1 Title: Assessment of a germplasm bank for the autochthonous cattle breed Asturiana de

2 la Montaña: Extender (Biociphos vs. BIOXCell) affected sperm quality but not field

3 fertility

4 Running title: Asturiana de la Montaña bull semen cryobank

5 Carolina Tamargo1, Carlos O. Hidalgo1, J. Néstor Caamaño1, Amer Salman2, Carmen Fueyo1,

6 Cristina Arija2, Ángel Fernández1, María J. Merino1, Felipe Martínez-Pastor2,3

1
7 Selección y Reproducción Animal-SERIDA, Principado de Asturias, Gijón, Spain
2
8 INDEGSAL and IMAPOR, Universidad de León, León, Spain
3
9 Dept. of Molecular Biology (Cell Biology), Universidad de León, León, Spain

10

11 Corresponding author:

12 Felipe Martínez-Pastor

13 felipe.martinez@unileon.es

14

1
15 Abstract

16 Semen banking is critical to preserving rare and autochthonous breeds. However, protocols

17 can change with time, leaving heterogeneous semen batches. The objective of this study was

18 to assess differences in sperm quality and field fertility. We report differences between

19 batches frozen with the Biociphos and BIOXCell extenders in the Asturiana de la Montaña

20 cryobank (autochthonous and endangered breed, Northern Spain). Doses from 48 bulls were

21 analyzed by CASA and flow cytometry. The 85-d non-return rates from AI records were used

22 to assess the fertility of 23853 AI. BIOXCell showed higher quality post-thawing. Differences

23 increased after a 5-h incubation at 37 °C and Biociphos yielded doses with lower resilience.

24 Field fertility did not differ between extenders (Biociphos: 57.4%±1.2; BIOXCell:

25 56.6%±3.0), possibly because of AI protocols compensating for differences inquality.

26 However, this needs to be confirmed by controlled intervention studies. In conclusion,

27 batches frozen with Biociphos may require specific strategies for compensating for the lower

28 sperm quality. Regular surveys and evaluation of cryobanks procedures may be useful to

29 characterizing stored batches and defining strategies to guaranteeing success in their future

30 use.

31

32 Keywords: autochthonous breed, bull, cryopreservation, extender, germplasm banking,

33 fertility

34

2
35 1 Introduction

36 Germplasm banking is a crucial strategy for preserving the genetics of endangered breeds,

37 supporting conservation programs (Doekes, Veerkamp, Bijma, Hiemstra, & Windig, 2018).

38 This report is based on a survey of the Asturiana de la Montaña (AM) cryobank in the CBA-

39 SERIDA (Centro de Biotecnología Animal, Gijón, Asturias, Spain) (Tamargo et al., 2009).

40 AM is an endangered and officially protected cattle breed (National Official Breed Catalog),

41 with high social and cultural importance.

42 The cryobank initially used the Biociphos extender (IMV, L'Aigle, France), and then changed

43 to BIOXCell (IMV). Both are soybean-based extenders extensively used for freezing bull

44 semen (Layek, Mohanty, Kumaresan, & Parks, 2016). There is only one report comparing

45 them (Pileckas, Riškevičiene, & Jomantas, 2014), with some advantage for BIOXCell. Thus,

46 we hypothesized that the batches frozen with Biociphos and still stored in the cryobank could

47 yield lower quality and fertility. IMV no longer produce Biociphos, but there are still

48 thousands of batches frozen with this extender, and this study could be useful for cryobanks

49 worldwide.

50 2 Materials and methods

51 The cryobank contained doses from 48 AM bulls, 23 frozen with Biociphos (1996–2006) and

52 25 with BIOXCell (2006–2016). The protocol (Muiño, Tamargo, Hidalgo, & Peña, 2008)

53 consisted of collecting semen from from adult bulls (median age 30 months) by artificial

54 vagina, extension to 92×106/ml, cooling to 4 °C, 3-h equilibration, packaging into 0.25-ml

55 straws, and freezing with a Digit-cool biofreezer (IMV). Batches were used for artificial

56 insemination (AI) in farms belonging to the breeders' association (ASCOL), following

57 standard protocols. Fertility was assessed by 85-d non-return rates (NRR) from historical

58 records provided by ASCOL. The records included information from 23853 AI for Biociphos

3
59 (1586 in heifers and 22267 in cows) and 1529 for BIOXCell (211 in heifers and 1318 in

60 cows). This study was approved by the Ethics Committee of the University of León.

61 This study was carried out using solely the samples stored in the cryobank, therefore no ethics

62 clearance was required. The cryobank survey (106 Biociphos and 128 BIOXCell batches, 2 to

63 4 per bull) was carried out by CASA and flow cytometry (details in the Supplementary

64 Material) after thawing (37 °C, 30 s) and after a 5-h incubation at 37 °C. CASA (Proiser,

65 Spain) consisted of an E400 Nikon microscope (10X phase contrast, 37 °C stage, Basler

66 A302fs digital camera) and the ISAS software. Data were clustered to obtain subpopulations

67 (Ledesma et al., 2017). Flow cytometry (CyAn ADP; Beckman Coulter; Brea, CA) yielded

68 viability, apoptosis, capacitation, mitochondrial activity and free radicals (Crespo-Félez et al.,

69 2017). Samples were stained (ThermoFisher; Waltham, MA) for 15 min at 37 °C with probes

70 at: 4.5 μM Hoechst 33342 and 33258, 100 nM YO-PRO-1, 1.5 μM propidium iodide, 1 μg/ml

71 PNA-FITC, 5 µM H2DCFDA, 1 µM MitoSOX, 100 nM Mitotracker Deep red, 2 μM M540.

72 Sperm chromatin was analyzed by SCSA (Martínez-Pastor et al., 2009), obtaining %DFI

73 (DNA fragmentation) and %HDS (chromatin compaction). Data were analyzed by linear

74 mixed-effects models (R statistical environment), with extender as fixed factor and bull as

75 grouping factor within the random part of the models.

76 3 Results and Discussion

77 Advances on extender formulation have improved both the fertility and the sanitary status of

78 cryopreserved semen (Layek et al., 2016). Biociphos was one of the earliest based on soybean

79 lecithin, showing variable performance (Gil et al., 2000; Muiño, Fernández, & Peña, 2007;

80 Thun, Hurtado, & Janett, 2002), but allowing standardization and microbial control (Bousseau

81 et al., 1998). BIOXCell superseded it, achieving extensive use (Akhter et al., 2010; Celeghini

82 et al., 2008; Lymberopoulos & Khalifa, 2010). Only one small study compared them

83 (Pileckas et al., 2014), finding lower motility after post-thawing incubation for Biociphos.

4
84 Our survey enabled a clear advantage for BIOXCell post-thawing to be detected, with

85 differences increasing after the incubation (tables 1 and 2 and Supplementary Material). The

86 presence of a subpopulation with fast spermatozoa was also favored by BIOXCell (Table 1).

87 Post-incubation results are critical, suggesting a higher vulnerability for spermatozoa frozen

88 with Biociphos. Differences in chromatin status variables were below 1%, but significant for

89 %DFI (Table 2).

90 There were no differences in NRR (Biociphos: 57.4%±1.2; BIOXCell: 56.6%±3.0), which

91 suggests that the AI protocols may have compensated for this lower quality by using a high

92 enough number of spermatozoa (Flowers, 2013). Indeed, the analyses showed that

93 uncompensable defects (chromatin) were well below the fertility thresholds for cattle

94 (Waterhouse et al., 2006). Nevertheless, the higher presence of compensable defects in the

95 Biociphos doses should be considered when using limited numbers of spermatozoa (e.g., low-

96 dose AI), since their effects on fertility could then become apparent. As a word of caution,

97 this fertility study was based on retrospective data and should be tested by intervention

98 designs in controlled environments.

99 Therefore, the survey disclosed a vulnerability regarding the Biociphos batches, advising to

100 adjust the cryobank strategic plan accordingly. These results may extrapolate to other

101 cryobanks, where surveys could disclose similar vulnerabilities.

5
102 TABLE 1 Extender comparison for total motility and sperm subpopulations (%, mean±SEM;

103 P-values show the extender effect significance within each analysis point).

Post-thawing 5-h incubation

Biociphos BIOXCell P-value Biociphos BIOXCell P-value
Total motility 31.3±1.5 37.1±1.5 0.009 8.2±1.1 23.4±1.3 <0.001
Slow erratic 4.6±0.4 5.1±0.4 0.093 7.4±0.9 5.3±0.4 0.010
Rapid linear 44.8±2.4 49.8±2.5 0.034 19.8±3.0 38.6±3.2 <0.001
Slow linear 50.7±2.5 45.1±2.5 0.036 72.8±3.0 56.1±3.3 <0.001
104

105 TABLE 2 Extender comparison for flow cytometry parameters (% except for cytoplasmic

106 ROS as mean fluorescence intensity; mean±SEM; P-values show the extender effect

107 significance within each analysis point).

Post-thawing 5-h incubation

Biociphos BIOXCell P-value Biociphos BIOXCell P-value
Viability 51.5±1.5 55.7±1.8 0.021 39.4±1.6 44.7±1.6 0.004
Apoptotic† 12.4±0.7 17.9±1.8 0.904 8.4±0.8 8.6±1.2 0.198
Acrosomal
21.1±1.1 19.6±1.1 0.147 49.6±1.3 49.3±1.5 0.367
damage
Capacitated† 1.1±0.2 1.5±0.2 0.024 0.5±0.1 1.3±0.1 <0.001
Active
46.9±1.5 45.9±1.8 0.616 36.6±1.6 38.9±1.7 0.329
mitochondria
Cytoplasmic
7.2±0.3 6.7±0.2 0.222 9.3±0.3 8.5±0.2 0.024
ROS
Superoxide+† 2.3±0.2 1.4±0.1 <0.001 6.0±0.6 3.7±0.3 <0.001
%DFI 1.5±0.2 2.0±0.1 0.018 1.5±0.2 2.1±0.2 0.012
%HDS 4.9±0.2 5.6±0.2 0.075 4.8±0.2 5.4±0.2 0.120
†
108 Ratio within viable spermatozoa.

109 Acknowledgements

110 The authors thank Beatriz de Arriba, Lucía Tejerina, ASCOL and ASEAMO. Supported by

111 grant RZP2013-00006-00-00 (MINECO, Spain). Collaboration enhanced by PIVEV network

112 (AGL2016-81890-REDT, MINECO).

6
113 Conflict of interest

114 None declared.

115 Data Availability Statement

116 The data that support the findings of this study are available from the corresponding author

117 upon reasonable request.

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180

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 Assessment of a germplasm bank for the autochthonous cattle breed Asturiana de la
Montaña: Extender (Biociphos vs. BIOXCell) affected sperm quality but not field
fertility

Supplementary material

Carolina Tamargo1, Carlos O. Hidalgo1, J. Néstor Caamaño1, Amer Salman2, Carmen Fueyo1,
Cristina Arija2, Ángel Fernández1, María J. Merino1, Felipe Martínez-Pastor2,3
1
Selección y Reproducción Animal-SERIDA, Principado de Asturias, Gijón, Spain
2
INDEGSAL and IMAPOR, Universidad de León, León, Spain
3
Dept. of Molecular Biology (Cell Biology), Universidad de León, León, Spain

Materials and methods

Sperm motility analysis

An aliquot of each sample was diluted in warm PBS at 20×106 ml-1. A 5-µl drop was
observed in a Makler chamber (10 µm depth; Haifa Instruments, Israel) on a phase contrast
microscope (Nikon E400; Nikon, Tokio, Japan), with a warmed stage at 37 °C and 10×
negative contrast optics). At least 5 fields and 200 cells were recorded at 53 frames/s for 1 s,
with a Basler A302fs digital camera (Basler Vision Technologies, Ahrensburg, Germany).
The images were processed with the ISAS software v. 1.19 (Proiser, Valencia, Spain), with 25
to 80 µm for head area; VCL (curvilinear path velocity) > 10 µm/s to classify a spermatozoon
as motile. The following variables were extracted from the raw motility data by using R
scripts (R Core Team, 2018): Total motility (MOT), progressive motility (PROG,
VAP>25 µm/s and STR>80%), VCL, VSL (straight path velocity), VAP (average path
velocity according to the average —smoothed— path; µm/s), LIN (linearity), STR
(straightness), WOB (wobble), ALH (amplitude of the lateral displacement of the sperm
head), and BCF (frequency of the flagellar beat).
We carried out a two-step procedure for clustering the raw motility data (Fernández-Gago et
al., 2017; Gallego et al., 2015; Ledesma et al., 2017; Felipe Martínez-Pastor, Tizado, Garde,
Anel, & de Paz, 2011). Two successive hierarchical clustering steps were performed on the
data (R Core Team, 2018), the first one for each analysis and the second one using the median
values of the first set of clusters. We ended up with a set of 3 clusters (Table S1), using the
proportions of spermatozoa in each cluster for subsequent analyses as described previously.

Sperm physiology analysis by flow cytometry

Flow cytometry analyses (Fernández-Gago, Domínguez, & Martínez-Pastor, 2013) were
carried out with a CyAn ADP (Beckman Coulter; Brea, CA), equipped with three diode lasers
(violet at 405 nm, blue at 488 nm and red at 635 nm). Samples were added to 300 µl of PBS
with 0.5% BSA at 106/ml and stained with different probes (ThermoFisher; Waltham, MA)
for 15 min at 37 °C, with probes (final concentration provided): Hoechst 33342 (H342, 4.5
µM) for discriminating debris; Hoechst 33258 (H258, 4.5 µM) or propidium iodide (PI; 1.5
µM) for viability; YO-PRO-1 (100 nM) for apoptotic-like changes; PNA-FITC (PNA; 1
µg/ml) for assessing the acrosomal status; H2DCFDA (CFDA; 5 µM) for detecting
1
cytoplasmic reactive oxygen species (ROS); merocyanine 540 (MC; 2 µM) for assessing
capacitation-like changes; MitoSOX (MX; 1 µM) for detecting mitochondria-produced
superoxide; Mitotracker Deep red (MT; 100 nM) for assessing mitochondrial activity. These
probes were combined as H342/YO-PRO-1/M540/PI/MT (viability, apoptosis, capacitation,
mitochondrial activity), H342/PNA/PI (viability and acrosomal status) and
H258/CFDA/MitoSOX (viability, cytoplasmic ROS and mitochondrial superoxide). The
fluorescence was collected by photodetectors provided with filters 450/50 (violet line, blue
fluorescence: H342 and H258), 530/40 (blue line, green fluorescence: YO-PRO-1, PNA-
FITC, H2DCFDA), 575/25 (blue line, orange fluorescence: Merocyanine 540), 613/20 (blue
line, red fluorescence: PI, MitoSOX) and 665/20 (red line, red fluorescence: Mitotracker deep
red). At least, 5000 spermatozoa were collected per sample, with a flow rate of 200 cells/s.
The analysis of the flow cytometry data was carried out using Weasel v. 3.4 (WEHI,
Melbourne, Australia). Figure S1 shows representative cytograms for these techniques.

Figure S1. Representative cytograms for the flow cytometry analyses: a) YO-PRO-1/PI
(viable/apoptotic/dead populations), b) YO-PRO-1/Merocyanine 540 (for capacitation
assessment), c) YO-PRO-1/Mitotracker (for mitochondrial assessment), d)
H2DCFDA/Hoechst 33258 (cytoplasmic ROS assessment) and e) MitoSOX/Hoechst 33258
(mitochondrial superoxide assessment). These techniques have been reviewed elsewhere (F
Martínez-Pastor et al., 2010).

2
Sperm chromatin structure assay
Chromatin stability was assessed by SCSA (Sperm Chromatin Structure Assay) (Evenson &
Jost, 2000). The technique is based on the denaturalization of broken DNA and on the
properties of acridine orange (AO), whose fluorescence shifts from green (dsDNA) to red
(ssDNA) depending on the degree of DNA denaturation. Samples were diluted in TNE buffer
(0.01 M Tris-HCl, 0.15 M NaCl and 1 mM disodium EDTA, pH 7.4) to a final sperm
concentration of 2×106 mL-1, and stored at -80 °C. For analysis, the samples were thawed on
crushed ice, 200 µL of sample were pipetted into a flow cytometry tube, and immediately
mixed with 0.4 mL of the acid-detergent solution (0.08 M HCl, 0.15 M NaCl, and 0.1%
Triton X-100, pH 1.2). After 30 s, 1.2 mL of the staining orange solution (6 µg/mL AO in
0.1 M citric acid, 0.2 M Na2HPO4, 1 mM disodium EDTA, and 0.15 M NaCl, pH 6.0) was
added to the tube. The tube was kept on ice for 3 min before flow cytometry analysis and run
through a FACScalibur flow cytometer (Becton Dickinson) controlled by the acquisition
software CellQuest v. 3. We analyzed 5000 spermatozoa per sample, exciting the acridine
orange with the Ar-ion 488 nm laser and using a 530/30 filter for the green fluorescence of
dsDNA-bound acridine orange (AO), and a 650 long-pass filter for the red fluorescence of
ssDNA-bound AO. Data were saved in flow cytometry standard (FCS) v. 2 files, which were
processed using the R statistical environment (R Core Team, 2018). We calculated the DNA
Fragmentation Index (DFI) for each spermatozoon as the ratio of red fluorescence to total
fluorescence (red+green). From the DFI values, we obtained the percentage of spermatozoa
with high fragmentation index (%DFI, DFI>25%), and the percentage of spermatozoa with
high DNA stainability (%HDS), defined as those events with green fluorescence above
channel 650.

Results
Table S1 shows the characteristics of the three subpopulations obtained from the cluster
analysis of the motility data. Table S2 describes the individual variables collected by CASA
(median values per sample). Tables S3 and S4 show the comparison between extenders
regarding the longevity of the sperm samples, defined as the ratio between the analysis after
the incubation and after thawing.

TABLE S1 Properties of the sperm subpopulations (clusters) obtained from the analysis of
the raw motility data (median ± median absolute deviation).
VCL VSL VAP LIN STR WOB ALH BCF
Subpopulation
(µm/s) (µm/s) (µm/s) (%) (%) (%) (µm) (Hz)
Slow erratic 41.6±14.2 3.8±3.0 17±6.1 9.5±7.1 22.6±17.4 43.0±12.7 2.3±0.6 4.0±3.0
Rapid linear 93.9±34.7 50.5±22.5 57.7±24.5 55.6±15.4 91.1±10.1 63.9±15.8 3.3±0.9 11.0±4.4
Slow linear 44.3±21.8 24.8±20.9 28.4±18.8 60.1±23.9 87.6±13.6 69.2±19.7 1.8±0.6 7.0±4.4

3
TABLE S2 Extender comparison (P-values for extender within sampling time) for
progressive motility and median motility parameters (mean ± SEM).
Post-thawing 5-h incubation
Biociphos BIOXCell P-value Biociphos BIOXCell P-value
PROG (%) 20.4±1.3 23.7±1.2 0.061 5.8±0.9 17.7±1 <0.001
VCL (µm/s) 90.4±4.2 101.9±4.5 0.104 44.0±2.1 73.2±3.8 <0.001
VAP (µm/s) 72.6±4.4 81.5±4.8 0.284 27.6±1.9 54.3±3.9 <0.001
VSL (µm/s) 55.3±2.5 59.4±2.7 0.348 23.7±1.8 46.4±3.0 <0.001
LIN (%) 63.8±0.9 62.8±0.7 0.337 56.6±2.1 63.3±1.4 0.010
STR (%) 86.6±0.9 86.1±0.9 0.764 88.4±0.8 91.5±0.3 <0.001
WOB (%) 78.1±1.6 77.3±1.5 0.717 65.5±1.9 71.1±1.7 0.030
ALH (%) 2.4±0.1 2.7±0.1 0.001 1.9±0.1 2.4±0.1 <0.001
BCF (%) 8.9±0.3 9.3±0.3 0.249 6.9±0.3 8.9±0.3 <0.001
PROG: Progressive motility; VCL: Curvilinear velocity; VAP: Average path velocity; VSL:
Straight path velocity; LIN: Linearity; STR: Straightness; WOB: Wobble; ALH: Amplitude
of the lateral displacement of the sperm head; BCF: Frequency of the flagellar beat.

TABLE S3 Spermatozoa longevity regarding CASA variables (mean ± SEM of the ratio
post-incubation / post-thawing as %).
Variable Biociphos BIOXCell P-value
MOT 27.0±2.8 63.7±2.7 <0.001
PROG 30.1±3.5 77.5±3.7 <0.001
VCL 57.0±4.1 77.3±3.6 <0.001
VAP 48.8±4.2 74.7±4.4 <0.001
VSL 50.6±4.3 81.9±4.2 <0.001
LIN 90.6±3.3 101.3±2.2 0.005
STR 101.9±1.5 106.8±1.1 0.006
WOB 86.5±2.2 93.3±1.9 0.021
ALH 83.9±2.1 92.8±2.1 0.005
BCF 79.5±2.9 97.5±2.4 <0.001
MOT: Total motility; PROG: Progressive motility; VCL: Curvilinear velocity; VAP: Average
path velocity; VSL: Straight path velocity; LIN: Linearity; STR: Straightness; WOB: Wobble;
ALH: Amplitude of the lateral displacement of the sperm head; BCF: Frequency of the
flagellar beat.

4
TABLE S4 Spermatozoa longevity regarding cytometry variables (mean ± SEM of the ratio
post-incubation / post-thawing as %).
Variable Biociphos BIOXCell P-value
Viability 76.8±2.1 81.9±1.5 0.267
Apoptotic† 73.6±5.8 84.4±16.9 0.609
Acrosomal damage 244.5±9.7 257.3±8.3 0.283
Capacitated† 79±4.9 120.0±9.7 0.107
Active mitochondria 91.5±2.0 96.4±1.5 0.774
ROS, MFI 129.4±4.2 126.5±3.9 0.499
Superoxide+† 209.9±11.3 190.5±8.2 0.087
%DFI 101.7±3.4 104.8±2.7 0.907
%HDS 98.0±2.2 96.9±2.6 0.791
†
Ratio within viable spermatozoa.

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