4 38.4 32.6 79.7 59.3 31.6 40.0 51.1 42.8 38.7 16.2 57.9

94.0 72.0 91.0 74.0 85.0 84.0 92.0 48.0 77.0 64.0 95.0 85.0

61.6 33.6 58.4 − 5.7 25.7 52.4 52.1 − 3.1 34.2 25.4 78.8 27.1

79.3 94.1 60.8 46.9 56.5 93.4 59.4 82.1 27.6 50.0 90.0 49.3

59.4 93.9 58.5 70.9 75.6 77.8 54.1 50.3 40.8 40.0 94.7 61.7

− 19.9 − 0.2 − 2.3 24.0 19.1 − 15.6 − 5.2 − 31.7 13.2 − 10.0 4.7 12.4

CASA – computer assisted sperm analysis. a Boars 1–3 identified as least HA change and boars 4–6 identified as most HA change. b Excluded from study, did not meet standard collection procedures in August.

were; Calcium-regulated heat stable protein 1-like (CARHSP), Calcium and integrin binding 1 (aka Calmyrin; CIB1), Ferritin, light polypeptide (FTL), Rab acceptor 1 (RABAC1), Ribosomal protein L10a (RPL10A), Ribosomal protein L8 (RPL8), Ribosomal protein S20 (RPS20), Sperm mitochondria-associated cysteine-rich protein (SMCP), and Spermatogenesis-associated 3 (SPATA3). From the nine transcripts that were detected among samples, two transcripts tended to have relative abundance differences. Relative quantity of CARHSP transcript tended (P = 0.0829) to be greater from June collection in comparison to August collection (2.3 ± 1.10 and

Methylation pathway genes. Reference/housekeeping genes. RT2 PCR Profiler Assay plate control genes.

4

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buildings. The boars in the current study were housed in buildings with evaporative cooling systems, which contribute to maintaining ambient temperatures, but cannot always accommodate extreme temperature changes. However it is likely that additional factors, outside of heat stress, may be influencing semen quality from summer periods. The most predominant effects of cryopreservation on sperm are loss of viability, reduction in motility, morphologic changes, and alteration in chromatin structure (Watson, 2000; Thomson et al., 2009). These same changes are also observed as a result of heat stress (Gibbs et al., 2013) and seasonality (Smital, 2009), to varying degrees. Gross semen characteristics (morphology, motility, and viability) were affected by season of collection as well as semen preparation methods. In the current study, the differences between motility and viability between both preparation and season are consistent with previous studies, albeit, the reduction in motility for cryopreserved semen was more severe than previously reported (Barranco et al., 2013; Daigneault et al., 2015). All semen, cooled-extended or cryopreserved, in this study; were commercially obtained from the same boar stud and manufacturer's recommendations for handling were followed. However, frozen-thawed semen was only incubated for 10 min prior to semen quality evaluation, which likely limited motile sperm found, but had no impact on viability, morphology or FHA evaluation. As a means to characterize potential differences among semen collection season and storage techniques, 32 motile-rich sperm transcripts were tested within our study (Yang et al., 2010) as well as transcripts within the methylation activity pathway. Sperm RNA has become a credible contributor to transgenerational acquired inheritance either directly influencing the sperm genome or occurring shortly after fertilization influencing the early embryo (Lalancette et al., 2008; Miller, 2014). Calcium-regulated heat-stable protein contains two RNA binding motifs and a cold-shock domain (Pfeiffer et al., 2011). Proteins with cold-shock domains regulate ribosome transition, mRNA degradation, and transcript termination rate (Online Mendelian Inheritance in Man, OMIM (TM), n.d.). In the current study, relative transcript abundance of CARHSP was greater from sperm collected in June than that of August. Others have suggested that CARHSP also plays a role in cellular oxidative stress response through dynamic and temporal associations with stress granules and processing bodies (Hou et al., 2011). Although temperature was not extreme within the current study, a seasonal response was detected suggesting possible fluctuations in responsive transcript activity. CARHSP is also a known mRNA stability enhancer for TNF-α (Li et al., 2016). Enhancement and stabilization of TNF-α leads to cellular inflammation (Li et al., 2016). The reduction of CARHSP transcript from the August collection may be a cytoprotective response to seasonal stress in boar sperm. In the current study, motile-rich sperm FTL transcript fluctuated by season, but only in boars with least sperm head shape change from June to August. Ferritin, light polypeptide subunit is a component of a cytosolic metal binding antioxidant protein, ferritin, which acts to restrict the availability of iron to participate in the conversion of hydrogen peroxide into toxic hydroxyl radicals through the Fenton reaction, thereby serving as a cytoprotective compound (Finazzi and Arosio, 2014). During a heat event, cytosolic iron is released from ferritin thereby increasing transition metal ions, which can donate an electron to O2, producing superoxide anions or hydrogen peroxide (Belhadj Slimen et al., 2016). Testicular concentration of iron in boars has been negatively associated with daily sperm production (Wise et al., 2003). Furthermore, Wise and others (Wise et al., 2003) observed that young boars with smaller testes had greater testicular concentrations of iron and ferritin, both of which decreased as testicular size increased with age. Others have also reported that sperm mitochondrial ferritin negatively correlated with elevated sperm DNA damage (Behrouzi et al., 2013). Ferritin, light polypeptide likely reflects ferritin, and may be serving to protect sperm from iron fluctuations. Therefore, boars with most sperm head change and static FTL expression, may not be able to

Table 4 Effect of semen preparation (cooled extended or frozen-thawed) on semen quality parameters.a Semen preparation

Morphology (%) Viability (%)

b

ExT

FrZ

67.9 ± 3.8 73.0 ± 2.1

76.4 ± 2.4* 22.7 ± 1.9**

a Values are the lsmeans ± standard error. Values between columns differ: *P < 0.05, **P < 0.0001. b Morphology expressed as percent normal.

Table 5 Effect of season (June or August) and preparation (ExT or FrZ) on motility.1 Season

Preparation

Percent motility2

SE

June August June August

ExT ExT FrZ FrZ

80.36a 43.43b 7.94c 7.56c

4.401 5.236 1.544 1.018

1 2

Values are lsmeans ± standard error. Values with different superscript (i.e., a, b, or c) differ by P < 0.05.

0.5 ± 0.36 RQ, respectively). A tendency for an interaction (P = 0.0821) between semen collection season and sperm head shape change was detected for the transcript, FTL. Motile-rich semen from June with least head shape change (10.1 ± 8.76 RQ) tended to have a greater relative transcript abundance of FTL than that of semen collected in August with least head shape change (1.6 ± 1.07 RQ). Both June and August collected semen from boars with most sperm head shape change were intermediate to the least head shape change boars regardless of season (P ≥ 0.1229) and similar (P = 0.5000) to each other (June/Most; 3.7 ± 2.49 RQ and August/Most; 6.8 ± 4.64 RQ). Pearson correlation estimates between sperm parameters and transcript levels indicated a marginal correlation of SMCP (0.48119, P = 0.0960) and SPATA3 (−0.59102, P = 0.0555) with sperm morphology. The transcript, RPLP1, tended (P = 0.0738) to be negatively correlated with sperm viability (− 0.62176). Sperm motility tended (P = 0.0604) to be negatively related to B2M (− 0.64548). However, regression analyses failed to elicit any significant relationships among these transcripts and sperm parameters. 4. Discussion In the present study, we evaluated the impact of seasonality and semen storage conditions on transcript expression for seasonality and methylation associated genes from corresponding sperm. It is important to point out that the current study was performed during the second coolest summer on record in the last 10 years (US Climate data). The ten year average (2005–2014) for July within 15 miles of the boar stud was 28.1 °C, while the 2014 average was only 25.6 °C. However, during the period of time known to impact spermatozoan development, 19–33 d prior to collection, (Parrish et al., 2017) thermal increases were present for the current study. It is also important to note that modern grow-finish pigs have between 6 and 41% greater heat production than earlier reported genetics (Brown-Brandl et al., 2011) and the maximum thermal neutral zone for growing pigs ranges between 17 °C and 23 °C (Brown-Brandl et al., 2013). Therefore, it appears newer genetics have yielded pigs with greater thermal sensitivities and even though the summer season was cooler, a thermal stress could have occurred. Furthermore, Barranco and others (Barranco et al., 2013) reported that semen collected during summer months (July–September) was less likely to withstand cryopreservation autonomous of the sperm quality of the ejaculate in comparison to winter (January–March) or spring (April–June) collections from boars housed in temperature-regulated 5

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Gibbs, K.M., Schindler, J.R., Parrish, J.J., 2013. Determining the effect of scrotal insulation on sperm production in the boar. In: ADSA-ASAS Joint Annual Meeting, Journal of Animal Science, Indianapolis, IN, USA, pp. 591. Hou, H., Wang, F., Zhang, W., Wang, D., Li, X., Bartlam, M., Yao, X., Rao, Z., 2011. Structure-functional analyses of CRHSP-24 plasticity and dynamics in oxidative stress response. J. Biol. Chem. 286, 9623–9635. Kunavongkrit, A., Suriyasomboon, A., Lundeheim, N., Heard, T.W., Einarsson, S., 2005. Management and sperm production of boars under differing environmental conditions. Theriogenology 63, 657–667. Lalancette, C., Miller, D., Li, Y., Krawetz, S.A., 2008. Paternal contributions: new functional insights for spermatozoal RNA. J. Cell. Biochem. 104, 1570–1579. Li, X., Kong, D., Chen, H., Liu, S., Hu, H., Wu, T., Wang, J., Chen, W., Ning, Y., Li, Y., Lu, Z., 2016. miR-155 acts as an anti-inflammatory factor in atherosclerosis-associated foam cell formation by repressing calcium-regulated heat stable protein 1. Sci. Rep. 6, 21789. http://dx.doi.org/10.1038/srep21789. (11 pp). Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2 (− Delta Delta C(T)) method. Methods 25, 402–408. Maxwell, W.M., Johnson, L.A., 1997. Membrane status of boar spermatozoa after cooling or cryopreservation. Theriogenology 48, 209–219. McNitt, J.I., First, N.L., 1970. Effects of 72-hour heat stress on semen quality in boars. Int. J. Biometeorol. 14, 373–380. Miller, D., 2014. Sperm RNA as a mediator of genomic plasticity. Adv. Biol. 179701. http://dx.doi.org/10.1155/2014/179701. (13pp). Nikkilä, M.T., Stalder, K.J., Mote, B.E., Rothschild, M.F., Gunsett, F.C., Johnson, A.K., Karriker, L.A., Boggess, M.V., Serenius, T.V., 2013. Genetic associations for gilt growth, compositional, and structural soundness traits with sow longevity and lifetime reproductive performance. J. Anim. Sci. 91, 1570–1579. Online Mendelian Inheritance in Man, OMIM (TM). Johns Hopkins University, Baltimore, MD. (MIM Number: 616885: 29.03.03). http://www.ncbi.nlm.nih.gov/omim/. Ostermeier, G.C., Sargeant, G.A., Yandell, B.S., Evenson, D.P., Parrish, J.J., 2001. Relationship of bull fertility to sperm nuclear shape. J. Androl. 22, 595–603. Ostermeier, G.C., Sargeant, G.A., Yandell, T.B.S., Parrish, J.J., 2001. Measurement of bovine sperm nuclear shape using Fourier harmonic amplitudes. J. Androl. 22, 584–594. Parrish, J.J., Ostermeier, C., Schindler, J., Willenburg, K.L., Enwall, L., Kaya, A., 2014. Quantifying Sperm Nuclear Shape with Fourier Harmonic Analysis and Relationship to Spermatogenesis and Fertility. Association for Applied Animal Andrology, International Veterinary Information Service, Newcastle, Australia, pp. 30–49. Parrish, J.J., Willenburg, K.L., Gibbs, K.M., Yagoda, K., Krautkramer, M.M., Loether, T.M., de Melo, F.C.S.A., 2017. Scrotal insulation and sperm production in the boar. Mol. Reprod. Dev. http://dx.doi.org/10.1002/mrd.22841. (10pp). Pfeiffer, J.R., McAvoy, B.L., Fecteau, R.E., Deleault, K.M., Brooks, S.A., 2011. CARHSP1 is required for effective tumor necrosis factor alpha mRNA stabilization and localizes to processing bodies and exosomes. Mol. Cell. Biol. 31, 277–286. Rasband, W.S., 1997–2016. ImageJ. U.S. National Institutes of Health, Bethesda, Maryland, USA. https://imagej.nih.gov/ij/. Saacke, R.G., 2008. Sperm morphology: its relevance to compensable and uncompensable traits in semen. Theriogenology 70, 473–478. SAS, 2011. Base SAS 9.3 Procedures Guide. SAS Institute Inc., Cary, NC, USA. Smital, J., 2009. Effects influencing boar semen. Anim. Reprod. Sci. 110, 335–346. St-Pierre, N.R., Cobanov, B., Schnitkey, G., 2003. Economic losses from heat stress by US livestock industries. J. Dairy Sci. 86, E52–E77. Thomson, L.K., Fleming, S.D., Aitken, R.J., De Iuliis, G.N., Zieschang, J.A., Clark, A.M., 2009. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum. Reprod. 24, 2061–2070. Trudeau, V., Sanford, L.M., 1986. Season and social environment influence the membrane integrity of ejaculated boar spermatozoa as assessed by ouabain sensitivity. Can. J. Physiol. Pharmacol. 64, 1407–1412. U.S. Climate Data Weather History Reports for the U.S. http://www.usclimatedata.com/ iowa/united-stated/3185 (web archive link, 26 October 2015)(accessed 26.10.15). Watson, P.F., 2000. The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60-61, 481–492. Willenburg, K.L., 2008. Characterization of Boar Nuclear Shape Using Fourier Harmonic Analysis, Animal Sciences (Thesis (Ph.D.)). University of Wisconsin, Madison, pp. 156. Wise, T., Lunstra, D.D., Rohrer, G.A., Ford, J.J., 2003. Relationship of testicular iron and ferritin concentrations with testicular weight and sperm production in boars. J. Anim. Sci. 81, 503–511. Yang, C.C., Lin, Y.S., Hsu, C.C., Tsai, M.H., Wu, S.C., Cheng, W.T., 2010. Seasonal effect on sperm messenger RNA profile of domestic swine (Sus Scrofa). Anim. Reprod. Sci. 119, 76–84. Yildiz, C., Ottaviani, P., Law, N., Ayearst, R., Liu, L., McKerlie, C., 2007. Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse. Reproduction 133, 585–595.

protect the sperm from seasonal-induced increases in free-iron concentrations. 5. Conclusions Semen quality was influenced by season as well as preservation methods. Transcript activity of genes known to be influenced by semen collection period tended to be altered in two genes with affiliation to cytoprotection, which could greatly influence proper sperm viability and/or fertility. These data support that season, even in the absence of extreme heat, and to a lesser extent, sperm head shape change; may have a deeper molecular influence on seasonal infertility within sperm. Conflicts of interest None of the authors (M.M. Krautkramer, T.M. Loether, J.R. Miles, J.J. Parrish, L.A. Rempel) has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the current paper. Acknowledgements The authors would like to thank Shanda Watts and Elane WrightJohnson for technical assistance in the processing of samples and Linda Parnell for secretarial assistance. Work herein was funded by the National Pork Board (Grant #14-052) and a HATCH grant awarded to Dr. Parrish. References Barranco, I., Ortega, M.D., Martinez-Alborcia, M.J., Vazquez, J.M., Martinez, E.A., Roca, J., 2013. Season of ejaculate collection influences the freezability of boar spermatozoa. Cryobiology 67, 299–304. Behrouzi, B., Kenigsberg, S., Alladin, N., Swanson, S., Zicherman, J., Hong, S.H., Moskovtsev, S.I., Librach, C.L., 2013. Evaluation of potential protein biomarkers in patients with high sperm DNA damage. Syst. Biol. Reprod. Med. 59, 153–163. Belhadj Slimen, I., Najar, T., Ghram, A., Abdrrabba, M., 2016. Heat stress effects on livestock: molecular, cellular and metabolic aspects, a review. Anim. Phys. Anim. Nutr. 100, 401–412. Brown-Brandl, T.M., Eigenberg, R.A., Purswell, J.L., 2013. Using thermal imaging as a method of investigating thermal thresholds in finishing pigs. Biosys. Eng. 114, 327–333. Brown-Brandl, T.M., Neienaber, J.A., Eigenberg, R.A., Xin, H., 2011. Heat and moisture production of growing-finishing gilts as affected by environmental temperature. In: Agricultural and Biosystems Engineering Conference Proceedings and Presentations. vol. 174 American Society of Agricultural and Biological Engineers, Louisville, KY, USA. http://lib.dr.iastate.edu/abe_eng_conf/174. Cerolini, S., Maldjian, A., Pizzi, F., Gliozzi, T.M., 2001. Changes in sperm quality and lipid composition during cryopreservation of boar semen. Reproduction 121, 395–401. Ciereszko, A., Ottobre, J.S., Glogowski, J., 2000. Effects of season and breed on sperm acrosin activity and semen quality of boars. Anim. Reprod. Sci. 64, 89–96. Daigneault, B.W., McNamara, K.A., Purdy, P.H., Krisher, R.L., Knox, R.V., Rodriguez-Zas, S.L., Miller, D.J., 2015. Enhanced fertility prediction of cryopreserved boar spermatozoa using novel sperm function assessment. Andrology 3, 558–568. Didion, B.A., Braun, G.D., Duggan, M.V., 2013. Field fertility of frozen boar semen: a retrospective report comprising over 2600 AI services spanning a four year period. Anim. Reprod. Sci. 137, 189–196. D'Occhio, M.J., Hengstberger, K.J., Johnston, S.D., 2007. Biology of sperm chromatin structure and relationship to male fertility and embryonic survival. Anim. Reprod. Sci. 101, 1–17. FASS, Guide for the Care and Use of Agricultural Animals Used in Research and Teaching, 2010. Federation of Animal Science Societies. (Champaign, IL). Finazzi, D., Arosio, P., 2014. Biology of ferritin in mammals: an update on iron storage, oxidative damage and neurodegeneration. Arch. Toxicol. 88, 1787–1802. Garner, D.L., Johnson, L.A., 1995. Viability assessment of mammalian sperm using SYBR14 and propidium iodide. Biol. Reprod. 53, 276–284.

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