Effect of natural betaine on estimates of semen quality in mature AI boars during summer heat stress

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Effect of natural betaine on estimates of semen quality in mature AI boars during summer heat stress F.A. Cabezón a , K.R. Stewart a,∗ , A.P. Schinckel a , W. Barnes b , R.D. Boyd b , P. Wilcock c , J. Woodliff d a b c d

Department of Animal Sciences, Purdue University, West Lafayette, IN, USA The Hanor Company Inc., Franklin, KY, USA AB Vista, Marlborough, UK Flow Cytometry & Cell Separation Facility, Purdue University, West Lafayette, IN, USA

a r t i c l e

i n f o

Article history: Received 15 December 2015 Received in revised form 16 March 2016 Accepted 18 March 2016 Available online xxx Keywords: Betaine Homocysteine Boar sperm Semen analysis

a b s t r a c t This study evaluated the effect of supplemental dietary betaine at three concentrations (0.0%, 0.63% and 1.26%) on semen characteristics, quality and quality after storage on boars. The trial was conducted between 22 July and 1 October 2014 in a boar stud located in Oklahoma. Boars were blocked by age within genetic line and randomly allotted to receive 0% (CON, n (line T) = 22, n (line L) = 10), 0.63% (BET-0.63%, n (line T) = 21, n (line L) = 6) or 1.26% (BET-1.26%, n (line T) = 23, n (line L) = 7). The diets containing betaine were fed over 10 weeks, to ensure supplemental betaine product (96% betaine) daily intakes of 16.34 and 32.68 g, for the BET-0.63% and BET-1.26% diets, respectively. Serum homocysteine concentrations were less for animals with betaine treatments (P = 0.016). Rectal temperatures of the boars were unaffected by betaine diets. Betaine tended to increase total sperm in the ejaculates when collectively compared with data of the control animals (P = 0.093). Sperm morphology analysis indicated there was a greater percent of sperm with distal midpiece reflex (P = 0.009) and tail (P = 0.035) abnormalities in boars fed the BET-1.26% than boars fed the BET-0.63% diet. Betaine concentration in the seminal plasma was greater in boars with betaine treatments, with animals being fed the 0.63% and 1.26% diets having 59.2% and 54.5% greater betaine concentrations in seminal plasma as compared with boars of the control group (P = 0.046). In conclusion, betaine supplementation at 0.63% and 1.26% tended to increase sperm concentration in the ejaculates by 6% and 13%, respectively, with no negative impacts on semen quality when 0.63% of betaine was included in the diet. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Sows and boars are susceptible to negative impacts from high ambient temperatures and humidity. More than 60% of randomly sampled sow farm operations in the U.S.A. reported a decrease in fertility during the hot sum-

∗ Corresponding author. E-mail address: [email protected] (K.R. Stewart).

mer months (Knox et al., 2013). This seasonal infertility is the result of stress from increased ambient temperatures above the thermal neutral zone and subsequent metabolic changes. McNitt and First (1970) evaluated heat stress in boars and reported that 72 h of exposure to a 33 ◦ C environmental temperature and 50% relative humidity resulted in a reduced sperm concentration and increased sperm abnormalities in ejaculates. Furthermore, Wettemann et al. (1979), found that boars exposed to 34.5 ◦ C for 8 h each day and 31 ◦ C for 16 h during 11 weeks, had less percent

http://dx.doi.org/10.1016/j.anireprosci.2016.03.009 0378-4320/© 2016 Elsevier B.V. All rights reserved.

Please cite this article in press as: Cabezón, F.A., et al., Effect of natural betaine on estimates of semen quality in mature AI boars during summer heat stress. Anim. Reprod. Sci. (2016), http://dx.doi.org/10.1016/j.anireprosci.2016.03.009

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Table 1 Boar age mean and standard deviation for treatment and genetic lines. Treatment

Genetic linea

Number of boars

CON BET 0.63% BET 1.26%

L L L

10 6 7

23.5 21.0 19.3

15.0 9.0 9.1

CON BET 0.63% BET 1.26%

T T T

22 21 23

22.7 21.5 22.9

10.3 9.1 10.5

a

Age mean

Age SD

L: Large White line; T: Terminal line.

Fig. 1. Mean weekly maximum and minimum temperatures in the housing area during the study, where week 0 started on 13 July 2014.

motile sperm for 5 weeks after heat stress exposure ended in contrast with boars exposed to 23 ◦ C. Environmental temperatures may not have to greatly exceed the thermal neutral zone to have detrimental effects on reproduction (Flowers, 1997) and the impact of heat stress can vary within and between genetic lines (Flowers, 2008). Betaine is a methylamine naturally occurring in plant and animal tissues and is also available as a feed additive. A methyl group is donated by betaine to convert homocysteine into methionine (Kidd et al., 1997). Betaine also functions as an osmo-protectant when the animal is stressed by disease, metabolic challenges, or environmental conditions (reviewed by Odle (1995) and Lipinski et al. (2012)) due to its solubility in water and dipolar zwitterion properties (Chambers and Kunin, 1985; Eklund et al., 2005). van Wettere et al. (2012) provided additional support for the concept that the effects of betaine may be enhanced during times of stress in animals. It was reported that there was an increase in litter size in sows at parity 3 or greater when betaine was supplemented during gestation in the summer months. Additionally, there may be some evidence for enhanced reproductive performance when pregnant sows are supplemented with additional methyl donors (NRC-42, 1976). A potential explanation for these effects could be the greater ability to regulate homocysteine concentrations in animals treated with betaine. Approximately 18% of men

seeking medical treatment for infertility have a mutation in the enzyme methylenetetrahydrofolate reductase (MTHFR, Bezold et al., 2001), which increases homocysteine concentrations. Knockout mice deficient in the MTHFR enzyme have elevated homocysteine concentrations, oligospermia and infertility. When betaine was supplemented to the mothers of MTHFR knockout mice during gestation and lactation, male offspring had a short-term improvement in fertility (Kelly et al., 2005). When the MTHFR knockout males were fed betaine following weaning, fertility improved. Feeding supplemental betaine to rabbit bucks during the period of summer seasonal infertility resulted in increased sperm concentrations, sperm motility, and ejaculate volume (Hassan et al., 2012). Collectively, these studies suggest that dietary betaine supplementation may improve reproductive performance in males, especially during summer infertility. To our knowledge, no research has been performed to evaluate the impact of betaine supplementation on boar reproduction during times of heat stress. The objective of this trial was, therefore, to assess the effects of betaine supplementation on boar sperm characteristics and number during the summer months.

2. Materials and methods The Purdue University Institutional Animal Care and Use Committee approved all procedures involving animals.

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Boars were blocked by age within genetic line and randomly allotted to receive 0% (CON, n (line T) = 22, n (line L) = 10), 0.63% (BET-0.63%, n (line T) = 21, n (line L) = 6) or 1.26% (BET-1.26%, n (line T) = 23, n (line L) = 7) of

0.001 0.002 2.6

Genetic × Blood test day Blood test day Genetic Treatment

0.027 0.199 0.016 2.7 66 40.2 23 30.2 30 35.0ab

BET Differencef CON v.s. BETe BET 1.26%

SE

T L

SE

P-Valueg

Superscripts (a,b) denote significant difference between treatments based on Tukey-Kramer method. c L: Large White line; T: Terminal line. d Blood test represents the measurements on days 9, 45 and 73 after the initiation of betaine-supplemented diets. e CON compared with BET: represents the orthogonal contrast between control and both betaine diets combined. f BET Difference: represents the orthogonal contrast between the betaine 0.63% and 1.26%. g Treatment by genetic line interaction (P = 0.62).

2.2. Experimental design

BET 0.63%

The study was conducted at a commercial boar stud in Ames, Oklahoma. The study was performed over 10 weeks from 22 July and 1 October 2014. Two genetic lines were used, a PIC terminal line (T, n = 66) and a pure PIC Large White line (L, n = 23) with an average age of 22.2 ± 10.3 months (Table 1). High and low temperatures (Fig. 1) and relative humidity in the boar housing facility were recorded daily using generic thermometers. During the trial, the overall, maximum and minimum mean relative humidity were 56.5%, 71.5% and 41.9%, respectively.

CON

2.1. Animals and housing

Genetic linec

a Diets were formulated to exceed NRC (2012) requirements and contained 2.42 g standardized ileal digestible Lys/Mcal ME. b Supplied per kg of complete diet: Zn, 125 mg; Fe, 100 mg; Mn, 50 mg; Cu, 24.98 mg; I, 0.7 mg; Se, 0.3 mg; vitamin A, 11023 IU; vitamin D3 , 1763.7 IU; vitamin E, 50.7 IU; vitamin K, 4.4 mg; vitamin B12 , 0.044 mg; riboflavin, 8.8 mg; d-pantothenate, 26.5 mg; niacin 55.1 mg; thiamine, 3.3 mg; pyridoxine, 3.3 mg; folic acid, 1.21 mg; biotin, 0.28 mg, and chromium, 0.4 mg/kg. c Used to bind toxins and to inhibit mold growth in feed (Kemin, Des Moines, IA, USA). d Phyzyme (Danisco A/S, Copenhagen, Denmark, USA). e Calculated values based on NRC (2012).

27 30.7b

3.18 16.61 0.89 0.77 0.59 0.16 0.27 0.23 0.66 0.57 2.62 0.80 0.52 650.91 0.30 1020.00

32 39.8a

58.79 15.00 10.00 9.84 2.50 1.25 1.05 0.46 0.40 0.24 0.13 0.20 0.10 0.04

Treatment

Calculated compositione ME, Mcal/kg Crude protein, % Total Lysine, % SID Lysine, % SID Threonine, % SID Tryptophan, % SID Methionine, % SID Cysteine, % SID Valine, % SID Isoleucine, % Linoleic acid, % Ca, % Total P, % Choline, mg/kg Selenium, ppm Phytase, FTU/kg

Dietary treatment

Blood Testd

Ingredients, % Corn, 8.5% CP Soybean meal, 46.5% CP Wheat middlings Soybean hulls Fish meal, menhaden select Corn oil Limestone Monocalcium phosphate, 21% P Salt Vitamin and mineral premixb Choline chloride, 60% Kemin KallSilc Kemin AmmoCurbc Phytased

Table 3 Overall homocysteine serum concentration for treatments and genetic lines.

Item

N Homocysteine (␮mol/l)

Table 2 Composition of the experimental diet, as-fed basisa .

3

0.114

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betaine product dietary inclusion (96% betaine, VistabetTM , AB Vista, Marlborough, UK) in the diets. Boars were fed 2.27–2.72 kg/day of feed one time per day to meet or exceed NRC 2012 requirements (Table 2). Betaine was included in a top dressed premix, which consisted in 59.05% of fine ground corn, 40.85% of betaine product and 0.10% of red synthetic dye. For boars in BET-0.63% and BET-1.26% treatment groups, 40 g and 80 g of premix were fed daily, respectively. This allowed a daily betaine product inclusion of 16.34 g for BET-0.63% treatment group and 32.68 g for BET-1.26% treatment group. Betaine supplementation in diets began on 16 July, one week before the start of semen collection. 2.3. Homocysteine serum concentrations Blood samples were collected from 89 boars at days 9, 45 and 73 after betaine supplementation started. The collection times were standardized to 3 h following morning feeding. Blood was collected in serum separator tubes from the saphenous vein in the leg and allowed to clot on ice overnight during shipment to Purdue University, West Lafayette, IN. Tubes containing the blood were centrifuged at 3000 r.p.m. for 20 min to separate serum. Serum was stored at −20 ◦ C until the determination of homocysteine concentration was performed with a solid-phase extraction immunoassay machine (IMMULITE® 1000Homocysteine, SIEMENS, Germany). The assay was performed according to manufacturer’s standard procedures (IMMULITE® 1000 Homocysteine, 2013, SIEMENS, Germany). 2.4. Rectal temperatures Rectal temperatures were recorded in a subset of 68 boars two times per week at 2–3 pm, from the third week of the trial until the end of betaine supplementation using a high-speed digital thermometer with a 12 cm rectal probe (Welch Allyn Thermometer, Mohawk Medical Mall® , NY) inserted into the rectum a minimum of 2.5 cm. 2.5. Ejaculate and betaine seminal plasma evaluation Weekly ejaculates from 89 boars were collected and evaluated. Upon collection the ejaculate was diluted 1:1 with pre-warmed semen extender (PreservTM Xtra semen extender Repro Quest, ReproQuest, Inc. Fitchburg, WI). Volume was determined using a volumetric glass and gram scale and concentration was estimated using light spectrophotometry (Metro Sperm). Total sperm production was calculated by multiplying the volume and concentration of each ejaculate. A different set of 18 boars (six boars for each treatment; six boars for genetic line L and 12 boars for genetic line T) was used to quantify the concentration of betaine in seminal plasma on weeks 1, 5 and 10 of the study. The seminal fluid was stored in −80 ◦ C prior to the time of extraction and analysis. An aliquot of 100 ␮L was measured and placed in a 1.7 mL centrifuge tube. To this, 1000 ng of d3 -betaine was added as an internal standard (CDN Isotopes, Pointe-Clare, Quebec, Canada). Protein was precipitated by the addition

of 0.5 mL of methanol. The tubes were briefly vortexed and centrifuged at 15,000 r.p.m. for 10 min. The supernatant was transferred to another tube. The supernatants were then dried in a rotary evaporation device and stored at −20 ◦ C until analysis. An Agilent 1200 Rapid Resolution liquid chromatography (LC) system coupled to an Agilent 6460 series QQQ mass spectrometer (MS) was used to analyze betaine in each sample. An Intrada Amino Acid 2.1 mm × 150 mm, 3 ␮m column (Imtakt, Portland, OR) was used for LC separation. The buffers were (A) 95:5 acetonitrile:water + 10 mM ammonium acetate + 0.6% acetic acid and (B) 5:95 acetonitrile:water + 10 mM ammonium acetate + 0.6% acetic acid. The linear LC gradient was as follows: time 0 min, 0% B; time 1 min, 0% B; time 8 min, 100% B; time 11 min, 100% B; time 12 min, 0% B; time 15 min, 0% B. The flow rate was 0.3 mL/min. The retention time of betaine was 6.6 min. Multiple reaction monitoring was used for MS analysis. The data were acquired in positive electrospray ionization (ESI) mode by monitoring the following transitions: Betaine 118.0–58.0 (30 V); d3 -Betaine 121.0–61.0 (30 V). The jet stream ESI interface had a gas temperature of 325 ◦ C, gas flow rate of 10 L/min, nebulizer pressure of 35 psi, sheath gas temperature of 250 ◦ C, sheath gas flow rate of 7 L/min, capillary voltage of 3500 V, and nozzle voltage of 1000 V. 2.6. Sperm motility and morphology examination Sperm motility and morphology analysis was performed for each boar every other week. Five ml of the 1:1 diluted semen was added to approximately 10 mL of additional semen extender in a 15 mL conical tube that was shipped overnight in Polar Tech Insulated Coolers with ice packs to maintain temperature during the transit to Purdue University, West Lafayette, IN. Upon arrival, sperm concentration was determined using Minitube’s SpermaCue device on the diluted samples. Samples were further extended (PreservTM Xtra semen extender Repro Quest, ReproQuest, Inc. Fitchburg, WI) to make the storage concentration of 1 billion sperm cells in 30 mL. Semen samples were stored in temperature controlled units (Minitube 70.79 L) at 17 ◦ C. Extended semen (1 mL) was allowed to warm in a 37 ◦ C warming block set on a rocker for 25 min. Two 3 ␮L samples of this pre-warmed extended semen were loaded into a Leja slide chamber and five fields were recorded for each sample. A computer assisted semen evaluation system (Ceros II by IMV Technologies, LEJA Fixed Volume Slides for Analysis) was used to record all semen mobility estimates. Mobility measurements included the sperm motion variables: amplitude of lateral head displacement (ALH), average path velocity (VAP), beat-cross frequency (BCF), curvilinear velocity (VCL), distance average path (DAP), distance curvilinear (DCL), distance straight line (DSL), linearity (LIN), straight-line velocity (VSL), wobble (WOB), percent of static cells and total and progressive motility. After sperm motility analysis was completed, 100 ␮L of 10% formalin (365 mL PBS, 135.14 mL Formaldehyde) was added to the conical tube and then the tube was capped for morphology evaluation. Semen (10 ␮L) was placed

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Fig. 2. Weekly rectal temperatures in boars; Data are presented as the LS mean +/− SE (Control: n = 23, Betaine 0.63%: n = 24 and Betaine 1.26%: n = 21); There was no treatment difference (P = 0.44) or treatment by week interaction (P = 0.95). Table 4 Homocysteine serum concentration for treatments by blood test daya . Blood test dayb

Treatment CON

9 days 45 days 73 days a b

34.47 42.28 42.69

BET 0.63% 28.22 30.68 33.09

BET 1.26% 30.52 35.20 39.25

SE 2.91 2.90 2.96

P-Value 0.237 0.009 0.043

Treatment by blood test day interaction (P = 0.41). Blood test represents the measurements on days 9, 45 and 73 after the initiation of betaine-supplemented diets.

on a microscope slide. Morphological abnormalities were evaluated for 200 randomly selected sperm cells using bright-field microscopy on a phase-contrast microscope at 40×. The categories for general abnormalities were: number of normal sperm, percent of sperm with proximal droplet, distal droplet, distal midpiece reflex (DMR), and tail abnormalities. After general sperm morphology evaluation was complete, acrosome morphology was evaluated with 100× on the phase-contrast scope. For normal and abnormal acrosome morphology 100 cells were evaluated. In addition, remaining semen samples were stored for 7 days to evaluate changes in the estimates of semen quality over typical storage times. Motility was analyzed 1, 3 and 7 days after collection and sperm morphology was evaluated on days 1 and 7. All previously described assays were performed with the stored semen samples. 2.7. Flow cytometry analysis Ejaculate samples from 89 boars were analyzed every other week at days 1 and 7 after collection. Sperm viability was determined using the Live/Dead Sperm Viability Kit (Molecular Probes, L-7011) utilizing a nucleic acid stain, SYBR-14 and propidium iodide (PI). The assay was performed according to manufacturer’s standard procedures (Thermo Fisher Scientific, 2001). The cells were analyzed on an FC500 flow cytometer (Beckman Coulter, Miami, FL). Events (n = 20,000) were collected for each sample and the

data were analyzed using Flowjo flow cytometry analysis software (Flowjo LLC, Ashland, OR).

2.8. Statistical analysis Data of boars (n = 7) were removed from the original data set, because of poor ejaculate characteristics and quality before and during the trial. Any individual observation that was greater than ±3.5 standard deviations from the mean was considered an outlier for each variable and removed from the data set. The model for rectal temperatures and variables that were recorded only at the time of semen collection (volume, concentration and total sperm), included betaine treatment, genetic line, and week as fixed effects, age as a covariate and boars nested within genetic line as a random variable. Blood test sampling for homocysteine serum concentration assessment was only performed three times during the trial instead of weekly. The model for variables that were recorded after semen arrived at Purdue University and following storage at 17 ◦ C (motility, morphology and flow cytometry variables), included betaine treatment, genetic line, week and days in storage as fixed effects, age as a covariate and boars nested within genetic line as a random variable. Because these variables were analyzed for each boar once every 2 weeks, blocks were defined as a 2-week period of time.

Please cite this article in press as: Cabezón, F.A., et al., Effect of natural betaine on estimates of semen quality in mature AI boars during summer heat stress. Anim. Reprod. Sci. (2016), http://dx.doi.org/10.1016/j.anireprosci.2016.03.009

N Rectal ◦ C a

BET 0.63%

BET 1.26%

23 38.50

24 38.55

21 38.56

SE

L

T

SE

CON vs. BETb

BET Differencec

Treatment

Genetic

Week

Genetic × Week

0.04

22 38.46

46 38.61

0.04

0.203

0.869

0.439

0.002

0.001

0.091

L: Large White line; T: Terminal line. CON compared with BET: represents the orthogonal contrast between control and both betaine diets combined. BET Difference: represents the orthogonal contrast between the betaine 0.63% and 1.26%. Treatment by genetic interaction (P = 0.50).

Table 6 Overall ejaculate characteristics and betaine seminal plasma concentration for treatments and genetic lines. Ejaculate

Genetic linesa

Treatment CON

BET 0.63%

BET 1.26%

N Volumeb (ml) Concentrationb (108 /ml) Total sperm (109 )

32 446.4 1.99 80.4

27 423.6 2.26 85.2

N Betaine seminal plasmac (ug/ml)

6 148.1

6 235.9

a b c d e f g

P-Valuef , g

SE

L

T

SE

30 471.7 2.05 90.8

26.10 0.12 4.22

23 404.3 2.48 92.0

66 490.1 1.73 79.0

66 26.8 0.12 3.8

6 228.8

6 32.2

6 227.4

12 181.1

12 31.4

CON vs. BETd

BET Differencee

Treatment

Genetic line

Week

Genetic × Week

0.964 0.196 0.093

0.159 0.175 0.336

0.368 0.183 0.143

0.007 0.001 0.005

0.001 0.001 0.001

0.041 0.003 0.001

12 0.046

12 0.877

12 0.126

12 0.248

12 0.001

12 0.001

L: Large White line; T: Terminal line. Volume and concentration are reported with 1:1 dilution. Betaine concentration in seminal plasma was recorded from a different set of boars on week 1, 5 and 10 of the study. CON compared with BET: represents the orthogonal contrast between control and both betaine diets combined. BET Difference: represents the orthogonal contrast between the betaine 0.63% and 1.26%. No treatment by genetic line interaction was found significant for volume, concentration or total sperm in the ejaculate (P > 0.17). No treatment by genetic line interaction was found for betaine seminal plasma concentration (P = 0.70).

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c d

CON

P-Valued

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Genetic linesa

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Table 5 Overall rectal body temperature for treatments and genetic lines.

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Time period (week or biweek) or days of betaine feeding were also evaluated as linear covariates. The model included the fixed effects of betaine treatment, genetic line, days in storage and linear effect of the time on betaine and interaction of betaine treatment by time. If the betaine treatment by time interaction was significant, separate linear regressions were evaluated for each betaine treatment. All variables were analyzed with a full model. Terms with P > 0.20 were removed from the model also with evaluation of the AIC values. Higher-level interaction terms were removed first. Lower-level non-significant interaction terms were removed only if higher-level interaction terms were already removed. All analyses were performed using MIXED procedure in SAS (version 9.4, SAS Institute Inc., Cary, NC). A multiple comparison of means was performed using Tukey-Kramer method. Also an orthogonal contrast between CON and both BET diets combined (CON v/s BET) and between BET treatments 0.63% and 1.26% (BET Difference) were performed. The slice option in SAS was used to evaluate betaine effect for each blood test day, biweek and genetic line after a treatment by blood test day or treatment by biweek or treatment by genetic interaction was found significant. For all analyses P < 0.05 was considered significant and P < 0.10 was considered a trend.

3. Results 3.1. Homocysteine serum concentrations The results for the analysis of serum homocysteine concentration in boars are shown in Table 3. Serum homocysteine concentrations were reduced 22.86% and 12.06% for the BET-0.63% and BET-1.26% treatment groups, respectively, compared to the CON group (P = 0.016). There were no significant differences between treatments on serum homocysteine concentration after 9 days of betaine feeding (Table 4). However, after 45 and 73 days of betaine supplementation, serum homocysteine concentrations were reduced in the BET-0.63% and BET-1.26% treatment groups relative to the CON treatment (P = 0.009 and P = 0.043, respectively). Moreover, serum homocysteine concentrations increased in a positive linear fashion from day 9 to 73 (b = 0.145 ± 0.02, P < 0.001). The boars of the L genetic line had lesser mean serum homocysteine concentrations than boars of the T line (P = 0.002). There were no treatment by genetic line or genetic line by blood test day interactions (P = 0.62 and P = 0.11, respectively).

3.2. Rectal temperature Rectal temperatures did not differ among betaine treatment groups (P = 0.44, Table 5). Rectal temperature was greater in boars of T than L line (P = 0.002). There were variations in rectal temperatures by week (Fig. 2, P < 0.001) and these tended to be different by genetic line (P = 0.091). There were no treatment by genetic line interactions (P = 0.50).

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3.3. Ejaculate and betaine seminal plasma evaluation Semen volume and sperm concentration are reported with the 1:1 semen dilution (Table 6). No statistical differences were found for ejaculate volume or concentration among the treatment groups (P = 0.37, P = 0.18, respectively). There was a tendency for total sperm produced in the ejaculate to increase with betaine supplementation (P = 0.093) as evidenced with the BET-0.63% treatment where there was a 5.97% increase and for the BET-1.26% group a 12.94% increase in total sperm as compared with values for control animals. Total sperm and volume of the ejaculate increased over the 10 week period (b = 0.890 ± 0.25, P < 0.001 and b = 10.58 ± 1.45, P < 0.001, respectively), and sperm concentration decreased over the same time (b = −0.014 ± 0.006, P = 0.014). There were no treatment by genetic line interactions for any variable (P > 0.17). Boars of the BET-0.63% and BET-1.26% treatment groups had 59.28% and 54.49% greater betaine seminal plasma concentrations than boars of the control group (P = 0.046). There were no treatment by genetic line interactions for betaine concentration in seminal plasma (P = 0.70). 3.4. Sperm motility and morphology The results for the analysis of sperm mobility variables are shown in Table 7. Overall, sperm mobility variables did not differ among treatments. However, there were several biweekly period by treatment interactions. Distance curvilinear (DCL) differed among treatments for biweekly period 3, where the CON group was 29.31 ± 1.38, BET-0.63% group was 31.73 ± 1.65 and BET-1.26% was 34.70 ± 1.51 (P = 0.003). Furthermore, treatments tended to differ on biweekly period 2 for LIN, where the CON group was 52.94 ± 1.35, BET-0.63% group was 51.18 ± 1.53 and BET-1.26% group was 48.78 ± 1.43 (P = 0.095). Treatments tended to differ on biweekly period 5 for total motility, where the CON was 39.46 ± 3.73, BET-0.63% group was 52.94 ± 4.41 and BET-1.26% group was 47.82 ± 3.98 (P = 0.054), progressive motility, where the CON group was 32.54 ± 3.49, BET-0.63% group was 44.66 ± 4.13 and BET-1.26% group was 39.84 ± 3.72 (P = 0.069) and static, where the CON group was 60.54 ± 3.73, BET-0.63% group was 46.88 ± 4.41 and BET-1.26% group was 52.20 ± 3.97 (P = 0.050). All sperm motion variables varied by biweekly period (P < 0.001), except BCF. The values of some semen characteristics increased linearly with increased biweekly period including: ALH (b = 0.125 ± 0.026, P < 0.001), VCL (b = 1.143 ± 0.536, P = 0.033) and static (b = 0.888 ± 0.368, P = 0.016). The values of some semen characteristics decreased linearly with increased biweekly period including: DSL (b = −0.225 ± 0.093, P = 0.015), LIN (b = −0.813 ± 0.211, P < 0.001), STR (b = −0.665 ± 0.158, P < 0.001), WOB (b = −0.524 ± 0.154, P < 0.001), progressive motility (b = −0.744 ± 0.341, P = 0.029) and total motility (b = −0.865 ± 0.367, P = 0.019). Days in storage influenced all the motion variables (P < 0.001), except for BCF and LIN. There was a genetic line by day interaction for several sperm mobility variables (P < 0.05, Table 7). There was no treatment by genetic line or treatment by day two-way

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a

BET 0.63%

BET 1.26%

32 4.3 47.5 32.3 84.2 18.0 32.0 13.8 51.7 79.5 36.5 62.3 41.0 34.3 59.0

27 4.5 50.6 32.9 89.1 19.0 33.6 14.8 51.0 79.0 39.4 61.8 47.1 40.0 52.9

30 4.5 49.2 32.7 89.5 18.7 34.1 14.3 50.2 78.7 37.4 60.9 43.3 36.4 56.7

SE

L

T

SE

1

3

7

SE

0.2 2.1 0.4 3.7 0.8 1.4 0.7 1.2 0.8 1.8 0.9 3.4 3.2 3.4

23 4.8 50.4 32.6 94.7 18.7 35.5 13.7 47.1 76.1 37.0 59.0 43.0 35.2 57.0

66 4.1 47.8 32.7 80.6 18.4 31.0 14.9 54.8 82.0 38.5 64.3 44.6 38.6 55.4

0.1 1.9 0.3 3.4 0.8 1.3 0.7 1.1 0.8 1.7 0.8 3.1 2.9 3.1

4.6 52.3 32.4 100.1 22.4 39.5 17.3 50.8 79.6 44.4 61.5 56.5 48.7 43.5

4.5 38.0 32.1 93.0 20.0 35.8 15.8 50.5 79.7 41.2 60.6 47.0 39.9 53.0

4.2 37.1 32.7 69.7 13.3 24.5 9.8 51.7 77.8 27.7 62.8 27.9 22.1 72.2

0.1 1.3 0.3 2.3 0.5 0.9 0.4 0.8 0.6 1.1 0.6 2.0 1.8 2.0

P-Valueg CON vs. BETe

BET Differencef

Treatment

Genetic line

Biweekc

Dayb

Genetic × Biweek

0.245 0.298 0.192 0.201 0.312 0.225 0.375 0.377 0.463 0.338 0.338 0.247 0.257 0.247

0.845 0.626 0.579 0.937 0.769 0.784 0.605 0.626 0.795 0.434 0.491 0.414 0.409 0.415

0.491 0.528 0.381 0.435 0.581 0.452 0.601 0.588 0.733 0.482 0.482 0.381 0.389 0.382

0.001 0.241 0.836 0.001 0.711 0.004 0.134 0.001 0.001 0.449 0.001 0.647 0.305 0.654

0.001 0.001 0.153 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001

0.001 0.001 0.197 0.001 0.001 0.001 0.001 0.242 0.001 0.001 0.001 0.001 0.001 0.001

0.001 0.005 0.001 0.032 0.002

Treatment × Biweek

0.018 0.031 0.021

0.001 0.022 0.072 0.001 0.012 0.032 0.012

Day × Biweek 0.001 0.001 0.001 0.001 0.024 0.011 0.001 0.001 0.012 0.001 0.001 0.001 0.001

Genetic × Day

Age 0.046

0.043 0.013 0.033 0.026

0.039 0.012

0.009 0.015 0.010

L: Large White line; T: Terminal line. Day represents the storage time of semen after collection. c Biweek represents blocks of 2-week period of time because these variables were analyzed for each boar once every 2 weeks. d Amplitude of Lateral Head Displacement (ALH), Average Path Velocity (VAP), Beat-Cross Frequency (BCF), Curvilinear Velocity (VCL), Distance Average Path (DAP), Distance Curvilinear (DCL), Distance Straight Line (DSL), Linearity (LIN), Straight-Line Velocity (VSL) and Wobble (WOB). e CON compared with BET: represents the orthogonal contrast between control and both betaine diets combined. f BET Difference: represents the orthogonal contrast between the betaine 0.63% and 1.26%. g No treatment by genetic line or treatment by day interaction was found for any variable (P > 0.39 and P > 0.12, respectively). b

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ALH (␮m) VAP (␮m/s) BCF (Hz) VCL (␮m/s) DAP (␮m) DCL (␮m) DSL (␮m) LIN (%) STR (%) VSL (␮m/s) WOB (%) Total Motility (%) Progressive Motility (%) Static (%)

CON

Dayb

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N ALH (␮m) VAP (␮m/s) BCF (Hz) VCL (␮m/s) DAP (␮m) DCL (␮m) DSL (␮m) LIN (%) STR (%) VSL (␮m/s) WOB (%) Total motility (%) Progressive motility (%) Static (%) Motion Variablesd

Genetic linea

Treatment

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Table 7 Overall sperm motility evaluation for treatments, genetic lines and storage time of semen.

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interaction for any semen motility measurements (P > 0.39 and P > 0.12, respectively). The results for the analysis of morphological characteristics of sperm are shown in Table 8. The percent of cells with a distal mid-piece reflex (DMR) was 71% greater for boars of the BET-1.26% than for boar of the BET-0.63% group (P = 0.009). For L line boars, the percent of sperm with DMR was 87.2% and 165% greater for the BET-1.26% treatment group than the CON and BET-0.63% treatment groups, respectively (P = 0.004, Table 9). There was no difference in DMR among treatments in the T line boars (P = 0.92). Betaine supplementation at 1.26% resulted in a greater DMR in boars of the L line than those of the T line (P < 0.001). Sperm tail abnormalities tended to be greater in the boars fed the BET-1.26% treatment compared with boars of the BET-0.63% group (Table 8, P = 0.035) and these differences occurred in biweekly periods 4 and 5 with tendencies in biweekly period 3 (Table 10). There was an effect of biweekly period for all the sperm morphological variables analyzed (P < 0.001). Percent normal sperm increased at greater rate over time for boars of the BET-1.26% treatment group than the boars of the BET-0.63% and CON treatment groups (P = 0.003, b = 2.394 ± 0.29, P < 0.001, b = 1.455 ± 0.40, P = 0.020, b = 1.112 ± 0.39, P = 0.001, respectively). The interaction of the linear effect of biweekly period by treatment on sperm tail abnormalities was significant (P = 0.015). The boars of the BET-1.26% treatment group had a greater increase in sperm tail abnormalities over time than the boars of the CON group, with boars in the BET-0.63% group being intermediate (BET-1.26% b = 0.547 ± 0.10, P < 0.001, BET-0.63% b = 0.383 ± 0.14, P = 0.26, CON b = 0.145 ± 0.14, P = 0.004) in sperm abnormalities. There was an interaction of the linear effect of biweekly period by treatment for normal acrosome percentage (P = 0.009). Normal acrosome percentage had no linear change over time for boars of the BET-1.26% treatment group (b = −0.005 ± 0.22, P < 0.98). The boars of the BET-0.63% group tended to have greater increases in acrosome damage (b = 0.557 ± 0.31, P = 0.072) and the boars of the CON group had a linear increase in acrosome damage over time (b = 0.929 ± 0.30, P = 0.002). A negative linear change occurred over biweekly periods for proximal droplet percentage (b = −0.431 ± 0.008, P < 0.001). There was an interaction of the linear effect of biweekly period by treatment on distal droplet percentage (P = 0.011). The boars fed the BET-1.26% diet had a greater decrease in distal cytoplasmic droplets than boars fed the CON diet. There were, however, no differences among betaine treatment groups in number of cytoplasmic droplets (BET-1.26% b = −1.619 ± 0.17, P < 0.001, BET0.63% b = −1.279 ± 0.23, P = 0.15, CON b = −0.937 ± 0.23, P = 0.003). There was an interaction of the linear effect of biweekly period by treatment for abnormal acrosome percentage (P = 0.004). There was no linear change in abnormal acrosome percentage over biweekly periods for the BET-1.26% group (BET-1.26% b = 0.114 ± 0.23, P < 0.62). The linear change in abnormal acrosome percentage over biweekly period for the boars of the BET-1.26% group tended to be different than for the boars of the BET-0.63% group (b = −0.429 ± 0.32, P = 0.093), and was different from that of the CON group (b = −0.948 ± 0.32, P < 0.001). Sperm

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tail abnormalities increased across all treatment groups between 1 and 7 days in storage (P = 0.039). Percentage of normal acrosomes, however, decreased (P < 0.001) over days of storage. Percentage of normal sperm tended to decrease across treatments between 1 and 7 days in storage, however, DMR tended to increase (P = 0.058 and P = 0.061, respectively). There was no treatment by genetic line two-way interaction for any variable (P > 0.37), except for DMR (P = 0.008). There was no treatment by day or genetic line by day interaction for any variable (P > 0.46 and P > 0.64, respectively). There was a treatment by biweekly period interaction for distal droplet and tail abnormalities (P = 0.013, and P < 0.001, respectively). There were biweekly period by day interactions for percent distal droplets (P < 0.001), normal acrosomes (P = 0.007) and abnormal acrosomes (P = 0.038). There was a linear effect of age for distal droplets (P = 0.002) and there was a tendency for a linear effect on percent of normal sperm and DMR (P = 0.091 and P = 0.050, respectively). 3.5. Flow cytometry The overall results for the flow cytometric evaluations are shown in Table 11. There were no differences between dietary treatments for the assay performed using flow cytometry Live/Dead (P = 0.59). There was a treatment by biweekly period interaction for this variable (P = 0.006), which indicates that some of the differences between treatments differed for each of the five biweekly periods (Table 12). Moreover, six additional days of storage decreased the percent of live sperm by 3.83% irrespective of treatment (P < 0.001). The percent of live sperm in an ejaculate decreased across biweekly periods of the study (P < 0.001, b = −0.755 ± 0.298, P = 0.012). There was no treatment by genetic line, and treatment by day or genetic line by day interaction (P = 0.40, P = 0.90 and P = 0.64, respectively). 4. Discussion The present study examined the effect of different inclusion rates of betaine supplementation on semen characteristics at collection and for up to 7 days in cold storage for boars collected during summer months. To the best of author’s knowledge there are no previous studies available in the literature, evaluating betaine supplementation in boars. The total sperm motility was less than the expected range for 1-day-old extended semen. This is likely an artifact of overnight shipment of the semen samples from Oklahoma to Indiana during the summer months. Japanese quail on which heat stress was imposed had greater homocysteine serum concentrations than birds kept at thermo-neutral temperature (Sahin et al., 2003). In the present study, betaine supplementation reduced serum homocysteine concentrations. The current results are consistent with previous studies in which homocysteine concentrations were reduced by dietary betaine supplementation in mice (Ji and Kaplowitz, 2003; Song et al., 2008) and humans (Schwab et al., 2002). Betaine is a methyl donor compound involved in the conversion of homocysteine into methionine. This reaction is

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Table 8 Overall morphology evaluation for treatments, genetic lines and storage time of semen. Morphology

Genetic linec

Treatment

N Normal (%) Proximal Droplet (%) Distal Droplet (%) Distal Midpiece Reflex (%) Tail Abnormalities (%) Normal Acrosome (%) Abnormal Acrosome (%)

CON

BET 0.63%

BET 1.26%

32 69.6 6.94 10.8 7.86ab 3.64 75.3 25.1

27 72.4 6.09 12.0 6.55b 3.03 78.2 22.6

30 63.2 6.85 12.3 11.3a 4.94 75.7 24.9

Dayd

SE

L

T

SE

1

7

SE

4.1 1.5 2.0 1.3 0.7 1.4 1.5

23 69.6 5.65 10.2 10.2 4.23 74.7 25.9

66 67.3 7.60 13.2 6.9 3.51 78.1 22.5

3.8 1.4 1.9 1.2 0.6 1.3 1.3

68.9 6.61 11.8 8.32 3.68 77.8 22.9

67.9 6.64 11.7 8.80 4.06 75.0 25.6

2.2 0.8 1.1 0.7 0.4 0.8 0.8

P-Valueh , i

Morphology

Normal (%) Proximal Droplet (%) Distal Droplet (%) Distal Midpiece Reflex (%) Tail Abnormalities (%) Normal Acrosome (%) Abnormal Acrosome (%)

CON vs. BETf

BET Differenceg

Treatment

Genetic

Biweeke

Dayd

Genetic × Biweek

0.685 0.780 0.554 0.450 0.628 0.296 0.383

0.108 0.721 0.903 0.009 0.035 0.223 0.260

0.243 0.905 0.829 0.022 0.091 0.291 0.377

0.600 0.239 0.163 0.016 0.307 0.034 0.031

0.001 0.001 0.001 0.001 0.001 0.001 0.001

0.058 0.899 0.786 0.061 0.039 0.001 0.001

0.011

Treatment × Biweek

0.013 0.001 0.004 0.001

0.001

Superscripts (a,b) denote significant difference between treatments based on Tukey-Kramer method. c L: Large White line; T: Terminal line. d Day represents the storage time of semen after collection. e Biweek represents blocks of 2-week period of time because these variables were analyzed for each boar once every 2 weeks. f CON compared with BET: represents the orthogonal contrast between control and both betaine diets combined. g BET Difference: represents the orthogonal contrast between the betaine 0.63% and 1.26%. h There was no treatment by genetic line interaction for any variable (P > 0.37), except for distal midpiece reflex (P = 0.008). i There was no treatment by day or genetic line by day interaction for any variable (P > 0.46 and P > 0.64, respectively). Table 9 Evaluation of distal midpiece reflex in treatments by genetic linesf . Genetic linee

Distal Midpiece Reflex (%) CON

BET 0.63%

ab

L T

BET 1.26%

b

8.58 7.13

ac

6.07 7.02

16.07 6.49d

SE

P-Value

2.27 1.21

0.004 0.917

Superscripts (a,b) denote significant difference between treatments based on Tukey-Kramer method. Superscripts (c,d) denote significant difference between genetic lines at each betaine level based on Tukey-Kramer method. e L: Large White line; T: Terminal line. f Treatment by genetic interaction (P = 0.008). Table 10 Evaluation of distal droplet, distal midpiece reflex and tail abnormalities in treatments by biweekly periodd . Biweekc

1 2 3 4 5

Distal droplet (%)

Tail abnormalities (%)

CON

BET 0.63%

BET 1.26%

SE

P-Value

CON

BET 0.63%

BET 1.26%

SE

P-Value

11.65 13.00 10.90 9.98 8.66

14.61 13.37 11.50 10.81 9.57

16.12 13.62 11.75 10.68 9.39

2.12 2.10 2.10 2.11 2.10

0.223 0.972 0.945 0.944 0.934

2.59 4.12 3.98 3.53ab 4.00

2.04 2.69 3.45 3.31b 3.65

2.31 4.22 5.52 6.73a 5.93

0.76 0.73 0.72 0.74 0.74

0.853 0.222 0.089 0.001 0.039

Superscripts (a,b) denote difference between treatments based on Tukey-Kramer method. c Biweek represents blocks of 2-week period of time because these variables were analyzed for each boar once every 2 weeks. d Treatment by biweek interaction for distal droplet (P = 0.013) and for tail abnormalities (P < 0.001).

mediated by betaine-homocysteine methyl-transferase (BHMT). Some studies have shown that BHMT activity rate was increased after betaine supplementation (Emmert et al., 1998; Wang et al., 2000). In mice, dietary supplementation of betaine induced BHMT enzyme activity, and homocysteine concentrations were reduced by approximately 40% (Pajares and Pérez-Sala, 2006).

Rectal temperatures were different among boars of the two genetic lines (P = 0.002) in the present study. A potential explanation could be that genetic lines have different metabolic and heat production rates; different sensitivity to heat stress, or different coping and adaptation abilities such increased respiration rates. Flowers (2008) reported that the impact of heat stress could vary within and between genetic lines.

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0.003

Day × Biweek

1.27

0.005

f

e

c

d

L: Large White line; T: Terminal line. Day represents storage time of semen after collection. Biweek represents blocks of 2-week period of time because this variable was analyzed for each boar once every 2 weeks. CON compared with BET: represents the orthogonal contrast between control and both betaine diets combined. BET Difference: represents the orthogonal contrast between the betaine 0.63% and 1.26%. There was no treatment by genetic line, treatment by day or genetic line by day interaction (P = 0.40, P = 0.90 and P = 0.64, respectively). b

Dayb

0.001 0.001

Biweekc Genetic

0.015 0.590

Treatment BET Differencee CON vs. BETd

0.416

2.32 P-Valuef

0.509 SYBR Live/Dead (%)

23 57.12 Flow cytometry

a

Genetic × Biweek

0.011

Treatment × Biweek

58.28 62.11 2.14

7 1 SE T SE

L

30 59.45

BET 1.26% BET 0.63%

27 62.04

CON

32 59.10 n SYBR Live/Dead (%)

Dayb Genetic linea Treatment Flow cytometry

Table 11 Overall flow cytometry evaluation for treatments, genetic lines and storage time of semen.

66 63.28

SE

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Total sperm production, the product of sperm concentration times semen volume, tended to increase with betaine supplementation with a positive linear response as betaine supplementation increased. Total concentration and semen volume were, however, not increased in the present study. Spermatogonial cells are prone to oxidation due to the greater concentration of unsaturated fatty acids in the plasma membrane. An antioxidant effect may exist as a result of betaine treatment (Alirezaei et al., 2012), which could explain how betaine functions to increase the number of sperm cells in the ejaculate by sparing oxidative cell loss. Betaine is a small and highly water-soluble molecule and, therefore, is expected to cross the bloodtestes barrier and be present in the testes. Transporters from SLC6 family involved in betaine translocation across the plasma membrane have been identified in testes cells (Chen et al., 2004). Also, greater betaine concentrations have been observed in mice testes. Testicular fluid concentrations were, however, poorly correlated (r = 0.38) with betaine plasma concentrations (Slow et al., 2009). Therefore, it is possible that betaine could be synthetized within testes cells. Furthermore, BHMT mRNA has been identified in Sertoli cells within testes (Edgard et al., 2002; Slow et al., 2009). Some of the accumulated testicular betaine could be utilized for the production of S-adenosyl methionine, which is needed for the synthesis of testicular creatine, a chemical required for sperm function and motility (Lee et al., 1998). Percent of sperm with the distal midpiece reflex abnormality was greater in the BET-1.26% than the BET-0.63% treatment group. This morphological abnormality could be associated with the exposure of sperm to hypotonic medium in the epididymis (Barth and Oko, 1989). In bulls, DMR results as sperm migrate to the distal portion of the epididymis as a consequence of environmental changes, probably in association with abnormal ionic concentration (Barth and Oko, 1989; Brito et al., 2003). The quantification of the concentration of betaine in seminal plasma showed that the concentration of betaine was increased with betaine supplementation, but no differences in betaine concentration were detected among animals with betaine supplemented diets. Further study is required to quantify the concentration and understand the potential function of betaine in seminal plasma. In general, the genetic line by week two-way interactions are difficult to interpret because these interactions can be considered as a genetic by environmental interaction, and are also associated with the increased duration of betaine supplementation. In the present study, the environment has been considered as a fixed factor to test the interactions, but in reality week or biweekly period is a random-environmental factor. The problem with sperm related variables, such as semen quality, sperm morphology and sperm motility is that not only the environment at the time of collection could affect these variables, but also by the environment present several weeks prior to the time of collection may impact these variables (Wettemann et al., 1979). With some variables, for which typical semen storage times were evaluated, there were genetic-line by day two-way interactions. In these cases, it is advisable to be

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Table 12 Evaluation of SYBR Live/Dead percent of sperm in treatments by biweekly periodb . Biweeka

1 2 3 4 5 a b

SYBR Live/Dead (%) CON

BET 0.63%

BET 1.26%

SE

P-Value

61.88 61.65 54.49 56.59 60.87

67.78 60.44 54.75 62.65 64.59

60.14 59.36 56.55 58.55 62.66

2.62 2.62 2.62 2.61 2.61

0.088 0.778 0.802 0.202 0.547

Biweek represents blocks of 2-week period of time because this variable was analyzed for each boar once every 2 weeks. Treatment by biweek interaction (P = 0.006).

conservative and use the semen earlier due to the inconsistency of the effect of storage time. Temperatures in Oklahoma during summer of 2014 were less than expected. Maximum temperature recordings inside the housing facility for the boars did not exceed 30 ◦ C. This could be important because dietary betaine is more effective when feed intake is restricted or the animal is under stressful conditions (Casarin et al., 1997). 5. Conclusions Betaine tended to increase total sperm production at summer months in boars. The 1.26% betaine dietary supplementation increased the percent of sperm cells with DMR. Overall, for most variables there was consistently positive change with 0.63% betaine supplementation but there was not a statistically significant impact due to the variation and limited sample size. Further studies should be conducted using a larger sample size to detect differences in variables with high variability. Conflict of interest None. Acknowledgements The authors thank the staff of Hanor Company, Kelly Haymaker and Mark Mcculley and the staff of Purdue University, Mary Myers, Griffin Nichols, Amber Jannasch and Margaret Yeadon for their assistance during the study. The financial support for this experiment was provided by Dr. P. Wilcock of AB Vista and is gratefully acknowledged. References Alirezaei, M., Jelodar, G., Ghayemi, Z., 2012. Antioxidant defense of betaine against oxidative stress induced by ethanol in the rat testes. Int. J. Pept. Res. Ther. 18, 239–247. Barth, A., Oko, R., 1989. Abnormal Morphology of Bovine Spermatozoa. Iowa State University Press, Ames, IA. Bezold, G., Lange, M., Peter, R.U., 2001. Homozygous methylenetetrahydrofolate reduces C677T mutation and male infertility. N. Engl. J. Med. 344 (15), 1172–1173. Brito, L.F., Silva, A.E., Barbosa, R.T., Unanian, M.M., Kastelic, J.P., 2003. Effects of scrotal insulation on sperm production, semen quality, and testicular echotexture in Bos indicus and Bos indicus x Bos taurus bulls. Anim. Reprod. Sci. 79, 1–15. Casarin, A., Forat, M., Zabaras-Krick, B.J., 1997. Interrelationships between betaine (Betafin-BCR) and level of feed intake on the performance parameters and carcass characteristics of growing-finishing pigs. J. Anim. Sci. 75 (Suppl. 1), 75 (Abstr.).

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Please cite this article in press as: Cabezón, F.A., et al., Effect of natural betaine on estimates of semen quality in mature AI boars during summer heat stress. Anim. Reprod. Sci. (2016), http://dx.doi.org/10.1016/j.anireprosci.2016.03.009