Influences on thermo-resistance of boar spermatozoa

Accepted Manuscript Influences on thermo-resistance of boar spermatozoa

M. Schulze, U. Jakop, M. Jung, F. Cabezón PII:

S0093-691X(18)30973-7

DOI:

10.1016/j.theriogenology.2018.12.022

Reference:

THE 14814

To appear in:

Theriogenology

Received Date:

16 October 2018

Accepted Date:

13 December 2018

Please cite this article as: M. Schulze, U. Jakop, M. Jung, F. Cabezón, Influences on thermoresistance of boar spermatozoa, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.12.022

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ACCEPTED MANUSCRIPT Influences on thermo-resistance of boar spermatozoa

M. Schulzea,*, U. Jakopa, M. Junga, F. Cabezónb a

Institute for Reproduction of Farm Animals Schönow, Bernauer Allee 10, D-16321 Bernau,

Germany b

Department of Animal Sciences, Purdue University, West Lafayette, 270 S Russel Street, IN

47907, USA

*Corresponding

author. Tel.: +49 3338-709822; fax: +49 3338-709810;

E-mail address: [email protected] (M. Schulze)

1

ACCEPTED MANUSCRIPT 1

ABSTRACT

2

The aim of this study was to investigate the influence of semen production management

3

in 25 European boar studs on thermo-resistance (TRT) of boar spermatozoa. TRT is an

4

important semen characteristic, easy to determine, and is used to explain variations in pig

5

fertility. During a study period from 2013 to 2018, 905 ejaculates were analyzed for TRT on

6

day 7 of semen storage. Statistical models showed that around 40% of the total variability in

7

TRT could be explained by production management factors. Significant selected predictors

8

were year, month, boar stud, boar age, dilution steps, mitochondrial activity and membrane

9

integrity of spermatozoa, bacterial contamination, arrival temperature of ejaculates in the

10

analysis laboratory, sperm concentration, ejaculate volume, sperm output, dose/ejaculate, and

11

electrical conductivity of pure water for extender preparation. TRT increased during the study

12

period. No effects were observed for breed, dilution rate, morphologically intact spermatozoa,

13

and refractive index of extender on TRT. A holistic view of the requirements in the field of boar

14

semen production is of great importance for future developments of individualized Hazard

15

Analysis and Critical Control Point concepts, prognosis models for boar semen quality, and

16

could help to improve the efficiency of AI organizations.

17 18

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1. Introduction

20

Artificial insemination (AI) is a tool for efficient distribution of high quality genetics and

21

efficient running of genetic programs [1]. Pig reproduction efficiency has dramatically increased

22

in the last decade in the major pork-producing countries [2]. At the same time, customer

23

demands for semen quality have become increasingly important [3]. High production intensity

24

leads to higher levels of technology for boar semen processing. Increased automation requires

25

well-trained laboratory staff to ensure correct boar semen operation procedures and ejaculates

26

need to be handled in a controlled manner to avoid sperm damage. Temperature [4], dilution

27

[5], and hygiene management [6] are identified factors influencing sperm quality in stored AI

28

doses.

29

Today, it is well known that fertilization is a complex process involving a large number

30

of events and several new technologies have been developed to assess sperm functionality

31

and in vitro fertility in recent years [7]. Among other parameters, thermo-resistance of boar

32

spermatozoa could be used to explain variations in pig fertility [8]. Thermo-resistance test

33

(TRT) simulates the time of spermatozoa in the female genital tract by exposure to 38 °C for a

34

long time (e.g. 300 min) and concomitantly sperm motility after this heat stress is measured.

35

Although spermatozoa are transported rapidly to the oviduct, where they wait for ovulation for

36

several hours. After this prolonged time, only spermatozoa with a functional metabolism are

37

motile. Solely spermatozoa showing adequate motility after this long time are able to fertilize,

38

which is simulated by TRT. A long storage time of spermatozoa combined with a long

39

incubation time at 38 °C enables the finding of even small differences between high quality

40

ejaculates from commercial boar studs. Even though TRT is relatively time consuming due to

41

long incubation, it is easy to perform with a short hands-on time. TRT is also one of the

42

important predictors of conception rates in bulls [9].

43

While many data sources exist for benchmarking based on classical sow herd

44

performance [10], there are limited sources available for identifying critical control points in

45

boar studs. Nevertheless, considerable variations exist in management practices among

46

European boar studs [11]. The aim of the present study was to identify factors explaining

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ACCEPTED MANUSCRIPT 47

variations in TRT of boar spermatozoa in 25 European boar studs during six years from 2013

48

to 2018. Possible influencing factors were simultaneously collected during a standardized

49

quality control audit. Based on the results described in this manuscript, several boar and

50

semen related characteristics and their effect on TRT of boar spermatozoa are discussed. This

51

allows boar studs to optimize their production management and reduce risks during semen

52

production. The identification of practices employed may be useful for discussing which

53

management tools are valuable for advancing semen production management in the future.

54 55

2. Materials and methods

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2.1. Data acquisition

57

Field investigations were performed over a six-year period (2013 to 2018) in 25 boar

58

studs (size 80 to 500 boars) from nine different organizations as part of an external quality

59

control program of the Institute for Reproduction of Farm Animals Schönow (IFN). Boar studs

60

belonging to the Association for Bioeconomy Research (FBF) were visited every two years

61

(mean: 2.4 ± 0.8 visits per study period; range between 1 to 4). In total, 60 quality control audits

62

were carried out.

63

The boar studs (1 to 25) were located throughout Germany (n = 21), Austria (n = 2),

64

and Switzerland (n = 2). From each of the 25 boar studs, between 11 and 20 arbitrarily selected

65

ejaculates from 905 boars (one ejaculate/boar) were analyzed for production, temperature,

66

and dilution management and subsequently for semen quality. Boars were between 8.5 and

67

60 months old. Four different breeds were considered in this study (Table 1).

68 69

2.2. Semen collection and processing

70

All boars were routinely used for semen collection and AI dose processing, received

71

commercial feed (pellets) for AI boars and were housed in individual pens equipped with nipple

72

drinkers according to the European Commission Directive for Pig Welfare. The collection

73

frequency of ejaculates did not exceed three collections within 2 weeks with at least 3 days of

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rest in between. Semen production protocols were used according to the general guidelines

75

for semen processing used in AI studs participating in a quality control audit of the IFN [3].

76

Ejaculates were collected by the gloved-hand method. The day of collection is specified

77

as Day 0 (d0). The pre-sperm phase of the ejaculate was discarded and the gel fraction of the

78

semen was removed by gauze filtration during collection. The volume and sperm concentration

79

of each ejaculate were recorded. The remaining ejaculate volume was used for semen dose

80

production.

81

The dilution of ejaculates was completed in one step at nine boar studs with 357

82

ejaculates, in two steps at 11 boar studs with 403 ejaculates, and in three steps at five boar

83

studs with 145 ejaculates with short-term (n = 780 samples) or long-term (n = 125 samples)

84

extenders as described previously [11]. Extender temperatures were monitored with a

85

pyrometer P 300 W TFA (TFA Dostmann, Wertheim, Germany) before first dilution of semen

86

(first dilution temperature).

87

None of the raw or pre-diluted semen samples were stored in a water bath or an

88

incubator during processing. Semen was filled in QuickTip Flexitubes® (n = 831, Minitüb,

89

Germany) or in GTB bags® (n = 74 samples, IMV, France). Immediately after filling, arbitrarily

90

chosen AI doses were selected for further analysis and were placed in a temperature-

91

controlled box at 21 °C for 90 min (controlled room temperature). The temperature was then

92

reduced to 17 °C and samples were transported to the control laboratory (overall cooling rate

93

4 °C/hour), where the samples were stored for 7 days at 17 °C during which they were

94

subjected to further analyses. Except for days on which analyses occurred, there was no

95

rotation of samples. Upon arrival in the laboratory, temperature of the sperm samples (arrival

96

temperature) was measured using a Flash III infrared thermometer (Hartenstein, Germany).

97 98

2.3. Chemicals

99

All chemicals used in this study were of analytical grade. Unless stated otherwise, the

100

chemicals were purchased from Merck (Darmstadt, Germany) and Roth (Karlsruhe, Germany).

101

Propidium iodide (PI) and Rhodamine 123 (R123) were obtained from Sigma-Aldrich

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(Steinheim, Germany), and fluorescein-isothiocyanate conjugated peanut agglutinin (FITC-

103

PNA) and Pisum sativum agglutinin (FITC-PSA) were purchased from Axxora (Lörrach,

104

Germany).

105 106

2.4. Evaluation of sperm quantity and quality

107

Volumes of ejaculates and sperm concentrations were reported by the boar studs and

108

used to calculate total sperm numbers (sperm output) in raw semen. The weight of the

109

ejaculate was used as the measure of volume. All other sperm parameters were evaluated in

110

the reference laboratory of the IFN. A NucleoCounter SP-100TM was used for measurement of

111

sperm concentration in every AI dose (total sperm number per dose) as specified by Revision

112

1.5 of the User Guide Manual No. 991-0100 (ChemoMetec A/S, Denmark).

113

For analysis of sperm morphology on d0, PBS-buffered formalin samples with a

114

concentration of 50-100 x 106 sperm per mL were used. Sperm morphology was evaluated by

115

counting 200 spermatozoa under phase contrast at a magnification of 800× (Jenaval, Carl

116

Zeiss Jena, Germany) and classified according to Schulze et al. [12].

117

Sperm viability and mitochondrial activity were assessed on d2 of semen storage by

118

double-staining with R123/PI and flow cytometry as described previously [8]. A triple-stain flow

119

cytometric method using PI, PNA, and PSA fluorescent dyes was used on d3 of semen storage

120

to discriminate between viable (intact plasma membrane) and dead spermatozoa and

121

characterize membrane integrity in the acrosomal region [13]. The sperm subpopulation with

122

intact plasma membranes and intact acrosomal membranes (PI neg., PNA neg. and PSA neg.)

123

was determined as percent from overall sperm population.

124

Additionally, on d7 of semen storage, a TRT test was performed as described

125

previously [5]. An aliquot of 10 mL was incubated at 38°C in an aerated water bath. At 300 min

126

of incubation, total motility was determined using the computer-assisted sperm analysis

127

(CASA) system SpermVision® (Minitüb, Germany; 2013-2016) and from 2016 to 2018 with

128

AndroVision® (Minitüb, Germany). Before changing to the new CASA system, it was

129

determined that they both delivered comparable results.

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2.5. Total aerobic cell count (bacterial contamination)

132

A dilution series of 10-2 to 10-6 with 0.9% saline solution was performed for each sperm

133

sample to determine the bacterial contamination (expressed as colony-forming units per

134

milliliter, CFU/mL). One hundred microliters of each dilution was plated on d2 onto two nutrient

135

agar plates (Oxoid-Thermo Fischer, Germany) and incubated at 37 °C under aerobic

136

conditions [14]. The cultures were inspected and bacterial growth was recorded after 48 hours

137

of incubation.

138 139

2.6. Evaluation of the purified water quality and boar semen extender preparation

140

Electrical conductivity of purified water for extender preparation was measured in each

141

boar stud with DIST 3® (Hanna Instruments, Kehl, Germany) according to the manual.

142

Refractive index (°Brix) of each extender in each boar stud was measured with a portable

143

refractometer (Ref. 24400/0160, Minitüb, Germany) according to the manual. By determining

144

the refractive index of the extender, the correct concentration of the prepared extender can be

145

monitored [15]. First, a calibration was done with purified water. Then, 30 µL of the prepared

146

extender was put on the measuring field prism. The cover plate was slightly pressed and after

147

30 second, the scale was read as described previously [15].

148 149

2.7. Statistical analysis

150

All analyses were performed using MEANS and GLM procedures in SAS (version 9.4,

151

SAS Institute Inc., Cary, NC, U.S.A.). All descriptive data are expressed as mean ± standard

152

deviation (SD). An ANCOVA test was used to estimate the impact of several categorical

153

explanatory (Table 1) and continuous explanatory variables (Table 2) on response variable

154

(TRT of boar spermatozoa). Best model was selected using GLMSELECT procedure with

155

SELECTION=STEPWISE (SELECT=SL CHOOSE=PRESS) option in the model statement.

156

The final selected model was the model with the smallest predicted residual sum of squares

157

(PRESS). When a significant effect was revealed within a categorical explanatory predictor, a

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multiple comparison of means was performed using the Tukey-Kramer method. Differences

159

were considered statistically significant when P ≤ 0.05.

160 161

3. Results

162

TRT was 43.9 ± 23.3% (range: 0 - 91.0%) on d7 of semen storage. The final selected

163

model for TRT is shown on Table 3. Model for TRT explained 39.5% of the total variability

164

(P < 0.001). The inclusion of boar stud term (given that all other terms were already in the

165

model) provided the largest type III sum of squares. The effects of number of dilution steps,

166

months, and bacterial contamination on TRT are shown in Figure 1, 2, and 3, respectively.

167

Dilution steps affected TRT (P = 0.020). However, TRT differences were found between one-

168

and three-step (P < 0.001) and between two- and three-step (P = 0.014) dilution procedures.

169

Month of production impacted (P < 0.001) and bacterial contamination affected TRT

170

(P = 0.027). Overall, TRT increased at a rate of 2.16 ± 0.50% per year (P < 0.001) and at a

171

rate of 0.12 ± 0.05% per month boar age (P = 0.011). As percent of mitochondrially active

172

spermatozoa and membrane-intact spermatozoa increased by one unit, TRT measurements

173

increased 0.87 ± 0.11% and 0.52 ± 0.12% (P < 0.001), respectively. As sperm concentration

174

increased by 0.1 × 109/mL spermatozoa in raw semen, TRT increased 2.36 ± 1.04%

175

(P = 0.024). The increase of electrical conductivity (b = -0.17 ± 0.04, P < 0.001), ejaculate

176

volume (b = -0.03 ± 0.01, P = 0.030), and arrival temperature (b = -1.21 ± 0.54, P = 0.024)

177

decreased TRT. Furthermore, the increase in sperm output increased (b = 0.20 ± 0.06,

178

P < 0.001) and doses per ejaculate decreased (b = -0.29 ± 0.08, P < 0.001) TRT.

179

Refractometer estimates did not affect TRT (P = 0.12) and percent morphologically intact

180

spermatozoa had no impact on TRT (P = 0.13), but were included in statistical models. The

181

total aerobic bacterial count in extended samples on d2 of semen storage averaged from 100

182

to 1,000 (range: 0 – 16,000) CFU per mL. A total of 11.5% (104 of 905 samples) tested positive

183

for bacterial contamination. For final packing, 91.8% sealed tubes and 8.2% bags were used

184

with a final packing volume of 86.8 + 4.3 mL. Packing process did not affect TRT.

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4. Discussion

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The estimates of effects presented in this paper were derived from model analyses that

188

were designed to account simultaneously for major production management factors potentially

189

affecting TRT of boar spermatozoa. Good laboratory practice standards that include quality

190

control procedures are becoming more and more established [16]. In the context of

191

globalization and with increasing competition between boar studs, inter-station comparability

192

of production management is of growing importance. Therefore, better knowledge and a

193

holistic approach to related factors influencing sperm quality could help to improve the

194

efficiency of AI organizations [17].

195

We could show that around 40% of the total variability in TRT of boar spermatozoa

196

could be explained by different management factors. The boar stud effect was not surprising,

197

since management of AI boars plays such an important role in efficient semen production [18].

198

Replacement rates in terminal sire lines are high, especially in Pietrains, which means many

199

young boars were included in the current study. It can be assumed that boar age was

200

confounded with season and boar stud with breed in the statistical model, since not all breeds

201

were represented in each boar stud. When both were in the model, boar stud explained more

202

variability than breed. Furthermore, TRT increased with increasing age of boar and showed a

203

monthly variability. The age effect can not be sufficiently explained at present time. We

204

hypothesize that older boars produces more seminal plasma components that have a

205

beneficial effect on stress resistance. This needs to be investigated in further studies. It could

206

also be observed that TRT increased during the whole study period. This could be an indication

207

of an improved production management in FBF boar studs during the last 6 years.

208

Usually, semen processing involves dilution and cooling of the boar ejaculate to a

209

storage temperature between 15 to 17 °C. Extenders, preservation processes, and

210

temperature changes are the main factors influencing sperm cell function [19]. After

211

ejaculation, sperm cells are sensitive to a fast temperature reduction. Processing of boar

212

semen during dilution might influence membrane function. Most boar studs in Europe use a

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two-step dilution procedure in which semen is first diluted (1:1) with a preheated extender [4].

214

Pre-dilution of ejaculates has been identified as a critical step during semen processing and,

215

if not performed within 30 min, may render spermatozoa more sensitive to damage during the

216

final dilution. A major impact on semen quality was the number of dilution steps with an

217

increasing number of dilution steps during processing impairing TRT. Multi-step dilution

218

procedures increase the risk of making a mistake during dilution [11].

219

Dilution per se presents a stress factor for spermatozoa due to a sudden change of the

220

surrounding milieu [20]. Protective seminal plasma components will enhance or reduce the

221

dilution effect depending on its concentration [21]. Surprisingly, higher dilution rates had no

222

effect on TRT in this study. However, there was an influence of volume of the ejaculate on

223

TRT. Consequently, the sperm concentration in the raw ejaculate seems to play an important

224

role affecting TRT. With increasing sperm concentration and decreasing ejaculate volume TRT

225

increased. Other studies in bulls [22, 23], humans [24], and stallions [25] reported that the final

226

sperm concentration alone was the primary factor for the dilution effect. As stated above, there

227

might be components in seminal plasma which are beneficial for maintenance of TRT [26, 27]

228

and these might be diluted by big volumes.

229

Poor results in semen quality as measured by TRT are also associated with bacterial

230

contamination as shown in our study. Consequences of bacterial contamination predominantly

231

reside in loss of sperm motility [28], and induction of sperm agglutination and membrane

232

damage [29], resulting in poor fertility and high economic losses in sow herds [30]. Remaining

233

ion load of the pure water for extender preparation is measured routinely in boar studs by

234

electrical conductivity. The current study clearly demonstrates that the electrical conductivity

235

has a significant impact on TRT. Residual ions may harm the spermatozoa and reduce motility

236

in TRT. If this stress to spermatozoa is f. e. due to a wrong osmotic balance or to sperm toxic

237

properties of the ions needs further investigation and depends strongly on the ions, which led

238

to the increase of conductivity from case to case.

239

Our current results reveal that there is an individual variation among boars concerning

240

TRT, and that there seems to be no such variation between the breeds investigated in this trial.

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ACCEPTED MANUSCRIPT 241

This is probably due to more than 86.5% of the boar population being Pietrain. Whether semen

242

from males of different breeds varies in storage tolerance has not been well studied so far. At

243

the end, no effects of the packing process were observed on TRT. The packing process as the

244

last step in boar semen processing in boar studs is done by automated systems and may not

245

damage spermatozoa [31]. Although time-consuming, analysis of TRT is a very sensitive

246

semen quality characteristic to uncover even small differences in semen quality during

247

improvement of boar semen processing in commercial boar studs.

248 249

5. Conclusions

250

Results confirmed that TRT of boar spermatozoa is a sensitive indicator for boar semen

251

quality. The impact of suboptimal semen handling procedures on semen quality has been

252

estimated in a six-year retrospective study in 25 European boar studs. In view of the dynamic

253

situation of semen production, processing of semen is not standardized among boar studs and

254

Hazard Analysis and Critical Control Point concepts must be specifically designed for individual

255

boar studs. A key tool for minimizing risk during boar semen processing is repeated training

256

and education of AI personnel at all levels. The scientific work has to be implemented through

257

knowledge transfer from theory into practice, usually over an extended period of time.

258 259

Acknowledgements

260

This research was partially supported by the Association for Bioeconomy Research

261

(FBF, Germany) and the AIF Inc. (grant no: ZF4276702SK6). We thank Anita Retzlaff for her

262

excellent technical assistance.

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contamination of boar semen and its relationship to sperm quality preserved in commercial

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extender containing gentamicin sulfate. Pol J Vet Sci 2016;19:451-9.

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[30] Kuster CE, Althouse GC. The impact of bacteriospermia on boar sperm storage and

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reproductive performance. Theriogenology 2016;85:21-6.

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[31] Knox R, Levis D, Safranski T, Singleton W. An update on North American boar stud

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practices. Theriogenology 2008;70:1202-8.

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Figure captions

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Figure 1. Effect of dilution steps on thermo-resistance of boar spermatozoa in 25 European

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boar studs during a six-year retrospective study period from 2013 to 2018. Different letters

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denote significant (P = 0.020) differences between dilution steps on thermo-resistance of boar

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spermatozoa using Tukey Kramer Method.

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Figure 2. Effect of month on thermo-resistance of boar spermatozoa in 25 European boar

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studs during a six-year retrospective study period from 2013 to 2018. Different letters denote

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significant (P < 0.001) differences between months on thermo-resistance of boar spermatozoa

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using Tukey Kramer Method.

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Figure 3. Effect of bacterial contamination on thermo-resistance of boar spermatozoa in 25

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European boar studs during a six-year retrospective study period from 2013 to 2018. Bacterial

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contamination was considered positive when concentration was ≥ 100 CFU/mL. Bacterial

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contamination showed a significant (P = 0.027) effect on thermo-resistance of boar

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spermatozoa.

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ACCEPTED MANUSCRIPT Table 1. Categorical explanatory variables used for ANCOVA Analysis (n = 905 ejaculates) Category Boar studs Breeds

Variables Boar stud 1-25 Pietrain Large White Duroc German Landrace Months January February March April May June July November December Dilution steps One-step Two-step Three-step Type of extender Short-term Long-term Bacterial contamination Negative Positive Bacterial contamination = positive defined as ≥ 100 CFU/mL

Number of samples (n) 25 783 46 46 30 203 119 197 123 53 46 63 59 42 357 403 145 780 125 801 104

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ACCEPTED MANUSCRIPT Table 2. Continuous explanatory variables used for ANCOVA Analysis (n = 905 ejaculates) Variables Dose volume, mL Total sperm number per dose, 109 Morphologically intact sperm, % Mitochondrially active sperm, % Membrane-intact sperm, % Arrival temperature at IFN, ºC Refractometry, °Brix Electrical conductivity, μS/cm First dilution temperature, °C Dilution rate Boar age, months Ejaculate volume, mL Sperm concentration, 109/mL Sperm output, 109 Dose/ejaculate

Mean 86.8 2.22 85.2 81.9 80.7 16.9 4.5 10.5 31.7 17.7 25.5 268.1 0.316 76.6 36.2

SD 4.3 0.56 10.1 6.7 8.1 1.8 0.3 30.8 2.4 18.6 14.6 99.9 0.147 30.4 16.6

Min 62.0 0.78 12.5 48.0 51.0 11.2 4.0 0 26.0 5.6 8.5 26.0 0.05 11.0 5.0

Median 87.0 2.12 88.0 83.0 82.0 16.8 4.4 0.9 31.8 12.0 21.0 259.0 0.289 72.0 33.0

Max 98.0 8.95 98.0 95.0 94.0 25.0 5.4 183 41.2 109.0 60.0 555.0 1.134 231.0 137.0

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ACCEPTED MANUSCRIPT Table 3. Influences of boar semen production and several covariates on thermo-resistance of boar spermatozoa at day 7 of semen storage in 25 European boar studs during retrospective study from 2013 to 2018 (ANCOVA analysis, Adj. R2 = 0.395, DF = 46 (855), SS = 208,855 (281,131), MS = 4,540 (329), F = 13.81, P < 0.001). Thermo-resistance was measured after 300 min incubation at 38 °C on day 7 of semen storage. Response variable

Thermoresistance

Input variables Intercept Boar stud Month Dilution steps Bacterial contamination, CFU/mL Year Morphologically intact sperm, % Mitochondrially active sperm, % Membrane-intact sperm, % Arrival temperature at IFN, ºC Electrical conductivity, μS/cm Boar age, months Ejaculate volume, mL Sperm concentration, 109/mL Sperm output, 109 Dose/ejaculate

Type III SS 69157 12753 2595 1625 6146 763 1959 6578 1692 6510 2158 1556 1677 3877 4134

Coefficient

SE

-15.53

14.78

2.16 -0.11 0.30 0.52 -1.21 -0.17 0.12 -0.03 23.58 0.20 -0.29

0.50 0.07 0.12 0.12 0.54 0.04 0.05 0.01 10.44 0.06 0.08

Fvalue -1.05 8.76 4.85 3.95 4.94 18.69 2.32 5.96 20.01 5.15 19.8 6.56 4.73 5.10 11.79 12.57

Pvalue 0.290 <0.001 <0.001 0.020 0.027 <0.001 0.128 0.015 <0.001 0.024 <0.001 0.011 0.030 0.024 <0.001 <0.001

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ACCEPTED MANUSCRIPT Highlights 

40% of the variability in TRT could be explained by production management factors.



Boar stud effect had the biggest impact on TRT.



TRT increased during the study period.

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HACCP concepts for boar studs could be improved.