Effect of boar seminal dose type (cervical compared with post-cervical insemination) on cooling curve, sperm quality and storage time

Journal Pre-proof Effect of boar seminal dose type (cervical compared with post-cervical insemination) on cooling curve, sperm quality and storage time ´ ´ R. C. Luongo, G Garrappa, PJ. Llamas-Lopez, E. Rodr´ıguez-Tobon, ´ ´ ´ ´ LopezUbeda, S. Abril-Sanchez, FA. Garc´ıa-Vazquez

PII:

S0378-4320(19)30688-8

DOI:

https://doi.org/10.1016/j.anireprosci.2019.106236

Reference:

ANIREP 106236

To appear in:

Animal Reproduction Science

Received Date:

30 July 2019

Revised Date:

31 October 2019

Accepted Date:

15 November 2019

´ ´ E, Please cite this article as: Luongo C, Garrappa G, Llamas-Lopez P, Rodr´ıguez-Tobon ´ ´ ´ ´ LopezUbeda R, Abril-Sanchez S, Garc´ıa-Vazquez F, Effect of boar seminal dose type (cervical compared with post-cervical insemination) on cooling curve, sperm quality and storage time, Animal Reproduction Science (2019), doi: https://doi.org/10.1016/j.anireprosci.2019.106236

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Effect of boar seminal dose type (cervical compared with post-cervical insemination) on cooling curve, sperm quality and storage time

Luongo C1, Garrappa G1,2, Llamas-López PJ1, Rodríguez-Tobón E1, López-Úbeda R1,3, AbrilSánchez S1, García-Vázquez FA1, 3*.

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Department of Physiology, Veterinary School, University of Murcia, Murcia, Spain. International

Excellence Campus for Higher Education and Research (Campus Mare Nostrum). 2Institute of

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Animal Research of the Semi-Arid Chaco (IIACS), Agricultural Research Center (CIAP), National Institute of Agricultural Technology (INTA), Tucuman, Argentina. 3Institute for Biomedical

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Research of Murcia (IMIB-Arrixaca), Murcia, Spain.

*Corresponding author: Francisco A. García-Vázquez, Dept. of Physiology, Faculty of Veterinary

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Science, University of Murcia, Campus de Espinardo. Murcia 30100, Spain (e-mail:

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[email protected]).

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ABSTRACT

Seminal doses used for cervical and post-cervical artificial insemination (CAI and PCAI,

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respectively) vary in volume, the number of spermatozoa and packaging. The aim was to evaluate the outcomes when there was use of routine processing procedures for CAI- and PCAI-doses. Two

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different types of seminal doses were processed: 1) CAI: 2.7 x 109 sperm/80ml; 2) PCAI: 1.5 x 109 sperm/45ml. In Experiment 1, the cooling curve of seminal doses during processing occurred in two phases: 1st) At room temperature (23.4 ± 0.5 ºC) from 0 (just after packaging) to 120 minutes; 2nd) At refrigeration (15.7 ± 0.8 ºC) from 121 to 240 minutes. For the PCAI-doses, the time required to reach room temperature was 47 min compared to 107 min for CAI-doses (decreasing velocity of 0.093 ºC/min and 0.048 ºC/min, respectively). During refrigeration, for the PCAI-doses the time required to reach the desired preservation temperature was 20 min less than for CAI-doses (PCAI: 1

90 min, 0.074 ºC/min; CAI: 110 min, 0.066 ºC/min). In Experiment 2, sperm motility, kinetic parameters and acrosome damage for both types of doses were evaluated at 0, 24, 48 and 72 h of refrigeration. Also, morphology, pH, and osmolality were assessed at 0 and 72 h. Values for all these did not differ between CAI- and PCAI-doses. In conclusion, PCAI-doses took less time than CAIdoses to reach the desired temperature, but sperm quality was similar for CAI- and PCAI-doses during storage. Nevertheless, the different cooling curves should be taken into consideration for further investigation.

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Keywords: Animal production; Boar semen; Ejaculate; Porcine; Preservation

1. Introduction

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Post-cervical artificial insemination (PCAI) of sows is the artificial insemination (AI) technique used to the greatest extent in countries with an intensive pig production industry (García-

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Vázquez et al., 2019). The PCAI technique requires the deposit of the semen directly in the uterus

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(uterine body) bypassing the barrier of the cervix (Watson and Behan, 2002; Hernández-Caravaca et al., 2012). Consequently, seminal doses of ~3.0 x 109 sperm cells in 80 to 100 ml volumes are commonly used with the cervical AI (CAI) technique whereas ~1.5 x 109 sperm cells in 45 ml used

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in PCAI (Hernández-Caravaca et al., 2012; Bortolozzo et al., 2015). This change results in an increase in the number of seminal doses produced per boar if the same reproductive performance is

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to occur, which could be translated to a more rapid genetic improvement and more homogeneous litters in addition to the large economic benefits (Watson and Behan, 2002; Rozeboom et al., 2004;

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Mezalira et al., 2005; Roberts and Bilkei, 2005; Hernández-Caravaca et al., 2012; García-Vázquez et al., 2019).

The number of doses obtained for PCAI per ejaculate may be two to three times greater

compared to CAI (reviewed by García-Vázquez et al., 2019). The preparation of this type of seminal dose, therefore, should be more precise with regard to protocols for processing than that for CAI seminal doses; nevertheless, some aspects concerning processing PCAI seminal doses are not standardized to the extent that occurs with CAI. 2

Different AI practices may result in differences in semen quality among swine AI centers (Waberski et al., 2008). Seminal dose production involves different phases, in which the temperature of preparation and preservation are important factors. The processing of the seminal doses involves two phases, the first occurs with a temperature decreases in the laboratory (room temperature), and a second phase occurs in the refrigerator, because it is recommended that there be refrigeration of seminal doses after the samples are initially processed at room temperature to reduce sperm metabolism (Dziekońska et al., 2009; Gączarzewicz et al., 2015). Boar spermatozoa are extremely sensitive to changes in temperature, so cold shock can compromise sperm motility and viability

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(Pursel et al., 1972; Althouse et al., 1998;). For the production of seminal doses, the ejaculate is collected at 37 to 38 ºC and diluted in warm extender (32 ºC) in one- or two-steps (Schulze et al., 2013). Subsequently, diluted semen is packaged in different containers (plastic bottles, bags or tubes)

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which may affect the cooling rate (Willenburg et al., 2011). Afterward, seminal doses have to be stored at ambient thermal conditions until the temperature gradually decreases to about 24 ºC

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(Schulze et al., 2013; Lopez-Rodriguez et al., 2017) before refrigeration of the doses occurs at 15 to 17 ºC (storage temperature) to avoid cold shock. In the case of seminal doses used for CAI, this

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period is estimated to be ~90 minutes (Schulze et al., 2013, 2019). Both insemination methods (CAI and PCAI) are often performed at the same farm, and PCAI seminal doses are currently prepared and

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stored using the same procedures that are used for doses when there is procedure of CAI which probably results in compromised fertility when there is use of PCAI, at least in some situations (Lopez-Rodriguez et al., 2017).

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After refrigeration occurs, seminal doses can be stored for several days until use (reviewed

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by Yeste, 2017). Storage of diluted semen at 15 to 17 ºC induces a reduction in sperm metabolism which is essential for seminal dose preservation (Lopez-Rodriguez et al., 2017). The preservation of semen doses at 15 to 17 ºC for as long as 6 days when there is use of CAI does not impair sperm quality or reproductive outcomes (Kuster and Althouse, 1999; Vyt et al., 2004; Riesenbeck, 2011; Karunakaran et al., 2017). Although the storage times can vary depending on the type of extender used from 3 to 10 days, commonly doses at the farm are used when there is no longer than 3 days of storage (Lopez-Rodriguez et al., 2017). Furthermore, packaging types for CAI and PCAI seminal 3

doses vary in shape, volume, and surface area, and although the packaging process is in 80 to 90 ml sealed tubes or bags does not affect thermo-resistance during preservation of boar sperm (Schulze et al., 2019), it is unknown if there is an effect of storage container size on temperature of the semen during storage or the seminal characteristics. The main objective of this study, therefore, was to provide fundamental knowledge about the processing and storage of PCAI seminal doses (1.5 x 109 sperm/45 ml) compared to CAI seminal doses (2.7 x 109 sperm/80 ml). 2. Material and methods

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2.1. Ethics of experimentation The ejaculates were obtained for AI purposes from a private company, and seminal doses obtained were used. There were no specific ethical approvals necessary to perform this research,

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because there were non-production animal manipulations that were needed to conduct this study. 2.2. Animals

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Boars housed in a commercial boar stud were included in this study. The age distribution (average ± SD) of the boars was 15.5 ± 0.7 months (range: 7-17). Animals were housed in individual

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pens (sawdust or straw bed) within the same building were fed a commercial feed (pellets). From 7 months of age, the basal feed ration (2 kg) was increased based on the body condition of the boars

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(maximal feed ration of 3.5 kg/day). Boars were dewormed twice per year and there were parvovirus and erysipelas vaccinations. The frequency of semen collection was ~1.5 times per week with an

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interval of 4 to 6 days between collections.

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2.3. Ejaculate collection and seminal doses preparation The rich fraction of the ejaculates from eight proven fertility boars (Adespolorca AI Center,

Lorca, Spain) was collected by the gloved-hand method (one ejaculate per boar). Semen was immediately diluted in a ratio 1:1 (v:v) during the collection process in commercial extender at 32 ºC (Zoosperm ND5, Import-Vet S.A., Barcelona, Spain) followed by a final dilution of extender at the same temperature (32 ºC). The criteria of the AI center for boar selections that would be used for ejaculates used for AI were a minimum of 70% normal spermatozoa morphology and sperm 4

progressive motility of at least 70%. Two different sizes of plastic bags were used for semen storage (Fig. 1): 1) CAI doses: 80 ml containing 2.7 x 109 spermatozoa with a total plastic surface of 23,800 mm2 (133.44 x 103 sperm/mm2); 2) PCAI doses: 45 ml containing 1.5 x 109 spermatozoa with total plastic surface of 18,000 mm2 (83.33 x 103 sperm/mm2). For both types of doses, an automatic system was used for semen packaging. 2.4. Experimental design 2.4.1. Experiment 1: Cooling curve during the preparation and refrigeration of seminal doses

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The stability of the temperature within the seminal doses (CAI compared with PCAI) was studied immediately after packaging. A total of six ejaculates from five boars were used (Duroc and Landrace). Sperm doses from the same boar were aliquoted in the same proportion for the two experimental groups (CAI and PCAI) to avoid boar effects. A Tinytag data logger computerized

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thermometer (Tinytag Plus 2, TGP-4020, Gemini Data Loggers, United Kingdom) modified with a probe extension (PT1000 Probe, PB-7006-1M5, Gemini Data Loggers, United Kingdom) was

(avoiding contact with the package wall).

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vertically positioned during the analysis to record the temperature in the middle of the seminal dose

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Furthermore, a Tinytag data logger computerized thermometer (Tinytag Ultra 2, TGU-4017, Gemini Data Loggers, United Kingdom) was used to record the room and refrigerator temperatures.

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During the study the temperature was recorded every minute for 240 min with this time period being divided into two phases: 1) From 0 min (just after packaging) to 120 min while seminal doses were

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stored on an open laboratory bench (23.4 ± 0.5 ºC) (mean ± SEM); 2) From 121 to 240 min while seminal doses were refrigerated (15.7 ± 0.8 ºC; mean ± SEM). During both phases semen storage,

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the temperatures of the laboratory and within the refrigerator were also recorded during the analysis period. Data were uploaded to a Microsoft Excel spreadsheet for subsequent analysis. For each type of seminal dose, the magnitude of the temperature decrease was calculated in

both phases of the experiment. This magnitude of temperature change was calculated as the difference between initial temperature [immediately after packaging at room temperature (1st phase) or at the time of initiation of refrigeration (2nd phase)] and for final temperature [when seminal doses

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reached the average temperature on the open laboratory (1st phase) or at refrigeration (2nd phase)] was divided by the time (min) required to reach the average temperature for each phase. 2.4.2. Experiment 2: Effect of seminal dose type on sperm quality during preservation Eight different males (Duroc, Pietrain, Rekor, and PIC) of proven fertility (one ejaculate per boar) were used in this experiment. From each ejaculate, two types of seminal doses were evaluated (CAI and PCAI). Doses were stored for 120 min at room temperature after packaging and storage at ~16 ºC. Values for sperm motility (total and progressive), kinetic variables and acrosome integrity

morphology were evaluated at 0 and 72 h of refrigeration.

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were evaluated at 0, 24, 48 and 72 h of refrigeration. Osmolality and pH in seminal doses and sperm

For sperm motility analysis, a Computer Assisted Semen Analysis (CASA) was used (ISAS® software, PROiSER R+D S.L., Valencia, Spain) coupled to phase-contrast microscopy

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(negative-pH 10x objective; Leica DMR, Wetzlar, Germany) and a digital camera (Basler Vision, Ahrensburg, Germany). Each sample was warmed at 38 °C for 10 min before evaluation (heat block

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CH100, Biosan Laboratories, Inc., MI, USA). A 4 µl drop of the sample was subsequently placed in a pre-warmed (38 ºC) chamber (20-micron Spermtrack® chamber, Proiser R+D, SL; Paterna, Spain)

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and evaluated using phase-contrast microscopy. The variables analyzed were the following: Total motility (%), progressive motility (%), curvilinear line velocity (VCL, µm/s), average path velocity

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(VAP, µm/s), straight line velocity (VSL, µm/s), amplitude of lateral head displacement (ALH, µm), percentage linearity (LIN, ratio of VSL/VCL, %), percentage straightness (STR, ratio of VSL/VAP,

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%), percentage oscillation (WOB, %) and beat-cross frequency (BCF, Hz). At least three different fields per sample were analyzed. A spermatozoon was considered to be motile when there was a

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VAP >10 µm/s. Progressive motility was considered to exist when there was a straightness (STR) >45%. Motility determinations were made at 25 frames per second for one second (25 images). For acrosome status and spermatozoa morphology evaluation, wet mounts of semen fixed in

buffered 2% glutaraldehyde solution were prepared. Spermatozoa were evaluated using a phasecontrast microscope (100x objective, Nikon®, Eclipse E2000). Spermatozoa were classified according to the morphology into one of the following categories: normal morphology, cells with cytoplasmic droplet, folded tail and coiled tail. Spermatozoa were classified according to the 6

acrosome status as follows: 1) normal apical ridge: when acrosomes had a crescentic apical ridge; 2) damaged apical ridge: when spermatozoa had an irregular apical shape or was absent. At least 100 spermatozoa per sample were evaluated for both, morphology and acrosome status. Osmolality in seminal doses was determined by a freezing point-based osmometer (MicroOsmometer 13DR; Hermann Roebling Messtechnik, Berlin, Germany). The results are expressed in mOsm/kg. The osmometer was calibrated each day using a 300 mOsm/kg standard supplied by the manufacturer. The pH in seminal doses was evaluated using a pH-meter (Crison, micro-pH 2002). The pH-meter was calibrated each day of analysis using 4.01 and 7.00 standard buffers (pH-Crison

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instruments, Hach, Spain) with a tolerance of the pH values of ± 0.02. 2.5. Statistical analysis

The statistical analysis was performed using the statistical software SAS University Edition

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(SAS, 2016). Fitting sphericity for repeated measurement was performed using the restricted likelihood ratio test between Huynh-Feldt (H-F) and unstructured (UN) covariance structures. If the

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difference between these (distributed under the null hypothesis as a χ2 with the difference between the degree of freedom, df) was greater than χ2 df, it was considered that there was sphericity of data.

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Progressive motility, VSL and VAP spermatozoa variables from the two experiments indicated nonsphericity of data. These non-sphericity parameters were compared using the F test of ANOVA

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option and the maximum likelihood method by mixed model procedure, simulating the nonparametric tests previously described by Akritas et al. (1997) and Brunner et al. (2002). The model

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included the seminal doses type of packaging (CAI or PCAI), time related to experimental groups and the interaction as the main effects. The UN covariance was used to correct degrees of freedom

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using the Greenhouse-Geisser adjustment. The Tukey post-hoc analysis was applied between experimental groups. The differences resulting from cooling rate with use of CAI compared with PCAI in Experiment 1, and total motility, VCL, LIN, STR, WOB, BCF, normal morphology, osmolality and pH of seminal doses from Experiment 2 were analyzed using Proc Mixed procedures. For cooling rate, the model included the seminal dose type of packaging, the interaction between CAI and PCAI at different time points as the main effect, and external temperature (room or refrigeration temperature) and seminal doses as a random effect. For the other variables of 7

Experiment 2, the model included the seminal dose type (CAI and PCAI), the time related to treatment and the interaction as the main effect, and seminal doses as the random effect. With all of these analyses, a first-order autoregressive covariance structure (AR1) was used to adjust the difference in data according to the differences over time, and the Tukey post-hoc analysis was applied to detect differences between experimental groups. Data are presented as mean ± SEM. Differences were considered statistically significant when P < 0.05. 3. Results 3.1. Experiment 1: Cooling curve during the preparation and refrigeration of seminal doses.

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The cooling curves of decreasing temperature during seminal dose preparations (CAI compared with PCAI) at room temperature (1st phase) and during the initial steps of refrigeration (2nd phase) are represented in Figure 2. For the PCAI-doses, the time required to reach room temperature

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was 47 min compared to 107 min for CAI-doses (decreasing velocity of 0.093 ºC/min and 0.048 ºC/min, respectively). Furthermore, the magnitude of temperature change for PCAI seminal doses

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was greater than for CAI doses from 3 to 51 minutes of storage at room temperature (P < 0.05). During refrigeration, for the PCAI-doses the time required to reach the desired preservation

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temperature was 20 min less than for CAI-doses (PCAI: 90 min, 0.074 ºC/min; CAI: 110 min, 0.066 ºC/min). Likewise, the temperature decrease during refrigeration was more rapid for PCAI- than

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CAI-doses being 166 and 179 min, respectively (P < 0.05). 3.2. Experiment 2: Effect of seminal dose type (CAI compared with PCAI) on sperm quality during

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preservation

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Data for results of sperm quality of CAI and PCAI seminal doses during 72 h of storage are summarized in Table 1 and Figure 3. Total and progressive motility of spermatozoa and values for kinetic variables did not differ between CAI and PCAI groups (Table 1) and there were no interactions between time and groups (Fig. 3A). Furthermore, some sperm velocity parameters were different during the storage period. There was a greater rate of decrease in progressive motility and STR between 24 to 48 h (P < 0.0001), which continued until 72 h of storage (P = 0.0002 and P = 0.002, respectively), though between 48 to 72 h the rate of decrease was not significant (Fig. 3A, C). 8

The VAP was slightly greater at 48 h as compared with the VAP at the 0 h (P = 0.004), and there was even a greater value for this variable at 72 h as compared with 24 h of storage (P = 0.03; Fig. 3B). There was a greater increase in VCL from 24 to 48 h (P = 0.0002), and 72 h (P < 0.0001) (Fig. 3B). The VSL did not vary at different times during storage (Fig. 3B). The LIN and WOB were less from 0 to 24 h (P = 0.02 and P = 0.0007, respectively), and there were further decreases in the values for these variables from 24 to 48 h of storage (P < 0.0001, both of them), and there was no change in values for these variables after 72h (P < 0.0001, both storage procedures; Fig. 3D, E). The increase in values for ALH was greater from 0 to 24 h (P = 0.002), however, there was a continued increase

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from 24 to 48 h (P = 0.006) and 72 h (P = 0.007), though between 48 to 72 h the difference in values for this variable was not significant (Fig. 3F). There was only an increase (P = 0.009) in BCF from 24 to 48 h of storage with there being no change in the values for this variable after 48 h of storage

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(Fig. 3F).

Regarding acrosome status, there were no differences between experimental groups (Table

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1) and there were no interactions between time and group (Fig. 3G). Acrosome integrity changed over time (P < 0.0001) with there being a continued decrease in values for this variable from hour 0

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to 72 of storage (P < 0.0001, for each dose type; Fig. 3G).

The percentage of spermatozoa with normal morphology and with cytoplasmic droplet was

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similar in CAI- and PCAI-doses (Table 1), without there being an interaction between time and seminal dose type (Fig. 3H). Although there were no differences between the two types of dose packaging on values for these variables, percentage of sperm with normal morphology and a

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cytoplasmic droplet was less at 72 than 0 h (P < 0.0001, both dose types; Fig. 3H) while the

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percentage of spermatozoa with a folded and coiled tail increased when the values at 0 and 72 h of storage were compared (P < 0.0001). Osmolality, and pH of seminal doses did not differ between doses types and did not change during the storage period (Table 1). 4. Discussion The evolution of AI in the pork production industry through the decades has resulted in the use of different types of seminal doses depending on the AI technique used. With the onset of the use of PCAI, there was not only supposed to be a change in the way to perform the AI technique per 9

se (García-Vázquez et al., 2019), but also changes related to the seminal dose type used. There has been little attention given to the different factors related to PCAI seminal doses such as handling and preservation of semen. The results of the present study indicate there are significant differences in magnitude of temperature changes during processing and management after packaging of CAI and PCAI semen doses used for swine AI. After ejaculation, boar semen processing is initiated with dilution of the ejaculate contents in a commercial extender (~32 ºC) and further preservation commonly in plastic bags at 15 to 17 ºC (Fig. 1). Boar spermatozoa, however, are particularly temperature-sensitive (Pursel et al., 1972) and

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rapid cooling from the temperature changes during dilution to refrigeration leads to a significant loss of sperm functions (Johnson et al., 2000). It, therefore, is worth noting that handling protocols for PCAI or CAI seminal doses should differ because with CAI-doses more time is required to reach the

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room and preservation temperatures than with PCAI doses both in changes that occur during the processing at room temperature and in the second phase of temperature changes after the sperm that

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are at room temperature are placed in a refrigerator. The results of this study indicate the protocols used during the cooling curve should be different depending on the type of doses used (CAI or PCAI).

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Considering the results of the present study, the times required for temperature changes during preservation of seminal doses used for CAI requires are longer than for seminal doses used for PCAI.

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In animal production the protocols applied to CAI or PCAI seminal doses, however, are similar. If seminal doses are stored for a longer than necessary time at room temperature (greater temperature than refrigerated), this could result in activation of metabolic pathways and lead to impairment of

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sperm viability during refrigerated preservation. The 25 ºC (room temperature) temperature could

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induce premature activation of stored sperm resulting from a lack of inhibition of metabolic processes (Gączarzewicz et al., 2015) leading to changes that would impair sperm quality (Petrunkina et al., 2005). A temperature for sperm storage that is greater than the refrigeration temperature results in a reduction in mitochondrial oxido-reductive capacity (Gączarzewicz et al., 2015) and induces AMPactivated kinase which functions as a regulator of boar sperm metabolism affecting sperm functions during storage (Hurtado de Llera et al., 2012; Martín-Hidalgo et al., 2013). The results of the present study indicate that the time to process seminal doses used for PCAI should at least get further 10

consideration. The plastic bags in which semen is stored has a considerable insulating temperature effect on the semen samples (Bailey and Buhr, 1995). Nevertheless, in the present study the temperature of PCAI seminal doses decreased to temperatures that were less than that of CAI seminal doses when both cooling curves were assessed (at room temperature or during refrigeration) after stabilization (reaching average temperature) which may indicate there is a different insulating effect of the plastic bags for the two types of seminal doses. This effect may be explained by the different plastic area surface exposed to the air temperature for the two types of semen doses (Fig. 1). The area of the bag for CAI-doses is 23,800 mm2, and the area of the bag for PCAI-doses is 18,000 mm2 (Fig.

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1). Thus, it is possible that there is a greater contact of sperm with the bag surface in the case of CAIdoses (113 x 103 sperm/mm2) than PCAI-doses (83 x 103 sperm/mm2), affecting sperm quality during storage. The results, however, indicated there was a similar spermatozoa quality for both types of

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seminal doses used, independent of the number of sperm in contact with the surface of the plastic bag containing the sperm (Fig. 1). Migration of some components of the packaging materials may

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have marked effects on sperm quality and subsequently the fertility (Nerin et al., 2014). The PCAI packaging used for the present study allows for a lesser number of spermatozoa per mm2 to be in

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contact with the packaging surface than with the CAI packaging at a similar sperm concentration (33.3 x 106 sperm/ml), so with the type of storage in the present study some toxic packaging

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component migrations could be less for the PCAI in comparison with CAI doses. After the semen is at room temperature, boar seminal doses are refrigerated until use in AI programs, which may take several days. The effect of the storage of seminal doses on sperm quality

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and fertility has been well documented for CAI seminal doses (Martín-Hidalgo et al., 2013; Pinart et

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al., 2015; Bielas et al., 2017) but not for PCAI-doses. Results from sperm quality evaluations indicate there were no differences between the two types of doses when stored at 16 ºC for 72 h. Furthermore, the results of the present study indicated that, in general, the quality of sperm in seminal doses changed over time. It is apparent the use of proper preservation procedures for boar semen in liquid storage may not correspond to optimal reproductive performance when this semen is used for AI. The main concern is a fact that conventional spermiogram parameters do not reflect the decrease in the fertilizing capacity during liquid storage at 15 to 17 ºC (Yeste et al., 2017). Other factors, 11

therefore, have to be taken into account, such as the response of liquid-stored sperm to bicarbonate (capacitation inductor necessary for fertilization) (Henning et al., 2014) or an extended period of storage beyond 3 days to ascertain any detrimental effect in in vitro (sperm function) and/or in vivo conditions (reproductive performance). In conclusion, with both types of seminal doses there were similar values related to sperm viability indicating there was maintenance of indicators of high quality sperm during 72 h of storage (~16 ºC), although the cooling curves of PCAI seminal doses in both phases (room temperature and refrigeration) were different from CAI seminal doses, indicating there was less time required for

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cooling to stabilized temperatures. The results of the present study may indicate that the handling of both types of doses could be conducted for similar time frames using similar procedures, although the different cooling curves for the two dose types should be taken into consideration during further

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investigations.

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Conflict of interest

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The authors have not conflict of interest to declare.

Acknowledgments

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The authors would like to thank Adespolorca AI Center (Lorca, Spain) and workers for providing the seminal doses for the study.

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Figures Caption

Fig. 1. Illustration of packaging used for CAI and PCAI seminal doses; CAI-dose presents a total surface area (blue area) of the plastic bag layer of 23,800 mm2 corresponding to 133.44 x 103 sperm/mm2; PCAI-dose presents a total surface area of the plastic bag layer of 18,000 mm2 corresponding to 83.33 x 103 sperm/mm2.

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Fig. 2. Cooling curve of seminal doses (CAI represented by a solid line and PCAI represented by a dashed line) during processing: 1) 1st phase at room temperature (white area from 0 to 120 min); 2) 2nd phase at refrigeration (shaded area from 121 to 240 min) (mean ± SEM); The fringe bounded by * means statistical differences (P < 0.05) between seminal dose type (CAI compared with PCAI); ■ indicates average room temperature and ♦ indicates the temperature at refrigeration (mean ± SEM)

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Fig. 3. Total motility and progressive motility (a), curvilinear line velocity (VCL), average path velocity (VAP) and straight line velocity (VSL) (b), percentage straightness (STR) (c), percentage linearity (LIN) (d), percentage oscillation (WOB) (e), beat cross frequency (BCF) and amplitude of lateral head displacement (ALH) (f), acrosome integrity (g) of spermatozoa from CAI (--●‐ ‐ ) and PCAI (–■–) seminal doses stored for as long as 72 h (mean ± SEM); normal morphology and cytoplasmic droplet of spermatozoa (h) from CAI ( ) and PCAI ( ) seminal doses incubated during 72 h (mean ± SEM); a,b,c,d Mean differences during storage (P < 0.05)

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Fig 2

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Fig 3

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Table 1 Variables of spermatozoa quality with refrigeration using CAI and PCAI packaging for seminal doses stored for 72 h (analyzed at 0, 24, 48 and 72 h) at a refrigeration temperature of ~16 ºC; Data are provided as mean ± SEM

Treatments CAI

PCAI

P-value

Pooled SEM

Total motility (%)

90.2

90.5

0.8

1.2

Progressive motility (%)

63.6

63.5

0.9

1.5

VCL (µm/s)

86.9

85.5

0.6

4.7

VSL (µm/s)

29.1

29.0

VAP (µm/s)

48.5

47.7

LIN (%)

35.6

35.3

STR (%)

61.7

61.5

WOB (%)

56.8

56.6

BCF (Hz)

7.6

Acrosome damage

6.7

Normal

Folded tail

pH

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Coiled tail

2.2

0.7

1.5

0.8

1.6

0.8

1.0

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0.6

0.7

0.2

8.0

0.8

0.8

87.8

0.7

1.4

3.2

3.5

0.4

0.4

3.4

3.4

1.0

0.5

5.6

5.7

0.8

1.2

6.9

6.9

0.9

0.02

307.1

304.8

0.2

2.9

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Osmolality (mOsm/kg)

0.9

88.1

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Cytoplasmatic droplet

0.9

7.6

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Morphology

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Variables

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