Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box

Abstract

Today, doses of refrigerated stallion semen are stored, sent, and transported in single-use polystyrene transport boxes. To address a growing concern for ecological and sustainable development, a new transport box made of recycled material, eco-responsible, called ECOOL Box, has been developed to ensure the transport and conservation of doses. Thermal performance to maintain the temperature between + 2°C and + 15°C for 48 hours, cooling kinetics during the first hour of storage, and biological parameters relating to motility, velocity and sperm viability were analyzed and compared to a control box. In total, up to 10 ejaculates were diluted, packaged in syringes (20.10 6 spermatozoa/mL, 10 mL per dose) and stored in transport boxes for 48 hours. The boxes were placed in a thermal chamber mimicking the daily temperature variations during a temperate winter or summer climate. Temperature readings were taken every 30 seconds using thermocouples. The quality of the semen was analyzed by a computer-assisted semen analyzer at the day of collection and after the 48 hours of storage. Sperm viability at the end of the 48 hours was analyzed using flow cytometry. No significant difference between the two boxes was observed on all thermal and biological parameters. The ECOOL Box allows the cooling of semen doses at a rate of-0.13°C/min, with no detrimental effect. In winter conditions, the ECOOL Box allows to maintain the doses between + 2.7°C and + 4.0°C for 48 hours. In summer condition, the ECOOL Box maintains the doses between + 7.2°C and + 15°C for 42 hours. The total sperm motility is reduced by only 5% compared to the day of collection. However, it remains above 70% in both conditions at the end of the experiment, which is in line with the recommendations of the French National Studs. The ECOOL Box thus shows its thermal and biological efficiency and allows the transport of equine doses in a safe and eco-responsible way.

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Research Article
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Abstract
Charlène Rouillon1*, Guy Delhomme1*, Frederic de Geoffroy2, Clothilde Gourtay2 and Eric Schmitt1
1IMV Technologies, Z.I. N°1 Est, Saint-Ouen-Sur-Iton, France
2Jumenterie du Haras du Pin, Institut Français du Cheval et de l’Equitation, Exmes, France
*Corresponding Author: Charlène Rouillon and Guy Delhomme, IMV Technologies, Z.I. N°1 Est, Saint-Ouen-Sur-Iton, France.
Received: June 07, 2021
Today, doses of refrigerated stallion semen are stored, sent, and transported in single-use polystyrene transport boxes. To address
a growing concern for ecological and sustainable development, a new transport box made of recycled material, eco-responsible,
called ECOOL Box, has been developed to ensure the transport and conservation of doses. Thermal performance to maintain the
-
ing to motility, velocity and sperm viability were analyzed and compared to a control box. In total, up to 10 ejaculates were diluted,
packaged in syringes (20.106 spermatozoa/mL, 10 mL per dose) and stored in transport boxes for 48 hours. The boxes were placed in
a thermal chamber mimicking the daily temperature variations during a temperate winter or summer climate. Temperature readings
were taken every 30 seconds using thermocouples. The quality of the semen was analyzed by a computer-assisted semen analyzer at


cooling of semen doses at a rate of - 0.13°C/min, with no detrimental effect. In winter conditions, the ECOOL Box allows to maintain
the doses between + 2.7°C and + 4.0°C for 48 hours. In summer condition, the ECOOL Box maintains the doses between + 7.2°C and
+ 15°C for 42 hours. The total sperm motility is reduced by only 5% compared to the day of collection. However, it remains above 70%
in both conditions at the end of the experiment, which is in line with the recommendations of the French National Studs. The ECOOL

Keywords: Stallion; Chilled Semen; Transport; Motility; Viability; Temperature
Abbreviations
     
Velocity on a Straight Line
Introduction
-
ing industry [24]. Usually, the trend for AI technologies is linked to some common advantages with chilled and with frozen semen. They
avoid animal transport and physical contact between mares and stallions, thus limiting transport costs and disease transmission. The use

Chilled semen is generally more resistant and requires less heavy and accurate insemination protocols. Indeed, using chilled semen at a
time of AI results in better pregnancy rates than frozen semen [15,22,23]. This is mainly due to large differences in initial ejaculate quality
[6,17,20] and ejaculate tolerance for chilled and cooled storage between stallions [5,8]. The composition of their seminal plasma does not

Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
ensure the protection of the sperm membranes during the freezing process [1,27]. Therefore, the composition and quality of the sperm
can help us to predict the success of fertility. Before chilling or freezing semen, semen quality must be evaluated on several discriminating
parameters including motility, viability, or acrosome integrity [2].

     
proper management of the stallion collection. The handling of semen must follow precise guidelines especially to avoid urine and bacterial
contamination, or osmolarity shock [10]. Once properly collected, the semen quality, based on the normal morphology rate and the total
motility of spermatozoa, must be validated to ensure the packaging and transport of good quality sample. This step allows to eliminate
from the procedure the ejaculate that may be damaged during transport and whose quality is not maintained. Semen packaging, especially
the dilution process and the dilution media, is necessary to decrease sperm concentration and maintain a good motility during the stor-

Technologies, L’Aigle, France) is one of the media that can ensure optimal semen storage, conservation and maintain the fertility potential
up to 48 hours post-collection [28].
Chilled semen is packaged into syringes doses, shipped the day of the semen collection, and received the following day for the insemi-
nation. A delay of up to 48 hours can thus elapse between the sending and the moment of insemination. Current transport methods tend
   
         -
semination [3,4,18,23,25,28]. Indeed, the metabolism of spermatozoa stored at body temperature (+ 35°C) is high [21] and induces the
formation of toxic products for the semen such as lactic acid and oxygen free radicals. This leads to a loss of sperm integrity, motility, and
    
semen quality. Indeed, it has been shown that by storing semen at + 4°C, sperm metabolism is reduced by only 7%. In the same manner,

The kinetics of decrease temperature is also an important factor to consider, as well as the risk of a thermal shock. The temperature range



Consequently, the choice of semen container for cooling and shipment is critical. The cooling rate and isolation of chilled semen transport
boxes must be maintained under rough external environmental conditions. Today, chilled semen is sent out in reusable boxes. However,
these boxes are bulky, heavy, and expensive. They must be systematically returned to the semen collection center. This adds transport
costs and brings sanitary risks as the boxes can carry pathogens. Some chilled semen is sent in disposable single-use boxes, which are
cheaper. In addition, these boxes are made from polystyrene, an environmentally unfriendly material.
In the context of sustainable development, the objective of this study was to develop an eco-responsible disposable chilled semen
transport box, called the ECOOL Box. The use of recycled polystyrene as a raw material appeared as a challenge. First, we studied the
thermal performance of the box and compared it to single-use box already available to breeders on the market. Next, we characterized the
semen conservation capacity of the box by assessing different sperm quality parameters. This study then allowed us to conclude on the
effectiveness of this eco-responsible box and on its capacity to maintain the quality of doses.
Materials and Methods
Study location, animals, and semen packaging

sexually mature stallions (French Saddle and Trotter, Hanoverian) aged from 6 to 21 years old, originating from the French Horse and
 

Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box

production (4 times a week) were collected at different moments of the study. A total of 10 to 12 ejaculates samples were collected on 5
to 6 different experiments.
      -
 6 spermatozoa/mL

microscope. All ejaculates with more than 70% of motile spermatozoa after collection were used in this study.
Ten mL of diluted semen were packaged in 20 mL sterile syringes without air exposure. The syringes were placed in different environ-
mental conditions: directly in a fridge at +4°C (Fridge), in a climatic chamber inside the eco-friendly transport box ECOOL Box (ECB) or in
the non-eco-responsible Control transport Box (CB), or in the climatic chamber outside the boxes (Outside).
A programmable freezer (Mini Digitcool, IMV Technologies, L’Aigle, France), connected to a liquid nitrogen tank was used as a climatic
chamber. The boxes were placed in the programmable freezer, and temperature programs mimicking the daily temperature variations

and 2).
Figure 1: Evolution of the outside temperature programmed in winter conditions according to the AFNOR standard (Outside, green dotted
line), and evolution of the internal box compartment (A) and syringe (B) temperatures in the Control Box (CB, grey line) and in the ECOOL
Box (ECB, red line), during 48 hours post-collection. Temperature curves of the CB and ECB represent the average of 5 experiments.

Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
Two syringes (for 2 ejaculates) were placed in each box. Furthermore, in dedicated compartment of the boxes, two eutectic plates were
placed. For summer conditions, two eutectics were at - 20°C and for winter conditions, one eutectic was at - 20°C and another was at + 4°C.
The temperatures in the inner compartment of the boxes and in the syringes were measured to follow the temperature evolution. One
measure every 30 seconds was recorded during the 48 hours of the experiment, using type K thermocouples (Mesurex, Saint-Arnoult-en-
Yvelines, France) connected to a graphic recorder (mini LOGGER GL200A, GraphTec, Irvine, USA).
Motility parameters analysis
The quality of the ejaculate was analyzed, following semen dilution and at the end of the 48 hours of storage, with a computer-assisted
semen analyzer (IVOS II, Hamilton Thorne, Beverly, USA). Diluted semen from the doses was incubated for 10 min at + 37°C. Then, 3 µl
of semen were placed in a Leja slide (Leja, IMV Technologies group, GN Nieuw-Vennep, Netherlands). Sperm motility and concentration
were checked. For each sample, the percentage of total motile spermatozoa, the percentage of progressive spermatozoa (rapid sperm
µm/sec), the curvilinear velocity (VCL), and the velocity on a straight
line (VSL) were calculated. Two chambers per samples were analyzed (frame rate = 60, frame acquired = 30, with a minimum of 8 frames
per chamber).
Viability and acrosomal integrity analysis


plate, a total of 57 000 spermatozoa had been dispensed in each analysis wells, and diluted with EasyBuffer B (023862, IMV Technolo-

measured were the percentage of viable and non-viable (dead) spermatozoa.
Figure 2: Evolution of the outside temperature programmed in summer conditions according to the AFNOR standard (Outside, green
dotted line), and evolution of the internal box compartment (A) and syringe (B) temperatures in the Control Box (CB, grey line) and in the
ECOOL Box (ECB, red line), during 48 hours post-collection. Temperature curves of the CB and ECB represent the average of 5 experiments.

Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
In addition, acrosome integrity assay was performed with the EasyKit:Viability and Acrosome integrity (IMV Technologies, L’Aigle,
       -

40 000 spermatozoa were dispensed in each analysis wells, and diluted with EasyBuffer B (023862, IMV Technologies) (qsp 200 µL).

percentages of viable or non-viable spermatozoa, and intact or disrupted acrosome.
Statistical analysis
Statistical analysis was performed on R software, version 1.1.463 (R Core Team, 2014). Results were presented as bar plots, with the
mean and standard deviation (SD). For multiple comparison analysis, Kruskall Wallis non-parametric and Tukey post-hoc test was done.

Results
Temperature evolution in the boxes and syringes
The temperature variation in the internal compartment in the boxes and in the syringes were studied in both Control Box (CB) and
ECOOL Box (ECB). In winter condition, the internal temperature curve of the ECOOL Box was on average slightly below the CB (- 0.62°C)

The temperature of both boxes remains within the adequate range of temperature for semen storage during the 48 hours of experiment

   

during all experiment (CB: 6.57

         



-
spite this difference of about 1°C, the minimum temperature reached in ECB remains above the minimum temperature of + 2°C required
for semen conservation. Regarding the maximum temperature recorded in both boxes, it was reached at the end of the 48 hours of experi-

-
   



-



Semen motility evaluation



Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
Figure 3: Motility parameters analysis after semen collection (0h) and after 48 hours of storage at +4°C (Fridge), in the ECOOL box (ECB),
in the Control Box (CB) or at external temperature (Outside) during winter condition. (A) Percentage of motile sperm and (B) percentage
of progressive sperm were analyzed using an IVOS II, after incubation of the semen samples at +37°C for 10 min (n = 10 semen samples, 5
experiments).
After 48 hours of experiment in winter condition, no difference in total motility was observed between doses stored in ECB (74.2%


-


(Table 1).
Winter conditions 48h
0h Fridge ECB CB Outside P-value
Motile VAP (µm/sec)
Mean (SD)  100 (11.8)    
Motile VSL (µm/sec)
Mean (SD) 72.3 (16.7) 81.2 (11.0) 77.0 (14.7)  73.6 (5.44) 0,314
Motile VCL (µm/sec)
Mean (SD)   201 (28.3)  185 (23.0) 0,240
Progressive VAP (µm/sec)
Mean (SD)  103 (11.1)    0,328
Progressive VSL (µm/sec)
Mean (SD) 82.1 (18.1)   86.6 (12.8)  0,260
Progressive VCL (µm/sec)
Mean (SD) 183 (27.8)    182 (18.4) 0,541
Table 1: Table of velocity parameters evaluated on motile and progressive sperm after semen collection (0h) and after 48 hours of storage
at +4°C (Fridge), in the ECOOL box (ECB), in the Control Box (CB) or at external temperature (Outside) in winter condition. Three different
velocities were analyzed using an IVOS II, after incubation of the semen samples at +37°C for 10 min (n = 10 semen samples, 5 experiments)
: VAP means velocity average pathway, VSL means straight- line velocity, and VCL means curvilinear velocity. The P-value indicates the dif-
ference between all the groups.

Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
-

    
4A and 4B). Nevertheless, the motility was 30% lower compared to doses stored in boxes or at +4°C, when it was placed in a summer
        -
tions (ECB: 41.7%        
      
motile sperm velocity parameters (Table 2). General significant difference was observed on average straight-line and curvilinear velocity
of progressive sperm between all five groups (-
ticomparison Tukey test.
Figure 4: Motility parameters analysis after semen collection (0h) and after 48 hours of storage at +4°C (Fridge), in the ECOOL box (ECB),
in the Control Box (CB) or at external temperature (Outside) during summer condition. (A) Percentage of motile sperm and (B) percentage
of progressive sperm were analyzed using an IVOS II, after incubation of the semen samples at +37°C for 10 min (n = 12 semen samples, 6
experiments).
Summer conditions 48h
0h Fridge ECB CB Outside P-value
Motile VAP (µm/sec)
Mean (SD) 85.3 (24.2)    87.4 (17.8) 0,172
Motile VSL (µm/sec)
Mean (SD) 64.2 (18.0)  68.4 (15.4) 67.4 (14.4)  0,232
Motile VCL (µm/sec)
Mean (SD)   188 (20.6) 186 (21.8)  0,051
Progressive VAP (µm/sec)
Mean (SD)     84.4 (33.3) 
Progressive VSL (µm/sec)
Mean (SD) 75.0 (18.5) 88.0 (12.5) 82.1 (12.0) 80.4 (14.1)  
Progressive VCL (µm/sec)
Mean (SD) 167 (31.8)  178 (15.8)  175 (72.5) 
Table 2: Table of velocity parameters evaluated on motile and progressive sperm after semen collection (0h) and after 48 hours of storage
at +4°C (Fridge), in the ECOOL box (ECB), in the Control Box (CB) or at external temperature (Outside) in summer condition. Three different
velocities were analyzed using an IVOS II, after incubation of the semen samples at +37°C for 10 min (n = 12 semen samples, 6 experiments):
VAP means velocity average pathway, VSL means straight- line velocity, and VCL means curvilinear velocity. The P-value indicates the differ-
ence between all the groups.

Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
Viability and acrosomal integrity tests
In winter condition, sperm viability results following 48 hours of storage under all three conditions were similar, both after analysis
-

Figure 5: Viability analysis after 48 hours of storage at in the ECOOL box (ECB), in the Control Box (CB) or at external temperature (Out-
side) during summer and winter conditions. Percentage of viable sperm was analyzed using the EasyKit Viability and concentration and the

(A) Winter Conditions ECB CB Outside P-value
Viable (intact + disrupted) (%)
Mean (SD) 77.4 (11.3) 78.0 (6.05) 74.7 (8.15) 0,304
Viable intact (%)
Mean (SD) 74.1 (10.7)  71.4 (7.48) 0,683
Viable disrupted (%)
Mean (SD) 3.28 (1.23) 3.30 (1.54) 3.28 (1.71) 
Dead intact (%)
Mean (SD)    0,276
Dead disrupted (%)
Mean (SD) 3.20 (1.70) 2.43 (0.618) 3.43 (1.48) 
(B) Summer Conditions ECB CB Outside P-value
Viable (intact + disrupted) (%)
Mean (SD)    0,077
Viable intact (%)
Mean (SD) 71.6 (13.6) 73.2 (10.3)  0,174
Viable disrupted (%)
Mean (SD) 2.33 (0.846) 3.85 (2.45) 1.68 (1.11) 0,242
Dead intact (%)
Mean (SD) 23.0 (11.3)  50.5 (27.5) 
Dead disrupted (%)
Mean (SD) 3.15 (2.82)  2.63 (1.23) 
Table 3: Acrosome integrity and viability analysis after 48 hours of storage in the ECOOL box (ECB), in the Control Box (CB) or at external
temperature (Outside) during winter (A) and summer (B) conditions. Percentage of viable or dead sperm with intact or disrupted acrosome
-
ference between all the groups.

Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
In summer condition, the sperm viability following 48 hours of storage in the ECB, CB or outside the boxes were identical (ECB: 72.1%

-

   
 
three conditions. The multicomparison test showed this percentage tended to be higher when the doses were placed outside the boxes,
than in the boxes, especially in the CB (p = 0.076).
The acrosomal integrity analysis table also shows no trend between the three conditions in either summer or winter. The proportion
of semen with damaged acrosome (viable + non-viable sperm) changed between 4.3% and 7.8% (Table 3).
Discussion
Despite the complexity of the development of a fresh semen transport box in recycled material, the challenge of combining thermal
performance and biological requirements was attempted with the ECOOL Box. This box has demonstrated its thermal insulation capacity
in all temperate seasonal conditions to maintain semen doses in the adequate range of + 2°C to + 15°C for 48 hours of storage.
-
sible that these thermal conditions may be maintained for several more hours before exceeding the recommended maximum + 15°C.
Quality was assessed based on several parameters of motility and viability. Although the proportion of total and progressive sperm has
been reduced by 2 to 5% after 48 hours of storage, the ECOOL Box allows to maintain a high sperm quality with more than 70% of motile
spermatozoa. According to the standard protocol applied by the French National Studs [27], with doses at a minimum concentration of
120.106 spermatozoa/ml and a motility at collection greater than 70%, the motility after 24 hours of storage at + 4°C must be greater
than 40% to ensure good fertility results. The proportion of progressive sperm in the refrigerated and transported doses of semen was
also shown to affect the pregnancy rate of mares [13]. The ECOOL Box has been shown to maintain the progressive sperm content during
long storage. Semen quality (motility and viability) was preserved in the ECOOL Box as well as in a constant temperature of + 4°C. These
results suggest that the box ensures good conservation of sperm quality in temperate winter conditions. Although the box continued to be
in the correct temperature range beyond the 48 hours of storage, and the effects of prolonged storage on the semen was not investigated,
it is not recommended to store the semen in the box for any longer period of time. It was reported that the stress induced by storing chilled
semen beyond 48 hours induced the activation of apoptotic pathways in the nucleus of sperm cells and led to DNA fragmentation. This is

In summer conditions, the box maintains the required temperature window for up to 42 hours. Beyond this time, the temperature of

and conditioned anaerobically, has shown that the parameters of sperm motility and gestation rates were as good as a conservation at
+ 4°C, we do not know the effects of conservation beyond this temperature [28]. It was shown that an incubation of only 1 hour at + 37°C
of stallion semen, previously chilled at + 4°C for an hour, induces DNA fragmentation [16]. We already demonstrate in our results that the
-
ture can thus induce a resumption of the metabolic activity of the cells. This would generate oxidative stress through the production of

[7]. However, even though the semen was stored for 48 hours, i.e. 6 hours at a temperature above the recommended + 15°C (from 42h to
48h), the quality of the doses was not affected (motility, velocity, viability). The total motility was lower by 5.4% in the ECOOL Box after 48
hours of storage compared to the day of collection, but still follows the standard protocol of the French National Studs [27]. Therefore, the
percentage of progressive motile sperm was not affected by the storage. The eco-designed transport box therefore presents no difference
with the control box and remains just as effective, as if the semen was stored diluted at constant + 4°C for 48 hours.

Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
The quality of the ECOOL Box is also based on its cooling kinetics. Although it is recommended to have a maximum temperature drop

ECOOL Box did not induce a thermal shock of the semen. As our results showed no impact of storage on semen quality, we can conclude
that this kinetics was not deleterious to the semen.
Beyond the thermal and biological performances that the ECOOL Box brings, we must also underline the important environmental

The integration of recycled plastics like this one, brings a real environmental gain. It allows, per ton of recycled plastics, a reduction of the
greenhouse effect with less 3 000 kg of CO2 equivalent produced, a reduction of energy and water consumption (13,000 kWh and 5.2 m3


Conclusion
In conclusion, this study showed the thermal performance of the ECOOL Box despite the use of recycled material. We also showed the
-
pact the quality of semen, our study was conducted using a standardized protocol including the dilution of semen in a validated conserva-

it possible to safely send doses of standard semen in an eco-responsible way.
Acknowledgements
The authors thank the staff of the French Horse and riding Institute (IFCE), for their valuable help in managing the stallions and col-
lecting the ejaculates. FDG collected the ejaculates. GD and CR designed the study, wrote the manuscript, contributed to the acquisition of



Funding Support

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Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
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Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
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Citation: Charlène Rouillon., et al. “Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box”. EC Veterinary Science
6.7 (2021).
Development of a New Transport Box for Chilled Stallion Semen, ECOOL Box
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Theriogenology
Volume 6 Issue 7 July 2021
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